忘记密码购物车Aetna considers any of the following serum tumor Markers for the stated indication medically necessary: Prostate-specific antigen (PSA) for prostate cancer screening (seeCPB 0521 - Prostate Cancer Screening), staging, monitoring response to therapy, and detecting disease recurrence; Carcinoembryonic antigen (CEA) for any of the following: As a preoperative prognostic indicator in members with known colorectal carcinoma or mucinous appendiceal carcinoma when it will assist in staging and surgical treatment planning; or Pancreatic cyst fluid CEA for distinguishing mucinous from non-mucinous malignant pancreatic cysts; or To detect asymptomatic recurrence of colorectal cancer after surgical and/or medical treatment for the diagnosis of colorectal cancer (not as a screening test for colorectal cancer); or To monitor response to treatment formetastaticcolorectal cancer; or For cholangiocarcinoma, gallbladder cancer, lung cancer, medullary thyroid cancer,metastatic breast cancer, mucinous ovarian cancer, and occult primary; or For evaluation of jaundice, abnormal liver function tests (LFTs) or for obstruction/abnormality of the bile duct on liver imaging; 1p19q codeletion molecular cytogenetic analysis for astrocytomasandgliomas; 5-hydroxyindoleacetic acid (5-HIAA) forneuroendocrine tumors; ALK gene fusion as a molecular biomarker in non-small cell lung cancer; ALK gene rearrangementfor diffuse large B cell lymphoma, anaplastic thyroid carcinoma, primary cutaneous CD30+ T-cell lymphoproliferative disorders, and post-transplant lymphoproliferative disorder; ALK expression for pancreatic adenocarcinoma, pediatric Hodgkin\'s lymphoma, inflammatory myofibroblastic tumor (IMT) with ALK translocation, breast implant-associated ALCL, peripheral T-cell lymphoma, and uterine sarcoma; APC for familial adenomatous polyposis when criteria are met in CPB 0140 - Genetic Testing; and for desmoid fibromatosis; experimental for other indications; Afirma Thyroid FNA analysis for assessingfine needle aspiration samples from thyroid nodules that are indeterminate; experimental for other indications. Repeat testing is considered experimental and investigational; Alpha fetoprotein (AFP) for the following indications: hepatocellular carcinoma; mediastinal mass; ovarian cancer; pelvic mass; testicular cancer; testicular mass; thymic carcinoma; and thymoma; Alfa fetoprotein (AFP) for testing forhepatocellular carcinomain hepatitis B carriers,or forpersons with cirrhosis and one or more of the following risk factors: alcohol use; alpha-1 antitrypsin deficiency; Asian female at least 50 years of age; Asian male at least 40 years of age; family history of HCC; genetic hemochromatosis; hepatitis C; nonalcoholic steatohepatitis; and stage 4 primary biliary cirrhosis; Alpha fetoprotein (AFP): serial measurementsto diagnose germ cell tumors in members with adenocarcinoma, or carcinoma not otherwise specified, involving mediastinal nodes; or the diagnosis and monitoring of hepatocellular carcinoma (e.g., before considering liver transplantation); Androgen receptor splice variant 7 (AR-V7) in circulating tumor cells to select therapy in metastatic castrate-resistant prostate cancer after progression on abiraterone or enzalutamide; BCL2 and BCL6 for the diagnosis of non-Hodgkin’s lymphoma and Castleman\'s disease; BCR/ABL fluorescent in situ hybridization (FISH) for lymphoblastic lymphoma, acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, and suspected myeloproliferative neoplasm; experimental for other indications; Beta-2 microglobulin (B2M) for multiple myeloma,non-Hodgkin\'s lymphoma and Waldenström\'s macroglobulinemia/ lymphoplasmacytic lymphoma; BIRC3 and MALT1 for gastric MALT lymphoma, non-gastric MALT lymphoma, nodal marginal zone lymphoma, and splenic marginal zone lymphoma; BRAF V600 mutationfor indeterminate thyroid nodules, hairy cell leukemia; gastrointestinal stromal tumors; colorectal cancer, Lynch syndrome; non-small cell lung cancer; thyroid carcinoma; infiltrative glioma, pancreatic adenocarcinoma, and melanoma (seeCPB 0715 - Pharmacogenomic and Pharmacodynamic Testing); or Lynch syndromefor persons meeting criteria in CPB 0140 - Genetic Testing; and colorectal cancer if KRAS nonmutated; experimental for other indications; Breast Cancer IndexFootnote2**to assess necessity of adjuvant chemotherapy or adjuvant endocrine therapy in females or males with recently diagnosed breast tumors, where all of the following criteria are met: Breast cancer is nonmetastatic (node negative); and Breast tumor is estrogen receptor and/or progesterone receptor positive; and Breast tumor is HER2 receptor negative; and Adjuvant therapy is not precluded due to any other factor (e.g., advanced age and/or significant co-morbidities); and Member and physician (prior to testing) have discussed the potential results of the test and agree to use the results to guide therapy; BTK (Bruton\'s tyrosine kinase) for chronic lymphocytic leukemia/small lymphocytic lymphoma; Cancer antigen 125 (CA 125) levels for any of the following: As a preoperative diagnostic aid in women with ovarian masses that are suspected to be malignant, such that arrangements can be made for intraoperative availability of a gynecological oncologist if the CA 125 is increased; or As a screening test for ovarian cancer when there is a family history of hereditary ovarian cancer syndrome (a pattern of clusters of ovarian cancer within two or more generations), where testing is performed concurrently with transvaginal ultrasoundandprophylactic salpingo-oophorectomy has not been performed. For this indication, screening is considered medically necessary every six monthsbeginning at 30 years of age or 10 years before the earliest age of the first diagnosis of ovarian cancer in the family; or Diagnosis of ovarian cancer in women with new symptoms (bloating, pelvic or abdominal pain, difficulty eating or feeling full quickly, or urinary frequency and urgency) that have persisted for three or more weeks, where the clinician has performed a pelvic and rectal examination and suspects ovarian cancer; or In members with adenocarcinoma of unknown primary, to rule out ovarian cancer; or In members with known ovarian cancer, as an aid in the monitoring of disease, response to treatment, detection of recurrent disease, or assessing value of performing second-look surgery; CA 15-3: Serial measurements of CA 15-3 (also known as CA 27-29 or Truquant RIA) in following the course of treatment in women diagnosed with breast cancer, especially advanced metastatic breast cancer (an increasing CA 15-3 level may suggest treatment failure); CA 19-9 for the following indications: to monitor the clinical response to therapy or detect early recurrence of disease in members with known gastric cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, ovarian cancer, small bowel adenocarcinoma, or adenocarcinoma of the ampulla of Vater; or to rule out cholangiocarcinomain persons with primary sclerosing cholangitis undergoing liver transplantation; or For evaluation of jaundice, abnormal liver function tests (LFTs) or obstruction/abnormality on imaging; or As a tumor marker for mucinous appendiceal carcinoma; CALCA (calcitonin) expression for medullary thyroid cancer or for adenocarcinoma or anaplastic/undifferentiated tumors of the head and neck; CALB2 (calretinin)expression for lung cancer and occult primary; CBFB for acute myeloid leukemia; CCND1 (cyclin D1) for B-cell lymphomas, primary cutaneous B-cell lymphomas, chronic lymphocytic leukemia/small lymphocytic lymphoma, and hairy cell leukemia; CD 20, for determining eligibility for anti-CD20 treatment (rituximab)- seeCPB 0314 - Rituximab; CD 25, for determining eligibility for denileukin diftitox (Ontak) treatment; CD 31 immunostaining, for diagnosis of angiosarcoma; CD 33, for lymphoblastic lymphoma and fordetermining eligibility for anti-CD33 (gemtuzumab, Mylotarg) treatment; CD 52, for post-transplant lymphoproliferative disorder, T-cell prolymphocytic leukemia, and for determining eligibility for anti-CD52 (alemtuzumab, Campath) treatment; CD117 (c-kit), for acute myeloid leukemia, cutaneous melanoma, gastrointestinal stromal tumors and systemic mastocytosis; CHGA (Chromogranin A) expressionfor neuroendocrine tumors, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma and occult primary; DecipherFootnote3***for the following indications: post biopsy in men with NCCN very-low-risk, low-risk, and favorable intermediate-risk prostate cancer who have a greater than 10 year life expectancy who have not received treatment for prostate cancer and are candidates for active surveillance or definitive therapy; or post biopsy in men with intermediate-risk prostate cancer when deciding whether to add androgen-deprivation therapy to radiation; or men with an undetectable PSA after prostatectomy for prostate cancer, to determine adjuvant versus salvage radiation therapy or to determine whether to initiate systemic therapies; DecisionDx-UM (Castle Biosciences, Phoenix, AZ)for risk stratification of persons with localized uveal melanoma; EndoPredict (also known as 12-gene score)Footnote2**to assess necessity of adjuvant chemotherapy in females or males with recently diagnosed breast tumors, where all of the following criteria are met: Breast cancer is nonmetastatic (node negative); and Breast tumor is estrogen receptor positive; and Breast tumor is HER2 receptor negative; and Adjuvant chemotherapy is not precluded due to any other factor (e.g., advanced age and/or significant co-morbidities); and Member and physician (prior to testing) have discussed the potential results of the test and agree to use the results to guide therapy; Epidermal growth factor receptor (EGFR) mutation testing for predicting response to EGFR-targeting tyrosine kinase inhibitors (erlotinib (Tarceva), gefitinib (Iressa), afatinib (Gilotrif), osimertinib (Tagrisso)) in non-small cell lung cancer; FIP1L1-PDGFRA fusion oncogene for systemic mastocytosis with peripheral blood eosinophilia; FIP1L1-PDGFRA gene rearrangements for myeloid/lymphoid neoplasms with peripheral blood eosinophilia and tyrosine kinase fusion genes; FLT3 gene mutation testing for acute lymphoblastic leukemia, acute myeloid leukemia (AML), myelodysplastic syndromes, myeloproliferative neoplasms, and myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase fusion genes; Human chorionic gonadotropin (HCG),serial measurementto diagnose germ cell tumors in members with adenocarcinoma, or carcinoma not otherwise specified, involving mediastinal nodes, or to monitor treatment in members with known trophoblastic tumors (invasive hydatidiform moles and choriocarcinomas) and germinal cell tumors (teratocarcinoma and embryonal cell carcinoma) of the ovaries or testes, or to monitor for relapse after remission is achieved; Human chorionic gonadotropin, beta (beta-HCG) for mediastinal mass; ovarian cancer; pelvic mass; testicular mass; testicular cancer; thymoma; or thymic carcinoma; Human epidermal growth factor receptor 2 (HER2) (ERBB2) evaluation in breast, gastric, colorectal, esophageal, esophageal gastric junction, salivary gland tumors, and non-small cell lung cancer - seeCPB 0313 - Trastuzumab (Herceptin and biosimilars), Trastuzumab and Hyaluronidase-oysk (Herceptin Hylecta); IGH@ (Immunoglobulin heavy chain locus), gene rearrangement analysis to detect abnormal clonal population(s)in non-Hodgkin’s lymphomas, chronic lymphocytic leukemia, hairy cell leukemia, and post-transplant lymphoproliferative disorder; IGK@ (Immunoglobulin kappa light chain locus), gene rearrangement analysis, evaluation to detect abnormal clonal population(s)for non-Hodgkin’s lymphoma, systemic light chain amyloidosis; Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genemutation for AML, chondrosarcomas, myelodysplastic syndromes, myeloproliferative neoplasms, orgliomas and glioblastomas; INHA (inhibin) expressionfor ovarian cancer orpelvic mass; Liquid biopsy (up to 50 genes) (e.g.,Resolution ctDx Lung) for persons with non-small cell lung cancer who are not medically fit for invasive sampling, or there is insufficient tissue for molecular analysis and follow-up tissue-based analysis will be done if an oncogenic driver is not identified;large liquid biopsy panels (greater than 50 genes) are considered experimental and investigational for non-small cell lung cancer;for Guardant360CDx non-small cell lung cancer and FoundationOne Liquid CDx for non-small cell lung cancer and prostate cancer- see CPB 0715 - Pharmacogenetic and Pharmacodynamic Testing; Lactate dehydrogenase (LDH) for acute lymphoblastic leukemia (ALL), bone cancer, kidney cancer, kidney mass, lung cancer, multiple myeloma, non-Hodgkin\'s lymphoma, pelvic mass, ovarian cancer, testicular cancer, or testicular mass; K-ras (KRAS) mutation analysis, with BRAF reflex testing, to predict non-response to cetuximab (Erbitux) and panitumumab (Vectibix) in the treatment of anal adenocarcinoma, metastatic colorectal cancer and small bowel adenocarcinoma; K-ras (KRAS) mutation analysis to predict non-response to erlotinib (Tarceva) in the treatment of non-small cell lung cancer; experimental for all other indications; KRASfor metastatic colorectal cancer, myelodysplastic syndromes, non-small cell lung cancer, pancreatic adenocarcinoma, and uterine sarcoma; MammaprintFootnote2**to assess necessity of adjuvant chemotherapy in females or males with recently diagnosed breast tumors, where all of the following criteria are met: Breast cancer is nonmetastatic (node negativeFootnote1*) or with 1-3 involved ipsilateral axillary lymph nodes; and Breast tumor is estrogen receptor positive or progesterone receptor positive; and Breast tumor is HER2 receptor negative(Rationale: adjuvant chemotherapy with trastuzumab (Herceptin) is considered to be medically necessary regardless of Mammaprint score for HER2 receptor positive lesions); and Member is determined to be at \"high clinical risk\" of recurrence usingAdjuvant! Online (see page 20 ofMINDACT study supplement for definitions of high clinical risk); and Adjuvant chemotherapy is not precluded due to any other factor (e.g., advanced age and/or significant co-morbidities); and Member and physician (prior to testing) have discussed the potential results of the test and agree to use the results to guide therapy; Measurement of estrogen and progesterone receptors breast cancers, occult primary, ovarian cancer, endometrial carcinoma, and uterine sarcoma; Mismatch repair (MSI/dMMR)(MLH1, MSH2, MSH6, PMS2) tumor testing (somatic mutations)for breast cancer,ovarian cancer, colorectal cancer, small bowel adenocarcinoma, esophageal cancer, esophagogastric junction cancer, gastric cancer,pancreatic cancer, cholangiocarcinoma, gallbladder cancer,pancreatic adenocarcinoma, cervical cancer, uterine cancer, prostate cancer, testicular cancer, penile cancer, myelodysplastic syndromes,Ewing sarcoma, and occult primary; for medical necessity of screeningof germline mutationsfor HNPCC/Lynch Syndrome with MLH1, MSH2, MSH6, see CPB 0140 - Genetic Testing; MLH1 tumor promoter hypermethylation for endometrial cancer; Murine double minute 2 (MDM2) for uterine sarcoma and soft tissue sarcoma; Mycosis fungoides, diagnosis: polymerase chain reaction (PCR) for T-cell receptor gamma chain gene rearrangementas an adjunct to the histopathologic diagnosis of mycosis fungoides; MYD88 (myeloid differentiation primary response 88) to differentiate Waldenstrom\'s macroglobinemia (WM) versus marginal zone lymphoma (MZL) if plasmacytic differentiation present forgastric MALT lymphoma, nodal marginal zone lymphoma, nongastric MALT lymphoma, and splenic marginal zone lymphoma; and for multiple myeloma; MyMRD NGS Panel for comprehensive prognostic assessment in individuals with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS); Next generation sequencing of tumor DNA (e.g., ClonoSeq)to detect or quantify minimal residual disease inpersons with multiple myeloma oracute lymphocytic leukemia; Myeloperoxidase (MPO) immunostaining, FLT3-ITD, CEBPA mutation, NPM1 mutation, and KIT mutation for diagnosis of acute myeloid leukemia; NPM1 in acute myeloid leukemia (AML) and myelodysplastic syndromes; experimental for other indications; NRAS for colorectal cancer, myelodysplastic syndrome, and blastic plasmacytoid dendritic cell neoplasm (BPDCN); NTRK for all solid tumors; Oncotype DX ProstateFootnote3*** for the following indications post biopsy: men with NCCN very-low-risk, low-risk, and favorable intermediate-risk prostate cancer who have greater than 10 year life expectancy and who have not received treatment for prostate cancer and are candidates for active surveillance or definitive therapy; or men with intermediate-risk prostate cancer when deciding whether to add androgen-deprivation therapy to radiation; PAM50 Risk of Recurrence (ROR)Score (also known as Prosigna Breast Cancer Prognostic Gene Signature Assay)Footnote2** to assess necessity of adjuvant chemotherapy in females or males with recently diagnosed breast tumors, where all of the following criteria are met: Breast cancer is nonmetastatic (node negative); and Breast tumor is estrogen receptor positive; and Breast tumor is HER2 receptor negative; and Adjuvant chemotherapy is not precluded due to any other factor (e.g., advanced age and/or significant co-morbidities); and Member and physician (prior to testing) have discussed the potential results of the test and agree to use the results to guide therapy; PDGFRA for gastrointestinal stromal tumors (GIST) and forpediatric acute lymphoblastic leukemia (see also entry above forFIP1L1-PDGFRA gene rearrangements and fusions); PDGFRB testing for myelodysplastic syndromes (MDS),dermatofibrosarcoma protuberans, acute lymphoblastic leukemia,and for myeloid/lymphoid neoplasms with peripheral blood eosinophilia and tyrosine kinase fusion genes; Phosphatidylinositol-4,5-bisphosphonate 3-kinase, catalytic subunit alpha polypeptide gene (PIK3CA) for breast cancer and uterine sarcoma; PLCG2 (phospholipase C gamma 2) for chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL); PML/RARA for acute promyelocytic leukemia; experimental for all other indications; ProlarisFootnote3*** for the following indications post-biopsy: men with NCCN very-low-risk, low-risk, and favorable intermediate-risk prostate cancer who have greater than 10 year life expectancy and who have not received treatment for prostate cancer and are candidates for active surveillance or definitive therapy; or men with intermediate-risk prostate cancer when deciding whether to add androgen-deprivation therapy to radiation; ProMarkFootnote3***for the following indications post-biopsy: men with NCCN very-low-risk, low-risk men, and favorable intermediate risk prostate cancer who have greater than 10 year life expectancy and who have not received treatment for prostate cancer and are candidates for active surveillance or definitive therapy; or men with intermediate-risk prostate cancer when deciding whether to add androgen-deprivation therapy to radiation; PTEN for uterine sarcoma and for persons meeting Cowden syndrome testing criteria in CPB 0140 - Genetic Testing; experimental for all other indications; Placental alkaline phosphatase (PLAP), to diagnose germ cell seminoma and non-seminoma germ cell tumors in unknown primary cancers; Quest Diagnostics Thyroid Cancer Mutation Panel for assessing fine needle aspiration samples from thyroid nodules that are indeterminate; experimental for other indications. Repeat testing is considered experimental and investigational; ROS-1 to predict response to crizotinib (Xalkori) for the treatment of non-small cell lung cancer (NSCLC); RUNX1 for acute myeloid leukemia, myelodysplastic syndrome, and systemic mastocytosis; Steroid hormone receptor status in both pre-menopausal and post-menopausal members to identify individuals most likely to benefit from endocrine forms of adjuvant therapy and therapy for recurrent or metastatic breast cancer; Targeted hematologic genomic sequencing panel (5-50 genes) for acute lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) (e.g., MedFusion myeloid malignancy analysis panel); Targeted solid organ genomic sequencing panel (5-50 genes) for colorectal cancer, cutaneous melanoma, pancreatic cancer, prostate cancer and non-small cell lung cancer (including Oncomine Dx Target Test (Thermo Fisher Scientific, Carlsbad, CA)); T-cell receptor gene rearrangements (TRA@, TRB@, TRD@, TRG@) for T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, nasal type extranodal NK/T-cell lymphoma, hepatosplenic gamma-delta T-cell lymphoma, peripheral T-cell lymphoma, primary cutaneous CD30+ T-cell lymphoproliferative disorders, myelodysplastic syndromes, Castleman\'s disease, mycosis fungoides/Sezary syndrome and myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase fusion genes; ThyGeNext/ThyGenX (formerly Mirinform Thyroid) for assessingfine needle aspiration samples from thyroid nodules that are indeterminate; experimental for other indications; repeat testing is considered experimental and investigational; ThyraMIR as a reflex test following ThyGenX for assessing fine needle aspiration samples from thyroid nodules that are indeterminate; experimental for other indications;repeat testing is considered experimental and investigational; Thymidine kinase for chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL); Thyroglobulin antibodies for thyroid cancer; Thyroglobulin (TG) expression for thyroid cancer, occult primary, and adenocarcinoma or anaplastic/undifferentiated tumors of the head and neck Thyroid transcription factor-1 (TTF-1) for lung cancer or neuroendocrine tumors; Thyroseq for assessingfine needle aspiration samples from thyroid nodules that are indeterminate; experimental for other indications. Repeat testing is considered experimental and investigational; TP53 for non-small cell lung cancer, peripheral T-cell lymphomas, chronic lymphocytic leukemia/small lymphocytic lymphoma, mantle cell lymphoma, splenic marginal zone lymphoma, acute myeloid leukemia, occult primary, and myelodysplastic syndromes; OncotypeDx Breast (also known as 21 gene RT-PCR test) to assess necessity of adjuvant chemotherapy in females or males with recently diagnosed breast tumors, where all of the following criteria are met: Breast cancer is nonmetastatic (node negativeFootnote1*) or with 1-3 involved ipsilateral axillary lymph nodes; and Breast tumor is estrogen receptor positive; and Breast tumor is HER2 receptor negative or breast tumor is HER2 receptor positive and less than 1 cm in diameter. (Rationale: adjuvant chemotherapy with trastuzumab (Herceptin) is considered to be medically necessary regardless of an Oncotype Dx Breast score for HER2 receptor positive lesions 1 cm or more in diameter); and Adjuvant chemotherapy is not precluded due to any other factor (e.g., advanced age and/or significant co-morbidities); and Member and physician (prior to testing) have discussed the potential results of the test and agree to use the results to guide therapy (i.e., member will forgo adjuvant chemotherapy if Oncotype Dx Breast score is low); Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1)Footnote2**to assess necessity of adjuvant chemotherapy in females or males with recently diagnosed breast tumors, where all of the following criteria are met: Breast cancer is nonmetastatic (node negative); and Breast tumor is estrogen receptor positive; and Breast tumor is HER2 receptor negative; and Adjuvant chemotherapy is not precluded due to any other factor (e.g., advanced age and/or significant co-morbidities); and Member and physician (prior to testing) have discussed the potential results of the test and agree to use the results to guide therapy; In addition, urokinase plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1) is considered medically necessary for the determination of prognosis in persons with newly diagnosed, node negative breast cancer; Vascular endothelial growth factor (VEGF) expression for Castleman\'s disease; Veristrat proteomic testing for patients with advanced NSCLC, whose tumors were without EGFR and anaplastic lymphoma kinase (ALK) mutations, who had progressed after at least one chemotherapy regimen), and for whom erlotinib was considered an appropriate treatment; WT-1 gene expression for desmoplastic round cell tumors,ovarian clear cell carcinomas, non-small cell lung cancer and occult primary; ZAP-70, for assessing prognosis and need for aggressive therapy in persons with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Footnote1* Either standard node dissection negative by hematoxylin and eosin (H E) staining or sentinel node negative by H E staining (if sentinel node is negative by H E, but immunoassay is positive, then still considered node negative for this purpose). In addition, women with isolated tumor cells in lymph nodes (micrometastases) are considered node negative. More than oneOncotype Dx test may be medically necessary for persons with breast cancerwho have two or morehistologically distinct tumors that meet medical necessity criteria. Repeat Oncotype Dx testing or testing of multiple tumor sites in the same person has no proven value for other indications. Oncotype Dx is considered experimental and investigational for ductal carcinoma in situ (OncotypeDx DCIS), colon cancer (OncotypeDx Colon),and all other indications other than breast cancer and prostate cancer. Footnote2**Aetna considers use of more than one type oftest to determine necessity of adjuvant therapy in breast cancer (Oncotype Dx Breast, Breast Cancer Index, EndoPredict, PAM50,Mammaprint, or uPA and PAI-1)experimental and investigational. Footnote3***Aetna considers repeat testing or use of more than one type of test to assess risk of prostate cancer progression (Oncotype Dx Prostate, Decipher, Prolaris, or ProMark) experimental and investigational. Aetna considers urinary biomarkers (e.g., bladder tumor antigen (BTA) (e.g., BTA Stat and BTA TRAK),nuclear matrix protein (NMP22) test, the fibrin/fibrinogen degradation products (Aura-Tek FDfP) test, or fluorescence in situ hybridization (FISH) (e.g.,Pathnostics Bladder FISH test, UroVysion Bladder Cancer test medically necessary in any of the following conditions: Follow-up of treatment for bladder cancer; or Monitoring for eradication of bladder cancer; or Recurrences after eradication. Aetna considers the BTA Stat test, the NMP22 test, the Aura-Tek FDP test, or the UroVysion fluorescent in situ hybridization (FISH) test experimental and investigational for screening of bladder cancer, evaluation of hematuria, and diagnosing bladder cancer in symptomatic individuals, and all other indications. Aetna considers the use offluorescence immunocytology (e.g., ImmunoCyt/uCyt) medically necessary as an adjunct to cystoscopy or cytology in the monitoring of persons with bladder cancer. Aetna considers the ImmunoCyte/uCyt immunohistochemistry test experimental and investigational in the evaluation of hematuria, diagnosingbladder cancer, or for screening for bladder cancer in asymptomatic persons. Aetna considers genetic testing for Janus Kinase 2 (JAK2) mutations inpersons with chronic myeloproliferative disorders (CMPDs) medically necessary for the following indications: qualitative assessment of JAK2-V617F sequence variant using methods with detection thresholds of up to 5% for initial diagnostic assessment of adult patients presenting withsymptoms of CMPD; diagnostic assessment of polycythemia vera in adults; and differential diagnosis of essential thrombocytosis and primary myelofibrosis from reactive conditions in adults. Aetna considers genetic testing for Janus Kinase 2 (JAK2) mutations inpersons with chronic myeloproliferative disorders (CMPDs) experimental and investigational for any other indication including: diagnostic assessment of myeloproliferative disorders in children; quantitative assessment of JAK2-V617F allele burden subsequent to qualitative detection ofJAK2-V617F. Aetna considers EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) medically necessary for the workup of myelodysplastic syndrome (MDS), and myeloproliferative neoplasms (MPN) to evaluate for higher-risk mutations associated with disease progression in members with primary myelofibrosis (PMF). Aetna considers EZH2 experimental and investigational for all other indications including diffuse large B-cell lymphomas. Aetna considers TERT (telomerase reverse transcriptase) medically necessary for the workup of gliomas (i.e.,infiltrative supratentorial astrocytoma/oligodendroglioma, anaplastic gliomas/glioblastoma), and myelodysplastic syndrome (MDS). Aetna considers TERT experimental and investigational for all other indications including thyroid carcinoma. CEAused for all other indications not notedaboveincluding any of the following: As a screening test for colorectal cancer; or As a sole determinant to treat a colorectal cancer member with adjuvant therapy or systemic therapy for presumed metastatic disease; or For diagnosis of esophageal carcinoma; or For screening, diagnosis, staging or routine surveillance of gastric cancer; Afirma Xpression Atlas; AFP for the diagnosis of trophoblastic tumors and oncologic indications other than those listed above; Assaying for loss of heterozygosity (LOH) on the long arm of chromosome 18 (18q) or deleted in colon cancer (DCC) protein (18q-LOH/DCC) for colorectal cancer; BBDRisk Dx; Biodesix BDX-XL2 test for distinguishing benign from malignant lung nodules; Biomarker Translation (BT) test for breast cancer and other indications; BioSpeciFx, including Comprehensive Tumor Profiling for any indication; BRAF and EGFR for esophageal carcinoma; Breast Cancer Gene Expression Ratio (HOXB13:IL17BR); BreastSentry; CA 125 for all other indications including use as a screening test for colorectal cancer or ovarian cancer (other than as indicated above) or for differential diagnosis of members with symptoms of colonic disease; CA 19-9 for all other indications including breast, colorectal, esophageal, gastro-esophageal, liver, or uterine cancer; ovarian cyst, NUT midline carcinoma of the nasal cavity, prediction of prognosis or treatment effect in persons with bladder (urothelial) cancer, screening persons with primary sclerosing cholangiitis without signs or symptoms of cholangiocarcinoma; or screening persons with primary sclerosing cholangitis for development of cholangiocarcinoma; Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) (e.g., Benign Diagnostics Risk Test) for breast atypical hyperplasia and forpredicting the risk of breast cancer; Carcinoembryonic antigen cellular adhesion molecule-7 (CEACAM-7) expression as a predictive marker for rectal cancer recurrence; Caris Molecular Intelligence/Caris Target Now Molecular Profiling Test; CDH1 and TP53 for ovarian cancer; CDX2 as a prognostic biomarker for colon cancer; CEA, Cyfra21-1 (a cytokeratin 19 fragment), p53, squamous cell carcinoma antigen (SCC-Ag) and vascular endothelial growth factor C (VEGF-C) for diagnosis of esophageal carcinoma; Circulating cell-free nucleic acids in colorectal cancer; Circulating tumor cell (CTC) assays (e.g., CellSearch assay) for all indications, including, but not limited to metastatic breast, colorectal, melanoma, and prostate cancers; CK5, CK14, p63, and Racemase P504S testing for prostate cancer; c-Met expression for predicting prognosis in persons with advanced NSCLC and colorectal cancer, and other indications; Cofilin (CFL1) as a prognostic and drug resistance marker in non-small cell lung cancer; ColonSentry test for screening of colorectal cancer; ColoPrint, CIMP, LINE-1 hypomethylation, and Immune cells for colon cancer; Colorectal Cancer DSA (Almac Diagnostics, Craigavon, UK); ConfirmMDx for prostate cancer; CxBladder test for bladder cancer; Cyclin D1 and FADD (Fas-associated protein with death domain) for head and neck squamous cell carcinoma; Decision DX-Melanoma (Castle Biosciences, Phoenix, AZ); DCIS Recurrence Score; Decipher Bladder; Des-gamma-carboxy prothrombin (DCP) (also known as \"prothrombin produced by vitamin K absence or antagonism II\" [PIVKA II]) for diagnosing and monitoring hepatocellular carcinoma (HCC)and other indications; EarlyCDT-Lung test; EGFR gene expression analysis for transitional (urothelial) cell cancer; EGFRVIII for glioblastoma multiforme; EML4-ALK as a diagnostic tool for stage IV non-small-cell lung cancer; Envisia Genomic Classifier; Excision repair cross-complementation group 1 protein (ERCC1) for persons with NSCLC, colon or with gastric cancer who are being considered for treatment with platinum-based chemotherapy, and other indications; ExoDx Prostate/ExosomeDx Prostate(IntelliScore); Fibrin/fibrinogen degradation products (FDP) test (e.g., DR-70 or Onko-Sure) for colorectal cancer; FoundationOne, FoundationOneCDxand FoundationOne Heme (except where FoundationOne CDx is used as a companion diagnostic test for somatic/tumor BRCA testing, seeCPB 0227 - BRCA Testing, Prophylactic Mastectomy, and Prophylactic Oophorectomyand CPB 0715 - Pharmacogenetic and Pharmacodynamic Testing); Galectin-3 for breast cancer, myelodysplastic syndrome, osteosarcoma, ovarian cancer, pancreatic cancer, and prostate cancer; Gene hypermethylation for prostate cancer; GeneKey (GeneKey Corp., Boston, MA); GeneSearch Breast Lymph Node (BLN) assay; Glutathione-S-transferase P1 (GSTP1) for screening, detection and management of prostate cancer; Guanylyl cyclase c (GCC or GUCY2C) (e.g., Previstage GCC Colorectal Cancer State Test) for colorectal cancer; HeproDx; HER2 testing of appendiceal cancer; HERmark testing for breast cancer and other indications; HMGB1 and RAGE in cutaneous malignancy (e.g., basal cell carcinoma, melanoma, and squamous cell carcinoma); Human epididymis protein 4 (HE4) (e.g., Elecsys HE4 assay)for endometrial cancer, ovarian cancer, or evaluation of pelvic mass, including to assist in the determination of referral for surgery to a gynecologic oncologist or general surgery,and for other indications; IHC4 (e.g., NexCourse IHC4 by AQUA Technology) for breast cancer; Immunoassay using magnetic nanosensor for diagnosis of lung cancer; Insight DX Breast Cancer Profile; Insight TNBCtype; Ki67 for breast cancer; Ki-67 in upper tract urinary carcinoma; 4Kscore; Lectin-reactive alpha-fetoprotein (AFP-L3) for liver cancer; Liquid biopsy (e.g., CancerIntercept, Colvera, GeneStrat, FoundationACT, FoundationOne Liquid, Guardant360, NeolabProstate) for any indication (other than small panels (less than 50 genes) for non-small cell lung cancer), including, but not limited to,breast cancer, colorectal cancer, melanoma, ovarian cancer or prostate cancer (For EGFRliquid biopsyfor non-small cell lung cancer (e.g., cobas EGFR Mutation Test v2) and PIK3CA testing (therascreen PIK3CA RGQ PCR Kit) for breast cancer, see CPB 0715 - Pharmacogenetic and Pharmacodynamic Testing); Long non-coding RNA in gallbladder cancer; Lymph2CX and Lymph3Cx Lymphoma Molecular Classification Assay to distinguish between primary mediastinal B-cell lymphoma (PMBCL) and diffuse large B-cell lymphoma (DLBCL); Mammostrat; Mass spectrometry-based proteomic profiling for indeterminate pulmonary nodules; MatePair targeted rearrangements (whole genome next-generation sequencing) for hematolymphoid neoplasia and solid organ neoplasia; Mayo Clinic Laboratories Urinary Steroid Profile for the management of adrenal malignancies; Measurement of circulating tumor cells (e.g., CellMax Life and FirstSightCRC) for screening of colorectal cancer; Merkel SmT Oncoprotein Antibody Titer; Merkel Virus VP1 Capsid Antibody; MI Cancer Seek; Microarray-based gene expression profile testing using the MyPRS test for multiple myeloma; Micro-RNAs (miRNAs) miRview mets and miRview mets2 (Rosetta Genomics Laboratories, Philadelphia, PA; Rosetta Genomics Ltd., Rehovot, Israel); Mi-Prostate Score (MiPS), an assay of TMPRSS2:ERG (T2:ERG) gene fusion, post-DRE urine expression of PCA3, and serum PSA (KLK3); miR-31now; Molecular Intelligence Services, including MI Profile and MI Profile X (formerly Target Now Molecualr Profiling Test, including Target Now Select and Target Now Comprehensive); Molecular subtyping profile (e.g., BluePrint) for breast cancer; mRNA gene expression profiling for cutaneous melanoma; MSK-IMPACT; MUC1 in gastric cancer; Mucin 4 expression as a predictor of survival in colorectal cancer; Mucin 5AC (MUC5AC) as serum marker for biliary tract cancer; My Prognostic Risk Signature (MyPRS) (Signal Genetics LLC, New York, NY); MyAML Next Generation Sequencing Panel; Myriad myPath Melanoma; NantHealth GPS Cancer Panels; Natera Signatera Molecular Monitoring (MRD) for breast cancer; NeoLAB Prostate Liquid Biopsy; NETest; NF1, RET, and SDHB for ovarian cancer; NRAS mutation for selecting persons with metastatic colorectal cancer who may benefit from anti-VEGF antibody bevacizumab; to predict disease prognosis and select persons with melanoma who may benefit from tyrosine kinase inhibitor therapies, and other indications; OncoOmicDx Targeted Proteomic Assay; OncoTarget/OncoTreat; Oncotype MAP PanCancer Tissue Test; OncoVantage; OVA1/Overa test; OvaCheck test; OvaSure; OncInsights (Intervention Insights, Grand Rapids, MI); OmniSeq Advance DNA and RNA sequencing (OmniSeq and LabCorp); PanGIA Prostate for determining if an individual should undergo a prostate biopsy; Pathwork Tissue of Origin Test/ResponseDx Tissue of Origin Test; Percepta Bronchial Genomic Classifier; Phosphatidylinositol-4,5-bisphosphonate 3-kinase, catalytic subunit alpha polypeptide gene (PIK3CA) for predicting disease prognosis and selecting individuals with metastatic colorectal cancer who are being considered for treatment with EGFR antagonists cetuximab and panitumumab, and indications other than breast cancer and uterine sarcoma; PLCG2 (phospholipase C gamma 2) for all indications other thanchronic lymphocytic leukemia (CLL); PreciseDx Breast Cancer Test; PreOvar test for the KRAS-variant to determine ovarian cancer risk; ProOnc TumorSourceDx test (Prometheus Laboratories, San Diego, CA) to identify tissue or origin for metastatic tumor; Prostate core mitotic test; Prostate Px and Post-Op Px for predicting recurence of prostate cancer; Prostate Cancer Risk Panel (FISH analysis by Mayo Clinic); Proveri prostate cancer assay (PPCA); PSA for screening women with breast cancer or for differentiating benign from malignant breast masses; PTEN gene expression for non-small cell lung cancer; Ras oncogenes (except KRAS, NRASand BRAF); ResponseDx Colon; Ribonucleotide reductase subunit M1 (RRM1) for persons with NSCLC who are being considered for treatment with gemcitabine-based chemotherapy, and other indications; ROMA (Risk of Ovarian Malignancy Algorithm) for ovarian cancer; Rotterdam Signature 76-gene panel; SelectMDx for prostate cancer; Sentinel Prostate Test for prostate cancer screening and determining the risk level of the disease; Serum amyloid A as a biomarker for endometrial endometrioid carcinoma to monitor disease recurrence and target response to therapy; Signatera for individuals with stage II/III colorectal cancer who are considering adjuvant chemotherapy and/or who are being monitored for relapse post-treatment; TargetPrint gene expression test for evaluation of estrogen receptor, progesterone receptor, and HER2receptor status in breast cancer; The 41-gene signature assay; Theros CancerType ID (bioTheranostics Inc., San Diego, CA); Thymidylate synthase; TMPRSS fusion genes for prostate cancer; Topographic genotyping (Pancragen (formerly PathFinderTG)); Total (whole) gene sequencing for cancer; TP53 mutation analysis for ovarian cancer; UroCor cytology panels (DD23 and P53)for bladder cancer; Vascular Endothelial Growth Factor (VEGF) except for Castleman\'s disease; Vascular endothelial growth factor receptor2 (VEGFR2) expression for identifying persons with colorectal cancer that is likely to respond to VEGF inhibition, and other indications; Whole exome sequencing (somatic mutations)(e.g., EXaCT-1Whole Exome Testing) for cancer; Any of the following circulating tumor markers also is considered experimental and investigational for screening asymptomatic subjects for cancer, diagnosis, staging, routine surveillance of cancer and monitoring the response to treatment: Background A tumor marker is a substance such as a protein, antigen or hormone in the body that may indicate the presence of cancer. Generally, these markers are specific to certain types of cancer and can be detected in blood, urine and tissue samples. The body may produce the marker in response to cancer or the tumor itself may produce the marker. The detection of tumor markers may be used to determine a diagnosis or as an indicator of disease (cancer) progression. It can also be used to document clinical response to treatment. Tumor markers include, but may not be limited to, alpha- fetoprotein (AFP), CA 15-3/CA 27.29, CA 19-9, CA-125, carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA). Tumor markers are normally produced in low quantities by cells in the body. Detection of a higher-than-normal serum level by radioimmunoassay or immunohistochemical techniques usually indicates the presence of a certain type of cancer. Currently, the main use of tumor markers is to assess a cancer\'s response to treatment and to check for recurrence. In some types of cancer, tumor marker levels may reflect the extent or stage of the disease and can be useful in predicting how well the disease will respond to treatment. A decrease or return to normal in the level of a tumor marker may indicate that the cancer has responded favorably to therapy. If the tumor marker level rises, it may indicate that the cancer is spreading. Finally, measurements of tumor marker levels may be used after treatment has ended as a part of follow-up care to check for recurrence. However, in many cases the literature states that measurements of tumor marker levels alone are insufficient to diagnose cancer for the following reasons: tumor marker levels can be elevated in people with benign conditions; tumor marker levels are not elevated in every person with cancer, especially in the early stages of the disease; and many tumor markers are not specific to a particular type of cancer; and the level of a tumor marker can be elevated by more than one type of cancer. Examples of Tumor Markers Include 5-Hydroxyindoleacetic acid (5-HIAA) – the main metabolite of serotonin, used as a marker in the evaluation of carcinoid tumors; Beta-2-Microglobulin (B2M) – A protein found on the surface of many cells. High levels of B2M are an indicator of certain kinds of cancer, including chronic lymphocytic leukemia, non-Hodgkin\'s lymphoma and multiple myeloma or kidney disease; Beta Human Chorionic Gonadotropin (beta HCG) – A type of tumor marker that may be found in higher than normal amounts in individuals with some types of cancer; Calcitonin – Hormone secreted by the thyroid that lowers blood calcium; Calretinin – A calcium-binding protein that is used as a marker in the evaluation of lung cancer and other diseases. Chromogranin A – A protein found inside neuroendocrine cells, which releases chromogranin A and other hormones into the blood. Chromogranin A may be found in higher than normal amounts in individuals with certain neuroendocrine tumors, small cell lung cancer, prostate cancer and other conditions Guanylyl cyclase c (GCC) – An enzyme that may be expressed only in the cells that line the intestine from the duodenum to the rectum. Inhibin – One of two hormones (designated inhibin-A and inhibin-B) secreted by the gonads (by Sertoli cells in the male and the granulosa cells in the female) and inhibits the production of follicle-stimulating hormone (FSH) by the pituitary gland; Lactate Dehydrogenase (LDH) – Marker used to monitor treatment of testicular cancer; Mucin-1 (MUC-1) – Carbohydrate antigen elevated in individuals with tumors of the breast, ovary, lung and prostate as well as other disorders; Napsin A – Protein used as a marker in the evaluation of lung cancer; Prealbumin – Marker of nutritional status and a sensitive indicator of protein synthesis. Also referred to as transthyretin; Prostate Specific Antigen (PSA) – Substance produced by the prostate gland. Levels of PSA in the blood often increase in men with prostate cancer. Thyroglobulin – Protein found in the thyroid gland. Some thyroglobulin can be found in the blood and this amount may be measured after thyroid surgery to determine whether thyroid cancer has recurred; Thyroid Transcription Factor-1 (TTF-1) – A protein that is used as a tumor marker in the evaluation of lung cancer; Transferrin – A protein in blood plasma that carries iron derived from food intake to the liver, spleen and bone marrow. Tumors may be evaluated with histology, which involves examination of the structure, especially the microscopic structure, of organic tissues.Methods of detecting tumor markers include, but are not limited to: Fluorescence in Situ Hybridization (FISH) – Laboratory technique used to detect small deletions or rearrangements in chromosomes.Immunohistochemical (IHC) Analysis – Laboratory process of detecting an organism in tissues with antibodies. Gene mutation testing can purportedly be used to find somatic mutations in cancerous cells that are not inherited. Some examples of genes that may have somatic mutations include: IDH1 and IDH2 genes (associated with acute myeloid leukemia [AML], gliomas and chondrosarcomas);NPM1 and FLT3 genes (associated with AML). Individualized molecular tumor profiling is a laboratory method of testing a panel of tumor markers, which may include genetic as well as biochemical markers, to establish a personalized molecular profile of a tumor to recommend treatment options. Mass spectrometry based proteomic profiling (eg, Veristrat, Xpresys Lung) is a multivariate serum protein test that uses mass spectrometry and proprietary algorithms to analyze proteins in an individual’s serum. The Xpresys is no longer on the market. Next-generation sequence (NGS) tests use select genes to purportedly identify molecular growth drivers for improved risk stratification and targeted therapies. Examples include: FoundationOne and OncoVantage for solid tumor cancers;FoundationOne Heme for hematological cancers and sarcomas; andThyGenX for indeterminate thyroid nodules. Liquid biopsy refers to serum testing for DNA fragments that are shed by cancer cells and released into the bloodstream. This method is purportedly used for screening, diagnosis and/or monitoring of cancer cells that may otherwise require a tissue sample. Multianalyte assays with algorithmic analyses (MAAAs) are laboratory measurements that use a mathematical formula to analyze multiple markers that may be associated with a particular disease state and are designed to evaluate disease activity or an individual’s risk for disease. The laboratory performs an algorithmic analysis using the results of the assays and sometimes other information, such as sex and age and converts the information into a numeric score, which is conveyed on a laboratory report. Generally, MAAAs are exclusive to a single laboratory which owns the algorithm. MAAAs have been proposed for the evaluation of pelvic masses, including assisting in the determination of referral for surgery to a gynecologic oncologist or to a general surgeon. Topographic genotyping (eg, PathFinderTG) is a test that examines a panel of 15 to 20 genetic markers in tissue biopsy or other tissue specimens to purportedly aid in the determination of indeterminate or equivocal cancer diagnoses. Prostate Specific Antigen (PSA)is a substance produced by the prostate gland. Levels of PSA in the blood often increase in men with prostate cancer. Elevated levels of Prostate-Specific Antigen (PSA) may also be found in the blood of men with benign prostate conditions, such as prostatitis and benign prostatic hyperplasia (BPH). While PSA does not allow distinction between benign prostate conditions and cancer, an elevated PSA level may indicate that other tests are necessary to determine whether cancer is present. PSA levels have been shown to be useful in monitoring the effectiveness of prostate cancer treatment, and in checking for recurrence after treatment has ended. Use of PSA for screening remains very controversial. Although researchers are in the process of studying the value of PSA along with digital rectal exams for routine screening of men ages 55 to 74 for prostate cancer; and the literature does not show at this time whether using PSA to screen for prostate cancer actually does reduce the number of deaths caused by this cancer. The American Cancer Society recommends clinicians and patients consider screening with PSA and digital rectal exam for African American men and men with familial tendency age 40 or older and all men age 50 or older. Cancer Care Ontario guidelines on active surveillance of prostate cancer(Morash, et al., 2015) state that the active surveillance protocol should include the following tests: PSA test every 3 to 6 months; digital rectal examination every year, anda 12- to 14-core confirmatory transrectal ultrasound (TRUS) biopsy (including anterior directed cores) within 6 to 12 months, then serial biopsy a minimum of every 3 to 5 years thereafter. The guidelines state that \"[c]urrent evidence shows that PSA kinetics does not reliably predict disease stability or reclassification to higher risk state. There was conflicting evidence whether PSA is a good predictor of disease progression or reclassification. Differences were also found in the ability of different measures of PSA, such as PSA velocity, PSA density, and PSA doubling time for predicting progression or reclassification. PSA monitoring is considered a necessary component of an AS protocol, but a rising PSA may be best viewed as a trigger for reappraisal (e.g., MRI, repeat biopsy) rather than a trigger for intervention.\" Prostate cancer antigen3(PCA3, also known as DD3) is a gene that has been found to be highly overexpressed in prostate cancer. This gene has been investigated as a potential diagnostic marker for prostate cancer. However, there are no published clinical outcome studies of the effectiveness of the PCA3 gene in screening, diagnosis or management of prostate cancer. Prostate cancer antigen 3 (PCA3) (Progensa, Gene-Probe, Inc.) encodes a prostate-specific mRNA. It is one of the most prostate cancer-specific genes identified, with over-expression in about 95% of cancers tested. The PCA3 urine assay is an amplified nucleic acid assay, which uses transcription-mediated amplification (TMA) to quantify PCA3 and PSA mRNA in prostate cells found in urine samples. The PCA3 score is calculated as the ratio between PCA3 and PSA mRNA. The main target population of this non-invasive test is men with raised PSA but a negative prostate biopsy. Other target groups include men with a slightly raised PSA, as well as men with signs and symptoms suggestive of prostate cancer. van Gils and colleagues (2007) stated that PCA3 is a promising prostate cancer marker. These investigators performed a multi-center study to validate the diagnostic performance of the PCA3 urine test established in an earlier single-institution study. The first voided urine after digital rectal examination (DRE) was collected from a total of 583 men with serum PSA levels between 3 and 15 ng/ml who were to undergo prostate biopsies. These researchers determined the PCA3 score in these samples and correlated the results with the results of the prostate biopsies. A total of 534 men (92 %) had an informative sample. The area under the receiver-operating characteristic curve, a measure of the diagnostic accuracy of a test, was 0.66 for the PCA3 urine test and 0.57 for serum PSA. The sensitivity for the PCA3 urine test was 65 %, the specificity was 66 % (versus 47 % for serum PSA), and the negative predictive value was 80 %. The authors concluded that the findings of this multi-center study validated the diagnostic performance of the PCA3 urine test in the largest group studied thus far using a PCA3 gene-based test. Marks and associates (2007) examined the potential utility of the investigational PCA3 urine assay to predict the repeat biopsy outcome. Urine was collected after DRE (3 strokes per lobe) from 233 men with serum PSA levels persistently 2.5 ng/ml or greater and at least one previous negative biopsy. The PCA3 scores were determined using a highly sensitive quantitative assay with TMA. The ability of the PCA3 score to predict the biopsy outcome was assessed and compared with the serum PSA levels. The RNA yield was adequate for analysis in the urine samples from 226 of 233 men (i.e., the informative specimen rate was 97 %). Repeat biopsy revealed prostate cancer in 60 (27 %) of the 226 remaining subjects. Receiver operating characteristic curve analysis yielded an area under the curve of 0.68 for the PCA3 score. In contrast, the area under the curve for serum PSA was 0.52. Using a PCA3 score cutoff of 35, the assay sensitivity was 58 % and specificity 72 %, with an odds ratio of 3.6. At PCA3 scores of less than 5, only 12 % of men had prostate cancer on repeat biopsy; at PCA3 scores of greater than 100, the risk of positive biopsy was 50 %. The authors concluded that in men undergoing repeat prostate biopsy to rule out cancer, the urinary PCA3 score was superior to serum PSA determination for predicting the biopsy outcome. The high specificity and informative rate suggest that the PCA3 assay could have an important role in prostate cancer diagnosis. Groskopf et al (2007) reported that the PCA3 score is independent of prostate volume and was highly correlated with the risk of positive biopsy. The PCA3 test was performed on 529 men scheduled for prostate biopsy. Overall, the PCA3 score had a sensitivity of 54% and a specificity of 74%. A PCA3 score of less than 5 was associated with a 14% risk of positive biopsy, while a PCA3 score of greater than 100 was associated with a 69% risk of positive biopsy. Haese et al (2007) presented preliminary results from a European multicenter study of PCA3. Enrolled patients had a PSA level of less than or equal to 2.5 ng/mL, had 1 or 2 previous negative biopsies, and were scheduled for repeat biopsy. The specificity of the PCA3 score (cutoff 35) was found to be 78%, and the sensitivity was 67%. Patients with a PCA3 score of greater than or equal to 35 had a 33% probability of a positive repeat biopsy, compared to a 6% probability for those with a PCA3 score of less than 35. In a review on biomarkers for prostate cancer detection, Parekh, et al. (2007) stated that prostate stem cell antigen, alpha-methyl coenzyme-A racemase, PCA3, early prostate cancer antigen, hepsin and human kallikrein 2 are promising markers that are currently undergoing validation. An assessment by the BlueCross BlueShield Association Technology Evaluation Center (BCBSA, 2008) found that, in general,PCA3 assay results to date are preliminary; interpretation of results has not been standardized and clinical utility studies of decision-making for initial biopsy, repeat biopsy or treatment have not been reported. Tosoian et al (2010) evaluated the relationship between PCA3 and prostate biopsy results in men in a surveillance program. Urine specimens were obtained from 294 men with prostate cancer enrolled in the Johns Hopkins surveillance program. The follow-up protocol included semi-annual free and totalPSA measurements, digital rectal examination and annual surveillance prostate biopsy. Cox proportional hazards regression was used to evaluate the association between PCA3 results and progression on surveillance biopsy (defined as Gleason pattern 4 or 5, more than 2 positive biopsy cores or more than 50% involvement of any core with cancer). Patients with progression on biopsy (12.9%) had a mean PCA3 score similar to that of those without progression (60.0 versus 50.8, p = 0.131). Receiver operating characteristics analysis suggested that PCA3 alone could not be used to identify men with progression on biopsy (area under the curve =0.589, 95% CI 0.496 to 0.683, p = 0.076). After adjustment for age and date of diagnosis PCA3 was not significantly associated with progression on biopsy (p = 0.15). The authors concluded that in men with low risk prostate cancer who were carefully selected for surveillance the PCA3 score was not significantly associated with short-term biopsy progression. They stated that further analysis is necessary to assess the usefulness of PCA3 in combination with other biomarkers or in selected subsets of patients undergoing surveillance. While there are studies examining the positive and negative predictive values of the PCA3 urine assay, there is currently a lack of evidence of the effect of this test on management of individuals with or suspected of prostate cancer. The PCA3 urine assay shows promise as a prostate cancer diagnostic tool, however, more research is needed to ascertain the clinical value of this assay for screening and diagnostic purposes. An assessment of PCA3 prepared for the Agency for Healthcare Research and Quality (2013) concluded: \"For diagnostic accuracy, there was a low strength of evidence that PCA3 had better diagnostic accuracy for positive biopsy results than tPSA elevations, but insufficient evidence that this led to improved intermediate or long-term health outcomes. For all other settings, comparators, and outcomes, there was insufficient evidence.\" The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group (2013)found insufficient evidence to recommend prostate cancer antigen 3 (PCA3) testing to inform decisions for when to re-biopsy previously biopsy-negative patients for prostate cancer or to inform decisions to conduct initial biopsies for prostate cancer in at-risk men (e.g., previous elevated prostate-specific antigen test or suspicious digital rectal examination). The EGAPP Working Group found insufficient evidence to recommend PCA3 testing in men with cancer-positive biopsies to determine if the disease is indolent or aggressive in order to develop an optimal treatment plan. The EGAPP Working Group concluded that,based on the available evidence, the overall certainty of clinical validity to predict the diagnosis of prostate cancer using PCA3 is deemed \"low.\" The EGAPP Working Group discouraged clinical use for diagnosis unless further evidence supports improved clinical validity. The EGAPP Working Group also found that, based on the available evidence, the overall certainty of net health benefit is deemed \"low.\" The EGAPP Working Group discourages clinical use unless further evidence supports improved clinical outcomes. Guidelines from the European Association of Urology (2015) state that \"[b]iological markers, include urine markers such as PCA3, the TMPRSS2: ERG fusion gene or PSA isoforms such as the Phi index,appear promising as does genomics on the tissue sample itself. However, further study data will be needed before such markers can be used in standard clinical practice.\" A Cancer Care Ontario Guideline on prostate cancer surveillance (Morash, et al., 2015), which has been endorsed by the American Society for Clinical Oncology (2016), did not include PCA3 level in their recommendation becauseevidence of PCA3to predict disease reclassification in prostate cancer was lacking. National Institute for Health and Care Excellence (NICE)’s clinical practice guideline on \"Diagnosing prostate cancer: PROGENSA PCA3 assay and Prostate Health Index\" (2015) stated that \" The PROGENSA PCA3 assay and the Prostate Health Index are not recommended for use in people having investigations for suspected prostate cancer, who have had a negative or inconclusive transrectal ultrasound prostate biopsy\". The assessment cited studies finding that adding the PCA3 score to clinical assessment and MRI had very little effect on the size of the reported area under the curve, with minimal change in derived sensitivity and specificityfor clinical assessment with MRI compared with clinical assessment using MRI and the PCA3 assay. In a Lancet review of prostate cancer, Attard, et al. (2016) stated that \"[s]everal studies have so far proven inconclusive as to whether PCA3 is useful to selectively detect aggressive prostate cancers.\" Hutchinson et al (2005) stated that in tissue-based assays, thymosin beta15 (B15) has been shown to correlate with prostate cancer and with recurrence of malignancy. To be clinically effective, it must be shown that thymosin B15 is released by the tumor into body fluids in detectable concentrations. These researchers developed a quantitative assay that can measure clinically relevant levels of thymosin B15 in human urine. Sixteen antibodies were raised against recombinant thymosin B15 and/or peptide conjugates. One antibody, having stable characteristics over the wide range of pH and salt concentrations found in urine and minimal cross-reactivity with other beta thymosins, was used to develop a competitive enzyme-linked immunosorbent assay (ELISA). Urinary thymosin B15 concentration was determined for control groups; normal (n = 52), prostate intraepithelial neoplasia (PIN, n = 36), and patients with prostate cancer; untreated (n = 7) with subsequent biochemical failure, radiation therapy (n = 17) at risk of biochemical recurrence. The operating range of the competition ELISA fell between 2.5 and 625 ng/ml. Recoveries exceeded 75%, and the intra- and inter-assay coefficients of variability were 3.3% and 12.9%, respectively. No cross-reactivity with other urine proteins was observed. A stable thymosin B15 signal was recovered from urine specimens stored at -20 degrees C for up to 1 year. At a threshold of 40 (ng/dl)/microg protein/mg creatinine), the assay had a sensitivity of 58% and a specificity of 94%. Relative to the control groups, thymosin B15 levels were greater than this threshold in a significant fraction of patients with prostate cancer (p 0.001), including 5 of the 7 patients who later experienced PSA recurrence. The authors concluded that an ELISA that is able to detect thymosin B15 at clinically relevant concentrations in urine from patients with prostate cancer has been established. They noted that the assay will provide a tool for future clinical studies to validate urinary thymosin B15 as a predictive marker for recurrent prostate cancer. Carcinoembryonic antigen (CEA) is a normal cell product that is over-expressed by adenocarcinomas, primarily of the colon, rectum, breast, and lung. It is normally found in small amounts in the blood of most healthy people, but may become elevated in people who have cancer or some benign conditions. CEA is an oncofetal glycoprotein present in the gastrointestinal tract and body fluids of the embryo and fetus (Chin, et al., 2006). It is also present in certain adult gastrointestinal cells, including the mucosal cells of the colorectum, and small amounts are present in blood. Blood levels are often elevated in patients with disseminated cancers and in some patients with nonmalignant disease. According to the available literature, the primary use of CEA is in monitoring colorectal cancer, especially when the disease has metastasized. CEA is also used after treatment to check for recurrence of colorectal cancer. However, the literature indicates a wide variety of other cancers can produce elevated levels of this tumor marker, including melanoma; lymphoma; and cancers of the breast, lung, pancreas, stomach, cervix, bladder, kidney, thyroid, liver, and ovary. Elevated CEA levels can also occur in patients with non-cancerous conditions, including inflammatory bowel disease, pancreatitis, and liver disease. The American Society of Clinical Oncology (ASCO)\'s update of recommendations for the use of tumor markers in gastrointestinal cancer (Gershon, et al., 2006) stated that post-operative CEA levels should be performed every 3 months for stage II and III disease for at least 3 years if the patient is a potential candidate for surgery or chemotherapy of metastatic disease. CA-125 Cancer antigen 125(CA-125) is a test that evaluates ovarian cancer treatment. CA-125 is a protein that is found more in ovarian cancer cells than in other cells. CA-125 is expressed by 80 percent of non-mucinous ovarian epithelial neoplasms (Chin et al, 2006). Approximately half of women with metastatic ovarian cancer have an elevated CA-125 level. The Gynecologic Cancer Foundation, the Society of Gynecologic Oncologists, and the American Cancer Society have issued a consensus statement to promote early detection of ovarian cancer, which recommends that women who have symptoms, including bloating, pelvic or abdominal pain, difficulty eating or feeling full quickly, and urinary frequency and urgency, are urged to see a gynecologist if symptoms are new and persist for more than three weeks (ACS, 2007; SGO, 2007). Ovarian cancer is among the deadliest types of cancer because diagnosis usually comes very late, after the cancer has spread. If the cancer is found and surgically removed before it spreads outside the ovary, the five year survival rate is 93%. However, only 19% of cases are detected early enough for that kind of successful intervention. It is estimated that 22,430 new cases and 15,280 deaths will be reported in 2007 (ACS, 2007). The consensus statement recommendations are based on studies that show the above symptoms appeared in women with ovarian cancer more than in other women (Goff, et al., 2004; Daly Ozols, 2004). The recommendations acknowledge that there is not consensus on what physicians should do when patients present with these symptoms. According to a consensus statement issued by the Gynecologic Cancer Foundation, pelvic and rectal examination in women with the symptoms is one first step. If there is a suspicion of cancer, the next step may be a transvaginal ultrasound to check the ovaries for abnormal growths, enlargement, or telltale pockets of fluid that can indicate cancer. Testing for CA-125 levels should also be considered. There is no evidence available that measurement of CA-125 can be effectively used for widespread screening to reduce mortality from ovarian cancer, nor that the use of this test would result in decreased rather than increased morbidity and mortality. According to the available literature, not all women with elevated CA 125 levels have ovarian cancer. CA 125 levels may also be elevated by cancers of the uterus, cervix, pancreas, liver, colon, breast, lung, and digestive tract. Non-cancerous conditions that can cause elevated CA 125 levels include endometriosis, pelvic inflammatory disease, peritonitis, pancreatitis, liver disease, and any condition that inflames the pleura. Menstruation and pregnancy can also cause an increase in CA 125. However, according to the available literature, changes in CA 125 levels can be effectively used in the management of treatment for ovarian cancer. In women with ovarian cancer being treated with chemotherapy, the literature suggests a falling CA 125 level generally indicates that the cancer is responding to treatment and increased survival is expected. Increasing CA 125 levels during or after treatment, on the other hand, may suggest that the cancer is not responding to therapy or that residual cancer remains. According to the available literature, failure of the CA 125 level to return to normal after three cycles of chemotherapy indicates residual tumor, early treatment failure and decreased survival. Under accepted guidelines, CA 125 levels can also be used to monitor patients for recurrence of ovarian cancer. Although an elevated CA 125 level is highly correlated with the presence of ovarian cancer, the literature suggests a normal value does not exclude residual or recurrent disease. Aetna\'s preventive services guidelines are based on the recommendations of leading primary care medical professional organizations and federal public health agencies. None of these organizations recommend routine screening of average-risk, asymptomatic women with serum CA-125 levels for ovarian cancer. These organizations have concluded that serum CA-125 levels are not sufficiently sensitive or specific for use as a screening test for ovarian cancer, and that the harms of such screening outweigh the benefits. The American College of Obstetricians and Gynecologists (2002) has stated that \"[u]nfortunately, there is no screening test for ovarian cancer that has proved effective in screening low-risk asymptomatic women. Measurement of CA 125 levels and completion of pelvic ultrasonography (both abdominal and transvaginal) have been the two tests most thoroughly evaluated.... Data suggest that currently available tests do not appear to be beneficial for screening low-risk, asymptomatic women because their sensitivity, specificity, positive predictive value, and negative predictive value have all been modest at best. Because of the low incidence of disease, reported to be approximately one case per 2,500 women per year, it has been estimated that a test with even 100% sensitivity and 99% specificity would have a positive predictive value of only 4.8%, meaning 20 of 21 women undergoing surgery would not have primary ovarian cancer. Unfortunately, no test available approaches this level of sensitivity or specificity.\" The National Cancer Institute (2004) has stated: \"There is insufficient evidence to establish that screening for ovarian cancer with serum markers such as CA 125 levels, transvaginal ultrasound, or pelvic examinations would result in a decrease in mortality from ovarian cancer. A serious potential harm is the false-positive test result, which may lead to anxiety and invasive diagnostic procedures. There is good evidence that screening for ovarian cancer with the tests above would result in more diagnostic laparoscopies and laparotomies than new ovarian cancers found. Unnecessary oophorectomies may also result.\" The U.S. Preventive Services Task Force (2004) recommends against routine screening with serum CA-125 level for ovarian cancer. The Task Force concluded that the potential harms of such screening outweigh the potential benefits. Human Epididymis Protein 4 (HE4)is a secreted glycoprotein that is being studied as a potential marker for ovarian cancer. A variety of other tumor markers have been investigated for early detection of ovarian cancer as well as different combinations of tumor markers complementary to CA 125 that could potentially offer greater sensitivity and specificity than CA 125 alone. Preliminary studies on HE4 (human epididymis protein 4), a marker for ovarian cancer, reported similar sensitivity to CA 125 when comparing ovarian cancer cases to healthy controls, and a higher sensitivity when comparing ovarian cancer cases to benign gynecologic disease (Hellstrom, et al., 2003 2008; Moore, et al., 2008;) However, an assessment on genomic tests for ovarian cancer prepared by Duke University for the Agency for Healthcare Research and Quality (AHRQ, 2006) stated, \"Although research remains promising, adaptation of genomic tests into clinical practice must await appropriately designed and powered studies in relevant clinical settings.\" Further studies are needed to determine if HE4 significantly adds to the sensitivity of CA 125 while maintaining a high specificity. National Comprehensive Cancer Network (NCCN) guidelines (2016) state that data show that HE4 and several other markers do not increase early enough to be useful in detecting early-stage ovarian cancer. CA 15-3 Cancer antigen 15-3 (CA 15-3) is a serum cancer antigen that has been used in the management of patients with breast cancer. According to the available literature, its low detection rate in early stage disease indicates that CA 15-3 cannot be used to screen or diagnose patients with breast cancer. It has been widely used to monitor the effectiveness of treatment for metastatic cancer. Elevated serum CA 15-3 concentrations are found in 5 percent of stage I, 29 percent of stage II, 32 percent of stage III and 95 percent of stage IV carcinoma of the breast (Chin, et al, 2006). Most (96 percent) patients with a CA 15-3 increase of greater than 25 percent have disease progression. Most (nearly 100 percent) patients with a CA 15-3 decrease of greater than 50 percent are responding to treatment. Cancers of the ovary, lung, and prostate may also raise CA 15-3 levels. The literature indicates elevated levels of CA 15-3 may be associated with non-cancerous conditions, such as benign breast or ovarian disease, endometriosis, pelvic inflammatory disease, and hepatitis. Similar to the CA 15-3 antigen, CA 27-29 is found in the blood of most breast cancer patients. The literature indicates CA 27-29 levels may be used in conjunction with other procedures (such as mammograms and measurements of other tumor marker levels) to check for recurrence in women previously treated for stage II and stage III breast cancer. CA 27-29 levels can also be elevated by cancers of the colon, stomach, kidney, lung, ovary, pancreas, uterus, and liver. First trimester pregnancy, endometriosis, ovarian cysts, benign breast disease, kidney disease, and liver disease are non-cancerous conditions that can also elevate CA 27-29 levels. Elevated CA 27.29 levels are primarily associated with metastatic breast cancer, where it can be used to monitor the course of disease, response to treatment, and detect disease recurrence (Chin, et al., 2006). Elevated serum CA 27.29 concentrations are found in 95 percent of stage IV breast cancer. In addition, CA 27.29 has been found to be elevated in lung (43 percent), pancreas (47 percent), ovarian (56 percent), and liver (55 percent) cancer. CA 19-9 Cancer antigen 19-9 (CA 19-9) is a mucin-glycoprotein first identified from a human colorectal carcinoma cell line and is present in epithelial tissue of the stomach, gall bladder, pancreas and prostate (Chin, et al., 2006). Concentrations are increased in patients with pancreatic, gastric, and colon cancer as well as in some nonmalignant conditions. Increasing levels generally indicate disease progression, whereas decreasing levels suggest therapeutic response. Initially found in colorectal cancer patients, CA 19-9 has also been identified in patients with pancreatic, stomach, hepatocellular cancer, and bile duct cancer. In those who have pancreatic cancer, the literature indicates higher levels of CA 19-9 tend to be associated with more advanced disease. Although the sensitivity of the CA 19-9 level in patients with pancreatic cancer is relatively high, the specificity is lowered by elevations that occur in patients with benign pancreatic or liver disease. Non-cancerous conditions that may elevate CA 19-9 levels include gallstones, pancreatitis, cirrhosis of the liver, and cholecystitis. Although excellent correlations have been reported between CA 19-9 measurements and relapse in patients with pancreatic cancer who are followed after surgical resection, no patient has been salvaged by the earlier diagnosis of relapse, a fact that reflects the lack of effective therapy. Guidelines from the National Comprehensive Cancer Network (NCCN, 2010) state that measurement of CA 19-9 should be considered in evaluating patients with intrahepatic or extrahepatic cholangiocarcinoma and gallbladder cancer. The guidelines note that CA 19-9 is often elevated in persons with cholangiocarcinoma or gallbladder cancer, although this marker is not specific for these cancers. Nehls, et al. (2004) considered CA19-9 as one of the several new potential tumor markers for the diagnosis of cholangiocarcinoma. Levy, et al. (2005) aimed to characterize the test properties of CA 19-9 and of a change in CA 19-9 over time in predicting cholangiocarcinoma in patients with primary sclerosing cholangitis. Charts of 208 patients were reviewed. Fourteen patients had cholangiocarcinoma. Median CA 19-9 was higher with cholangiocarcinoma (15 versus 290 U/ml, p 0.0001). A cutoff of 129 U/ml provided: sensitivity 78.6%, specificity 98.5%, adjusted positive predictive value 56.6% and negative predictive value 99.4%. The median change over time was 664 U/ml in cholangiocarcinoma compared to 6.7 U/ml in primary sclerosing cholangitis alone (p 0.0001). A cutoff of 63.2 U/ml for change in CA 19-9 provided: sensitivity 90%, specificity 98% and positive predictive value 42%. CA 19-9 is produced by adenocarcinomas of the pancreas, stomach, gall-bladder, colon, ovary, and lung, and it is shed into the circulation. Although numerous studies have addressed the potential utility of CA 19-9 in adenocarcinoma of the colon and rectum, the sensitivity of CA 19-9 was always less than that of the CEA test for all stages of disease. Its use for screening asymptomatic populations has been hampered by a false-positive rate of 15% to 30% in patients with non-neoplastic diseases of the pancreas, liver, and biliary tract. Only a few studies have addressed the use of CA 19-9 in monitoring patients\' post-primary therapy. Significant postsurgical decreases are observed for CA 19-9, but these decreases have not been correlated with survival or disease-free interval. In monitoring response to treatment, decreases in CEA have been found to more accurately reflect response to therapy than did decreases of CA 19-9. Progressive increases of the marker may signal disease progression in 25% of the patients who express the CA 19-9 marker, but this monitoring provides only a minimal lead time of 1 to 3 months. Monitoring with CA 19-9 has not been shown to improve the management of patients with colorectal cancer. The serum CA 19-9 level does not add significant information to that provided by CEA, which is currently regarded as the marker of choice for this neoplasm. Sinakos and colleagues (2011) evaluated the long-term outcomes in Mayo Clinic patients presenting with primary sclerosing cholangitis (PSC) between 2000 and 2010 (n= 73) for incidence of cholangiocarcinoma (CCA). The results showed initial levels of CA 19-9 in patients without CCA were significantly lower than those from patients with CCA (p 0.0001). No factors known to affect CA 19-9 levels were identified in 33% of the patients without CCA; endoscopic treatment and recurrent bacterial cholangitis were associated with levels of CA 19-9 in 26% and 22% of these patients, respectively. Juntermanns (2011) prospectively analyzed a bile duct tumor database and retrieved records of 238 patients who underwent surgery between 1999 and 2008. Their findings included that pre-operative CA19-9 serum levels did not show a statically reliable differentiation between benign or malignant dignity. The authors concluded that current diagnostics cannot differentiate malignant from benign tumor masses in the hepatic hilum with required reliability. The authors further concluded that administration of CIK cells, thymus factor, IL-2 and IFN-alpha after AHSCT could improve the immunologic function of patients, and TH1/TH2 ratio may virtually reflect the immune status of patients, but that more information is required to make prognostic assessments of immune reconstruction and the long-term survival rate. Sarbia et al (1993) investigated 69 adenocarcinomas of the esophagogastric junction and found high rates of antigen expression were found for the \"intestinal\" markers CA 19-9 (between 55.5% and 100%) and BW 494 (between 42.9 and 86.7%). The authors concluded that these data, in combination with CK-20 expression, PGII, and 2B5 indicate that the distribution of adenocarcinomas with gastric and.or intestinal differentiation at the esophagogastric junction forms a continuum with out clear-cut borders. This study has not been replicated and NCCN guidelines for Esophageal and Esophagogastric Junction Cancers does not include recommendations for CA 19-9 testing for these indications (NCCN, 2011). The American Society of Clinical Oncology (ASCO)\'s update of recommendations for the use of tumor markers in gastrointestinal cancer (Gershon, et al., 2006) stated that for pancreatic cancer, CA 19-9 can be measured every 1 to 3 months for patients with locally advanced or metastatic disease receiving active therapy. Mucinous carcinoma of the appendix is a rare entity most commonly associated with primary tumors of the appendix and colon, and for which spread is generally confined to the abdominal cavity (Andreopoulou et al, 2007). Imaging assessment of these mucinous lesions is difficult, and recent studies have explored the use of tumor markers as clinical tools in evaluation of mucinous carcinoma of the appendix. Carmignani et al (2004) evaluated patients with synchronous systemic and intraperitoneal dissemination of appendix cancer treated with cytoreductive surgery and perioperative regional chemotherapy with a mean follow up time of 42.6 months. Results of this study indicated that patients with elevated CEA and CA 19-9 levels had a shorter median survival time (p=0.0083 and p = 0.0193, respectively). In a subsequent study, Carmingnani et al (2004) prospectively recorded tumor markers CEA and CA19-9 within 1 week prior to definitive treatment. The investigators found CEA elevated in 56% of 532 patients and CA19-9 elevated in 67.1% of those patients. They reported that \"although the absolute level of tumor marker did not correlate with prognosis, a normal value indicated an improved survival.\" Their findings included an elevated CEA in 35.2% of 110 patients determined to have recurrent disease and an elevated CA 19-9 in 62.9%, while 68.2% of patients had at least one of the tumor markers elevated. Current guidelines indicate that for liver transplantation for primary sclerosing cholangitis, stringent efforts should be made to detect superadded cholangiocarcinoma, including measurement of CA 19-9 (Devlin O\'Grady, 1999). Carmignani et al (2004a) conducted a study to report the role of combined treatments, including cytoreductive surgery and perioperative regional chemotherapy, in patients with synchronous systemic and intraperitoneal dissemination of appendix cancer. Study subjects were treated with cytoreductive surgery and perioperative regional chemotherapy and statistical analysis of variables utilized survival as an end point and included demographic characteristics, prior surgical score (PSS), tumor marker levels, peritoneal cancer index (PCI), and completeness of cytoreduction (CC). With a mean follow-up of 42.6 months, median survival time (MST) for 15 patients was 28 months and 5-year survival rate was 29.4 %. Female patients had a longer MST than male patients (p = 0.0199) and survival was better in patients with PSS 0 and 1 (p = 0.0277). Patients with elevated CEA and CA 19-9 levels had a shorter MST (p = 0.0083 and p = 0.0193, respectively) while PCI and CC comparisons did not show significant differences. The morbidity rate (n = 2) was 13.3 % and the mortality (n = 2) rate was also 13.3 %. The authors concluded that \"acceptable morbidity and mortality and a 29.4 % 5-year survival rate allows cytoreductive surgery and regional chemotherapy to be considered as a treatment option for selected patients with synchronous systemic and intraperitoneal dissemination of appendix cancer.\" Carmignani et al (2004b)in a further publication regarding gastrointestinal cancer, stated that carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) tumor markers have found selected clinical application. The authors remarked that the use of these tumor markers in mucinous epithelial tumors of the appendix has not been previously determined. Thus, the authors conducted a study in which, in patients with peritoneal dissemination of a mucinous epithelial malignancy of the appendix, tumor markers CEA and CA 19-9 were prospectively recorded preoperatively within 1 week prior to definitive treatment and if the appendiceal tumor recurred, the tumor marker was determined. The primary endpoint was the accuracy of these two tumor markers in the management of this disease for these two specific clinical situations. CEA was elevated in 56 % of 532 patients and CA 19-9 was elevated in 67.1 % of these patients. Although the absolute level of tumor marker did not correlate with prognosis, a normal value indicated an improved survival. CEA was elevated in 35.2 % of 110 patients determined to have recurrent disease and CA 19-9 was elevated in 62.9 %. At least one of the tumor markers was elevated in 68.2 % of patients. An elevated CEA tumor marker at the time of recurrence indicated a reduced prognosis and both CEA and CA 19-9 tumor markers were elevated in a majority of these patients. This should be a valuable diagnostic tool previously underutilized in this group of patients. These tumor markers were also of benefit in the assessment of prognosis in that a normal level indicated an improved prognosis. At the time of a reoperative procedure, CEA and CA 19-9 tumor markers gave information regarding the progression of disease and have practical value in the management of epithelial appendiceal malignancy with peritoneal dissemination. Andreopoulou et al (2007) stated thatmucinous carcinoma of the appendix isa rare entity with a distinct natural history that poses diagnostic and therapeutic challenges and that mucinous peritoneal carcinomatosis is most commonly associated with primary tumors of the appendix and colon. The authors stated that usually the spread remains confined to the abdominal cavity and that imaging assessment of these mucinous lesions is difficult, while tumor markers (CEA and CA19.9) may be surrogates for extent of disease. Recruitment for large scale studies given the rare nature of mucinous appendiceal carcinoma would be challenging. However, available evidence does illustrate a benefit to use of CA 19-9 in patients with mucinous appendiceal carcinoma. National Comprehensive Cancer Network’s clinical practice guideline on \"Hepatobiliary cancers” (Version 1.2021) states that CEA and Ca 19-9 are baseline tests, and should not be performed to confirm diagnosis of gallbladder cancer, or cholangiocarcinoma (extra-hepatic or intra-hepatic). An UpToDate review on \"Tumors of the nasal cavity” (Dagan et al, 2021) does not mention CA 19-9. Furthermore, National Comprehensive Cancer Network’s Biomarkers Compendium (2021) does not list NUT midline carcinoma tumor of the nasal cavity to be associated with CA 19-9 expression. Cathepsins This enzyme plays a critical role in protein catabolism and tissue remodeling (Chin, et al., 2006). Over-expression is associated with non-ductal carcinoma and metastasis at the time of breast cancer diagnosis. High levels may have clinical significance in predicting decreased metastasis-free survival and decreased overall survival in women with node-negative breast cancer. Svatek et al (2008) examined the role of urinary cathepsin B and L in the detection of bladder urothelial cell carcinoma. These investigators concluded that urinary cathepsin L is an independent predictor of bladder cancer presence and invasiveness in patients with a history of urothelial carcinoma of the bladder. They stated that further evaluation of this marker is necessary before its use as an adjunct to cystoscopy for urothelial carcinoma of the bladder. CD 20 CD 20 is used to determine eligibility for rituximab (Rituxan; anti-CD20) treatment in patients with B-cell non-Hodgkin\'s lymphomas (NHL) (Chin, et al., 2006). Rituximab is a genetically engineered, chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B-cell lymphocytes. Since non-Hodgkin\'s Lymphoma (NHL) subtypes may differ in their response to rituximab, determination of drug sensitivity is important for choosing therapy. CD 25 CD 25 is used to determine eligibility for denileukin diftitox treatment in patients with persistent or recurrent CTCL (Chin, et al., 2006). Denileukin diftitox (Ontak) is a cutaneous T-cell lymphoma (CTCL) therapy that targets the high-affinity interleukin-2 (IL-2) receptor. The IL-2 receptor may exist in a low-affinity form (CD25), an intermediate-affinity form (CD122/CD132), and a high-affinity form (CD25/CD122/CD132). Patients whose malignant cells express the CD25 component of the IL-2 receptor may respond to Ontak therapy. CD 33 CD 33 is used to determine eligibility for gemtuzumab (Mylotarg, anti-CD33) treatment in patients with acute myeloid leukemia (Chen, et al., 2006). Gemtuzumab consists of a recombinant, humanized IgG kappa antibody conjugated to a cytotoxic anti-tumor antibiotic, calicheamicin, which binds specifically to the CD33 antigen. This antigen is found on the surface of leukemic blasts and immature normal cells of myelomonocytic lineage, but not in normal hematopoietic stem cells. CD 52 CD 52 is used to determine eligibility for alemtuzumab (Campath, anti-CD52) treatment in patients with chronic lymphocytic leukemia (Chen, et al., 2006). CD52 is an antigen that can be expressed at high density on the surface of malignant CLL cells. Alemtuzumab is a humanized antibody targeted against CD52 and its binding is necessary for cell death and therapeutic response. CD 117, c-kit CD 117 is used to determine eligibility for treatment with imatinib mesylate in patients with c-kit-positive gastrointestinal stromal tumors (GISTs) (Chen, et al., 2006). The glycoprotein c-kit (CD117) is a member of the receptor tyrosine kinase subclass III family and has been implicated in a number of malignancies. Imatinib mesylate, a tyrosine kinase inhibitor, is effective in treating GISTs and other tumors that express c-kit. Human chorionic gonadotropin (HCG) is normally produced in increasing quantities by the placenta during pregnancy. Accepted guidelines provide that HCG levels can be used to screen for choriocarcinoma in women who are at high risk for the disease, and to monitor the treatment of trophoblastic disease. The literature states that elevated HCG levels may also indicate the presence of cancers of the testis, ovary, liver, stomach, pancreas, and lung. Accepted guidelines provide that alpha fetoprotein (AFP) and b-HCG measurements are valuable for determining prognosis and monitoring therapy in patients with non-seminomatous germ cell cancer. Because of the low incidence of elevated AFP and b-HCG levels in early-stage cancer, the literature suggests these markers have no value in screening for testicular cancer. However, the specificity of these markers is such that when determined simultaneously, at least one marker will be positive in 85% of patients with active cancer. The value of AFP and b-HCG as markers is enhanced by a low frequency of false-positive results and by the chemoresponsiveness of testicular cancer. The literature states that only rarely do patients with other types of cancer have elevated levels of AFP. Non-cancerous conditions that can cause elevated AFP levels include benign liver conditions, such as cirrhosis or hepatitis, ataxia telangiectasia, Wiscott-Aldrich syndrome, and pregnancy. Alpha-fetoprotein (AFP) isa protein that is normally elevated in pregnant women since it is produced by the fetus; however, AFP is not usually found in the blood of adults. In men and in women who are not pregnant, an elevated level of AFP may indicate liver, ovarian or testicular cancer. Alpha-fetoprotein is normally produced by a developing fetus. Alpha fetoprotein levels begin to decrease soon after birth and are usually undetectable in the blood of healthy adults, except during pregnancy. According to accepted guidelines, an elevated level of AFP strongly suggests the presence of either primary liver cancer or germ cell cancer of the ovary or testicle. As AFP is an established test for the diagnosis and monitoring of hepatoma, it is used as a screening tool to rule out the presence of a liver neoplasm before considering liver transplantation. This is especially pertinent in cases (e.g., cirrhosis) where there is an increased risk of developing a primary liver tumor. Elevated serum AFP levels are most closely associated with nonseminomatous testicular cancer and hepatocellular cancer (Chin, 2006). The rate of clearance from serum after treatment is an indicator of the effectiveness of therapy. Conversely, the growth rate of progressive disease can be monitored by serially measuring serum AFP concentrations over time. ER, PR Estrogen receptor (ER) and progesterone receptor (PR) predicts response to hormone therapy for women with advanced breast cancer and those receiving adjuvant treatment, and prognosticates the aggressiveness of a tumor (Chin, 2006). The estrogen receptor and progesterone receptor are intracellular receptors that are measured directly in tumor tissue. These receptors are polypeptides that bind their respective hormones, translocate to the nucleus, and induce specific gene expression. Breast cancers are dependent upon estrogen and/or progesterone for growth and this effect is mediated through ERs and progesterone receptors (ER/PR) (Chin, et al., 2006). Both receptors may be over-expressed in malignant breast tissue. Most oncologists have used the estrogen receptor and also the progesterone receptor not only to predict the probability of response to hormonal therapy at the time of metastatic disease, but also to predict the likelihood of recurrent disease, and to predict the need for adjuvant hormonal therapy or chemotherapy. Although these latter uses for estrogen and progesterone receptors are commonly accepted by most oncologists, the data on which these conclusions are based arecontroversial. Neuron-specific enolase (NSE) has been detected in patients with neuroblastoma, small cell lung cancer, Wilms\' tumor, melanoma, and cancers of the thyroid, kidney, testicle, and pancreas. However, studies of NSE as a tumor marker have concentrated primarily on patients with neuroblastoma and small cell lung cancer. According to the available literature, measurement of NSE level in patients with these diseases cannot be correlated to the extent of the disease, the patient\'s prognosis, or the patient\'s response to treatment because of the poor sensitivity of this marker. LASA is a complex marker that measures the amount of sialic acid in serum and can be elevated in serum from patients with any number of different neoplasms. Elevations in blood LASA levels have been reported in patients with mammary (63 percent), gastroenteric (65 percent), pulmonary (79 percent), and ovarian (94 percent) neoplasms as well as those with leukemia (86 percent), lymphoma (87 percent), melanoma (84 percent), sarcoma (97 percent), and Hodgkin disease (91 percent). As a result, this assay may not have high specificity or sensitivity necessary for cancer detection (Chen, et al., 2006). This serum cancer marker has not been widely accepted for use in the detection or prognosis of colorectal carcinoma. There is no practical information concerning outcome and the use of LASA in the medical literature. Although several articles describe the use of LASA in the diagnosis of colorectal cancer and its association with tumor-node-metastasis (TNM) stage, it has been shown that patients with colorectal polyps and colorectal carcinoma both have elevated LASA levels, and that the levels returned to baseline after removal of either polyps or carcinomas. p53 is a tumor suppressor gene on the short arm of chromosome 17 that encodes a protein that is important in the regulation of cell division. Although the full role of p53 in the normal and neoplastic cell is unknown, there is evidence that the gene product is important in preventing the division of cells containing damaged DNA. p53 gene deletion or mutation is a frequent event along with other molecular abnormalities in colorectal carcinogenesis. The literature on p53 abnormality and prognosis in colorectal cancer suffers from a paucity of reported data and the use of a variety of techniques in assay and statistical analysis in the small numbers of cases analyzed. For these reasons, the literature generally does not recommend p53 analysis as a routine approach to assisting in the management of patients with colorectal cancer. Guidelines from the American Society for Clinical Oncology (2016) recommend against the use of p53 to guide adjuvant chemotherapy in breast cancer. This is a moderate-strength recommendation based upon intermediate-quality evidence. Zap-70 Zeta-chain-associated protein kinase 70, which is used as a prognostic marker in (CLL). Zap-70 is indicated to assess prognosis and need for aggressive therapy in patients with chronic lymphocytic leukemia (CLL) (Chin, et al., 2006). ZAP-70 is a 70-kD member of the Syk family of protein tyrosine kinases. It is expressed primarily in T-cells and natural killer (NK) cells and is critical for signal transduction following T-cell receptor engagement. In CLL B-cells, elevated ZAP-70 expression appears to predict the need for therapy as effectively as IgVH mutation status. Although ZAP-70 expression is strongly correlated with IgVH mutation status, the combination of the two markers may provide greater prognostic value than either marker alone. Positive ZAP-70 results predict an aggressive disease course. The serine protease urokinase-type plasminogen activator (uPA) and its primary inhibitor, plasminogen activator inhibitor-1 (PAI-1), have shown promise for risk assessment and prediction of therapeutic response in primary breast cancer (Chin, et al., 2006). High levels of uPA or PAI-1 in primary tumor tissue are associated with an aggressive disease course and poor prognosis in both node-positive and node-negative breast cancer. A report by the Belgian Healthcare Knowledge Centre (KCE) (San Miguel, et al., 2015)found no studies reporting on the impact of uPA/PAI-1 on clinical management (clinical utility). Guidelines from the American Society for Clinical Oncology (2016) state: \"If a patient has ER/PgR-positive, HER2-negative (node-negative) breast cancer, the clinician may use urokinase plasminogen activator and plasminogen activator inhibitor type 1 to guide decisions on adjuvant systemic therapy.\" This is a weak recommendation based upon high-quality evidence.The ASCO guidelines recommend the use of urokinase plasminogen activator and plasminogen activator inhibitor type 1 to guide decisions on adjuvant systemic therapy in patients with HER2-positive breast cancer or TN breast cancer. IgVh Mutation Status Chronic lymphocytic leukemia (CLL) patients can be divided into two basic groups on the basis of the mutational status of the immunoglobulin heavy-chain variable-region (IgVH) gene in leukemic cells (Chin, 2006). Patients with IgVH mutations have longer survival than those without IgVH mutation. Thus, mutation analysis may be useful for planning management strategies. Kappa / Lambda Light Chain Elevated serum levels of monoclonal free light chains are associated with malignant plasma cell proliferation (e.g., multiple myeloma), primary amyloidosis, and light chain deposition disease (Chen et al, 2006). The appearance of higher levels of free light chains in the urine may be indicative of kidney disease or malignant lymphoproliferative disease such as multiple myeloma. These tests have been used for the detection of multiple myeloma. The ras proto-oncogenes are normal cellular components, which are thought to be important for transduction of signals required for proliferation and differentiation. The ras oncogene family has 3 members: H-ras, K-ras, and N-ras. Ras gene mutations can be found in a variety of tumor types, although the incidence varies greatly. The highest incidences are found in adenocarcinomas of the pancreas (90 %), colon (50 %), and lung (30 %); thyroid tumors (50 %), and myeloid leukemia (30 %). Investigators have established an association between some genotypes of K-ras (KRAS) oncogenes and response to treatment with cetuximab or panitumumab (Lievre et al, 2006and 2008; Di Fiore et al, 2007; Gonçalves et al, 2008; De Roock et al, 2008). Patients whose tumors express specific forms of the KRAS gene exhibit considerably decreased responses to cetuximab and panitumumab. It has been theorized that cetuximab and panitumumab do not target epidermal growth factor receptor (EGFR) associated with these specific KRAS mutations and thus are unable to block their activation. It has been suggested that KRAS genotype be considered as a selection factor for cancer patients who are candidates for treatment with cetuximab or panitumumab. Karapetis and colleagues (2008) stated that treatment with cetuximab improves overall survival (OS) and progression-free survival (PFS) and preserves the quality of life in patients with colorectal cancer that has not responded to chemotherapy. The mutation status of the K-ras gene in the tumor may affect the response to cetuximab and have treatment-independent prognostic value.These investigatorsanalyzed tumor samples, obtained from 394 of 572 patients (68.9 %) with colorectal cancer who were randomly assigned to receive cetuximab plus best supportive care or best supportive care alone, to look for activating mutations in exon 2 of the K-ras gene.They evaluated if the mutation status of the K-ras gene was associated with survival in the cetuximab and supportive-care groups. Of the tumors evaluated for K-ras mutations, 42.3 % had at least one mutation in exon 2 of the gene. The effectiveness of cetuximab was significantly associated with K-ras mutation status (p = 0.01 and p 0.001 for the interaction of K-ras mutation status with OS and PFS, respectively). In patients with wild-type K-ras tumors, treatment with cetuximab as compared with supportive care alone significantly improvedOS (median of9.5 versus 4.8 months; hazard ratio for death, 0.55; 95 % confidence interval [CI], 0.41 to 0.74; p 0.001) and PFS (median of3.7 months versus 1.9 months; hazard ratio for progression or death, 0.40; 95 % CI, 0.30 to 0.54; p 0.001). Among patients with mutated K-ras tumors, there was no significant difference between those who were treated with cetuximab and those who received supportive care alone with respect to OS(hazard ratio, 0.98; p = 0.89) or PFS (hazard ratio, 0.99; p = 0.96). In the group of patients receiving best supportive care alone, the mutation status of the K-ras gene was not significantly associated with OS (hazard ratio for death, 1.01; p = 0.97). The authors concluded that patients with a colorectal tumor bearing mutated K-ras did not benefit from cetuximab, whereas patients with a tumor bearing wild-type K-ras did benefit from cetuximab. The mutation status of the K-ras gene had no influence on survival among patients treated with best supportive care alone. The ASCO\'s provisional clinical opinion ontesting for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-EGFR monoclonal antibody therapy (Allegra et al, 2009) stated that based on systematic reviews of the relevant literature, all patients with metastatic colorectal carcinoma who are candidates for anti-EGFR antibody therapy should have their tumor tested for KRAS mutations in a CLIA-accredited laboratory. If KRAS mutation in codon 12 or 13 is detected, then patients with metastatic colorectal carcinoma should not receive anti-EGFR antibody therapy as part of their treatment. The KRAS oncogene mutation tests are intended to aid in the formulation of treatment decisions for patients who may be candidates for treatment of metastatic epithelial cancers with anti-EGFR therapies such as cetuximab or panitumumab. Several tests for KRAS mutation are currently available in the United States; however, at this time, no KRAS genotype test kits have been approved by the FDA. At the 2008 Annual Meeting of the American Society of Clinical Oncology (ASCO), data on 540 patients with metastatic colorectal cancer in the randomized, phase III CRYSTAL trial were presented. Among 192 patients with KRAS mutations, there was no improvement in overall responses or PFS from the addition of cetuximab to standard chemotherapy. In the patients with normal KRAS, the 1-year PFS rate was 43 % for patients receiving cetuximab versus 25 % for those receiving only standard chemotherapy, and the overall response rate was 59 % versus 43 %, respectively (van Cutsem, 2008). Also at the 2008 ASCO meeting, data from 233 metastatic colorectal cancer patients were presented that confirmed the correlation of KRAS status with patient response to anti-EGFR therapy. No benefit was found after addition of cetuximab to standard chemotherapy with FOLFOX (the combination of fluorouracil, leucovorin, and oxaliplatin) in patients with a mutated KRAS; however, addition of cetuximab to FOLFOX increased both response rate and PFS in patients with a wild-type (i.e., un-mutated) KRAS gene (Bokemeyer, 2008). Response to panitumumab was correlated to KRAS status in a published phase III trial. A total of 427 patients with metastatic colorectal cancer received either panitumumab or best supportive care. Panitumumab exhibited a 17% response rate among patients with normal KRAS, but 0% response among patients with KRAS mutations (Amado, 2008). A meta-analysis of results from 8 studies involving 817 patients with colorectal cancer found that the presence of KRAS mutation predicted lack of response to treatment with anti-EGFR monoclonal antibodies (e.g., panitumumab or cetuximab), whether as stand-alone therapy or in combination with chemotherapy (Linardou et al, 2008).This analysisalso provided empirical evidence that k-RAS mutations are highly specific negative predictors of response (de-novo resistance) to single-agent EGFR tyrosine-kinase inhibitors in advanced non-small cell lung cancer; and similarly to anti-EGFR monoclonal antibodies alone or in combination with chemotherapy in patients with metastatic colorectal cancer. The Blue Cross and Blue Shield Association (BCBSA, 2008) Technology Evaluation Center Medical Advisory Panel concluded that use of KRAS mutation analysis meets TEC criteria to predict non-response to anti-EGFR monoclonal antibodies cetuximab and panitumumab to treat metastatic colorectal cancer. The TEC assessment found that the evidence is sufficient to conclude that patients with mutated KRAS tumors in the setting of metastatic colorectal cancer do not respond to anti-EGFR monoclonal antibody therapy. The assessment explained that the data show that the clinical benefit of using EGFR inhibitors in treating metastatic colorectal cancer, either as monotherapy or in combination with other treatment regimens, is not seen in patients with KRAS-mutated tumors. The assessment found: \"This data supports knowing a patient\'s tumor mutation status before consideration of use of an EGFR inhibitor in the treatment regimen. Identifying patients whose tumors express mutated KRAS will avoid exposing patients to ineffective drugs, avoid exposure to unnecessary drug toxicities, and expedite the use of the best available alternative therapy.\" Colorectal cancer guidelines from the National Comprehensive Cancer Network (NCCN, 2010) recommend consideration of reflex BRAF testing in patients with wild type KRAS. The NCCN guidelines explain that several small studies suggest that patients with wild-type KRAS and a BRAF mutation are unlikely to respond toanti-EGFR therapies such as cetuximab and panitumumab. The guidelines explain that patients with a known BRAF mutation are unlikely to respond to anti-EGFR antibodies, although the data are somewhat inconsistent. Studies demonstrate that in patients with metastatic colorectal cancer, about 8 percent have mutations in the BRAF gene. Testing for the BRAF V600E mutation is performed by PCR amplification and direct DNA sequence analysis. Ratner et al (2010) stated that ovarian cancer (OC) is the single most deadly form of women\'s cancer, typically presenting as an advanced disease at diagnosis in part due to a lack of known risk factors or genetic markers of risk. The KRAS oncogene and altered levels of the microRNA (miRNA) let-7 are associated with an increased risk of developing solid tumors. In this study, these researchers investigated a hypothesized association between an increased risk of OC and a variant allele of KRAS at rs61764370, referred to as the KRAS-variant, which disrupts a let-7 miRNA binding site in this oncogene. Specimens obtained were tested for the presence of the KRAS-variant from non-selected OC patients in 3 independent cohorts, 2 independent ovarian case-control studies, and OC patients with hereditary breast and ovarian cancer syndrome (HBOC) as well as their family members. The results indicated that the KRAS-variant is associated with more than 25 % of non-selected OC cases. Furthermore, these researchers found that it is a marker for a significant increased risk of developing OC, as confirmed by 2 independent case-control analyses. Lastly, they determined that the KRAS-variant was present in 61 % of HBOC patients without BRCA1 or BRCA2 mutations, previously considered uninformative, as well as in their family members with cancer. These findings supported the hypothesis that the KRAS-variant is a genetic marker for increased risk of developing OC, and they suggested that the KRAS-variant may be a new genetic marker of cancer risk for HBOC families without other known genetic abnormalities. Hollestelle et al (2011) noted that recently, a variant allele in the 3\'UTR of the KRAS gene (rs61764370 T G) was shown to be associated with an increased risk for developing non-small cell lung cancer, as well as OC, and was most enriched inOC patients from HBOC families. This functional variant has been shown to disrupt a let-7 miRNA binding site leading to increased expression of KRAS in vitro. In the current study, these investigators genotyped this KRAS-variant in breast cancer index cases from 268 BRCA1 families, 89 BRCA2 families, 685 non-BRCA1/BRCA2 families, and 797 geographically matched controls. The allele frequency of the KRAS-variant was found to be increased among patients with breast cancer from BRCA1, but not BRCA2 or non-BRCA1/BRCA2 families as compared to controls. As BRCA1 carriers mostly develop ER-negative breast cancers, these researchers also examined the variant allele frequency among indexes from non-BRCA1/BRCA2 families with ER-negative breast cancer. The prevalence of the KRAS-variant was, however, not significantly increased as compared to controls, suggesting that the variant allele not just simply associates with ER-negative breast cancer. Subsequent expansion of the number of BRCA1 carriers with breast cancer by including other family members in addition to the index cases resulted in loss of significance for the association between the variant allele and mutant BRCA1 breast cancer. In this same cohort, the KRAS-variant did not appear to modify breast cancer risk for BRCA1 carriers. More importantly, results from the current study suggested that KRAS-variant frequencies might be increased among BRCA1 carriers, but solid proof requires confirmation in a larger cohort of BRCA1 carriers. Therascreen KRAS RGQ PCR Kit (Qiagen) is intended to detect 7 mutations in codons 12 and 13 of the KRAS gene (Raman, et al., 2013). The kit utilizes two technologies — ARMS and Scorpions — for detection of mutations in real-time PCR. The therascreen KRAS RGQ PCR kit is being developed as a companion diagnostic to aid clinicians, through detection of KRAS mutations, in the identification of patients with metastatic colorectal cancer (mCRC) who are more likely to benefit from cetuximab. PreOvar™ tests (Mira Dx) for the KRAS-variant, and will help identify ovarian cancer patients whose female relatives should also be evaluated for the KRAS-variant (Raman, et al., 2013). PreOvar™ may also help assess the relative risk of developing ovarian cancer for women who have a family history of ovarian cancer without a living proband (ancestor with the disease). The KRAS-Variant is present in 6-10% of the general population and 25% of non-selected women with epithelial ovarian cancer. Additionally, the KRAS-variant was identified in over 60% of Hereditary Breast and Ovarian Cancer (HBOC) patients that were previously classified as \"uninformative,\" or negative for other known genetic markers of ovarian cancer risk. The test determines if KRAS-variant may put someone at increased risk for developing ovarian cancer. The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group (EWG) (2013) found that, for patients with metastatic colorectal cancer (mCRC) who are being considered for treatment with cetuximab or panitumumab, there is convincing evidence to recommend clinical use of KRAS mutation analysis to determine which patients are KRAS mutation positive and therefore unlikely to benefit from these agents before initiation of therapy. The level of certainty of the evidence was deemed high, and the magnitude of net health benefit from avoiding potentially ineffective and harmful treatment, along with promoting more immediate access to what could be the next most effective treatment, is at least moderate. The EWG found insufficient evidence to recommend for or against BRAF V600E testing for the same clinical scenario (EGAPP, 2013). The level of certainty for BRAF V600E testing to guide antiepidermal growth factor receptor (EGFR) therapy was deemed low. The EWG encourages further studies of the potential value of testing in patients with mCRC who were found to have tumors that are wild type (mutation negative) for KRAS to predict responsiveness to therapy. Bladder Cancer: BTA-stat, NMP22, Urovysion, ImmunoCyt In the United States, bladder malignancy is the 4th commonest cancer in men and the 8th commonest in women. Patients usually present with urinary tract symptoms (e.g., gross or microscopic hematuria or irritative voiding symptoms such as frequency, dysuria, and urgency). Evaluations of these patients usually entail voided-urine cytology, cystoscopy, and upper urinary tract imaging such as intravenous pyelography, renal sonography, or retrograde pyelography. Most newly diagnosed bladder cancers are superficial (i.e., not invading beyond the lamina propria on histological examination), and are known as transitional cell carcinoma (TCC). These superficial bladder cancers are usually managed by transurethral resection. However, the literature shows that approximately 50to 75 % of treated TCC recur. Furthermore, 10 to 15 % of TCC progress to muscle-invasive bladder cancer. According to the literature, the prevalence of recurrence after initial treatment as well as the natural history of TCC necessitates long-term follow-up. Following treatment, accepted guidelines provide that patients who have previously been diagnosed with TCC should usually undergo urine cytology/cystoscopy every 3 months in the 1st year, every 6 months in the 2nd year, and once-yearly afterwards. Currently, urine cytology with confirmatory cystoscopy represents the cornerstone for the identification of bladder tumors. However, the subjectivity and low sensitivity of cytology led to the development of several urine-based tests as adjuncts to cytology/cystoscopy for the diagnosis and follow-up of patients with TCC. These tests include the BTA Stat test (Bard Diagnostic, Redmond, WA), the NMP22 test (Matritech, Newton, MA), the Aura-Tek FDP test (PerImmune, Rockville, MD), and the Vysis UroVysion FISH Test (Vysis, Inc., Downers Grove, IL). They are usually objective, qualitative (BTA Stat and Aura-Tek FDP), or quantitative (NMP22, UroVysion), and have higher sensitivity than cytology, but some have lower specificity. So far, no single bladder tumor marker has emerged as the generally accepted test of choice, and none has been established as a screening tool for bladder malignancy. Urine-based markers, such as proteins with increased cancer cell expression or chromosomal abnormalities in the urine, may be detected using a variety of laboratory methods to aid in the management of bladder cancer. The following markers/tests are currently available: Bladder tumor antigen (BTA) (eg, BTA stat and BTA TRAK) Fluorescence immunocytology (eg, ImmunoCyt/uCyt+) Fluorescence in situ hybridization (FISH) (eg, UroVysion) mRNA quantification by RT-qPRC testing (eg, Cxbladder) Nuclear matrix protein 22 (NMP22) (eg, NMP22 BladderChek and Matritech NMP22 Test) Urine-based markers have a role in the detection of bladder cancer recurrence in individuals with a history of bladder cancer and are used adjunctively with urinary cytology and cystoscopy. These tests have also been proposed for bladder cancer screening, diagnosis of bladder cancer in individuals symptomatic of bladder cancer and for the evaluation of hematuria. The UroVysion Bladder Cancer Kit (UroVysion Kit) (Baycare Laboratories) is designed to detect aneuploidy for chromosomes 3, 7, 17, and loss of the 9p21 locus via fluorescence in situ hybridization (FISH) in urine specimens from persons with hematuria suspected of having bladder cancer (Raman, et al., 2013). FISH analysis is used in conjunction with cystoscopy to monitor for recurrence among those with previously diagnosed bladder cancer. FISH analysis is a surveillance tool in established primary and secondary bladder adenocarcinoma. The ImmunoCyt is an immunocytochemistry assay for the detection of tumor cells shed in the urine of patients previously diagnosed with bladder cancer (Chen, et al., 2006). This test is intended to augment the sensitivity of cytology for the detection of tumor cells in the urine of individuals previously diagnosed with bladder cancer. The test has been used for detection of tumor cells in the urine of individuals previously diagnosed with bladder cancer, and foruse in conjunction with cytoscopy as an aid in the management of bladder cancer. Although urine cytology has been shown to be less accurate than urinary biomarker tests, familiarity with the method as well as ease of performance justify the continued routine use of the former by primary care physicians, especially in patients who have no history of bladder malignancy. The urine-based biomarker tests have been shown to be accurate in detecting low-grade bladder tumors. In particular, these tests may be of help in deciding the need for further diagnostic assessment of patients with a history of bladder cancer and negative results on urine cytology. For example, elevated levels of urinary bladder tumor markers in patients with a history of TCC may warrant earlier, rather than delayed, cystoscopic examination. On the other hand, consideration may be given to lengthening the intervals between cystoscopic investigations when values of these tumor markers are normal. An assessment by the Adelaide Health Technology Assessment (Mundy Hiller,2009)concluded that theNMP BladderCheck and UroVysion FISH assay, designed for the detection of bladder cancer in high risk patients, have poor sensitivity and poor positive predictive values.The assessmentrecommended that these assays notbe used in asymptomatic patients. The assessmentsuggested, however, that these tests may be useful in the monitoring of patients with transitional cell carcinoma between cytoscopies.The AHTArecommended that this technology not be assessed further. An assessment prepared for the Agency for Healthcare Research and Quality (Meleth, et al., 2014) found:\"Although UroVysion is marketed as a diagnostic rather than a prognostic test, limited evidence from two small studies (total N=168) rated as low or medium risk of bias supported associations between test result and prognosis for risk of recurrence. We found no studies that directly assessed the impact of a test of interest on both physician decision-making and downstream health outcomes to establish clinical utility. We attempted to construct an indirect chain of evidence to answer the overarching question, but we were unable to do so. Even in the cases where the tests seemed to add value in determining prognosis (i.e., evidence of clinical validity), we found no evidence that using the test was related to improved outcomes for patients.\" The American Urologic Association’s guideline on \"Diagnosis, evaluation and follow-up of asymptomatic microhematuria (AMH) in adults\" (Davis et al, 2012) stated that \"The use of urine cytology and urine markers (Nuclear Matrix Protein 22 [NMP22], bladder tumor antigen [BTA]-stat, and UroVysion fluorescence in situ hybridization assay [FISH]) is not recommended as a part of the routine evaluation of the asymptomatic microhematuria patient\". Chou et al (2015) systematically reviewed the evidence on the accuracy of urinary biomarkers for diagnosis of bladder cancer in adults who have signs or symptoms of the disease or are undergoing surveillance for recurrent disease. Data sources included Ovid MEDLINE (January 1990 through June 2015), Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and reference lists. A total of 57 studies that evaluated the diagnostic accuracy of quantitative or qualitative nuclear matrix protein 22 (NMP22), qualitative or quantitative bladder tumor antigen (BTA), FISH, fluorescent immunohistochemistry (ImmunoCyt [Scimedx]), and Cxbladder (Pacific Edge Diagnostics USA) using cystoscopy and histopathology as the reference standard met inclusion criteria; case-control studies were excluded. Dual extraction and quality assessment of individual studies were carried out; overall strength of evidence (SOE) was also assessed. Across biomarkers, sensitivities ranged from 0.57 to 0.82 and specificities ranged from 0.74 to 0.88. Positive likelihood ratios ranged from 2.52 to 5.53, and negative likelihood ratios ranged from 0.21 to 0.48 (moderate SOE for quantitative NMP22, qualitative BTA, FISH, and ImmunoCyt; low SOE for others). For some biomarkers, sensitivity was higher for initial diagnosis of bladder cancer than for diagnosis of recurrence. Sensitivity increased with higher tumor stage or grade. Studies that directly compared the accuracy of quantitative NMP22 and qualitative BTA found no differences in diagnostic accuracy (moderate SOE); head-to-head studies of other biomarkers were limited. Urinary biomarkers plus cytologic evaluation were more sensitive than biomarkers alone but missed about 10 % of bladder cancer cases. The authors concluded that urinary biomarkers miss a substantial proportion of patients with bladder cancer and are subject to false-positive results in others; accuracy is poor for low-stage and low-grade tumors. They stated that research is needed to understand how the use of these biomarkers with other diagnostic tests affect the use of cystoscopy and clinical outcomes. In an editorial that accompanied the afore-mentioned study, Abbosh and Plimack (2015) stated that \"Until urinary biomarkers become available that are sufficiently accurate to supplant the current recommended detection algorithms in biomarker-negative patients, they will not be a cost-effective addition to strategies to detect bladder cancer\". In summary, urine-based bladder tumor marker tests have been shown to be useful as an adjunct to urine cytology and cystoscopy in monitoring for recurrences of bladder cancer, but according to the available literature should not be used as a screening tool for bladder malignancy. The U.S. Preventive Services Task Force (USPSTF, 2004) has concluded that the potential harms of screening for bladder cancer using available tests, such as microscopic urinalysis, urine dipstick, urine cytology, or such new tests as bladder tumor antigen (BTA) or nuclear matrix protein (NMP22) immunoassay, outweigh any potential benefits. Cxbladder O\'Sullivan and colleagues (2012) examined if the RNA assay uRNA® and its derivative Cxbladder® have greater sensitivity for the detection of bladder cancer than cytology, NMP22™ BladderChek™ and NMP22™ ELISA, and whether they are useful in risk stratification. A total of 485 patients presenting with gross hematuria but without a history of urothelial cancer were recruited prospectively from 11 urology clinics in Australasia. Voided urine samples were obtained before cystoscopy. The sensitivity and specificity of the RNA tests were compared to cytology and the NMP22 assays using cystoscopy as the reference. The ability of Cxbladder to distinguish between low grade, stage Ta urothelial carcinoma and more advanced urothelial carcinoma was also determined. uRNA detected 41 of 66 urothelial carcinoma cases (62.1 % sensitivity, 95 % confidence interval [CI]: 49.3 to 73.8) compared with NMP22 ELISA (50.0 %, 95 % CI: 37.4 to 62.6), BladderChek (37.9 %, 95 % CI: 26.2 to 50.7) and cytology (56.1 %, 95 % CI: 43.8 to 68.3). Cxbladder, which was developed on the study data, detected 82 %, including 97 % of the high grade tumors and 100 % of tumors stage 1 or greater. The cut-offs for uRNA and Cxbladder were pre-specified to give a specificity of 85 %. The specificity of cytology was 94.5 % (95 % CI: 91.9 to 96.5), NMP22 ELISA 88.0 %, (95 % CI: 84.6 to 91.0) and BladderChek 96.4 % (95 % CI: 94.2 to 98.0). Cxbladder distinguished between low-grade Ta tumors and other detected urothelial carcinoma with a sensitivity of 91 % and a specificity of 90 %. The authors concluded that uRNA and Cxbladder showed improved sensitivity for the detection of urothelial carcinoma compared to the NMP22 assays. Stratification with Cxbladder provides a potential method to prioritize patients for the management of waiting lists. An UpToDate review on \"Clinical presentation, diagnosis, and staging of bladder cancer\" (Lotan and Choueiri, 2013) does not mention the use of mRNA biomarkers/PCR testing as a management tool for bladder cancer. Furthermore, NCCN’s clinical practice guideline on \"Bladder cancer\" (Version 1.2014) does not mention the use of mRNA biomarkers/PCR testing as a management tool for bladder cancer. An assessment of urinary biomarkers for diagnosis of bladder cancer prepared for the Agency for Healthcare Research and Quality (Chou, et al., 2016) identified only one study of Cxbladdermeeting inclusion criteria, graded as moderate quality, with an overall strength of evidence of \"low,\" OncotypeDx Breast Oncotype Dx (Genomic Health, Inc., Redwood City, CA) is a diagnostic laboratory-developed assay that quantifies the likelihood of breast cancer recurrence in women with newly diagnosed, stage I or II, node negative, estrogen receptor positive breast cancer, who will be treated with tamoxifen. The assay analyzes the expression of a panel of 21 genes, and is intended for use in conjunction with other conventional methods of breast cancer analysis. Together with staging, grading, and other tumor marker analyses, Oncotype Dx is intended to provide greater insight into the likelihood of systemic disease recurrence. Clinical studies have evaluated the prognostic significance of the Oncotype Dx multigene assay in breast cancer (Paik et al, 2004; Esteva et al, 2003). Oncotype Dxanalyses the patterns of 21 genes is being applied as a quantification tool for likelihood of breast cancer recurrence within 10 years of newly diagnosed, stage I or II, lymph node-negative, hormone receptor-positive breast cancer in women who will be treated with tamoxifen (Raman, et a., 2013). Oncotype is being applied as a quantification tool for likelihood of breast cancer recurrence in 10 years in women with newly diagnosed breast cancer. It is also intended to assist in making decisions regarding adjuvant chemotherapy based on recurrence likelihood. There currently is a lack of evidence from prospective clinical studies of the impact of this test on the management of women with breast cancer demonstrating improvements in clinical outcomes (Lopez, et al., 2010; Romeo, et al., 2010; Tiwana, et al., 2013; IETS, 2013), Bastand Hortobagyi (2004) commented that \"[b]efore use of the recurrence score [from the Oncotype Dx multigene assay] is applied to general patient care, however, additional studies are needed.\" The National Cancer Institute is sponsoring a prospective, randomized controlled clinical study, the TAILORx study, using the Oncotype Dx assay to help identify a group of patients with a mid-range risk of recurrence to determine whether treating patients with hormonal therapy only is equivalent to treating them with hormonal therapy in combination with adjuvant chemotherapy. However, there is indirect evidence of the clinical utility of the Oncotype Dx. Paik et al (2006) used banked tumor samples from previous clinical studies of tamoxifen and adjuvant chemotherapy in early breast cancer to assess the performance of the Oncotype Dx multigene assay in predicting response to adjuvant chemotherapy. These investigators examined tumor samples from subjects enrolled in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B20 trial to determine whether there is a correlation between the recurrence score (RS) determined by Oncotype Dx in tumor samples and subsequent response to adjuvant chemotherapy. A total of 651 patients were assessable (227 randomly assigned to tamoxifen and 424 randomly assigned to tamoxifen plus chemotherapy). The test for interaction between chemotherapy treatment and RS was statistically significant (p = 0.038). Patients with high-RS (RS greater than or equal to 31) tumors (ie, high risk of recurrence) had a large benefit from chemotherapy (relative risk, 0.26; 95% confidence interval 0.13 to 0.53; absolute decrease in 10-year distant recurrence rate: mean, 27.6%; standard error, 8.0%). Patients with low-RS (less than 18) tumors derived minimal, if any, benefit from chemotherapy treatment (relative risk, 1.31; 95% confidence interval, 0.46 to 3.78; absolute decrease in distant recurrence rate at 10 years: mean, -1.1%; standard error, 2.2%). The investigators found that patients with intermediate-RS tumors did not appear to have a large benefit, but the investigators concluded that the uncertainty in the estimate cannot exclude a clinically important benefit. One limitation of the study by Paik et al (2006) is that the NASBP B20 trial was conducted before the advent of important advances in breast cancer chemotherapy, including the introduction of trastuzumab (Herceptin), which has been demonstrated to improve overall and disease-free survival in breast cancer patients with HER2 positive tumors. Current guidelines recommend the use of trastuzumab adjuvant chemotherapy in women with metastatic HER2 positive breast cancer, and women with HER2 positive nonmetastatic breast cancers 1 cm or more in diameter. Thus, the Oncotype Dx score would not influence the decision to use adjuvant trastuzumab in women with HER2 positive tumors 1 cm or more in diameter. Commenting on an early report of this study by Paik et al, of the Oncotype Dx presented in abstract form, the BlueCross BlueShield Association Technology Evaluation Center assessment stated that \"additional studies in different populations are needed to confirm whether risk prediction is sufficiently accurate for physicians and patients to choose with confidence whether to withhold adjuvant chemotherapy.\" An international consensusgroup (Azim, et al., 2013)found the available evidence on the analytical and clinical validity of Oncotype Dx Breastto be convincing. However, neither the Oncotype Dx or none of the other genomic tests the evaluateddemonstrated robust evidence of clinical utility: they stated that it was not clear from the current evidence that modifying treatment decisions based on the results of a given genomic test could result in improving clinical outcome. The selection criteria for the TailorRx prospective trial of OncotypeDx state that candidates should have negative axillary nodes as determined by a sentinel lymph node biopsy and/or axillary dissection as defined by the American Joint Committee on Cancer6th Edition Staging System (NCI, 2009).The American Joint Committee on Cancer (AJCC) 6th Edition criteria redefined isolated tumor cells as node negative (the prior version of the criteria, AJCC 5th Edition,classified isolated tumor cells as node positive).\"Isolated tumor cells (single cells or cell deposits) will now be defined as tumor cell deposits no larger than 0.2 mm in diameter that may or may not (but usually do not) show histologic evidence of malignant activity. Pending further information, isolated tumor cells will be classified as node-negative, because it is believed that the unknown benefits of providing treatment for these small lesions would not outweigh the morbidity caused by the treatment itself.\" (Singletary, et al., 2002). However, the banked tumor samples used in the study by Paik, et al. (2006) to validate the OncotypeDx were classified based on AJCC 5th Ed. criteria. In addition, there is new evidence demonstrating that women with isolated tumor cells are at a significantly increased risk of breast cancer. Investigators from theNetherlandsfound anassociationbetween isolated tumor cells and micrometastases in regional lymph nodesand clinical outcome of breast cancer (de Boer, et al., 2009). These investigators identified all patients in the Netherlands who underwent a sentinel-node biopsy for breast cancer before 2006 and had breast cancer with favorable primary-tumor characteristics and isolated tumor cells or micrometastases in the regional lymph nodes. Patients with node-negative diseasewere randomly selected from the years 2000 and 2001. The primary end point was disease-free survival.The investigators identified 856 patients with node-negative disease who had not received systemic adjuvant therapy (the node-negative, no-adjuvant-therapy cohort), 856 patients with isolated tumor cells or micrometastases who had not received systemic adjuvant therapy (the node-positive, no-adjuvant-therapy cohort), and 995 patients with isolated tumor cells or micrometastases who had received such treatment (the node-positive, adjuvant-therapy cohort). The median follow-up was 5.1 years. The adjusted hazard ratio for disease events among patients with isolated tumor cells who did not receive systemic therapy, as compared with women with node-negative disease, was 1.50 (95% confidence interval [CI], 1.15 to 1.94); among patients with micrometastases, the adjusted hazard ratio was 1.56 (95% CI, 1.15 to 2.13). Among patients with isolated tumor cells or micrometastases, the adjusted hazard ratio was 0.57 (95% CI, 0.45 to 0.73) in the node-positive, adjuvant-therapy cohort, as compared with the node-positive, no-adjuvant-therapy cohort. The investigators concluded that isolated tumor cells or micrometastases in regional lymph nodes were associated with a reduced 5-year rate of disease-free survival among women with favorable early-stage breast cancer who did not receive adjuvant therapy. In patients with isolated tumor cells or micrometastases who received adjuvant therapy, disease-free survival was improved. The Medical Advisory Panel of the BlueCross BlueShield Association Technology Evaluation Center (BCBCA, 2014) concluded that use of Oncotype DX to determine recurrence risk for deciding whether to undergo adjuvant chemotherapy in women with unilateral, nonfixed, hormone receptor‒positive, lymph node‒negative breast cancer who will receive hormonal therapy meets the Blue Cross and Blue Shield Association Technology Evaluation Center (TEC) criteria. A technology assessment by the BlueCross BlueShield Association (2014)stated: \"Technical performance of the assay is well documented and is unlikely to be a major source of variability; rather, tissue sampling is likely the greatest source of variability. Retrospective epidemiologic analyses indicated strong, independent associations between Oncotype DX recurrence score (RS) result and distant disease recurrence or death from breast cancer. The evidence identified a subset of conventionally classified, high-risk patients who are at sufficiently low risk of recurrence by Oncotype DX that they might reasonably decide that the harms (toxicity) of chemotherapy outweigh the very small absolute benefit. Two studies of the original validation data, in which conventionally classified patients were reclassified by Oncotype DX result, indicated that the test provides significant recurrence risk information in addition to conventional criteria for individual patient risk classification. Additional evidence indicated that Oncotype DX results are significantly associated with breast cancer death in a community-based patient population, and that RS high-risk patients benefit from chemotherapy, whereas benefits for other RS categories were not statistically significant. Thus, the evidence was judged sufficient to permit conclusions regarding probable health outcomes.\" The Oncotype Dx has also been promoted for use in women with node-positive, ER-positivebreast cancer. An assessment by the BlueCross BlueShield Association (2010)concluded that it has not yet been demonstrated whether use of the Oncotype Dxfor selecting adjuvant chemotherapy in patients with lymph-node-positive breast cancer improves health outcomes. The report explained that theevidence for not selecting chemotherapy for women with low RS values is based on low event rates and wide confidence intervals that include the possibility of benefit from chemotherapy. Because the data allow for a possible benefit of chemotherapy in patients with low RS results, it is unknown if health outcomes would be improved, the same, or worse, if chemotherapy was withheld in these women. The report stated that, due to the lack of clear and sufficient information, there is a need for a second, confirmatory study.The report stated that the Fred Hutchinson Cancer Research Center will conduct a nationwide, NCI-sponsored,Phase III clinical trial to determine the predictive ability of the Oncotype Dx to identify which patients with lymph-node-positive breast cancer will benefit from chemotherapy. The clinical evidence base for OncotypeDX is considered to be the most robust. There was some evidence on the impact of the test on decision-making and to support the case that OncotypeDX predicts chemotherapy benefit; however, few studies were UK based and limitations in relation to study design were identified. OncotypeDX has a more robust evidence base, but further evidence on its impact on decision-making in the UK and the predictive ability of the test in an estrogen receptor positive (ER+), lymph node negative (LN-), human epidermal growth factor receptor 2 negative (HER2-) population receiving current drug regimens is needed. Guidelines from the National Comprehensive Cancer Network (NCCN, 2015) state that \"the 21-gene RT-PCR assay recurrence score can be considered in select patients with 1-3 involved ipsilateral axillary lymph nodes to guide the addition of combination chemotherapy to standard hormone therapy. A retrospective analysis of a prospective randomized trial suggests that the test is predictive in this group similar to its performance in node-negative disease.\" The NCCN guidelines (2015) explained:\"Unplanned, retrospective subset analysis from a single randomized clinical trial in post-menopausal, ALN-positive, ER-positive breast cancer found that the 21-gene RT-PCR assay may provide predictive information for chemotherapy in addition to tamoxifen [citing Albain, et al., 2010]. Patients with a high score in the study benefited from chemotherapy, whereas patients with a low score did not appear to benefit from the addition of chemotherapy regardless of the number of positive lymph nodes. Patient selection for assay use remains controversial.\" \"The RxPONDER trial will confirm the SWOG-8814 trial data for women with ER-positive, node-positive disease treated with endocrine therapy with or without chemotherapy based on risk scores.\" Guidance from the National Institute for Health and Care Excellence (2013) stated: \"Oncotype DX is recommended as an option for guiding adjuvant chemotherapy decisions for people with estrogen ER+, LN- and HER2- early breast cancer if: The person is assessed as being at intermediate risk; and information on the biological features of the cancer provided by Oncotype DX is likely to help in predicting the course of the disease and would therefore help when making the decision about prescribing chemotherapy; and the manufacturer provides Oncotype DX to National Health Service (NHS) organisations according to the confidential arrangement agreed with the National Institute for Health and Care Excellence (NICE). NICE encourages further data collection on the use of Oncotype DX in the NHS.\" An assessment by the Belgian Healthcare Knowledge Center (KCE)(San Miguel, et al., 2015) concluded that \"the evidence for Oncotype DX is more robust than the evidence for other tests.\" TheKCEReport noted, however, thatimportant evidence gaps are still present.TheKCE review mostlyidentified studies supporting the prognostic ability (clinical validity) of the test.The KCE judged thesestudies to be of moderate to high quality.The KCE foundno prospective studies reporting on the impact of Oncotype DX on long-term outcomes such as overall survival, while four studies indicated that Oncotype DX leads to changes in decision making. The KCE identifiedtwo studies on the predictive benefit of the test,one forlymph nodepatients. The KCE reported also noted that the first evidence relating to improvements in quality of life and reductions in patient anxiety as a result of using the test has been reported, but this is based on small patient numbers and further evidence is required. Guidelines from the American Society for Clinical Oncology (2016) state: \"If a patient has ER/PgR-positive, HER2-negative (node-negative) breast cancer, the clinician may use the 21-gene recurrence score (RS; Oncotype DX; Genomic Health, Redwood City, CA) to guide decisions on adjuvant systemic chemotherapy.\" This is a strong recommendation based upon high quality evidence. The ASCO guidelines recommend against OncotypeDx Breast to guide decisions on adjuvant systemic chemotherapy for patients with ER/PgR-positive, HER2-negative (node-positive) breast cancer.The guidelines also recommend against the use of OncotypeDx Breast in women with HER2-positive breast cancer or TN breast cancer. The guidelines recommended against the use ofOncotypeDx Breastto guide decisions on extended endocrine therapy forpatients with ER/PgR-positive, HER-2 negative (node-negative) breast cancer who have had 5 years of endocrine therapy without evidence of recurrence. Acceptance of 21-gene recurrence score assays as tools for clinical decision making in women or men with early stage breast cancer is controversial due to the lack of prospective validation studies, nevertheless, 2007 guidelines from an expert panel convened by ASCO on tumor markers in breast cancer concluded that multiparameter gene expression analysis (i.e., Oncotype Dx assay) can be used to predict the risk of recurrence in women with newly diagnosed, node-negative, ER-positive breast cancer. Although it is reasonable to consider the use of a 21-gene recurrence score assay in males, none of the data generated to date have been in men with breast cancer (Gradishar, 2010). A 2009 abstract that looked at cases of male breast cancer (BC) with Oncotype Dx, concluded, \"This large genomic study of male BC reveals a heterogeneous biology as measured by the standardized quantitative oncotype Dx breast cancer assay, similar to that observed in female BC. Some differences, which may reflect the differences in hormone biology between males and females, were noted and deserve further study.\" (Shak et al, 2009). MammaPrint MammaPrint a 70-gene profile that classifies breast cancer into Low Risk or High Risk of recurrence, by measuring genes representative of all the pathways of cancer metastases which were selected for their predictive relationship to 10-year recurrence probability (Raman, et al., 2013). MammaPrint is indicated for women who have stage I or II breast cancer, are lymph node positive or negative, are ER-positive or negative and tumor size of less than five centimeters. MammaPrint determines if the patient is a candidate for chemotherapy. In February 2007, the Food and Drug Administration (FDA) approved Mammaprint (Agendia, Amsterdam), a DNA microarray-based test used to predict whether women with early breast cancer might face the disease again. The test measures the activity of 70 genes, providing information about the likelihood that cancer will recur. It measures each of these genes in a sample of a woman\'s breast-cancer tumor and then uses a specific formula to produce a score that determines if the patient is deemed low-risk or high-risk for metastasis. In clinical trials, 1 in 4 women found to be at high risk by Mammaprint had recurrence of their cancer within 5 years. However, there are questions regarding the accuracy of this test. The positive predictive values at 5 and 10 years were 23 % and 29 %, respectively, while the corresponding negative predictive values were 95 % and 90 %, respectively. Mammaprint was tested on 307 patients under the age of 61 years who underwent surgery for stage I or stage II breast cancer, and who have tumor size equal to or less than 5 cm, and lymph node-negative. The study found that Mammaprint more than doubled physicians\' ability to predict breast cancer recurrence. Cardoso et al (2016)conducted a study to evaluate the clinical utility of the 70-gene signature test (MammaPrint). The study was exerpted from a phase III randomized trial. In this study, of 6693 enrolled women with early stage breast cancer, women with low clinical and genomic risk did not receive chemotherapy whereas those at high risk did receive chemotherapy. All study subjects had their genomic risk evaluated using MammaPrint. The authors noted that \"the primary goal was to assess whether, among patients with high-risk clinical features and a low-risk-gene-expression profile who did not receive chemotherapy, the lower boundary of the 95% confidence interval for the rate of 5-year survival without distant metastasis would be 92% (i.e. the noninferiority boundary, or higher). The number of women found to be at high clinical risk and low genomic risk was 1550. In this group, the 5 year survival rate without distant metastases was 94.7% among those not receiving chemotherapy. The authors concluded that among women with early-stage-breast cancer who were at high clinical risk and low genomic risk for recurrence, the receipt of chemotherapy on the basis of the 60 gene signature led to a 5-year survival rate without distant metastasis that was 1.5 percentage points lower than the rate with chemotherapy. Acomment by Hudis and Dickler (2016) stated that it can be challenging to convince practitioners that chemotherapy is not need in an otherwise healthy younger population. They further noted that the primary aim of the study on one study of a 70-gene signature test was to \"declare non-inferiority against a predefined benchmark of a 5 year metastasis-free survival rate in just one cohort: patients with a high clinical risk for whom a discordant low genomic risk led to the omission of otherwise standard chemotherapy.\" They concluded that although for select patients providers may wish to use the MammaPrint, the actions they will take as a result of this testing will be variable and may over time change as a result of further study. The study by Cardoso et al (2016) was a 5-year median follow-up results of the MINDACT trial, which is to follow subjects for 10 years. The authors noted that follow-up is ongoing to determine whether their findings remain valid for longer-term outcome. These investigators noted that \"In the critical group of patients at high clinical risk and low genomic risk, the use of adjuvant chemotherapy led to a trend toward a higher rate of the 5-year outcome than that with no chemotherapy, which included a rate of survival without distant metastasis that was 1.5 percentage points higher, a rate of disease-free survival that was 2.8 percentage points higher, and a rate of overall survival that was 1.4 percentage points higher with chemotherapy than with no chemotherapy in the intention-to-treat population and a rate of survival without distant metastasis that was 1.9 percentage points higher, a rate of disease-free survival that was 3 percentage points higher, and a rate of overall survival that was 1.5 percentage points higher with chemotherapy than with no chemotherapy in the per-protocol population. The study was not powered to assess the statistical significance of these differences. Some 50 % of the study patients were defined as being at low clinical risk. In this group, we did not find any meaningful difference in the 5-year rate of survival without distant metastasis between patients at high genomic risk who received chemotherapy and those who did not receive chemotherapy. On the basis of these data, the results for the 70-gene signature do not provide evidence for making recommendations regarding chemotherapy for patients at low clinical risk\". In an editorial that accompanied the afore-mentioned study, Hudis and Dickler (2016) stated that \"a difference of 1.5 percentage points, if real, might mean more to one patient than to another. Thus, the stated difference does not precisely exclude a benefit that clinicians and patients might find meaningful. An adequately powered randomization or a higher threshold for 5-year metastasis-free survival might have provided a more convincing result but would have raised other major challenges for the investigators\". A focused update by the American Society for Clinical Oncology (ASCO) (Kopp, et al., 2017) states that If a patient has hormone receptor–positive, human epidermal growth factor receptor 2 (HER2)–negative, node-negative breast cancer, the MammaPrint assay may be used in those with high clinical risk to inform decisions on withholding adjuvant systemic chemotherapy due to its ability to identify a good-prognosis population with potentially limited chemotherapy benefit.The guidelines state that, if a patient has hormone receptor–positive, HER2-negative, node-positive breast cancer, the MammaPrint assay may be used in patients with one to three positive nodes and a high clinical risk to inform decisions on withholding adjuvant systemic chemotherapy. However, such patients should be informed that a benefit from chemotherapy cannot be excluded, particularly in patients with greater than one involved lymph node.The guideline update was based upon an assessmentof data on clinical utility from the MINDACT trial plus other published literature. BluePrint Molecular subtyping profile or BluePrint is proposed for the evaluation of an individual’s prognosis when diagnosed with breast cancer. The multigene profile classifies breast cancer into basal type, luminal type and ERBB type (HER2/neu positive) molecular subclasses to stratify an individual’s risk to purportedly assist with treatment decisions. Agendia BluePrint has an 80-gene profile that classifies breast cancer into molecular subtypes (Raman, et al., 2013). The profile separates tumors into Basal-type, Luminal-type and ERBB2-type subgroups by measuring the functionality of downstream genes for each of these molecular pathways to inform the physician of the potential effect of adjuvant therapy. Krijgsman et al (2012) noted that classification of breast cancer into molecular subtypes maybe important for the proper selection of therapy, as tumors with seemingly similar histopathological features can have strikingly different clinical outcomes. Herein, these researchers reported the development of a molecular subtyping profile (BluePrint), which enables rationalization in patient selection for either chemotherapy or endocrine therapy prescription. An 80-Gene Molecular Subtyping Profile (BluePrint) was developed using 200 breast cancer patient specimens and confirmed on 4 independent validation cohorts (n = 784). Additionally, the profile was tested as a predictor of chemotherapy response in 133 breast cancer patients, treated with T/FAC neoadjuvant chemotherapy. BluePrint classification of a patient cohort that was treated with neoadjuvant chemotherapy (n = 133) shows improved distribution of pathological Complete Response (pCR), among molecular subgroups compared with local pathology: 56 % of the patients had a pCR in the Basal-type subgroup, 3 % in the MammaPrint low-risk, luminal-type subgroup, 11 % in the MammaPrint high-risk, luminal-type subgroup, and 50 % in the HER2-type subgroup. The group of genes identifying luminal-type breast cancer is highly enriched for genes having an Estrogen Receptor binding site proximal to the promoter-region, suggesting that these genes are direct targets of the Estrogen Receptor. Implementation of this profile may improve the clinical management of breast cancer patients, by enabling the selection of patients who are most likely to benefit from either chemotherapy or from endocrine therapy. An assessment by the National Institute for Health Research (Ward, et al., 2013) found the evidence for Blueprint was limited. Because of the limited available data identified forthis test,the NIHR was unable to draw firm conclusions about its analytical validity, clinical validity (prognostic ability) and clinical utility. The report stated that further evidence on the prognostic and predictive ability ofthis test was required. A report by the Belgian Healthcare Knowledge Centre (KCE) (San Miguel, et al., 2015) found thatlimited evidence for the prognostic ability (clinical validity) ofBluePrint. The KCE foundinsufficient evidence onthe impact of BluePrint on clinical management (clinical utility). Furthermore, there is no information regarding BluePrint/molecular subtyping from NCCN’s clinical practice guideline on \"Breast cancer\" (Version 2.2013). TargetPrint TargetPrint®, ER/PR/HER2 Expression Assay (Agendia) is a microarray-based gene expression test which offers a quantitative assessment of the patient’s level of estrogen receptor (ER), progesterone receptor (PR) and HER2/neu overexpression within her breast cancer (Raman, et al., 2013). TargetPrint is offered in conjunction with MammaPrint to provide the physician an even more complete basis for treatment decisions. TargetPrint delivers an added benefit to the diagnostic process. Immunohistochemistry provides a semi-quantitative positive or negative result, whereas the gene expression result provided by TargetPrint allows physicians to integrate the absolute level of ER, PR and HER2 gene expression into treatment planning. TargetPrint determines if the patient is a candidate for hormonal therapy. TargetPrint is a microarray-based gene expression test which offers a quantitative assessment of the patient’s level of estrogen receptor (ER), progesterone receptor (PR) and HER2/neu overexpression in breast cancer. The manufacturer states that TargetPrint is offered in conjunction with MammaPrint gene expression profiling to provide the physician an even more complete basis for treatment decisions. The manufacturer states that, as compared to Immunohistochemistry (IHC), TargetPrint provides additional information. Whereas IHC provides a semi-quantitative positive or negative result, the gene expression result provided by TargetPrint provides data on the absolute level of ER, PR and HER2 gene expression. Published information on the TargetPrint is limited to studies examining its correlation with measurements of ER, PR, and HER2 receptors (Gunven et al, 2011; Gevensleben et al, 2010; Roepman et al, 2009). There is a lack of evidence from published prospective clinical studies that demonstrates that quantification of ER, PR, and HER2 gene expression by TargetPrint alters management such that clinical outcomes are improved. Symphony Symphony(Agendia) provides complete tumor profiling and is used to support therapeutic choices for breast cancer (Raman, 2013). SYMPHONY includes four assays to support breast cancer treatment decisions: MammaPrint® determines the risk of recurrence. BluePrint™ determines molecular subtypes and TargetPrint® determines estrogen receptor (ER), progesterone receptor (PR), and HER2 status. TheraPrint™ identifies alternative types of therapy for metastatic disease. SYMPHONY provides genomic information assisting with therapeutic decisions even for cases that have been otherwise classified as indeterminate, such as grade 2, small tumors, HER2 and/or lymph node positive. MammaPrint® determines if the patient is a candidate for chemotherapy. TargetPrint® determines if the patient is a candidate for hormonal therapy. BluePrint® provides information on the sub-classification of the tumor which guides the choice of therapies and combinations of therapies. TheraPrint® identifies alternative types of therapy for metastatic disease. Rotterdam Signature 76-Gene Panel The Rotterdam Signature test (Veridex) is a 76-gene expression assay (Raman, 2013). Sixty genes are intended to evaluate estrogen-receptor positive samples and 16 genes to evaluate estrogen-receptor negative samples. In a validation study that tested the signature on samples from 148 women, 50 fell into the low-risk group and 98 into the high-risk group. The test had 88% specificity and 39% sensitivity for the low-risk group, with a hazard ratio for distant relapse within 5 years of 5.74 comparing the high-risk group to the low-risk group. The Rotterdam Signature identifies women at high and low risk of disease recurrence. The Rotterdam Signature 76-gene panel (Veridex, LLC) is a multivariate index assay that is intended to assist in assessing a patient’s risk of systemic recurrence of cancer following successful initial treatment of localized node-negative breast cancer with surgery and tamoxifen alone. This multigene assay is intended for use in lymph-node negative breast cancer patients. The Rotterdam Signature panel uses microarray processing to measure cellular concentrations of mRNA in fresh tissue samples. The Rotterdam Signature panel uses the Human Genome U133a GeneChip (Affymetrix, Inc.) to identify patients that have gene expression signatures associated with either a low or high risk of developing metastatic disease. A multicenter study investigated the ability of the Rotterdam 76-gene signature to identify patients at risk of distant metastases within 5 and 10 years of first diagnosis, using frozen tissue samples from 180 patients with node-negative breast cancer who had not received systemic chemotherapy (Foekens, et al., 2006). The Rotterdam 76-gene signature correctly identified 27 out of 30 cases of relapse within 5 years (90% sensitivity) and 75 out of 150 patients who did not relapse (50% specificity). An earlier summary of the same study (Foekens, et al., 2005) reported a hazard ratio for distant metastasis-free survival comparing favorable versus unfavorable signature = 7.41 (95% confidence interval 2.63-20.9); p = 8.5 x 10-6). The hazard ratio of overall survival comparing favorable versus unfavorable signature = 5.45 (95% confidence interval 1.62-18.3); p = .002. There are no published studies that have assessed the clinical utility of the Rotterdam 76-gene signature by monitoring the long-term outcomes of the patients selected and not selected for chemotherapy on the basis of assay results. Breast Cancer Gene Expression Ratio / Breast Cancer Index The Breast Cancer Gene Expression Ratio (HOXB13:IL17BR, also known as H/I) (AviaraDx, Inc., Carlsbad, CA) is intended to predict the risk of disease recurrence in women with estrogen receptor (ER)-positive, lymph node-negative breast cancer. The Breast Cancer Gene Expression Ratio is based on the ratio of the expression of two genes: the homeobox gene-B13 (HOXB13) and the interleukin- 17B receptor gene (IL17BR). In breast cancers that are more likely to recur, the HOXB13 gene tends to be over-expressed, while the IL-17BR gene tends to be under-expressed. Ma et al (2004) reported on the early validation of theHOXB13:IL17BR gene expression ratio. The investigatorsgenerated gene expression profiles of hormone receptor-positive primary breast cancers in a set of 60 patients treated with adjuvant tamoxifen monotherapy. An expression signature predictive of disease-free survival was reduced to a two-gene ratio, HOXB13 versus IL17BR, which outperformed existing biomarkers. The investigators concluded that ectopic expression of HOXB13 in MCF10A breast epithelial cells enhances motility and invasion in vitro, and its expression is increased in both preinvasive and invasive primary breast cancer. The investigators suggested thatHOXB13:IL17BR expression ratio may be useful for identifying patients appropriate for alternative therapeutic regimens in early-stage breast cancer. In an 852-patient retrospective study, Ma, et al (2006) found that the HOXB13:IL17BR ratio (H:I expression ratio) independently predicted breast cancer recurrence in patients with ER-positive, lymph-node negative cancer. The H:I expression ratio was found to be predictive in patients who received tamoxifen therapy as well as in those who did not. Expression of HOXB13, IL17BR, CHDH, estrogen receptor (ER) and progesterone receptor (PR) were quantified by real-time polymerase chain reaction (PCR) in 852 formalin-fixed, paraffin-embedded primary breast cancers from 566 untreated and 286 tamoxifen-treated breast cancer patients. Gene expression and clinical variables were analyzed for association with relapse-free survival (RFS) by Cox proportional hazards regression models. The investigators reported that, in the entire cohort, expression of HOXB13 was associated with shorter RFS (p = .008), and expression of IL17BR and CHDH was associated with longer RFS (p 0.0001 for IL17BR and p = 0.0002 for CHDH). In ER-positive patients, the HOXB13:IL17BR index predicted clinical outcome independently of treatment, but more strongly in node-negative patients. In multivariate analysis of the ER-positive node-negative subgroup including age, PR status, tumor size, S phase fraction, and tamoxifen treatment, the two-gene index remained a significant predictor of RFS (hazard ratio [HR] = 3.9; 95 % CI:1.5 to 10.3; p = .007). Thevalue of the Breast Cancer Gene Expression Ratio was also evaluated in a study by Goetz et al (2006). That study found that a high H:I expression ratio is associated with an increased rate of relapse and mortality in ER-positive, lymph node-negative cancer patients treated with surgery and tamoxifen. Goetz et al (2006) examined the association between the ratio of the HOXB13 to IL17BR expression and the clinical outcomes of relapse and survival in women with ER-positive breast cancer enrolled onto a North Central Cancer Treatment Group adjuvant tamoxifen trial (NCCTG 89-30-52). Tumor blocks were obtained from 211 of 256 eligible patients, and quantitative reverse transcription-PCR profiles for HOXB13 and IL-17BR were obtained from 206 patients. In the node-positive cohort (n = 86), the HOXB13/IL-17BR ratio was not associated with relapse or survival. In contrast, in the node-negative cohort (n = 130), a high HOXB13/IL-17BR ratio was associated with significantly worse RFS [HR, 1.98; p = 0.031], disease-free survival (DFS) (HR, 2.03; p = 0.015), and OS (HR, 2.4; p = 0.014), independent of standard prognostic markers. The Blue Cross and Blue Shield Association Technology Evaluation Center (BCBSA, 2007) announced that its Medical Advisory Panel (MAP) concluded that the use of the Breast Cancer Gene Expression Ratio gene expression profiling does not meet the TEC criteria. The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group(2009) found insufficient evidence to make a recommendation for or against the use of the H:I ratio test to improve outcomes in defined populations of women with breast cancer.EGAPP concluded that theevidence is insufficient to assess the balance of benefits and harms of the proposed uses of this test. The EWG encouraged further development and evaluation of these technologies. In a systematic review on gene expression profiling assays in early-stage breast cancer, Marchionni, et al. (2008) summarized evidence on the validity and utility of 3 gene expression-based prognostic breast cancer tests: Oncotype Dx, MammaPrint, and H/I. The authors concluded that gene expression technologies show great promise to improve predictions of prognosis and treatment benefit for women with early-stage breast cancer. However, more information is needed on the extent of improvement in prediction, characteristics of women in whom the tests should be used, and how best to incorporate test results into decision making about breast cancer treatment. Guidelines from the American Society for Clinical Oncology (Harris, et al., 2007) found that, in newly diagnosed patients with node-negative, estrogen-receptor positive breast cancer, the Oncotype Dx assay can be used to predict the risk of recurrence in patients treated with tamoxifen. The ASCO guidelines concluded that Oncotype Dx may be used to identify patients who are predicted to obtain the most therapeutic benefit from adjuvant tamoxifen and may not require adjuvant chemotherapy. The ASCO guidelines found, in addition, that patients with high recurrence scores appear to achieve relatively more benefit from adjuvant chemotherapy than from tamoxifen. ASCO found that there are insufficient data at present to comment on whether these conclusions generalize to hormonal therapies other than tamoxifen, or whether this assay applies to other chemotherapy regimens. Guidelines from the American Society for Clinical Oncology (Harris, et al., 2007) concluded that the precise clinical utility and appropriate application for other multiparameter assays, such as the MammaPrint assay, the Rotterdam Signature, and the Breast Cancer Gene Expression Ratio are under investigation. ASCO also found insufficient data to recommend use of proteomic patterns for management of patients with breast cancer. Sgori et al (2013) found that, in the absence of extended letrozole therapy, high H/I identifies a subgroup of ER-positive patients disease-free after 5 years of tamoxifen who are at risk for late recurrence. The investigators also found that, when extended endocrine therapy with letrozole is prescribed, high H/I predicts benefit from therapy and a decreased probability of late disease recurrence. Sgori, et al. conducted aprospective-retrospective, nested case-control design of 83 recurrences matched to 166 nonrecurrences from letrozole- and placebo-treated patients within MA.17 trial. Expression of H/I within primary tumors was determined by reverse-transcription polymerase chain reaction with a prespecified cutpoint. The investigators determined thepredictive ability of H/I for ascertaining benefit from letrozole using multivariable conditional logistic regression including standard clinicopathological factors as covariates. All statistical tests were two-sided. The investigators reported thathigh H/I was statistically significantly associated with a decrease in late recurrence in patients receiving extended letrozole therapy (odds ratio [OR] = 0.35; 95% confidence interval [CI] = 0.16 to 0.75; P = .007). In an adjusted model with standard clinicopathological factors, high H/I remained statistically significantly associated with patient benefit from letrozole (OR = 0.33; 95% CI = 0.15 to 0.73; P = .006). Reduction in the absolute risk of recurrence at 5 years was 16.5% for patients with high H/I (P = .007). The interaction between H/I and letrozole treatment was statistically significant (P = .03). BioTheranostics Breast Cancer Index (BCI) is a prognostic biomarker that provides quantitative assessment of the likelihood of distant recurrence in patients diagnosed with estrogen receptor-positive, lymph node-negative breast cancer (Raman, et al., 2013). In development and validation studies, BCI stratified about 50% of tamoxifen treated ER+, node-negative breast cancer patients into a low risk group for 10-year distant recurrence. BCI is a molecular assay developed from the combination of two indices: HOXB13:IL17BR and five cell cycle-associate gene index (BUB1B, CENPA, NEK2, RACGAP1, RRM2) that assesses tumor grade. The test is performed on a formalin-fixed, paraffin-embedded (FFPE) tissue block. Ma et al (2008) reported on the development and early validation of a five-gene reverse transcription PCR assay for molecular grade index (MGI) that has subsequently been incorporated into BCI and is suitable for analyzing routine formalin-fixed paraffin-embedded clinical samples. The investigators found that the combination of MGI and HOXB13:IL17BR outperformed either alone and identifies a subgroup (approximately 30%) of early stage estrogen receptor-positive breast cancer patients with very poor outcome despite endocrine therapy. From their previously published list of genes whose expression correlates with both tumor grade and tumor stage progression, the investigators selected five cell cycle-related genes to build MGI and evaluated MGI in two publicly available microarray data sets totaling 410 patients. Using two additional cohorts (n =323), the investigators developed a real-time reverse transcription PCR assay for MGI, validated its prognostic utility, and examined its interaction with HOXB13:IL17BR. The investigators reported that MGI performed consistently as a strong prognostic factor and was comparable with a more complex 97-gene genomic grade index in multiple data sets. In patients treated with endocrine therapy, MGI and HOXB13:IL17BR modified each other\'s prognostic performance. High MGI was associated with significantly worse outcome only in combination with high HOXB13:IL17BR, and likewise, high HOXB13:IL17BR was significantly associated with poor outcome only in combination with high MGI. Jerevall et al (2011) reported on the development of the Breast Cancer Index,a dichotomous index combining two gene expression assays, HOXB13:IL17BR (H:I) and molecular grade index (MGI),to assess risk of recurrence in breast cancer patients. The study objective was to demonstrate the prognostic utility of the combined index in early-stage breast cancer. In a blinded retrospective analysis of 588 ER-positive tamoxifen-treated and untreated breast cancer patientsfrom the randomized prospective Stockholm trial which was conducted during 1976 to 1990, H:I and MGI were measured using real-time RT-PCR. Association with patient outcome was evaluated by Kaplan-Meier analysis and Cox proportional hazard regression. A continuous risk index was developed using Cox modelling. The investigators found thatthe dichotomous H:I+MGI was significantly associated with distant recurrence and breast cancer death. The greater than 50% of tamoxifen-treated patients categorized as low-risk had less than 3% 10-year distant recurrence risk. A continuous risk model (Breast Cancer Index (BCI)) was developed with the tamoxifen-treated group and the prognostic performance tested in the untreated group was 53% of patients categorized as low risk with an 8.3% 10-year distant recurrence risk. Jankowitz et al (2011) reported on a study tovalidate the prognostic performance of BCI in estrogen-receptor positive, lymph node negative breast cancer patients. Theinvestigators found that, inthis characteristically low-risk cohort, BCI classified high versus low-risk groups with about a five-fold difference in 10-year risk of distant recurrence and breast cancer-specific death. Theinvestigators identified tumor samples from 265estrogen-receptor positive, lymph-node negativetamoxifen-treated patientsfrom a single academic institution\'s cancer research registry. They performed theBCI assayand assignedscoresbased on a predetermined risk model. The investigators assessed riskby BCI and Adjuvant Online! (AO) and correlated these to clinical outcomes in the patient cohort. The investigators found thatBCI was a significant predictor of outcome inthis cohort of estrogen-receptor positive, lymph-node negative patients (median age: 56-y; median follow-up: 10.3-y), treated with adjuvant tamoxifen alone or tamoxifen with chemotherapy (32%). BCI categorized 55%, 21%, and 24% of patients as low, intermediate and high-risk, respectively. The 10-year rates of distant recurrence were 6.6%, 12.1% and 31.9% and of breast cancer-specific mortality were 3.8%, 3.6% and 22.1% in low, intermediate, and high-risk groups, respectively. In a multivariate analysis including clinicopathological factors, BCI was a significant predictor of distant recurrence (HR for 5-unit increase = 5.32 [CI 2.18-13.01; P = 0.0002]) and breast cancer-specific mortality (HR for a 5-unit increase = 9.60 [CI 3.20-28.80; P 0.0001]). AO was significantly associated with risk of recurrence. In a separate multivariate analysis, both BCI and AO were significantly predictive of outcome. In a time-dependent (10-year) ROC curve accuracy analysis of recurrence risk, the addition of BCI and AO increased predictive accuracy in all patients from 66% (AO only) to 76% (AO+BCI) and in tamoxifen-only treated patients from 65% to 81%. The authors concluded that BCI and AO are independent predictors with BCI having additive utility beyond standard of care parameters that are encompassed in AO. The authors acknowledge that this study is limited by the fact that it was a retrospective, single-institution study and that results may have been biased on the basis of specimen availability and patterns of referral to the tertiary academic center. Mathieu et al (2012) assessed the performance of BCI topredict chemosensitivity based on pathological complete response (pCR) and breast conservation surgery (BCS). The authors performed the BCI assay ontumor samples from 150 breast cancer patients from a single institutiontreated with neoadjuvant chemotherapy. The authorsused logistical regression and c-index to assess predictive strength and additive accuracy of BCI beyond clinicopathologic factors. BCI classified 42% of patients as low, 35% as intermediate and 23% as high risk. Low BCI risk group had 98.4% negative predictive value (NPV) for pCR and 86% NPV for BCS. High versus low BCI group had a 34 and 5.8 greater likelihood of achieving pCR and BCS, respectively (P=0.0055; P=0.0022). BCI increased c-index for pCR (0.875-0.924; p=0.017) and BCS prediction (0.788-0.843; p=0.027) beyond clinicopathologic factors. The authors concluded thatBCI significantly predicted pCR and BCS beyondclinicopathologic factors. High NPVs indicate that BCI could be a useful tool to identify breast cancer patients who are not eligible for neoadjuvant chemotherapy. The authors concluded that \"these results suggest that BCI could be used to assess both chemosensitivity and eligibility for BCS.\" The authors stated that an important limitation of this study is that, in this retrospective analysis, patients were not selected based on ER or HER2 expression for the indications of neoadjuvant chemotherapy.The authors explained that thiscould have increased the predictive strength of BCI given that this biomarker was initially developed and validated in ER + node-negative patients Zhang et al (2013)examined the prognostic performance ofBCI for prediction of early (0-5 years) and late (more than 5 years) risk of distant recurrence in patients with estrogen receptor-positive (ER(+)), lymph node-negative (LN(-)) tumors. The BCI model was validated by retrospective analyses of tumor samples from tamoxifen-treated patients from a randomized prospective trial (Stockholm TAM, n = 317) and a multi-institutional cohort (n = 358). Within the Stockholm TAM cohort, BCI risk groups stratified the majority (approximately 65%) of patients as low risk with less than 3% distant recurrence rate for 0 to 5 years and 5 to 10 years. In the multi-institutional cohort, which had larger tumors, 55% of patients were classified as BCI low risk with less than 5% distant recurrence rate for 0 to 5 years and 5 to 10 years.Zhang and colleagues found that, for both cohorts, continuous BCI was the most significant prognostic factor beyond standard clinicopathologic factors for 0 to 5 years and more than five years. The authors concluded thatthe prognostic sustainability of BCI to assess early- and late-distant recurrence risk at diagnosis has clinical use for decisions of chemotherapy at diagnosis and for decisions for extended adjuvant endocrine therapy beyond five years. Sgori et al (2013) compared the prognostic ability of the BCI assay,the Oncotype DX Breast, and IHC4for both early and late recurrence in patients with estrogen-receptor-positive, node-negative (N0) disease who took part in the Arimidex, Tamoxifen, Alone or in Combination (ATAC) clinical trial. Inthis prospective comparison study,Sgori and colleaguesobtained archival tumor blocks from the TransATAC tissue bank from all postmenopausal patients with estrogen-receptor-positive breast cancer from whom theOncotype DX and IHC4 values had already been derived.The investigatorsdid BCI analysis in matched samples with sufficient residual RNA using two BCI models -- cubic (BCI-C) and linear (BCI-L)-using previously validated cutoffs.The prospectively-defined primary study objective was to evaluate overall (0–10y) prognostic performance of the BCI-C model for DR in ER+ N0 patients. Secondary objectives were: 1) assessment of the prognostic performance of the BCI-L model and its components, H/I and MGI, for overall (0–10y), early (0–5y) and late (5–10y) DR; 2) comparative performance of BCI-L versus the Oncotype DX RS and IHC4.To assess the ability of the biomarkers to predict recurrence beyond standard clinicopathological variables,the investigatorscalculated the change in the likelihood-ratio from Cox proportional hazards models.Suitable tissue was available from 665 patients with estrogen-receptor-positive, N0 breast cancer for BCI analysis. The primary analysis showed significant differences in risk of distant recurrence over 10 years in the categorical BCI-C risk groups (p 0·0001) with 6·8% (95% CI 4·4-10·0) of patients in the low-risk group, 17·3% (12·0-24·7) in the intermediate group, and 22·2% (15·3-31·5) in the high-risk group having distant recurrence. BCI-C analyzed as a continuous variable was not significantly associated with overall (0–10y) risk of DR when adjusted for CTS (inter-quartile HR=1·39; 95% CI, 0·99 to 3·70; LR-Δχ2=3·70; P=0·054). Comparison of the prognostic performance of BCI-L to BCI-C indicated that unlike BCI-C, BCI-L was a significant predictor of risk of recurrence as a continuous variable, and the HR after adjustment with CTS was 2·19 versus 4.86 between high- and low-risk groups for BCI-C and BCI-L, respectively. Thus, all subsequent analyses were performed utilizing BCI-L. The secondary analysis showed that BCI-L was a much stronger predictor for overall (0-10 year) distant recurrence compared with BCI-C (interquartile HR 2·30 [95% CI 1·62-3·27]; likelihood ratio (LR)-Δχ(2)=22·69; p 0·0001). When compared with BCI-L, theOncotype Dx breast score was less predictive (HR 1·48 [95% CI 1·22-1·78]; LR-Δχ(2)=13·68; p=0·0002) and IHC4 was similar (HR 1·69 [95% CI 1·51-2·56]; LR-Δχ(2)=22·83; p 0·0001). All further analyses were done with the BCI-L model. In a multivariable analysis, all assays had significant prognostic ability for early distant recurrence (BCI-L HR 2·77 [95% CI 1·63-4·70], LR-Δχ(2)=15·42, p 0·0001;Oncotype Dx Breastscore HR 1·80 [1·42-2·29], LR-Δχ(2)=18·48, p 0·0001; IHC4 HR 2·90 [2·01-4·18], LR-Δχ(2)=29·14, p 0·0001); however, only BCI-L was significant for late distant recurrence (BCI-L HR 1·95 [95% CI 1·22-3·14], LR-Δχ(2)=7·97, p=0·0048; 21-gene recurrence score HR 1·13 [0·82-1·56], LR-Δχ(2)=0·48, p=0·47; IHC4 HR 1·30 [0·88-1·94], LR-Δχ(2)=1·59, p=0·20). The authors concluded thatBCI-L was the only significant prognostic test for risk of both early and late distant recurrence and identified two risk populations for each timeframe.BCI-L could help to identify patients at high risk for late distant recurrence who might benefit from extended endocrine or other therapy. An important limitationis that the evaluation of BCI-L was a secondary objective of this study; the primary objective was evaluation of BCI-C. Aneditorial (Ignatiadis, 2013) accompanying the study by Sgroi, et al. stated that the BCI test is \"ready for prime time\" in treatment decision making for post-menopausal, estrogen-receptor positive women who have undergone 5 years of hormonal therapy. The editorial noted that there are other molecular diagnostic assays that also have been shown to predict late recurrence. For support, the editorial cited a study by Sestak, et al. (2013),which found that, in the last follow-up phase, Clinical Treatment Score (CTS) added most prognostic information for distant recurrence in years 5 to 10 forbreast cancer patientsin the ATAC trial.Sestak, et al.reported that, in amultivariate modelthat incorporated CTS,PAM50provided the strongest additional prognostic factorin the5 to 10 yearfollowup phase, followed by BCI, and with IHC4 and RS adding the leastprognostic information. A manufacturer funded study (Gustavsen, et al., 2014)reported ona model that foundBCIto becost savingfrom a third-party payer perspective, based upon assumptions about the impact of BCI on adjuvant chemotherapy use, extended endocrine therapy use, and endocrine therapy compliance.The authors developed two economic models to project the cost-effectiveness of BCI in a hypothetical population of patients with estrogen-receptor positive, lymph-node negative breast cancer compared withstandard clinicopathologic diagnostic modalities. The authors modeled costs associated withadjuvant chemotherapy, toxicity, followup, endocrine therapy, and recurrence over 10 years. The models examined cost utility compared with standard practice when used at diagnosis and in patients disease-free at 5 years post diagnosis. The authors reported thatuse of BCI was projected to be cost saving in both models. In the newly diagnosed population, net cost savings were $3803 per patient tested. In the 5 years post diagnosis population, BCI was projected to yield a net cost savings of $1803 per patient tested. The authors reported that sensitivity analyses demonstrated that BCI was cost saving across a wide range of clinically relevant input assumptions. Preliminary data suggest that molecular approaches including gene expression platforms such asBCI may add to classical clinical parameters including tumor size and node status at diagnosis, but further research is needed(Smith, et al., 2014; Bianchini Gianni, 2013; Ignatiadis and Sotiriou, 2013). The clinical utility of BCI and other molecular diagnostics inpredicting late recurrencehas yet to be established (Foukakis and Bergh, 2015).Italso remains to beestablished which of several molecular diagnostic tests in development are the most appropriate for detecting late recurrence (Sestak Kuzick, 2015). An assessment by the National Institute for Health Research (Ward, et al., 2013) found that, based on the limited available data, no firm conclusions can be drawn about the analytical validity, clinical validity (prognostic ability) and clinical utility of the Breast Cancer Index. The assessment stated that further evidence on the prognostic and predictive ability ofthis testis required. An assessment by IETS (2013) and a consensus statement (Azim, et al., 2013) reached similar conclusions. An assessment by the BlueCross BlueShield Association (2015) concluded that the evidence is insufficient to permit conclusionsabout the Breast Cancer Index on health outcomes. Although evidence supports the association of risk classes defined by the Breast Cancer Index and recurrence and survival outcomes, it remains to be shown whether the Breast Cancer Index adds incremental prognostic information to standard clinical risk classifiers. An assessment by the Belgian Healthcare Knowledge Centre (KCE) (San Miguel, et al.,2015) found that the evidence for the H/I ratio assay is limited to studies supporting the prognostic ability (clinical validity) of the test. They found insufficient evidence for theimpact of the H/I ratio assay on clinical management (clinical utility). A review published in the ASCO Educational Book (Smith, et al., 2014) reviewed the BCI and other currently available molecular diagnostics for selecting and determining the optimal duration of endocrine adjuvant therapy in women with early stage estrogen receptor positivebreast cancer:\"Further research into applying molecular features and gene expression scores to standard clinico-pathologic criteria for tailoring extended endocrine therapy is now a high priority.... An important research challenge is now to identify which patients are likely to benefit from this type of long-term therapy. Preliminary data suggest that molecular approaches including gene expression platforms such as ROR may add to classical clinical parameters including tumor size and node status at diagnosis.\" A Palmetto Medicare Local Coverage Determination (LCD) allows coverage of the Breast Cancer Index in certainpost-menopausalwomen with estrogen-receptor positive breast cancer, reasoning thatthe data defined benefit of the BCI test appears to be when a woman is having significant side effects or has other concerns regarding adjuvant tamoxifen therapy and is opposed to taking more than 5 years of tamoxifen or starting on an AI (letrazole) after tamoxifen (CMS, 2014).The LCD noted, however, that,there is anincrease in recurrence risk with increasing BCI score such that, \"at the 95% confidence interval (CI), the risk in some individuals categorized in the BCI-low group could be as high as 20%. Due to the data complexity, there is a significant possibility that a physician might consider all BCI-L patients at negligible risk, and thus not consider extended hormone therapy and consequently lead women from the NCCN recommended interventions. Given the low toxicity and low cost of extended therapy, the false sense of security could deny many women from lifesaving therapy.\" There is a lack of consensus among guidelines regarding the value of molecular assays in determining whether longer durations of adjuvant endocrine therapy beyond 5 yearsare clinically indicated.Guidelines from the American Society for Clinical Oncology (Burstein, et al., 2014) on adjuvant endocrine therapy for hormone-receptor positive breast cancerstate:\"Well-established clinical factors including tumor size; nodal status; ER, PgR, and HER2 biomarkers; and molecular diagnosticassays serve as prognostic factors for breast cancer recurrence. However, there are no robust specific clinical or biomarker measures that selectively predict early versus late recurrence, nor predict whether tamoxifen or AI therapy would be appropriate treatment, nor determine whether longer durations of adjuvant endocrine therapy are clinically indicated.\" The National Comprehensive Cancer Networkguidelines for breast cancer version 2, 2015 states: \"Multiple other multi-gene mor multi-gene expression assay systems have been developed. These systems are generally based upon small, retrospective studies, and the Panel believes that none are currently sufficiently validated to warrant inclusion in the guideline.\" The St. Gallen guideline panel (Coates, et al., 2015)found that Oncotype DX, MammaPrint, PAM-50 ROR score, EndoPredict and the Breast Cancer Index were all considered usefully prognostic for years 1-5, but only the Oncotype Dx commanded a majority in favor of its value in predicting the usefulness of chemotherapy.The Panel agreed thatthe PAM50 ROR score was clearly prognostic beyond five years, and that the Mammaprint was not prognostic beyond 5 years. The Panel was divided about the prognostic value of theBreast Cancer Index, the Oncotype DX, and EndoPredict in this time period. ESMO guidelines (Senkus, et al., 2013) state:\"Molecular signatures for ER-positive breast cancer such as OncotypeDx, EndoPredict, Breast Cancer Index or for all types of breast cancer (pNO-1) such as MammaPrint and Genomic Grade Index are commercially available, but none of them have proven robust clinical utility so far. In some cases of difficult decision, such as grade 2 ER-positive HER-2 negative and node-negative breast cancer, MammaPrint and Oncotype DX may be used in conjunction with all clinicopathological factors, to help in treatment decision-making.\" Guidelines from the American Society for Clinical Oncology (2016) state: \"If a patient has ER/PgR-positive, HER2-negative (node-negative) breast cancer, the clinician may use the Breast Cancer Index to guide decisions on adjuvant systemic therapy.\" This is a moderate strength recommendation based upon intermediate quality evidence. ASCO guidelines recommend use of the Breast Cancer Index to guide decisions on adjuvant systemic therapy in patients with ER/PgR=positive, HER2-negative (node-positive) breast cancer. The guidelines also recommend against the use of the Breast Cancer Index in HER2-positive breast cancer or TN breast cancer. The guidelines also recommended against the use ofThe Breast Cancer Indexto guide decisions on extended endocrine therapy forpatients with ER/PgR-positive, HER-2 negative (node-negative) breast cancer who have had 5 years of endocrine therapy without evidence of recurrence. Mammostrat Mammostrat (Clarient) is a novel test for estimating the risk for recurrence in hormone-receptor positive, early stage breast cancer that is independent of proliferation and grade (Raman, et al., 2013). Five biomarkers are combined with a defined mathematical algorithm resulting in a risk index. Mammostrat is clinically validated and has been studied on more than 4,500 total patients in numerous independent cohorts that include the NSABP B14 and B20 trials. Clinicians and patients are faced with difficult choices as to whether to add toxic adjuvant chemotherapy in addition to standard endocrine treatment. Mammostrat may help clinicians understand the inherent aggressiveness of the tumor and the likelihood of tumor recurrence. The Mammostrat is a prognostic immunohistochemistry (IHC) test that measures the risk of breast cancer recurrence in post-menopausal, node-negative, estrogen receptor-expressing breast cancer patients who will receive hormonal therapy and are considering adjuvant chemotherapy. The test analyzes five monoclonal antibody biomarkers and applies a diagnostic algorithm to assess whether patients have a high, moderate, or low risk of recurrence after they have had their breast cancer tumor surgically removed and have been treated with tamoxifen. Bartlett et al (2010) tested the efficacy of the Mammostrat in a mixed population of cases treated in a single center with breast-conserving surgery and long-term follow-up. Tissue microarrays from a consecutive series of 1,812 women managed by wide local excision and post-operative radiotherapy were collected. Of 1,390 cases stained, 197 received no adjuvant hormonal or chemotherapy, 1,044 received tamoxifen only, and 149 received a combination of hormonal therapy and chemotherapy. Median age at diagnosis was 57 years, 71% were post-menopausal, 23.9% were node-positive and median tumor size was 1.5 cm. Samples were stained using triplicate 0.6 mm2 tissue microarray cores, and positivity for p53, HTF9C, CEACAM5, NDRG1 and SLC7A5 was assessed. Each case was assigned a Mammostrat risk score, and distant recurrence-free survival (DRFS), relapse-free survival (RFS) and overall survival (OS) were analyzed by marker positivity and risk score. Increased Mammostrat scores were significantly associated with reduced DRFS, RFS and OS in ER-positive breast cancer (p 0.00001). In multivariate analyses the risk score was independent of conventional risk factors for DRFS, RFS and OS (p 0.05). In node-negative, tamoxifen-treated patients, 10-year recurrence rates were 7.6+/- 1.5% in the low-risk group versus 20.0+/- 4.4% in the high-risk group. Further, exploratory analyses revealed associations with outcome in both ER-negative and un-treated patients. The authors concluded that the Mammostrat can act as an independent prognostic tool for ER-positive, tamoxifen-treated breast cancer and the results of the study revealed a possible association with outcome regardless of node status and ER-negative tumors. There is insufficient evidence to determine whether the Mammostrat test is better than conventional risk assessment tools in predicting the recurrence of breast cancer. Furthermore, neither NCCN or ASCO have incorporated the test into their guidelines as amanagement tool. Guidance from the National Institute for Health and Clinical Excellence (NICE, 2013) states that the Mammostrat is\"only recommended for use in research in people with ER+, LN− and HER2− early breast cancer, to collect evidence about potentially important clinical outcomes and to determine the ability of the tests to predict the benefit of chemotherapy ... The tests are not recommended for general use in these people because of uncertainty about their overall clinical benefit and consequently their cost effectiveness.\" An assessment by the Belgian Healthcare Knowledge Centre (KCE) (San Miguel, et al., 2015) found that the evidence for Mammostrat is mainly limited to studies supporting the prognostic ability (clinical validity) of the test. The KCE stated that these studies include a large sample size and appear to be of reasonable quality. The KCEcitedone study reporting on clinical utility in terms of the predictive ability of the test by risk group. \"However, further evidence is required.\" Guidelines from the American Society for Clinical Oncology (2016) state: \"If a patient has ER/PgR-positive, HER2-negative (node-positive or node-negative) breast cancer, the clinician should not use the five-protein assay (Mammostrat; Clarient, a GE Healthcare company, Aliso Viejo, CA) to guide decisions on adjuvant systemic therapy.\" This is a moderate strength recommendation based upon intermediate-quality evidence. The ASCO guidelines recommend against the use of Mammostrat toguide decisions on adjuvant systemic therapy for patientswith HER2-positive or TN breast cancer. OvaChek The OvaCheck™ (Correlogic Systems, Inc.) is a proteomic analysis of blood for the early detection of ovarian cancer. A similar test, which involved a different molecular pattern, was the subject of a 2002 study of 216 women with ovarian cancer. That study showed that the proteomic test had a specificity of 100% and a sensitivity of 95%, with a positive predictive value of 94% (Petricoin, et al., 2002). While this study showed that a proteomic test detected ovarian cancers even where CA-125 levels were normal, this study included only women who had been detected with ovarian cancer by other means. There is inadequate evidence that this test will be effective for screening women with undetected ovarian cancer. In addition, there is concern, given the low prevalence of ovarian cancer, that this test is not sufficiently specific for use in screening. The National Cancer Institute explains that even an ovarian cancer test with a specificity of 99% means that 1% of those who did not have cancer would test positive, which is \"far too high a rate for commercial use\" (NCI, 2004). For a rare disease such as ovarian cancer, which has an approximate prevalence of 1 in 2,500 in the general population, a 99% specificity and 100% sensitivity translates into 25 women falsely identified for every one true cancer found. The OvaCheck™ test employs electrospray ionization (ESI) type of mass spectrometry using highly diluted denatured blood samples. This method differs from a matrix-assisted laser desorption ionization (MALDI) analysis of undiluted native sera samples that was used in the Lancet study and is currently under investigation by the National Cancer Institute and Food and Drug Administration (NCI, 2004). The NCI notes that \"[t]he class of molecules analyzed by these two approaches, and thus the molecules that constitute the diagnostic patterns, would be expected to be entirely different.\" Neither the NCI nor FDA has been involved in the design or validation of OvaCheck™ methodology. As the Ovacheck test is performed as a \"home-brewed\" test by two national laboratories instead of as a commercially available kit, FDA approval of the OvaCheck test may not be required. The Society for Gynecologic Oncologists (SGO, 2004) has reviewed the literature regarding OvaCheck and concluded that \"more research is needed to validate the test’s effectiveness before offering it to the public.\" Similarly, the American College of Obstetricians and Gynecologists (2004) has stated that \"more research is needed to validate the test\'s effectiveness before recommending it to the public.\" An assessment of the Ovacheck test and other genomic tests for ovarian cancer prepared for the Agency for Healthcare Research and Quality by the Duke Evidence-Based Practice Center (Myers, et al., 2006) reached the following conclusions: \"Genomic test sensitivity/specificity estimates are limited by small sample sizes, spectrum bias, and unrealistically large prevalence of ovarian cancer; in particular, estimates of positive predictive values derived from most of the studies are substantially higher than would be expected in most screening or diagnostic settings. We found no evidence relevant to the question of the impact of genomic tests on health outcomes in asymptomatic women. Although there is a relatively large literature on the association of test results and various clinical outcomes, the clinical utility of changing management based on these results has not been evaluated.\" Specifically regarding Ovacheck and other proteomic tests for ovarian cancer, the assessment found that, \"[a] lthough all studies reported good discrimination for the particular protein profile studied, there were several recurrent issues that limit the ability to draw inferences about potential clinical applicability,\" in particular technical issues with the assays themselves, variations in analytic methods used among studies, and an unrealistically high prevalence of ovarian cancer in the datasets compared to what would be expected in a normal screening population. OvaSure OvaSure is an ovarian cancer screening test that entails the use of 6 biomarkers (leptin, prolactin, osteopontin, insulin-like growth factor II, macrophage inhibitory factor and CA-125) to assess the presence of early stage ovarian cancer in high-risk women. Visintin et al (2008) characterized and validated the OvaSure for discriminating between disease-free and ovarian cancer patients. These researchers analyzed 362 healthy controls and 156 newly diagnosed ovarian cancer patients. Concentrations of leptin, prolactin, osteopontin, insulin-like growth factor II, macrophage inhibitory factor, and CA-125 were determined using a multiplex, bead-based, immunoassay system. All 6 markers were evaluated in a training set (181 samples from the control group and 113 samples from ovarian cancer patients) and a test set (181 sample control group and 43 ovarian cancer). Multiplex and ELISA exhibited the same pattern of expression for all the biomarkers. None of the biomarkers by themselves was good enough to differentiate healthy versus cancer cells. However, the combination of the 6 markers provided a better differentiation than CA-125. Four models with less than 2% classification error in training sets all had significant improvement (sensitivity 84 % to 98% at specificity 95%) over CA-125 (sensitivity 72% at specificity 95%) in the test set. The chosen model correctly classified 221 out of 224 specimens in the test set, with a classification accuracy of 98.7%. The authors noted that the OvaSure is the first blood biomarker test with a sensitivity of 95.3% and a specificity of 99.4% for the detection of ovarian cancer. Six markers provided a significant improvement over CA-125 alone for ovarian cancer detection. Validation was performed with a blinded cohort. They stated that this novel multiplex platform has the potential for efficient screening in patients who are at high risk for ovarian cancer. However, the Society of Gynecologic Oncologists (SGO, 2008) released an opinion regarding OvaSure, which stated that additional research is needed before the test should be offered to women outside the context of a research study. Moreover, SGO stated that it will \"await the results of further clinical validation of OvaSure with great interest\". Furthermore, according to the FDA’s web site, the FDA sent the Laboratory Corporation of America a warning letter stating that it is illegally marketing OvaSure to detect ovarian cancer. According to the FDA warning letter, their review indicates that this product is a device under section 201(h) of the Food, Drug, and Cosmetic Act (FDCA or Act), 21 U.S.C. 321(h), because it is intended for use in the diagnosis of disease or other conditions, or in the cure, treatment, prevention, or mitigation of disease. The Act requires that manufacturers of devices that are not exempt obtain marketing approval or clearance for their products from the FDA before they may offer them for sale. This helps protect the public health by ensuring that new devices are shown to be both safe and effective or substantially equivalent to other devices already legally marketed in this country for which approval is not required. According to the FDA warning letter, no such determination has been made for OvaSure. NCCN Guidelines Panel Members (NCCN, 2016) believe that the OvaSure screening test should not be used to detect ovarian cancer. The NCCN guidelines explain that the OvaSure test uses 6 biomarkers, including leptin, prolactin, osteopontin, insulin-like growth factor II, macrophae inhibitory factor, and CA-125. Thrombospondin-1 Thrombospondin-1 (THBS-1), an angiogenesis inhibitor, has been identified as a potential monitoring marker in gynecologic malignancies. In a randomized phase III study on the co-expression of angiogenic markers and their associations with prognosis in advanced epithelial ovarian cancer, Secord, et al. (2007) reported that high THBS-1 may be an independent predictor of worse progression-free and overall survival in women with advanced-stage EOC. However, the authors stated, \"A larger prospective study is warranted for validation of these findings.\" Previstage GCC Guanylyl Cyclase C (GCC or GUCY2C) (Diagnocure) a gene coding for a protein found in cells, lining the intestine from the duodenum to the rectum (Raman, et al., 2013). It is involved in water transport, crypt morphology and suppression of tumorigenesis. It is not normally found in tissue in other parts of the body, and therefore, GCC detected outside of the intestine, indicates presence of colorectal cancer metastases. Early studies have indicated that the presence of GCC in the blood may be an early indicator of micrometastases that would otherwise escape detection by the current standard methods of monitoring. Earlier detection provides an opportunity for more immediate treatment or surgical intervention to potentially improve patient outcomes and survival rates. This is a diagnostic test for recurrence by identification of micrometastasis in the blood. Guanyl cyclase C (GCC) is a receptor protein normally expressed in high concentrations on the luminal surface of the gastrointestinal epithelium. Expression of GCC persists on mucosal cells that have undergone malignant transformation. Thus, GCC has potential use as a marker to determine spread of colorectal cancer to lymph nodes. A retrospective study of 21 patients post surgical resection of colorectal cancer found that all 11 of 21 patients who were free of cancer for 5 years or more were negative for GCC in lymph nodes, whereas all 10 of 21 patients whose cancer returned within 3 years of surgery were positive for GCC. However, the value of the GCC marker test in the management of colorectal cancer needs to be evaluated in prospective clinical outcome studies. A large prospective study is currently being conducted to compare standard histological examination of lymph nodes to the GCC marker test. Previstage™ Guanylyl Cyclase C (GCC or GUCY2C) (Diagnocure) is a gene coding for a protein found in cells, lining the intestine from the duodenum to the rectum (Raman, et al., 2013). It is involved in water transport, crypt morphology and suppression of tumorigenesis. It is not normally found in tissue in other parts of the body, and therefore, GCC detected outside of the intestine, indicates presence of colorectal cancer metastases. GCC mRNA has shown to be highly accurate in detecting the spread and recurrence of colorectal cancer, respectively in lymph nodes and blood, thereby representing a significant improvement over traditional detection methods. Previstage is a predictive test for risk stratification of recurrence and prognostic marker for recurrence. Thymidylate Synthase Thymidylate synthase is a DNA synthesis related gene. According to Compton (2008), the prognostic value of this promising and potentially clinically applicable molecular marker has been studied in colorectal cancer. Compton found that the independent influence of this marker on prognosis remains unproven. Compton explained that \"[v]ariability in assay methodology, conflicting results from various studies examining the same factor, and the prevalence of multiple small studies that lack statistically robust, multivariate analyses all contribute to the lack of conclusive data.\" Compton concluded that before this marker can be incorporated into clinically meaningful prognostic stratification systems, more studies are required using multivariate analysis, well-characterized patient populations, reproducible and current methodology, and standardized reagents. In a special report on pharmacogenomics of cancer, the BlueCross and BlueShield Association\'s Technology Evaluation Center (TEC) (2007) described the results of a meta-analysis on thymidylate synthase protein expression and survival in colorectal cancer that stated low thymidylate synthase expression was significantly associated with better survival, but heterogeneity and possible bias prevented firm conclusions. Guidelines from the American Society for Colon and Rectal Surgeons (2004) stated: \"In the future, DNA analysis and the intratumoral expression of specific chemical substances\", including thymidylate synthase, \"may be used routinely to further assess prognosis or response to therapy.\" In addition, Shankaran et al (2008) stated in a review on the role of molecular markers in predicting response to therapy in patients with colorectal cancer, \"Although to date no molecular characteristics have emerged as consistent predictors of response to therapy, retrospective studies have investigated the role of a variety of biomarkers, including microsatellite instability, loss of heterozygosity of 18q, type II transforming growth factor beta receptor, thymidylate synthase, epidermal growth factor receptor, and Kirsten-ras (KRAS).\" Tumour angiogenesis is associated with invasiveness and the metastatic potential of various cancers. Vascular endothelial growth factor (VEGF), the most potent and specific angiogenic factor identified to date, regulates normal and pathologic angiogenesis. An evidence report from Cancer Care Ontario (Welch et al, 2008) on the use of the VEGF inhibitor bevacizumab in colorectal cancer explained that the increased expression of VEGF has been correlated with metastasis, recurrence, and poor prognosis in many cancers, including colorectal cancer. Guidelines from the National Institute for Health and Clinical Excellence (NICE, 2007) explained that bevacizumab (Avastin) is a recombinant humanised monoclonal IgG1 antibody that acts as an angiogenesis inhibitor. It targets the biological activity of VEGF, which stimulates new blood vessel formation in the tumour. However, neither the FDA approved labeling of bevacizumab or evidence-based guidelines recommend measurement of VEGF to diagnose colorectal cancer or to select patients for treatment. In a special report on pharmacogenomics of cancer, the BlueCross and BlueShield Association\'s Technology Evaluation Center (TEC) (2007) stated that pre-treatment VEGF levels do not appear to be predictive of response to anti-angiogenic therapy. Shin and colleagues (2013) evaluated inhibitory effects of bevacizumab on VEGF signaling and tumor growth in-vitro and in-vivo, and assessed phosphorylation of VEGF receptor 2 (VEGFR2) and downstream signaling in endothelial cells as pharmacodynamic markers using phospho-flow cytometry. These researchers also validated markers in patients with mCRC treated with bevacizumab-based chemotherapy. In in-vitro studies, bevacizumab inhibited proliferation of human umbilical vein endothelial cells in association with reduced VEGF signaling. Notably, bevacizumab inhibited VEGF-induced phosphorylation of VEGFR-2, Akt, and extra-cellular signal-regulated kinase (ERK). In-vivo, treatment with bevacizumab inhibited growth of xenografted tumors and attenuated VEGF-induced phosphorylation of Akt and ERK. The median percentages of VEGFR2 + pAkt + and VEGFR2 + pERK + cells, determined by phospho-flow cytometry, were approximately 3-fold higher in mCRC patients than in healthy controls. Bevacizumab treatment decreased VEGFR2 + pAkt + cells in 18 of 24 patients on day 3. The authors concluded that bevacizumab combined with chemotherapy decreased the number of VEGFR2 + pAkt + cells, reflecting impaired VEGFR2 signaling. Together, these data suggested that changes in the proportion of circulating VEGFR2 + pAkt + cells may be a potential pharmacodynamic marker of the effectiveness of anti-angiogenic agents, and could prove valuable in determining drug dosage and administration schedule. ProstatePx Donovan et al (2008) from Aureon, the manufacturer of Prostate Px, reported on the development and validation of their systems pathology model for predicting prostate cancer recurrence after prostatectomy. The clinical utility of defining high risk for failure after radical prostatectomy is to decide whether patients require closer follow-up than average or whether adjuvant radiotherapy, hormone therapy, or chemotherapy would be of benefit. In this analysis, the concordance index for the systems pathology approach used by Aureon was 0.83, but was only slightly better than a 10-variable model that used only the usual clinical parameters, with a concordance index of 0.80. The corresponding hazard ratios for clinical failure were 6.37 for the 10-variable clinical model, and 9.11 for the systems pathology approach. In an accompanying editorial, Klein, et al. (2009) questioned the clinical significance of these differences. They noted that \"[a]lthough the difference in concordance indices was statistically significant, the question is whether there is sufficient clinical relevance to justify the extra effort, expense, and clinical expertise needed for the systems approach ... In contemporary clinical practice, a patient with a hazard ratio of 6.37 generated by the model using easily derived, routinely reported clinical and pathological parameters is just as likely to be a candidate for closer monitoring or adjuvant therapy than one with a hazard ratio of 9.11 generated by the systems approach\". Sutcliffe et al (2009) provided an evidence-based perspective on the prognostic value of novel markers in localized prostate cancer and identified the best prognostic model including the 3 classical markers and investigated if models incorporating novel markers are better. Eight electronic bibliographic databases were searched. The reference lists of relevant articles were checked and various health services research-related resources consulted via the internet. The search was restricted to publications from 1970 onwards in the English language. Selected studies were assessed, data extracted using a standard template, and quality assessed using an adaptation of published criteria. Because of the heterogeneity regarding populations, outcomes and study type, meta-analyses were not undertaken and the results are presented in tabulated format with a narrative synthesis of the results. A total of 30 papers met the inclusion criteria, of which 28 reported on prognostic novel markers and5 on prognostic models. A total of 21 novel markers were identified from the 28 novel marker studies. There was considerable variability in the results reported, the quality of the studies was generally poor and there was a shortage of studies in some categories. The marker with the strongest evidence for its prognostic significance was PSA velocity (or doubling time). There was a particularly strong association between PSA velocity and prostate cancer death in both clinical and pathological models. In the clinical model the hazard ratio for death from prostate cancer was 9.8 (95 % CI 2.8 to 34.3, p 0.001) in men with an annual PSA velocity of more than 2 ng/ml versus an annual PSA velocity of 2 ng/ml or less; similarly, the hazard ratio was 12.8 (95 % CI 3.7 to 43.7, p 0.001) in the pathological model. The quality of the prognostic model studies was adequate and overall better than the quality of the prognostic marker studies. Two issues were poorly dealt with in most or all of the prognostic model studies: inclusion of established markers, and consideration of the possible biases from study attrition. Given the heterogeneity of the models, they can not be considered comparable. Only2 models did not include a novel marker, and1 of these included several demographical and co-morbidity variables to predict all-cause mortality. Only2 models reported a measure of model performance, the C-statistic, and for neither was it calculated in an external data set. It was not possible to assess whether the models that included novel markers performed better than those without. This review highlighted the poor quality and heterogeneity of studies, which render much of the results inconclusive. It also pinpointed the small proportion of models reported in the literature that are based on patient cohorts with a mean or median follow-up of at least 5 years, thus making long-term predictions unreliable. Prostate-specific antigen velocity, however, stood out in terms of the strength of the evidence supporting its prognostic value and the relatively high hazard ratios. There is great interest in PSA velocity as a monitoring tool for active surveillance but there is as yet no consensus on how it should be used and, in particular, what threshold should indicate the need for radical treatment. In an editorial on clinically relevant prognostic markers for prostate cancer, Gelmann and Henshall (2009) stated that \"[u]ntil we have sufficiently discriminating markers to inform treatment decisions, the problem of whom to treat will continue to grow exponentially as the number of cases of screening-detected low-risk cancer increases\". Circulating Tumor Cells (e.g., CellSearch) Circulating tumor cell (CTC) test, CellSearch, is a blood test that has been proposed as a method to determine prognosis, evaluate progression and assess treatment response in individuals with metastatic breast, colorectal and prostate cancers. CTC assays were developed to detect cells that break away from tumors and enter the blood stream. The CellSearch™ Epithelial Cell Kit, along with the CellSpotter™ Analyzer (Veridex, LLC, Warren, NJ) is a device designed to automate the detection and enumeration of circulating tumor cells (CTCs) of epithelial origin (CD45-, EpCAM+, and cytokeratins 8, 18+ and/or 19+) in whole blood in patients with advanced breast cancer (Ellery, et al., 2010; Raman, et al., 2011). It is intended for use in adjunctively monitoring and predicting cancer disease progression and response to therapy. The CellSearch Epithelial Cell Kit received FDA 510(k) clearance on January 21, 2004. The FDA concluded that the device is substantially equivalent to immunomagnetic circulating cancer cell selection and enumeration systems. These devices consist of biological probes, fluorochromes and other reagents, preservation and preparation devices and semi-automated analytical instruments to select and count circulating cancer cells in a prepared sample of whole blood. The CellSearch Epithelial Cell Kit quantifies CTCs by marking cancerous cells with tiny, protein-coated magnetic balls in whole blood. These cells are stained with fluorescent markers for identification and then dispensed into a cartridge for analysis where a strong magnetic field is applied to the mixture causing the magnetically marked cells to move to the cartridge surface. The cartridge is then analyzed by the CellSpotter Analyzer. A medical professional rechecks the CTCs and the CellSpotter Analyzer tallies the final CTC count. In a prospective, multicenter study, Cristofanilli et al (2004) used the CellSearch System on 177 patients with measurable metastatic breast cancer for levels of CTCs both before the patients started a new line of therapy and at follow-up. The progression of the disease or the response to treatment was determined with the use of standard imaging studies at the participating centers every nine to twelve weeks. Outcomes were assessed according to levels of CTCs at baseline, before the patients started a new therapy. In the first test, patients with 5 or more CTCs per 7.5 ml of blood compared to a group with fewer than 5 CTCs had a shorter median progression-free survival (2.7 months vs. 7.0 months) and shorter overall survival (10.1 months vs. greater than 18 months). At the follow-up visit, approximately three to four weeks after the initiation of therapy, the percentage of patients with more than 5 CTC was reduced from 49 percent to 30 percent, suggesting a benefit from therapy. The difference in progression-free survival between the two groups remained consistent (2.1 months for women with 5 or more CTCs vs. 7 months for women with less than 5 CTCs). Overall, survival in the women with more than 5 CTCs was 8.2 months compared to greater than 18 months in the cohort with less than 5 CTCs. Cristofanilli concluded that the number of CTCs before treatment was an independent predictor of progression-free survival and overall survival in patients with metastatic breast cancer. However, Cristofanilli also concluded that the results may not be valid for patients who do not have measurable disease or for those starting a new regimen of hormone therapy, immunotherapy, or both. He states, \"The prognostic implications of an elevated level of circulating tumor cells for patients with metastatic disease who are starting a new treatment may be an opportunity to stratify these patients in investigational studies\". Furthermore, the study did not address whether patients with an elevated number of circulating tumor cells might benefit from other therapies. Thus, this minimally invasive assay requires further evaluation as a prognostic marker of disease progression and response to therapy. The clinical application of quantifying CTCs in the peripheral blood of breast cancer patients remains unclear. Published data in the peer-reviewed medical literature are needed to determine how such measurements would guide treatment decisions and whether these decisions would result in beneficial patient outcomes (Kahn, et al., 2004; Abeloff, et al., 2004). An assessment of CellSearch by AETSA (2006) concluded \"In the current stage of development of this technology, there is no evidence that it provides any advantage over existing technology for CTC identification or indeed any additional clinical use.\" Guidelines from the American Society for Clinical Oncology (Harris, et al., 2007) found: \"The measurement of circulating tumor cells (CTCs) should not be used to make the diagnosis of breast cancer or to influence any treatment decisions in patients with breast cancer. Similarly, the use of the recently U.S. Food and Drug Administration (FDA)-cleared test for CTC (CellSearch Assay) in patients with metastatic breast cancer cannot be recommended until further validation confirms the clinical value of this test.\" An assessment by the Canadian Agency for Drugs and Technologies in Health (CADTH, 2012) found thatstudies indicatethat measurement of CTCs using the CellSearch system could be used as prognostic factors for progression of the disease and the potential treatment of patients with ovarian cancer. No economic studies were identified, therefore the cost-effectiveness of the CellSearch system could not be summarized. Although studies relate circulating tumor cells to prognostic indicators (see, e.g., Cohen, et al., 2008; De Giorgi, et al., 2009), there are a lack of published prospective clinical studies demonstrating that measurement of CTCs alters management such that clinical outcomes are improved. Such clinical outcome studies are currently ongoing. Current guidelines from the National Comprehensive Cancer Network (NCCN) make no recommendations for use of circulating tumor cells. Guidelines from the American Society for Clinical Oncology (2016) state: \"The clinician should not use circulating tumor cells to guide decisions on adjuvant systemic therapy.\" This is a strong recommendation based upon intermediate-quality evidence. Scher et al (2015) noted that clinical trials in castration-resistant prostate cancer (CRPC) need new clinical end-points that are valid surrogates for survival. These researchers evaluated circulating tumor cell (CTC) enumeration as a surrogate outcome measure. Examining CTCs alone and in combination with other biomarkers as a surrogate for OS was a secondary objective of COU-AA-301, a multi-national, randomized, double-blind phase III trial of abiraterone acetate plus prednisone versus prednisone alone in patients with metastatic CRPC previously treated with docetaxel. The biomarkers were measured at baseline and 4, 8, and 12 weeks, with 12 weeks being the primary measure of interest. The Prentice criteria were applied to test candidate biomarkers as surrogates for OS at the individual-patient level. A biomarker panel using CTC count and lactate dehydrogenase (LDH) level was shown to satisfy the 4 Prentice criteria for individual-level surrogacy; 12-week surrogate biomarker data were available for 711 patients. The abiraterone acetate plus prednisone and prednisone-alone groups demonstrated a significant survival difference (p = 0.034); surrogate distribution at 12 weeks differed by treatment (p 0.001); the discriminatory power of the surrogate to predict mortality was high (weighted c-index, 0.81); and adding the surrogate to the model eliminated the treatment effect on survival. Overall, 2-year survival of patients with CTCs less than 5 (low risk) versus patients with CTCs greater than or equal to 5 cells/7.5 ml of blood and LDH greater than 250 U/L (high risk) at 12 weeks was 46 % and 2 %, respectively. The authors concluded that a biomarker panel containing CTC number and LDH level was shown to be a surrogate for survival at the individual-patient level in this trial of abiraterone acetate plus prednisone versus prednisone alone for patients with metastatic CRPC. They stated that independent phase III clinical trials are needed to validate these findings. An assessment from the Institut National d’Excellence en Santé et Services Sociaux (INESSS) (Arsenault Le Blanc, 2016) concluded: \"Based on the scientific literature identified, the use of CellSearch tests as a predictive and prognostic biomarker in patients with early-stage breast cancer is not justified. The evidence is insufficient for establishing a concrete association between the presence of CTCs pre- and posttreatment and patient survival. In the case of patients with metastatic breast cancer, the examination of the scientific literature suggests that CTC enumeration prior to treatment could be a prognostic biomarker for patient survival. Despite the prognostic value of CTC enumeration, based on studies, its clinical utility has yet to be confirmed. For now, CellSearch tests should not be used outside the context of a clinical study. Further studies are needed to determine if the CellSearch test could play a clinically significant role in managing breast cancer patients.\" Her-2/neu Estrogen and progestin receptors are important prognostic markers in breast cancer, and the higher the percentage of overall cells positive as well as the greater the intensity, the better the prognosis. Estrogen and progesterone receptor positivity in breast cancer cells is an indication the patient may be a good candidate for hormone therapy. HER-2/neu is an oncogene. Its gene product, a protein, is over-expressed in approximately 20 to 30% of breast cancers. The over-expressed protein is present in unusually high concentration on the surface of some malignant breast cancer cells, causing these cells to rapidly proliferate. It is important because these tumors are susceptible to treatment with Herceptin (trastuzumab), which specifically binds to this over-expressed protein. Herceptin blocks these protein receptors, inhibiting continued replication and tumor growth. HER2/neu may also be expressed in ovarian, gastric, colorectal, endometrial, lung, bladder, prostate, and salivary gland (Chen, et al., 2006). HER-2/neu is an oncogene encoding a growth factor receptor related to epidermal growth factor receptor (EGFR) and is amplified in approximately 25-30 percent of node-positive breast cancers (Chin, et al. 2006). Overexpression of HER-2/neu is associated with decreased disease-free and overall survival. Over-expression of HER-2/neu may be used to identify patients who may be may benefit from trastuzumab (Herceptin™ ) and/or high dose chemotherapy. Trastuzumab is a humanized monoclonal antibody targeting the HER 2/neu (c-erbB-2) oncogene. Her-2 has been used to: assess prognosis of stage II, node positive breast cancer patients; predict disease-free and overall survival in patients with stage II, node positive breast cancer treated with adjuvant cyclophosphamide, doxorubicin, 5-fluorouracil chemotherapy; and determine patient eligibility for Herceptin treatment (Chen, et al., 2006). TheCollege of American Pathologists (CAP) recommends FISH as an optimal method for HER2/neu testing; therefore, positive IHC results are usually confirmed by FISH testing. There are additional tests that may be used in breast cancer cases, such as DNA ploidy, Ki-67 or other proliferation markers. However, most authorities believe that HER-2/neu, estrogen and progesterone receptor status are the most important to evaluate first. The other tests do not have therapeutic implications and, when compared with grade and stage of the disease, are not independently significant with respect to prognosis. Harris et al (2007) updated ASCO\'s recommendations for the use of tumor marker tests in the prevention, screening, treatment, and surveillance of breast cancer. Thirteen categories of breast tumor markers were considered, 6 of which were new for the guideline. The following categories showed evidence of clinical utility and were recommended for use in practice: CA 15-3, CA 27.29, CEA, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, urokinase plasminogen activator, plasminogen activator inhibitor 1, and certain multi-parameter gene expression assays. Not all applications for these markers were supported, however. The following categories demonstrated insufficient evidence to support routine use in clinical practice: DNA/ploidy by flow cytometry, p53, cathepsin D, cyclin E (fragments or whole length), proteomics, certain multi-parameter assays, detection of bone marrow micrometastases, and circulating tumor cells (e.g., CellSearch assay). These guidelines found present data insufficient to recommend measurement of Ki67, cyclin D, cyclin E, p27, p21, thymidine kinase, topoisomerase II, or other markers of proliferation to assign patients to prognostic groups. The guidelines also found insufficient data to recommend assessment of bone marrow micrometastases for management of patients with breast cancer. Guidelines from the American Society for Clinical Oncology (2016) recommend against the use of soluble HER2 levels to guide selection of type of adjuvant therapy in breast cancer. This is a moderate-strength recommendation based upon low-quality evidence. The guidelines also recommend against the use of HER2 gene coamplification to guide adjuvant chemotherapy selection in breast cancer. PancraGen (formerly PathFinderTG - Pancreas) PathFinderTG (RedPath Integrated Pathology, Pittsburgh, PA), also known as topographic genotyping, is described by the manufacturer as a quantitative genetic mutational analysis platform for resolving \"indeterminate, atypical, suspicious, equivocal and non-diagnostic specimen\" diagnoses from pathology specimens (RedPath, 2007). The manufacturer states that PathFinder TG \"focuses on acquired mutational damage rather than inherited genetic predisposition for certain diseases, although there are certain NIH recommended inherited conditions for which we do test.\" The manufacturer states that the temporal sequence of acquired mutational damage revealed by the PathFinderTG test is an earlier demonstration of tumor biological aggressiveness than current staging systems that rely on the depth of invasion already achieved by the tumor. Most available published evidence for topographic genotyping focuses on retrospective analyses of pathology specimens examining correlations of test results with tumor characteristics (e.g., Saad et al, 2008; Lin et al, 2008; Finkelstein et al, 2003; Pollack et al, 2001; Riberio et al, 1998; Kounelis et al, 1998; Finkelstein et al, 1998; Holst et al, 1998; Jones et al, 1997; Holst et al, 1997; Pricolo et al, 1997; Przygodzki et al, 1997; Finkelstein et al, 1996; Kanbour-shakir et al, 1996; Ribeiro et al, 1996; Pryzgodzki et al, 1996; Safatle-Ribeiro et al, 1996; Papadaki et al, 1996; Przygodzki et al, 1996; Pricolo et al, 1996; Finkelstein et al, 1994).There are no prospective clinical outcome studies on the use of topographic genotyping in guiding patient management.Current evidence-based guidelines from leading medical professional organizations and public health agencies do not include recommendations for topographic genotyping. In a review on molecular analysis of pancreatic cyst fluid, Shen and colleagues (2009) stated that a large study with validation of PathFinderTG molecular testing of pancreatic fluid will be needed before a firm conclusion can be drawn. A systematic evidence review of the PathFinderTG prepared for the Agency for Healthcare Research and Quality (Trikalinos, et al., 2010)reviewed evidence available at that time, and found that most studies on loss-of-heterozygosity based topographic genotyping with PathfinderTG were excluded because they only described the molecular profile of different tumors, without assessing the ability of the method to help make diagnosis, prognosis or treatment guidance. Thereview found no studies that directly measured whether using loss-of-heterozygosity based topographic genotyping with PathfinderTG improves patient-relevant clinical outcomes. The review reported that eligible studies on the diagnostic and prognostic ability of loss-of-heterozygosity based topographic genotyping with PathfinderTG were small in sample sizes and had overt methodological limitations. The review reported that important characteristics of their designs were not clearly reported. The report noted that loss-of-heterozygosity based topographic genotyping with PathfinderTG is claimed to be particularly useful in cases where conventional pathology is unable to provide a conclusive diagnosis. However, the included studies were not designed to address this question. Therefore, it is unclear if the findings of the reviewed studies are directly applicable to patients with the same cancers but with inconclusive diagnosis. A subsequent study by Panarelli et al (2012) comparing PathFinderTG to cytological examination, finding concordance in 35 percent of cases. The authors concluded that the PathfinderTG panel may aid the classification of pancreatic lesions, but is often inaccurate and should not replace cytologic evaluation of these lesions. The manufacturer has announced that the PathginderTG - Pancreas has been rebranded Pancragen. Al Haddad et al (2015) reported on amulticenter retrospective chart review study to determine the diagnostic accuracy ofintegrated molecular pathology (Pancragen)for pancreatic adenocarcinoma, and the utility of IMP testing under current guideline recommendations for managing pancreatic cysts.The authors found that Pancragenmore accurately determined the malignant potential of pancreatic cysts than a Sendai 2012 guideline management criteria model. Patients who had undergone previousPancragen testing as prescribed by their physician and for whom clinical outcomes were available from retrospective record review were included (n = 492). Performance was determined by correlation between clinical outcome and previousPancragen diagnosis (\"benign\"/\"statistically indolent\" vs. \"statistically higher risk [SHR]\"/\"aggressive\") or an International Consensus Guideline (Sendai 2012) criteria model for \"surveillance\" vs. \"surgery.\" The Cox proportional hazards model determined hazard ratios for malignancy. Benign and statistically indolentPancragen diagnoses had a 97 % probability of benign follow-up for up to 7 years and 8 months from initialPancragen testing. SHR and aggressive diagnoses had relative hazard ratios for malignancy of 30.8 and 76.3, respectively (both P   0.0001). Sendai surveillance criteria had a 97 % probability of benign follow-up for up to 7 years and 8 months, but for surgical criteria the hazard ratio was only 9.0 (P   0.0001). In patients who met Sendai surgical criteria, benign and statistically indolentPancragen diagnoses had a  93 % probability of benign follow-up, with relative hazard ratios for SHR and aggressive IMP diagnoses of 16.1 and 50.2, respectively (both P   0.0001). The authors concluded that Pancragen may improve patient management by justifying more relaxed observation in patients meeting Sendai surveillance criteria. Loren et al (2016)used registry datatodetermine if initial adjunctivePancragen testing influenced future real-world pancreatic cyst management decisions for intervention or surveillancerelative to 2012 International Consensus Guideline (ICG)recommendations, and if this benefitted patient outcomes. Usingdata from the National Pancreatic Cyst Registry, the investigators evaluatedassociations between real-world decisions (intervention vs. surveillance), ICG model recommendations (surgery vs. surveillance) and Pancragen diagnoses (high-risk vs. low-risk) using 2 × 2 tables. The investigators used Kaplan Meier and hazard ratio analyses to assess time to malignancy. Odds ratios (OR) for surgery decision were determined using logistic regression. Of 491 patients, 206 received clinical intervention at follow-up (183 surgery, 4 chemotherapy, 19 presumed by malignant cytology). Overall, 13% (66/491) of patients had a malignant outcome and 87% (425/491) had a benign outcome at 2.9years\' follow-up. When ICG andPancragen were concordant for surveillance/surgery recommendations, 83% and 88% actually underwent surveillance or surgery, respectively. However, when discordant,Pancragen diagnoses were predictive of real-world decisions, with 88% of patients having an intervention when ICG recommended surveillance butPancragen indicated high risk, and 55% undergoing surveillance when ICG recommended surgery butPancragen indicated low risk. These Pancragen-associated management decisions benefitted patient outcomes in these subgroups, as 57% had malignant and 99% had benign outcomes at a median 2.9years\' follow-up.Pancragen was also more predictive of real-world decisions than ICG by multivariate analysis: OR 11.4 (95% CI 6.0 - 23.7) versus 3.7 (2.4 - 5.8), respectively. Kowalski et al (2016)examined the utility of integrated molecular pathology (IMP) in managing surveillance of pancreatic cysts based on outcomes and analysis of false negatives (FNs) from a previously published cohort (n=492). In endoscopic ultrasound with fine-needle aspiration (EUS-FNA) of cyst fluid lacking malignant cytology, IMP demonstrated better risk stratification for malignancy at approximately 3 years’ follow-up than International Consensus Guideline (Fukuoka) 2012 management recommendations in such cases. The investigators reviewedpatient outcomes and clinical features of Fukuoka and IMP FN cases. Practical guidance for appropriate surveillance intervals and surgery decisions using IMP were derived from follow-up data, considering EUS-FNA sampling limitations and high-risk clinical circumstances observed. Surveillance intervals for patients based on IMP predictive value were compared with those of Fukuoka. Outcomes at follow-up for IMP low-risk diagnoses supported surveillance every 2 to 3 years, independent of cyst size, when EUS-FNA sampling limitations or high-risk clinical circumstances were absent. In 10 of 11 patients with FN IMP diagnoses (2% of cohort), EUS-FNA sampling limitations existed; Fukuoka identified high risk in 9 of 11 cases. In 4 of 6 FN cases by Fukuoka (1% of cohort), IMP identified high risk. Overall, 55% of cases had possible sampling limitations and 37% had high-risk clinical circumstances. Outcomes support more cautious management in such cases when using IMP. An American Gastroenterological Association Technical Review (Scheiman et al,2014) stated: \"Testing for molecular alterations in pancreatic cyst fluid is currently available and reimbursed by Medicare under certain circumstances. Case series have confirmed malignant cysts have greater number and quality of molecular alterations, but no study has been properly designed to identify how the test performs in predicting outcome with regard need to surgery, surveillance or predicts interventions leading to improved survival. This adjunct to fine-needle aspiration (FNA) may provide value in distinct clinical circumstances, such as confirmation of a serous lesion due to a lack of KRAS or GNAS mutation in a macrocystic serous cystadenoma, but its routine use is not supported at the present time.\" A guideline from the American Society for Gastrointestinal Endoscopy (Muthusamy, et al., 2016) stated:\"A more recent study demonstrated that integrated molecular analysis of cyst fluid (ie, combining molecular analysis with results of imaging and clinical features) was able to better characterize the malignant potential of pancreatic cysts compared to consensus guidelines for the management of mucinous cysts [citing Al Haddad, et al., 2015]. ... Molecular analysis (which requires only 200 mL of fluid) may be most useful in small cysts with nondiagnostic cytology, equivocal cyst fluid CEA results, or when insufficient fluid is present for CEA testing [citing Al Haddad, et al., 2014]. However, additional research is needed to determine the precise role molecular analysis of cyst fluid will play in evaluating pancreatic cystic lesions.\" Guidelines published in April 2015 by the American Gastroenterological Association (Vege, et al., 2015) have no recommendations for use of topographic genotyping for evaluating pancreatic cysts. Otherguidelines (NCCN,2015;Vege, et al., 2015; Del Chiaro, et al., 2013; Sahani, et al., 2013; Tanaka, et al., 2012) have no firm recommendations for topographic genotyping for assessing indeterminate pancreatic cysts. The International consensus guidelines for \"The management intraductal papillary mucinous neoplasm (IPMN) and mucinous cystic neoplasm (MCN) of the pancreas\" (Tanaka et al, 2012) stated that endoscopic ultrasound (EUS) is recommended for all cysts with worrisome features or for cysts greater than 3 cm without these features. Endoscopic US confirmation of a mural nodule, any features of main duct involvement (intraductal mucin or thickened main duct wall), or suspicious or positive cytology for malignancy is an indication for surgical resection. Cysts with high-risk stigmata should be resected in patients medically fit for surgery, although EUS is optional. Endoscopic US can be considered in smaller cysts without worrisome features but is not required. Endoscopic US analysis should include at least cytology, amylase level, and CEA. The guidelines stated that elevated CEA is a marker that distinguishes mucinous from non-mucinous cysts, but not benign from malignant cysts. Khalid et al (2004) noted that brush cytology of biliary strictures to diagnose pancreaticobiliary malignancy suffers from poor sensitivity. These researchers attempted to improve the diagnostic yield of pancreaticobiliary brush cytology through analysis of tumor suppressor gene linked microsatellite marker loss of heterozygosity (LOH) and k-ras codon 12 mutation detection. A total of 26 patients with biliary strictures underwent endoscopic retrograde cholangiography with brush cytology. A panel of 12 polymorphic microsatellite markers linked to 6 tumor suppressor genes was developed. Genomic DNA from cell clusters acquired from brush cytology specimens and micro-dissected surgical malignant and normal tissue underwent polymerase chain amplification reaction (PCR); PCR products were compared for LOH and k-ras codon 12 mutations. A total of 17 patients were confirmed to have pancreaticobiliary adenocarcinoma; 9 patients had benign strictures (8 proven surgically, 1 by follow-up). Cytomorphological interpretation was positive for malignancy (n = 8), indeterminate (n = 10), and negative for malignancy (n = 8). Selected malignant appearing cytological cell clusters and micro-dissected histological samples from cancer showed abundant LOH characteristic of malignancy while brushings from 9 cases without cancer carried no LOH (p 0.001); LOH and k-ras mutations profile of the cytological specimens was almost always concordant with the tissue samples. Presence of k-ras mutation predicted malignancy of pancreatic origin (p 0.001). The authors concluded that LOH and k-ras codon 12 mutation analysis of PCR amplified DNA from biliary brush cytology discriminated reactive from malignant cells, with 100 % sensitivity, specificity, and accuracy. Minor variations in LOH in brushings and in different sites within the same tumor likely reflect intra-tumoral mutational heterogeneity during clonal expansion of pre- and neoplastic lineages. Nodit et al (2006) noted that the clinical course of pancreatic endocrine tumor (PET) varies depending on tumor aggressiveness, disease extent, and possibly accumulated molecular alterations. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) results, although accurate in diagnosing PET, correlated poorly with PET outcome. The role of detecting key molecular abnormalities in predicting PET behavior and clinical outcome from EUS-FNA material remains unknown. In this study, patients with confirmed PET who underwent EUS-FNA during a 32-month period were included. Patient demographics and clinical data were recorded and follow-up information was obtained by contacting their physician to evaluate disease progression. Representative tumor cells were micro-dissected from the FNA material. DNA was harvested and amplified, targeting a panel of 17 polymorphic microsatellite markers on chromosomes 1p, 3p, 5q, 9p, 10q, 11q, 17p, 17q, 21q, and 22q. The polymerase chain reaction (PCR) products were subjected to fluorescent capillary gel electrophoresis to detect microsatellite loss. The fractional allelic loss (FAL) was calculated. A total of 25 patients were studied; 13 were classified histologically as benign PET limited to the pancreas and 12 as malignant PET (invasive or metastatic). The mean FAL in the benign and malignant PET was 0.03 and 0.37 (p 0.0001), respectively. In addition, the mean FAL was significantly greater in those with disease progression as compared with patients with stable disease (0.45 versus 0.09, respectively, p 0.0001). The authors concluded that micro-satellite loss analysis of EUS-FNA material from PET can be performed reliably and an FAL value of more than 0.2 is associated with disease progression . These researchers stated that this technique may have value in the pre-operative assessment and risk stratification of patients with PET. The authors stated that the small sample size and limited follow-up period were drawbacks of this study, which needed replication in larger prospective studies with longer follow-up periods. The impact of individual micro-satellite markers on the PET clinical course also required further study. In this study certain microsatellites (3p26, 5q23, 17q23, and 21q23) were lost only in malignant PETs, but with the small number of specimens studied the significance of this was unclear. Interestingly, both malignant PETs with a single allelic loss (5q23 and 17q23) each involved micro-satellites not lost in the benign PET. Finkelstein et al (2012) aimed to supplement microscopic examination of biliary cytobrush specimens to improve sensitivity by mutational profiling (MP) of: selected cells micro-dissected from cytology slides; and corresponding cell-free DNA (cfDNA) in residual supernatant fluid. From 43 patients with brushings of bile or pancreatic duct strictures, DNA was extracted from micro-dissected cells and 1 to 2 ml of cytocentrifugation supernatant fluid. Mutational analysis targeted 17 genomic sites associated with pancreaticobiliary cancer, including sequencing for KRAS point mutation and LOH analysis of micro-satellites located at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, and 22q. Mutations were found in 25/28 patients with malignancy, and no mutations were found in 5/5 patients with benign surgical results. The cell-free supernatant fluid generally contained higher levels and quality of DNA, resulting in increased detection of mutations in most patients. KRAS mutations only occurred in patients with pancreatic cancer; MP of supernatant fluid specimens resulted in high sensitivity and specificity for malignancy, improving the detection of malignancy over cytology alone. The authors concluded that In this study they had shown that neoplastic free DNA was present in the extracellular compartment even when a particular cytology sample lacked sufficient cellularity to afford a definitive diagnosis. Most importantly, the cell-free supernatant, available as a residual specimen after cytocentrifugation, should be regarded as a potentially valuable source of information due to its content of adequate amounts of free DNA for robust mutational analysis with the capacity to address issues related to sampling variation and to detect neoplasia at an early stage of development. The authors stated that this study had several limitations. First, the total number of test samples was relatively small and the results shown here require confirmation with additional specimens. In particular, the addition of more confirmed negative specimens would strengthen the findings around sensitivity. Second, this study was restricted to the use of Saccomanno’s fixation which, though commonly used, was not the only fixative used in cytology practice. While each fixative merits individual testing with respect to its capacity to deliver adequate levels of representative, intact supernatant DNA for MP, it was reasonable to expect favorable results with other methods of sample preparation as most cytology fixatives were alcohol-based and not expected to induce significant DNA degradation. Indeed, additional unpublished work in the authors’ laboratory involving testing of other supernatant fluids indicated that most common cytology fixatives yielded amplifiable DNA (with the notable exception of CytoRich Red). This was consistent with their prior experience in genotyping micro-dissected cytology slides, in which most slides yielded amplifiable DNA regardless of the cytology fixative used. While none of the supernatant specimens evaluated in this study failed to provide adequate DNA for MP, it was expected that a small proportion of markedly hypocellular specimens, likely from non-neoplastic states, would fail to meet the lower limit of DNA quantity for analysis. It should be noted that in this study, when micro-dissection alone was used, 2/18 cases proved to be false negative for mutation detection (1 cholangiocarcinoma and 1 pancreatic adenocarcinoma). While no false negative malignant stricture cases were seen in cohort 2A where both micro-dissection and supernatant fluid analysis were utilized, in 2 patients, the supernatant fluid manifested a lesser extent of mutational change than that present in the corresponding micro-dissected stained cytology cells. These findings emphasized that the sampling variation and other limitations may nevertheless be present in individual cases limiting or preventing the detection of cancer. It remained essential to integrate all of the information including clinical and imaging findings to optimize individual patient diagnosis. Finkelstein et al (2014) noted that diagnosis of fine-needle aspirations of pancreatic solid masses is complicated by many factors that keep its false-negative rate high. These researchers’ novel approach analyzes cell-free cytocentrifugation supernatant, currently a discarded portion of the specimen. Supernatant and cytology slides were collected from 25 patients: 11 cases with confirmed outcome [5 positive (adenocarcinoma) and 6 negative (inflammatory states)], plus 14 without confirmed outcomes. Slides were micro-dissected, DNA was extracted from micro-dissections and corresponding supernatants, and all were analyzed for KRAS point mutation and loss of heterozygosity. Notably, higher levels of free DNA were found in supernatants than in corresponding micro-dissected cells. Supernatants contained sufficient DNA for mutational profiling even when samples contained few to no cells. Mutations were present in 5/5 malignancies and no mutations were present in inflammatory states. The authors concluded that these findings supported using supernatant for mutational genotyping when diagnostic confirmation is needed for pancreatic solid masses. These researchers stated that the data presented suggested that supernatant fluid should be regarded as a valuable source of information that may address many diagnostic issues and may serve as a useful, complimentary tool for pathologists when microscopic examination is suboptimal. The authors stated that several limitations of this molecular analysis of cytocentrifugation supernatant were recognized. The total number of test samples was not large, and the promising results shown here need to be evaluated with a greater number of specimens. In addition, this study was restricted to the use of Saccomanno’s fixation. Ideally, each commonly used fixative should be individually tested for its capacity to deliver adequate levels of representative supernatant DNA for mutational profiling. It is reasonable, however, to expect favorable results with other methods of sample preparation since most cytology fixatives are alcohol based and are not expected to induce significant DNA degradation. Consistently, prior work has shown that cytology specimens based on micro-dissected stained cytology cells, are especially suitable for mutational analysis. Deftereos et al (2014) stated that FNA of pancreatic solid masses can be significantly impacted by sampling variation. Molecular analysis of tumor DNA can be an aid for more definitive diagnosis. These investigators evaluated how molecular analysis of the cell-free cytocentrifugation supernatant DNA can help reduce sampling variability and increase diagnostic yield. Atotal of 23-FNA smears from pancreatic solid masses were performed. Remaining aspirates were rinsed for preparation of cytocentrifuged slides or cell blocks. DNA was extracted from supernatant fluid and assessed for DNA quantity spectrophotometrically and for amplifiability by quantitative PCR (qPCR). Supernatants with adequate DNA were analyzed for mutations using PCR/capillary electrophoresis for a broad panel of markers (KRAS point mutation by sequencing, micro-satellite fragment analysis for loss of heterozygosity (LOH) of 16 markers at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, and 22q). In selected cases, micro-dissection of stained cytology smears and/or cytocentrifugation cellular slides were analyzed and compared. In all, 5/23 samples cytologically confirmed as adenocarcinoma showed detectable mutations both in the micro-dissected slide-based cytology cells and in the cytocentrifugation supernatant. While most mutations detected were present in both micro-dissected slides and supernatant fluid specimens, the latter showed additional mutations supporting greater sensitivity for detecting relevant DNA damage. Clonality for individual marker mutations was higher in the supernatant fluid than in micro-dissected cells. Cytocentrifugation supernatant fluid contains levels of amplifiable DNA suitable for mutation detection and characterization. The finding of additional detectable mutations at higher clonality indicated that supernatant fluid may be enriched with tumor DNA. The authors concluded that the findings of this study suggested how the supernatant fluid can be utilized as a source of molecular information and could become a powerful addition to standard cytology evaluation. Mutational profiling of DNA in normally discarded supernatant fluid may help resolve occasional diagnostic challenges and may serve as a useful, complementary tool for cytopathologists when microscopic examination yielded no conclusive diagnosis or when a specimen is suboptimal. Malhotra et al (2014) aimed to better understand the supporting role that MP of DNA from micro-dissected cytology slides and supernatant specimens may play in the diagnosis of malignancy in FNA and biliary brushing specimens from patients with pancreaticobiliary masses. Cytology results were examined in a total of 30 patients with associated surgical (n = 10) or clinical (n = 20) outcomes; MP of DNA from micro-dissected cytology slides and from discarded supernatant fluid was analyzed in 26 patients with atypical, negative or indeterminate cytology. Cytology correctly diagnosed aggressive disease in 4 patients. Cytological diagnoses for the remaining 26 were as follows: 16 negative (9 false negative), 9 atypical, 1 indeterminate. MP correctly determined aggressive disease in 1 false negative cytology case and confirmed a negative cytology diagnosis in 7 of 7 cases of non-aggressive disease. Of the 9 atypical cytology cases, MP correctly diagnosed 7 as positive and 1 as negative for aggressive disease. One specimen that was indeterminate by cytology was correctly diagnosed as non-aggressive by MP. When first-line malignant (positive) cytology results were combined with positive 2nd-line MP results, 12/21 cases of aggressive disease were identified, compared to 4/21 cases identified by positive cytology alone. The authors concluded that when 1st-line cytology results were uncertain (atypical), questionable (negative), or not possible (non-diagnostic/indeterminate), MP provided additional information regarding the presence of aggressive disease. When used in conjunction with 1st-line cytology, MP increased detection of aggressive disease without compromising specificity in patients that were difficult to diagnose by cytology alone. The authors stated that this study had several drawbacks including a small sample size that limited their ability to calculate the diagnostic performance of MP in pancreatic masses and associated biliary strictures. Although MP allowed these researchers to detect additional cases of aggressive disease, even when cytology and MP results were combined into one overall diagnosis, 9 cases of malignancy were missed. These falsely negative results were likely due to a combination of the less than perfect sensitivity of both tests as well as to sampling limitations related to FNA and brushing techniques. These investigators noted that despite such limitations, these promising findings do provide support for future larger scale studies, with the addition of supernatant analysis providing an opportunity to overcome some of these limitations. Gonda et al (2017) stated that it is a challenge to detect malignancies in biliary strictures. Various sampling methods are available to increase diagnostic yield, but these require additional procedure time and expertise. These investigators evaluated the combined accuracy of fluorescence in situ hybridization (FISH) and PCR-based DNA MP of specimens collected using standard brush techniques. These researchers performed a prospective study of 107 consecutive patients treated for biliary strictures by endoscopic retrograde cholangiopancreatography from June 2012 through June 2014. They carried out routine cytology and FISH analyses on cells collected by standard brush techniques, and analyzed supernatants for point mutations in KRAS and LOH mutations in tumor-suppressor genes at 10 loci (MP analysis was performed at Interpace Diagnostics). Strictures were determined to be non-malignant based on repeat image analysis or laboratory test results 12 months after the procedure. Malignant strictures were identified based on subsequent biopsy or cytology analyses, pathology analyses of samples collected during surgery, or death from biliary malignancy. These researchers determined the sensitivity and specificity with which FISH and MP analyses detected malignancies using the exact binomial test. The final analysis included 100 patients; 41 % had biliary malignancies. Cytology analysis identified patients with malignancies with 32 % sensitivity and 100 % specificity. Addition of FISH or MP results to cytology results increased the sensitivity of detection to 51 % (p 0.01) without reducing specificity. The combination of cytology, MP, and FISH analyses detected malignancies with 73 % sensitivity (p 0.001); FISH identified an additional 9 of the 28 malignancies not detected by cytology analysis, and MP identified an additional 8 malignancies; FISH and MP together identified 17 of the 28 malignancies not detected by cytology analysis. The authors concluded that these findings supported the use of both FISH testing and PCR-based MP of tumor-suppressor gene LOH and KRAS in evaluation of cytology-negative or indeterminate biliary strictures; MP allowed for increased diagnostic yield from each individual brush, given that normally discarded, cell-free supernatant material that contains DNA can be analyzed. Based on these findings, these researchers suggested using either FISH or MP as a 2nd-line diagnostic modality to 1st-line cytology. They stated that MP may be best prioritized to scenarios of low cellularity. Any case that is negative or indeterminate by 2 testing modalities should undergo a 3rd to increase the probability of detecting possible malignancy. To do so, normally discarded supernatant fluid should be retained for MP testing during the standard cytology cytocentrifugation preparation of cells for cytology. These researchers stated that additional studies may help to better understand the reflex order of sequential testing and the impact of this reflex on health economics. The authors stated that this study had several drawbacks that may have impacted generalized conclusions. A somewhat higher benign stricture rate was noted in their cases than in other prior series. There also were relatively few primary sclerosing cholangitis (PSC) patients included in this study. Prior studies have shown that there is a significant aneuploidy rate associated with pre-malignant lesions seen in PSC. Because of this, specificity of FISH for malignancy was expected to be lower in a cohort of PSC patients than the authors reported in their cohort. Less was known about detection of KRAS mutations in the progression of PSC to cholangiocarcinoma. However, based on this study cohort and prior studies, these findings likely were not generalizable to the PSC population. Khosravi et al (2018) stated that indeterminate cytology occurs in a significant number of patients with solid pancreaticobiliary lesion that undergo EUS-FNA or endoscopic retrograde cholangiopancreatography (ERCP) and can incur further expensive testing and inappropriate surgical intervention. Mutation profiling improves diagnostic accuracy and yield but the impact on clinical management is uncertain. These researchers determined the performance of MP in clinical practice and its impact on management in solid pancreaticobiliary patients with indeterminate cytology. Solid pancreaticobiliary patients with non-diagnostic, benign, atypical or suspicious cytology who had past MP testing were included. Mutation profiling examined KRAS mutation and a tumor suppressor gene associated loss of heterozygosity mutation panel covering 10 genomic loci. Two endo-sonographers made management recommendations without and then with MP results, indicating their level of confidence. Mutation profiling improved diagnostic accuracy in 232 patients with indeterminate cytology. Among patients with non-diagnostic cytology, low-risk MP provided high specificity and negative predictive value (NPV) for the absence of malignancy while high-risk MP identified malignancies otherwise undetected. Mutation profiling increased clinician confidence in management recommendations and resulted in more conservative management in 10 % of patients. Mutation profiling increased the rate of benign disease in patients recommended for conservative management (84 % to 92 %, p 0.05) and the rate of malignant disease in patients recommended for aggressive treatment (53 % to 71 %, p 0.05). The authors concluded that MP improved diagnostic accuracy and significantly impacted management decisions. Low-risk MP results increased recommendations for conservative management and increased the rate of benign outcomes those patients, helping to avoid unnecessary aggressive interventions and improve patient outcomes. These researchers stated that their study was limited by its retrospective nature. Moreover, they noted that although high-risk MP results were able to help confirm the presence of malignancy in cases in which cytology indicated a high suspicion of malignancy, low-risk results could not effectively exclude the possibility of malignancy in such cases. Kushnir et al (2019) noted that routine cytology of biliary stricture brushings obtained during ERCP has suboptimal sensitivity for malignancy. These investigators compared the individual and combined ability of cytology, FISH analysis and PCR-based MP to detect malignancy in standard biliary brushings. They performed a prospective study of patients undergoing ERCP using histology or 1 year follow-up to determine patient outcomes; MP was performed on free-DNA from biliary brushing specimens using normally discarded supernatant fluid. MP examined KRAS point mutations and tumor suppressor gene associated LOH mutations at 10 genomic loci; FISH examined chromosome specific gains or losses. A total of 101 patients were included in final analysis and 69 % had malignancy. Cytology had 26 % sensitivity and 100 % specificity for malignancy. Using either FISH or MP in combination with cytology increased sensitivity to 44 % and 56 %, respectively. The combination of all 3 tests (cytology, FISH, and MP) had the highest sensitivity for malignancy (66 %). There was no difference in the specificity of cytology, FISH or MP testing when examined alone or in combination; MP improved diagnostic yield of each procedure from 22 % to 100 %; FISH improved yield to 90 %; MP detected 21 malignancies beyond that identified by cytology; FISH detected an additional 13. The combination of FISH and MP testing detected an additional 28 malignancies. The authors concluded that both MP and FISH are complimentary molecular tests that could significantly increase detection of biliary malignancies when used in combination with routine cytology of standard biliary brush specimens. Gene Expression Profiling for Cancer of Unknown Primary Carcinoma of unknown primary (CUP) is a biopsy-proven metastatic solid tumor with no primary tumor identified and represents approximately 2% to 4% of all cancer cases. The diagnosis of CUP is made following inconclusive results from standard tests (e.g., biopsy, immunochemistry and other blood work, chest x-rays, and occult blood stool test). The absence of a known primary tumor presents challenges to the selection of appropriate treatment strategies. As a result, patients have a poor prognosis, and fewer than 25% survive 1 year from the time of diagnosis. A variety of tissue-biopsy testing techniques currently are used to determine the origin of the CUP, including immunochemistry; histological examination of specimens stained with eosin and hematoxylin, and electron microscopy. These techniques definitively identify the type of carcinoma in less than 20% to 30% of CUP. Gene expression profiling is a technique used to identify the genetic makeup of a tissue sample by characterizing the patterns of mRNA transcribed, or \"expressed\", by its DNA. Specific patterns of gene expression, reflected in unique configurations of mRNA, are associated with different tumor types. By comparing the gene expression profile (GEP) of an unknown tumor to the profiles of known primary cancers (\"referent\" profiles), it may be possible to determine the type of tumor from which the CUP originated. In July 2008, the FDA cleared for marketing the Pathwork Tissue of Origin test (Pathwork Diagnostics, Sunnyvale, CA), a gene expression profiling test that uses microarray processing to determine the type of cancer cells present in a tumor of unknown origin. The test uses the PathChip (Affymetrix Inc., Santa Clara, CA), a custom-designed gene expression array, to measure the expression from 1,668 probe sets to quantify the similarity of tumor specimens to 15 common malignant tumor types, including: bladder, breast, colorectal, gastric, germ cell, hepatocellular, kidney, non-small cell lung, non-Hodgkin\'s lymphoma, melanoma, ovarian, pancreatic, prostate, soft tissue sarcoma, and thyroid. The degree of correspondence between the tissue sample\'s GEP and a referent profile is quantified and expressed as a probability-based score. A multi-center, clinical validation study reported on comparisons of diagnoses based on GEP from 477 banked tissue samples of undifferentiated and poorly differentiated metastases versus standard of care pathology based diagnoses. Comparison of the GEP based diagnoses versus pathology based diagnoses yielded an 89 % agreement and the concurrence was greater than 92 % for 8 out of 15 types of primary tumors. The overall accuracy of the test was approximately 95 % and 98 % for positive and negative determinations, respectively (Monzon et al, 2007). Gene expression profiling is a promising technology in the management of CUP; however, there is insufficient evidence of its clinical utility compared to that achieved by expert pathologists using current standards of practice. A draft clinical guideline on metastatic malignant disease of unknown origin by the National Institute for Clinical Excellence (NICE, 2010) recommends against using gene expression profiling (e.g., Pathwork TOT,CupPrint, TherosCancerTypeID, miRview Mets) to identify primary tumors in patients with CUP. The guideline explained that currently there is no evidence that gene-expression based profiling improves the management or changes the outcomes for patients with CUP. Guidelines on occult primaryfrom the National Comprehensive Cancer Network (NCCN, 2010) state that, while gene expression profiling looks promising, \"prospective clinical trials are necessary to confirm whether this approach can be used in choosing treatment options which would improve the prognosis of patients with occult primary cancers.\" An assessment by the Andalusian Agency for Health Technology Assessment (AETSA, 2012) of microRNAs as a diagnostic tool for lung cancer found only two studies assessing the analytical validity of miRview in patients with non-small cell lung cancer. The sensitivity of the miRNA for the detection of carcinoma was between 96% and 100% and the specificity was between 90% and 100%. The area under the ROC curve was close to unity and the positive and negative probability ratios showed a high diagnostic accuracy (9.6 and 0.04, respectively). The assessment stated that, although the quality of the studies was moderate to high, the sensitivity of the diagnostic test may be overestimated as it is a case-control design. A technology assessment prepared for the Agency for Healthcare Research and Quality (Meleth et al, 2013) found that the clinical accuracy of the PathworkDx, miRview, and CancerTypeID are similar, ranging from 85 percent to 88 percent, and that the evidence that the tests contribute to identifying a tumor of unknown origin was moderate. The assessment concluded that we do not have sufficient evidence to assess the effect of the tests on treatment decisions and outcomes. The assessment noted that most studies of these tests were funded wholly or partially by the manufacturers of these tests, and that the most urgent need in the literature is to have the clinical utility of the tests evaluated by research groups that have no evidence conflict of interest. Monzon et al (2009) stated that malignancies found in unexpected locations or with poorly differentiated morphologies can pose a significant challenge for tissue of origin determination. Current histologic and imaging techniques fail to yield definitive identification of the tissue of origin in a significant number of cases. The aim of this study was to validate a predefined 1,550-gene expression profile for this purpose. Four institutions processed 547 frozen specimens representing 15 tissues of origin using oligonucleotide microarrays were used in this study. Half of the specimens were metastatic tumors, with the remainder being poorly differentiated and undifferentiated primary cancers chosen to resemble those that present as a clinical challenge. In this blinded multi-center validation study the 1,550-gene expression profile was highly informative in tissue determination. The study found overall sensitivity (positive percent agreement with reference diagnosis) of 87.8 % (95 % CI: 84.7 % to 90.4 %) and overall specificity (negative percent agreement with reference diagnosis) of 99.4 % (95 % CI: 98.3 % to 99.9 %). Performance within the subgroup of metastatic tumors (n = 258) was found to be slightly lower than that of the poorly differentiated and undifferentiated primary tumor subgroup, 84.5 % and 90.7 %, respectively (p = 0.04). Differences between individual laboratories were not statistically significant. The authors concluded that this study represents the first adequately sized, multi-center validation of a gene-expression profile for tissue of origin determination restricted to poorly differentiated and undifferentiated primary cancers and metastatic tumors. These results indicate that this profile should be a valuable addition or alternative to currently available diagnostic methods for the evaluation of uncertain primary cancers. Monzon and Koen (2010) stated that tumors of uncertain or unknown origin are estimated to constitute 3 % to 5 % of all metastatic cancer cases. Patients with these types of tumors show worse outcomes when compared to patients in which a primary tumor is identified. New molecular tests that identify molecular signatures of a tissue of origin have become available. The authors reviewed the literature on existing molecular approaches to the diagnosis of metastatic tumors of uncertain origin and discuss the current status and future developments in this area. Published peer-reviewed literature, available information from medical organizations (NCCN), and other publicly available information from tissue-of-origin test providers and/or manufacturers were used in this review. The authors concluded that molecular tests for tissue-of-origin determination in metastatic tumors are available and have the potential to significantly impact patient management. However, available validation data indicate that not all tests have shown adequate performance characteristics for clinical use. Pathologists and oncologists should carefully evaluate claims for accuracy and clinical utility for tissue-of-origin tests before using test results in patient management. The personalized medicine revolution includes the use of molecular tools for identification/confirmation of the site of origin for metastatic tumors, and in the future, this strategy might also be used to determine specific therapeutic approaches. Anderson and Weiss (2010) noted that pathologists use various panels of IHC stains to identify the site of tissue of origin for metastatic tumors, particularly poorly or undifferentiated cancers of unknown or uncertain origin. Although clinicians believe that immunostains contribute greatly to determining the probable primary site among 3 or more possibilities, objective evidence has not been convincingly presented. This meta-analysis reviews the objective evidence supporting this practice and summarizes the performance reported in 5 studies published between 1993 and 2007. A literature search was conducted to identify IHC performance studies published since 1990 that were masked, included more than 3 tissues types, and used more than 50 specimens. The 5 studies found in this search were separated into 2 subgroups for analysis: those, which included only metastatic tumors (n = 368 specimens) and the blended studies, which combined primary tumors and metastases (n = 289 specimens). The meta-analysis found that IHCs provided the correct tissue identification for 82.3 % (95 % CI: 77.4 % to 86.3 %) of the blended primary and metastatic samples and 65.6 % (95 % CI: 60.1 % to 70.7 %) of metastatic cancers. This difference is both clinically and statistically significant. The authors concluded that this literature review confirms that there is still an unmet medical need in identification of the primary site of metastatic tumors. It establishes minimum performance requirements for any new diagnostic test intended to aid the pathologist and oncologist in tissue of origin determination. GeneSearch BLN The presence of breast tumor cells in axillary lymph nodes is a key prognostic indicator in breast cancer. During surgery to remove breast tumors, patients often undergo biopsy of the sentinel (i.e., first) node(s) that receive lymphatic fluid from the breast. Excised sentinel lymph nodes are currently evaluated post-operatively by formalin-fixed paraffin-embedded Hematoxylin and Eosin (H E) histology andIHC. GeneSearch™ Breast Lymph Node (BLN) assay (Veridex, LLC, Warren, NJ) is a novel method to examine the extracted sentinel lymph nodes for metastases and can provide information during surgery within 30 to 40 minutes from the time the sentinel node is removed, potentially avoiding a second operation for some patients. The GeneSearch BLN assay received FDA pre-market approval on July 16, 2007 as a qualitative in vitro diagnostic test for the rapid detection of metastases larger than 0.2 mm in nodal tissue removed from sentinel lymph node biopsies of breast cancer patients. GeneSearch BLN assay uses real time reverse transcriptase polymerase chain reaction (RT-PCR) to detect the gene expression markers, mammaglobin (MG) and cytokeratin 10 (CI19), which are abundant in breast tissue but scarce in lymph node cells. In the clinical trial conducted by Veridex, which was submitted to the FDA, the sensitivity of the GeneSearch BLN Assay was reported to be 87.6 % and the specificity was 94.2 % (Julian et al, 2008). According to the product labeling, \"The GeneSearch™ Breast Lymph Node (BLN) assay may be used in conjunction with sentinel lymph node biopsy for a patient who has been counseled on use of this test and has been informed of its performance. False positive results may be associated with increased morbidity. False negative and inconclusive test results may be associated with delayed axillary node dissection. Clinical studies so far are inconclusive about a benefit from treatment based on findings of breast cancer micro-metastases in sentinel lymph nodes.\" Blumencranz et al (2007) compared the GeneSearch BLN assay with results from conventional histologic evaluation from 416 patients at 11 clinical sites and reported that the GeneSearch BLN assay detected 98 % of metastases greater than 2 mm in size and 57 % of metastasis less than 0.2 mm. False positives were reported in 4 % of the cases. However, there were several limitations of this study, including the lack of a description of patient recruitment, inadequate descriptions of several analyses performed, substantial variations in test performance across sites, and ad hoc comparison of the assay to other intra-operative techniques. Viale et al (2008) analyzed 293 lymph nodes from 293 patients utilizing the GeneSearch BLN assay. Using histopathology as the reference standard, the authors reported that the BLN assay correctly identified 51 of 52 macro-metastatic and 5 of 20 micro-metastatic sentinel lymph nodes (SLNs), with a sensitivity of 98.1 % to detect metastases larger than 2 mm, 94.7 % for metastases larger than 1 mm, and 77.8 % for metastases larger than 0.2 mm. The overall concordance with histopathology was 90.8 %, with a specificity of 95.0 %, a positive predictive value of 83.6 %, and a negative predictive value of 92.9 %. When the results were evaluated according to the occurrence of additional metastases to non-SLN in patients with histologically positive SLNs, the assay was positive in 33 (91.7 %) of the 36 patients with additional metastases and in 22 (66.6 %) of the 33 patients without further echelon involvement. The authors concluded that the sensitivity of the GeneSearch BLN assay is comparable to that of the histopathologic examination of the entire SLN by serial sectioning at 1.5 to 2 mm. Although treatment for metastases larger than 2.0 mm is widely accepted as beneficial, clinical studies have not yet provided data for a consensus on benefit from treatment based on very small breast cancer metastases (between 0.2 mm and 2.0 mm) in SLNs. False positive results may be associated with increased morbidity, usually due to effects of axillary node dissection surgery. Patients who undergo axillary lymph node dissection (ALND) have significantly higher rates of increased swelling in the upper arm and forearm (lymphedema), pain, numbness, and motion restriction about the shoulder when compared with patients who undergo only sentinel lymph node dissection (SLND). False negative and inconclusive test results may be associated with delayed axillary node dissection. Clinical studies so far are inconclusive regarding a benefit from treatment based on findings of breast cancer micro-metastases in SLNs. Preliminary data suggest that the GeneSearch BLN assay has high specificity and moderate sensitivity when only macro-metastases are included in the analysis. The clinical significance of micro-metastases is still being debated in the literature, thus, the failure of the GeneSearch BLN assay to perform adequately in the detection of micro-metastases is of unknown significance. A systematic evidence review by the BlueCross BlueShield Association Technology Evaluation Center (BCBSA, 2007) determined that the use of the GeneSearch BLN assay to detect sentinel node metastases in early stage breast cancer does not meet the TEC criteria. The assessment stated, \"There are several operational issues that add difficulty to the use of the GeneSearch assay, including the need for fresh specimens (rather than putting them in formalin for permanent fixation), the learning curve involved in reducing both the percentage of invalid results (from about 15% initially to 4 - 8% for more experienced technicians) and the time to perform the test compared to alternative intra-operative techniques (which take less than 15 minutes).\" Furthermore, the assessment stated \"The GeneSearch assay also provides less information for staging than other intra-operative procedures, since it cannot distinguish between micro- and macro-metases. Nor can it indicate the location of the metastasis (inside or outside the node). Post-operative histology is therefore required in all cases. It is less crucial when frozen section histology is performed, since pathologists can judge the size of the metastasis and its location from this test, although distortion is possible. To summarize, the data available is inadequate to assess the clinical utility of the GeneSearch assay compared to either post-operative histology alone or to the alternative intra-operative tests such as imprint cytology and frozen section histology. In addition, the balance of benefits versus harms may require higher specificity to avoid unnecessary ALNDs and their sequelae, whereas the GeneSearch design emphasizes sensitivity.\" A report byAdelaide Health Technology Assessmentstated that, if the GeneSearch BLN Assay is to play a role in reducing the mortality of breast cancer patients, it will be through more accurate diagnosis of breast cancer metastasis during SNB (Ellery, et al., 2010). The report noted, however, that, as yet thereare no data to indicate whether SNB itself lowers the mortality rate among breast cancer patients. Hence, it is unclear whether the GeneSearch BLN Assay would have any indirect effect on breast cancer mortality until further investigation into SNB concludes. Thus, there is insufficient evidence to make a conclusion about the effectiveness of the GeneSearch BLN assay. The FDA is requiring the manufacturer to conduct two post-approval studies. The primary objective of the first study is to estimate the positive predictive concordance between the GeneSearch BLN assay and histology as routinely practiced and the objectives of the second clinical study are determine the assay turn-around-time from the time of node removal to the report of the assay result to the surgeon and determine whether the assay result was or was not received in time to make an intra-operative decision and collect data in relation to other surgical procedures during the sentinel lymph node dissection/breast surgery to determine if the assay turn-around-time resulted in longer surgery time. BT Test Provista Life Sciences (Phoenix, AZ) has developed a laboratory test called the Biomarker Translation Test, or the BT Test, which is a test score based on a multi-protein biomarker analysis (i.e., IL-2, -6,-8,-12, TNFa, EGF, FGF, HGF, VEGF) and medical profile of an individual\'s risk factors for breast cancer. It is intended to be used as an adjunctive test along with other breast cancer detection modalities, however, there are no published studies of the effectiveness of this test. Bcl-2 Bcl-2 (B-cell CLL/lymphoma 2; BCL2) is a proto-oncogene whose protein product, bcl-2, suppresses programmed cell death (apoptosis), resulting in prolonged cellular survival without increasing cellular proliferation. Dysregulation of programmed cell death mechanisms plays a role in the pathogenesis and progression of cancer as well as in the responses of tumors to therapeutic interventions. Many members of the Bcl-2 family of apoptosis-related genes have been found to be differentially expressed in various malignancies (Reed, 1997). Salgia (2008) reviewed the evidence for detection of Bcl-2 in lung cancer. The author observed that Bcl-2 over-expression has been reported in 22 to 56% of lung cancers with a higher expression in squamous cell carcinoma as compared to adenocarcinoma histology. The author concluded, however, that the association of Bcl-2 expression and prognosis in non-small cell lung cancer is unclear. Multiple reports have demonstrated that Bcl-2-positive lung cancers are associated with a superior prognosis compared to those that are Bcl-2 negative. However, other studies have failed to demonstrate any survival impact with bcl-2 positivity, while over-expression has also been associated with a poorer outcome. A meta-analysis that included 28 studies examining the prognostic influence of Bcl-2 in non-small cell lung cancer concluded that over-expression of Bcl-2 was associated with a significantly better prognosis in surgically resected (hazard ratio 0.5, 95% CI 0.39-0.65). Compton (2008) recently reviewed the evidence on the Bcl-2 oncogene and other tumor markers in colon cancer. Compton explained that Bcl-2 is a gene related to apoptosis/cell suicide. Bcl-2 over-expression leading to inhibition of cell death signaling has been observed as a relatively early event in colorectal cancer development. The author concluded that the independent influence of the Bcl-2 oncogene on prognosis remains unproven, and explained that the variability in assay methodology, conflicting results from various studies examining the same factor, and the prevalence of multiple small studies that lack statistically robust, multivariate analyses all contribute to the lack of conclusive data. Compton concluded that before the Bcl-2 oncogene and certain other tumor markers can be incorporated into clinically meaningful prognostic stratification systems, \"more studies are required using multivariate analysis, well-characterized patient populations, reproducible and current methodology, and standardized reagents.\" Compton (2008) reviewed the evidence for intratumor microvessel density (MVD) and antibodies against CD31 in colorectal cancer. The author explained that intratumoral MVD is a reflection of tumor-induced angiogenesis. Microvessel density has been independently associated with shorter survival in some, but not all studies. A meta-analysis of all studies relating MVD expression to prognosis concluded that at least some of the variability could be explained by the different methods of MVD assessment. The author noted that there was a significant inverse correlation between immunohistochemical expression and survival when MVD was assessed using antibodies against CD31 or CD34, but not factor VIII. The author concluded, however, that there is a need for evaluation of MVD in large studies of prognostic factors using multivariate analysis; however, standard guidelines for staining, evaluation, and interpretation of MVD are lacking. In a review, Hayes (2008) reviewed the evidence for assessing angiogenesis factors in breast cancer. The author noted that, in an early report, MVD count (as indicated by IHC staining for endothelial cells, such as factor VIII-related antigen or CD31) was a statistically significant independent predictor of both disease-free and overall survival in women with both node-negative and node-positive breast cancer. The author noted, however, that subsequent data are conflicting, with some studies confirming and others refuting the initial findings. The author stated that, \"As with many of the other tumor marker studies, evaluation of angiogenesis is complicated by technical variation, reader inconsistency, and potential interaction with therapy.\" Burgdorf (2006) reviewed the use of CD31 in acquired progressive lymphangioma. The author stated that special staining techniques reveal that the cells are variably positive for CD31, but that the staining patterns are too variable to be of diagnostic importance. Some authorities have stated that CD31 staining may be useful for diagnosing angiosarcomas (Schwartz, 2008; Carsiand Sim, 2008; Fernandezand Schwartz, 2007; McMainsand Gourin, 2007). CD31 immunostaining can help confirm that the tumor originates from blood vessels. TOP2A Topoisonmerase II alpha is a protein encoded by the TOP2A gene and is proposed as a predictive and prognostic marker for breast cancer. It is also proposed as an aid in predicting response to anthracycline therapy in breast cancer. Two types of tests are available for topoisonmerase II alpha: topoisomerase II alpha protein expression testing by immunohistochemistry (IHC); andTOP2A gene amplification testing by FISH (eg, TOP2A FISH pharmDx Assay). The topoisomerase II alpha gene (TOP2A) is located adjacent to the HER-2 oncogene at the chromosome location 17q12-q21 and is either amplified or deleted (with equal frequency) in a great majority of HER-2 amplified primary breast tumors and also in tumors without HER-2 amplification. Recent experimental as well as numerous, large, multi-center trials suggest that amplification (and/or deletion) of TOP2A may account for both sensitivity or resistance to commonly used cytotoxic drugs (e.g., anthracyclines) depending on the specific genetic defect at the TOP2A locus. An analysis of TOP2A aberrations in the Danish Breast Cancer Cooperative Group trial 89D (Nielsen, et al., 2008) suggested a differential benefit of adjuvant chemotherapy in patients with primary breast cancer, favoring treatment with epirubicin in patients with TOP2A amplifications, and perhaps deletions; however, the authors concluded that, \"Additional studies are needed to clarify the exact importance of TOP2A deletions on outcome, but deletions have proven to be associated with a very poor prognosis.\" The National Comprehensive Cancer Network (NCCN, 2008) guideline on breast cancer does not address the use of TOP2A testing. Guidelines from the American Society for Clinical Oncology (2016) state: \"The clinician should not use TOP2A gene amplification or TOP2A protein expression by IHC to guide adjuvant chemotherapy selection.: This is a moderate-strength recommendation based upon high quality evidence. The guidelines also recommend against the use of TOP2A gene coamplification to guide adjuvant chemotherapy selection. TSP-1 Ghoneim et al (2008) explained that thrombospondin-1 (TSP-1) is a member of a family of five structurally related extracellular glycoproteins that plays a major role in cell-matrix and cell to cell interactions. Due to its multifunctional nature and its ability to bind to a variety of cell surface receptors and matrix proteins, TSP-1 has been identified as a potential regulator of angiogenesis and tumor progression. Data collected by Secord, et al. (2007) suggested that high THBS-1 levels may be an independent predictor of worse progression-free and overall survival in women with advanced-stage epithelial ovarian cancer. However, a phase II clinical trail (Garcia, et al., 2008) of bevacizumab and low-dose metronomic oral cyclophosphamide in recurrent ovarian cancer reported that levels of TSP-1 were not associated with clinical outcome. In a review on multidrug resistance in acute leukemia, List and Spier (1992) explained that the mdr1 gene or its glycoprotein product, P-glycoprotein, is detected with high frequency in secondary acute myeloid leukemia (AML) and poor-risk subsets of acute lymphoblastic leukemia. Investigations of mdr1 regulation in normal hematopoietic elements have shown a pattern that corresponds to its regulation in acute leukemia, explaining the linkage of mdr1 to specific cellular phenotypes. Therapeutic trials are now in progress to test the ability of various MDR-reversal agents to restore chemotherapy sensitivity in high-risk acute leukemias. In a phase III multi-center randomized study to determine whether quinine would improve the survival of adult patients with de novo AML, Soary et al (2003) reported that neither mdr1 gene or P-glycoprotein expression influenced clinical outcome. A phase I/II study of the MDR modulator Valspodar (PSC 833, Novartis Pharma) combined with daunorubicin and cytarabine in patients with relapsed and primary refractory acute myeloid leukemia (Gruber et al, 2003) reported that P-glycoprotein did not give an obvious improvement to the treatment results. MRP-1 Motility-related protein (MRP-1) is a glycoprotein with a sequence identical to that of CD9, a white blood cell differentiation antigen. The level of MRP-1/CD9 expression has been found in investigational studies to inhibit cell motility and low MRP-1/CD9 expression may be associated with the metastatic potential of breast cancer (Miyake et al, 1995). CD9 immuno-expression is also being investigated as a potential new predictor of tumor behavior in patients with squamous cell carcinoma of the head and neck (Mhawech et al, 2004) as well as other tumors (e.g., urothelial bladder carcinoma, colon cancer, lung cancer); however, prospective studies are needed to determine the clinical role of MRP-1/CD9 expression in tumors. The National Comprehensive Cancer Network\'s guideline on occult primary tumors includes placental alkaline phosphatase (PLAP) as a useful marker to assist in identifying germ cell seminoma and non-seminoma germ cell tumors in unknown primary cancer (NCCN, 2009). Myeloperoxidase (MPO), a blood protein, is a major component of azurophilic granules of neutrophils. Myeloperoxidase analysis has been used to distinguish between the immature cells in acute myeloblastic leukemia (cells stain positive) and those in acute lymphoblastic leukemia (cells stain negative). The National Comprehensive Cancer Network guidelines on acute myeloid leukemia (AML) includeMPO analysis in the classification of AML (NCCN, 2008). Matsuo et al (2003) examined the prognostic factor of the percentage of MPO-positive blast cells for AML. Cytochemical analysis of MPO was performed in 491 patients who were registered to the Japan Adult Leukemia Study Group (AML92 study). Patients were divided into two groups using the percentage of MPO-positive blast (high [ or = 50%] and low [ 50%]). Complete remission rates were 85.4% in the former and 64.1% in the latter (p = 0.001). The OS and DFS were significantly better in the high MPO group (48.3 versus 18.7% for OS, and 36.3 versus 20.1% for DFS, p 0.001, respectively). Multi-variate analysis showed that both karyotype and the percentage of MPO-positive blast cells were equally important prognostic factors. The high MPO group still showed a better survival even when restricted to the intermediate chromosomal risk group or the patients with normal karyotype (p 0.001). The OS of patients with normal karyotype in the high MPO group was almost equalto that of the favorable chromosomal risk group. The authors concluded that the percentage of MPO-positive blast cells is a simple and highly significant prognostic factor for AML patients, and especially useful to stratify patients with normal karyotype. The most commonly used marker for hepatocellular carcinoma (HCC) is the AFP level. Des-gamma-carboxy prothrombin (DCP) (also known as \"prothrombin produced by vitamin K absence or antagonism II\" [PIVKA II]) has also shown promise in the diagnosis of HCC (Toyoda et al, 2006; Ikoma et al, 2002; Nomura et al, 1996; Liebman et al, 1984). In one series of 76 patients with HCC, this marker was elevated in 69 patients with a mean serum concentration of 900 mcg/L. Much lower mean values were seen in patients with chronic active hepatitis, metastatic disease to the liver, and normal subjects (10 and 42 mcg/L and undetectable, respectively) (Liebman et al, 1984). Elevations in serum levels of DCP are less frequent in tumors less than 3 cm in size (Nakamura et al, 2006; Weitzand Liebman, 1993;). Aoyagi et al (1996) as well asWeitzand Liebman (1993) reported that abnormal prothrombin levels do not correlate well with serum AFP. Toyoda at al (2006) measured AFP, lens culinaris agglutinin A-reactive fraction of AFP (AFP-L3), and DCP for the evaluation of tumor progression and prognosis of patients with HCC (n = 685) at the time of initial diagnosis. Positivity for AFP 20 ng/dL, AFP-L3 10% of total AFP, and/or DCP 40 mAU/mL was determined. In addition, tumor markers were measured after treatment of HCC. Of the 685 patients, 337 (55.8%) were positive for AFP, 206 (34.1%) were positive for AFP-L3, and 371 (54.2%) were positive for DCP. In a comparison of patients positive for only 1 tumor marker, patients positive for AFP-L3 alone had a greater number of tumors, whereas patients positive for DCP alone had larger tumors and a higher prevalence of portal vein invasion. When patients were compared according to the number of tumor markers present, the number of markers present clearly reflected the extent of HCC and patient outcomes. The number of markers present significantly decreased after treatment. The authors concluded that tumor markers AFP-L3 and DCP appeared to represent different features of tumor progression in patients with HCC and that the number of tumor markers present could be useful for the evaluation of tumor progression, prediction of patient outcome, and treatment efficacy. The National Comprehensive Cancer Network\'s guideline on HCC (NCCN, 2008) does not include measurement of DCP among the surveillance test options for HCC. According to NCCN guidelines, proposed surveillance for the early detection of HCC among high-risk populations (e.g., chronic hepatitis C virus-infected patients) includes liver ultrasonography every 3 to 6 months and evaluation of alkaline phosphatase, albumin, and AFP. The guidelines stated, \"It is not yet clear if early detection of hepatocellular cancer with routine screening improves the percentage of patients detected with disease at a potentially curative stage, but high-risk chronic hepatitis C virus - infected patients should be considered for ongoing recurrent screening until these issues have been resolved. The level of des-gamma-carboxy-prothrombin protein induced by vitamin K absence (PIVKA-II) is also increased in many patients with hepatocellular carcinoma. However, as is true with AFP, PIVKA-II may be elevated in patients with chronic hepatitis.\" Furthermore, according to Sherman (2008), DCP has not been adequately studied as a screening test for HCC and cannot be recommended at this time. NMP66 Researchers at Matritech (Newton, MA) have detected the presence of nuclear matrix protein (NMP) in the blood of women at the early stage of breast cancer, which is absent in the blood of healthy women, as well as those with fibroadenoma. NMP66 has been selected as a marker for further development and clinical trials of a test for use in the detection and monitoring of women with, or at risk for, breast cancer have been initiated (Wright and McGechan, 2003). However, there are no published studies on the effectiveness of NMP66 testing at this time. HERmark HERmark Breast Cancer Assay ( biosciences monogram) is used to help determine prognosis and therapeutic choices for metastatic breast cancer (Raman, et al., 2013). Clinical practice guidelines recommend determining HER2 status in patients with all invasive breast cancer, but caution that current HER2 testing methods such as central immunohistochemistry and Fluorescence in situ Hybridization test may be inaccurate in approximately 20% of cases. According to the HERmark Web site, their method precisely quantifies HER2 total protein and HER2 homodimer levels in formalin-fixed, paraffin-embedded tissue sections and outperformed Fluorescence in situ Hybridization at determining patient outcomes in patients with metastatic breast cancer. HERmark testing has been proposed for a number of indications, including useto predict response to trastuzumab in the treatment of metastatic breast cancer. Monogram, the manufacturer of theHERmark test, claims that the testcan provide a more precise and quantitative measurement of the HER2 gene thanIHCand fluorescent in-situ hybridization (FISH) tests. The HERmark provides a quantitative measurement of HER2 total protein and HER2 homodimer levels, while conventional methods are an indirect measure of the HER2 gene,the manufacturerclaims. The HERmark test will be offered as a CLIA-validated assay through Monogram\'s CAP-certified clinical laboratory. Other proposed indications for HERmarkinclude determining the prognosis for breast cancer, and predicting treatment results in cancers other than breast cancer (e.g., ovarian prostate, head and neck, etc.). There are no current recommendations from leading medical professional organizations for the use of HERmark testing for breast cancer. Yardley et al (2015)compared quantitative HER2 expression by the HERmark Breast Cancer Assay (HERmark) with routine HER2 testing by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), and correlated HER2 results with overall survival (OS) of breast cancer patients in a multicenter Collaborative Biomarker Study (CBS). Two hundred and thirty-two formalin-fixed, paraffin-embedded breast cancer tissues and local laboratory HER2 testing results were provided by 11 CBS sites. HERmark assay and central laboratory HER2 IHC retesting were retrospectively performed in a blinded fashion. HER2 results by all testing methods were obtained in 192 cases. HERmark yielded a continuum of total HER2 expression (H2T) ranging from 0.3 to 403 RF/mm2 (approximately 3 logs). The distribution of H2T levels correlated significantly (P 0.0001) with all routine HER2 testing results. The concordance of positive and negative values (equivocal cases excluded) between HERmark and routine HER2 testing was 84% for local IHC, 96% for central IHC, 85% for local FISH, and 84% for local HER2 status. OS analysis revealed a significant correlation of shorter OS with HER2 positivity by local IHC (HR=2.6, P=0.016), central IHC (HR=3.2, P=0.015), and HERmark (HR=5.1, P 0.0001) in this cohort of patients most of whom received no HER2-targeted therapy. The OS curve of discordant low (HER2 positive but H2T low, 10% of all cases) was aligned with concordant negative (HER2 negative and H2T low, HR=1.9, P=0.444), but showed a significantly longer OS than concordant positive (HER2 positive and H2T high, HR=0.31, P=0.024). Conversely, the OS curve of discordant high (HER2 negative but H2T high, 9% of all cases) was aligned with concordant positive (HR=0.41, P=0.105), but showed a significantly shorter OS than concordant negative (HR=41, P 0.0001). Noon et al (2010) stated that renal cell carcinoma (RCC) is the most common type of kidney cancer and follows an unpredictable disease course. These researchers reviewed 2 critical genes associated with disease progression --p53 and murine double minute 2 (MDM2) --and provided a comprehensive summary and critical analysis of the literature regarding these genes in RCC. Information was compiled by searching the PubMed database for articles that were published or e-published up to April 1, 2009. Search terms included renal cancer, renal cell carcinoma, p53, and MDM2. Full articles and any supplementary data were examined; and, when appropriate, references were checked for additional material. All studies that described assessment of p53 and/or MDM2 in renal cancer were included. The authors concluded that increased p53 expression, but not p53 mutation, is associated with reduced overall survival/more rapid disease progression in RCC. There also was evidence that MDM2 up-regulation is associated with decreased disease-specific survival. Two features of RCC stood out as unusual and will require further investigation: increased p53 expression is tightly linked with increased MDM2 expression; and patients who have tumors that display increased p53 and MDM2 expression may have the poorest overall survival. Because there was no evidence to support the conclusion that p53 mutation is associated with poorer survival, it seemed clear that increased p53 expression in RCC occurs independent of mutation. The authors stated that further investigation of the mechanisms leading to increased p53/MDM2 expression in RCC may lead to improved prognostication and to the identification of novel therapeutic interventions. OVA1 is a blood testused to aid in the evaluation of pelvic masses for the likelihood of malignancy before surgery. OVA1 measures five biomarkers: apolipoprotein A1 (Apo A-1), beta-2 microglobulin (B2M), CA-125 prealbumin and transferrin. The results of these measurements are applied to an algorithm, resulting in a numerical score. The OVA1 Test (Vermillion Inc. and Quest Diagnostics)is a serum test that is intended tohelp physicians determine if a woman is at risk for a malignant pelvic mass prior to biopsy or exploratory surgery, when the physician’s independent clinical and radiological evaluation does not indicate malignancy (Mundy, et al., 2010).The OVA1 Testemploys an in vitro diagnostic multivariate index (IVDMIA)that combines the results of five immunoassays to produce a numerical score indicating a women\'s likelihood of malignancy. The OVA1 Testis intended to help physicians assess if a pelvic mass is benign or malignant in order to help determine whether to refer a woman to a gynecologic oncologist for surgery. The OVA1 Test was cleared by the FDA for use in women who meet the following criteria: over age 18, ovarian adnexal mass present for which surgery is planned, and not yet referred to an oncologist. The intended use of theOVA1 Test is an aid to further assess the likelihood that malignancy is present when the physician’s independent clinical and radiological evaluation does not indicate malignancy. According to the product labeling, the OVA1 Test is not intended as a screening or stand-alone diagnostic assay. There is a lack of evidence in the peer-reviewed published medical literature on the OVA1 Test. Ueland et al (2011) sought to compare the effectiveness of physician assessment with the OVA1 multivariate index assay in identifying high-risk ovarian tumors. The multivariate index assay was evaluated in women scheduled for surgery for an ovarian tumor in a prospective, multi-institutional trial involving 27 primary- care and specialty sites throughout the United States. Preoperative serum was collected, and results for the multivariate index assay, physician assessment, and CA 125 were correlated with surgical pathology. Physician assessment was documented by each physician before surgery. CA 125 cutoffs were chosen in accordance with the referral guidelines of the American College of Obstetricians and Gynecologists. The study enrolled 590 women, with 524 evaluable for the multivariate index assay and CA 125, and 516 for physician assessment. Fifty-three percent were enrolled by nongynecologic oncologists. There were 161 malignancies and 363 benign ovarian tumors. Physician assessment plus the multivariate index assay correctly identified malignancies missed by physician assessment in 70% of nongynecologic oncologists, and 95% of gynecologic oncologists. The multivariate index assay also detected 76% of malignancies missed by CA 125. Physician assessment plus the multivariate index assay identified 86% of malignancies missed by CA 125, including all advanced cancers. The investigators stated that the performance of the multivariate index assay was consistent in early- and late-stage cancers. Ware Miller et al (2011) sought to estimate the performance of the ACOG referral guidelines for pelvic mass with the OVA1 multivariate index assay. A prospective, multi-institutional trial included 27 primary care and specialty sites throughout the United States. The College guidelines were evaluated in women scheduled for surgery for an ovarian mass. Clinical criteria and blood for biomarkers were collected before surgery. A standard CA 125-II assay was used and the value applied to the multivariate index assay algorithm and the CA 125 analysis. Study results were correlated with surgical pathology. Of the 590 women enrolled with ovarian mass on pelvic imaging, 516 were evaluable. There were 161 malignancies (45 premenopausal and 116 postmenopausal). The College referral criteria had a modest sensitivity in detecting malignancy. Replacing CA 125 with the multivariate index assay increased the sensitivity (77-94%) and negative predictive value (87-93%) while decreasing specificity (68-35%) and positive predictive value (52-40%). Similar trends were noted for premenopausal women and early-stage disease. Bristow et al (2013) sought to validate the effectiveness of a multivariate index assay in identifying ovarian malignancy compared to clinical assessment and CA125-II, among women undergoing surgery for an adnexal mass after enrollment by non-gynecologic oncology providers. A prospective, multi-institutional trial enrolled female patients scheduled to undergo surgery for an adnexal mass from 27 non-gynecologic oncology practices. Pre-operative serum samples and physician assessment of ovarian cancer risk were correlated with final surgical pathology. A total of 494 subjects were evaluable for multivariate index assay, CA125-II, and clinical impression. Overall, 92 patients (18.6%) had a pelvic malignancy. Primary ovarian cancer was diagnosed in 65 patients (13.2%), with 43.1% having FIGO stage I disease. For all ovarian malignancies, the sensitivity of the multivariate index assay was 95.7% (95%CI=89.3-98.3) when combined with clinical impression. The multivariate index assay correctly predicted ovarian malignancy in 91.4% (95%CI=77.6-97.0) of cases of early-stage disease, compared to 65.7% (95%CI=49.2-79.2) for CA125-II. The multivariate index assay correctly identified 83.3% malignancies missed by clinical impression and 70.8% cases missed by CA125-II. Multivariate index assay was superior in predicting the absence of an ovarian malignancy, with a negative predictive value of 98.1% (95%CI=95.2-99.2). Both clinical impression and CA125-II were more accurate at identifying benign disease. The multivariate index assay correctly predicted benign pathology in 204 patients (50.7%, 95%CI=45.9-55.6) when combined with clinical impression. Longoria et al (2014) sought to analyze the effectiveness of the OVA1 multivariate index assay (MIA) in identifying early-stage ovarian malignancy compared to clinical assessment, CA 125-II, and modified American Congress of Obstetricians and Gynecologists (ACOG) guidelines among women undergoing surgery for an adnexal mass. Patients were recruited in 2 related prospective, multi-institutional trials involving 44 sites. All women had preoperative imaging and biomarker analysis. Preoperative biomarker values, physician assessment of ovarian cancer risk, and modified ACOG guideline risk stratification were correlated with surgical pathology. A total of 1016 patients were evaluable for MIA, CA 125-II, and clinical assessment. Overall, 86 patients (8.5%) had primary-stage I/II primary ovarian malignancy, with 70.9% having stage I disease and 29.1% having stage II disease. For all early-stage ovarian malignancies, MIA combined with clinical assessment had significantly higher sensitivity (95.3%; 95% confidence interval [CI], 88.6-98.2) compared to clinical assessment alone (68.6%; 95% CI, 58.2-77.4), CA 125-II (62.8%; 95% CI, 52.2-72.3), and modified ACOG guidelines (76.7%; 95% CI, 66.8-84.4) (P .0001). Among the 515 premenopausal patients, the sensitivity for early-stage ovarian cancer was 89.3% (95% CI, 72.8-96.3) for MIA combined with clinical assessment, 60.7% (95% CI, 42.4-76.4) for clinical assessment alone, 35.7% (95% CI, 20.7-54.2) for CA 125-II, and 78.6% (95% CI, 60.5-89.8) for modified ACOG guidelines. Early-stage ovarian cancer in postmenopausal patients was correctly detected in 98.3% (95% CI, 90.9-99.7) of cases by MIA combined with clinical assessment, compared to 72.4% (95% CI, 59.8-82.2) for clinical assessment alone, 75.9% (95% CI, 63.5-85.0) for CA 125-II, and 75.9% (95% CI, 63.5-85.0) for modified ACOG guidelines. Bristow et al (2013) assessed the impact on referral patterns of using the OVA1 Multivariate Index Assay, CA125, modified-American College of Obstetricians and Gynecologists referral guidelines, and clinical assessment among patients undergoing surgery for an adnexal mass after initial evaluation by nongynecologic oncologists. Overall, 770 patients were enrolled by nongynecologic oncologists from 2 related, multiinstitutional, prospective trials and analyzed retrospectively. All patients had preoperative imaging and biomarker analysis. The subset of patients enrolled by nongynecologic oncologists was analyzed to determine the projected referral patterns and sensitivity for malignancy based on multivariate index assay (MIA), CA125, modified-American College of Obstetricians and Gynecologists (ACOG) guidelines, and clinical assessment compared with actual practice. The prevalence of malignancy was 21.3% (n = 164). In clinical practice, 462/770 patients (60.0%) were referred to a gynecologic oncologist for surgery. Triage based on CA125 predicted referral of 157/770 patients (20.4%) with sensitivity of 68.3% (95% confidence interval [CI], 60.8-74.9). Triage based on modified-ACOG guidelines would have resulted in referral of 256/770 patients (33.2%) with a sensitivity of 79.3% (95% CI, 72.4-84.8). Clinical assessment predicted referral of 184/763 patients (24.1%) with a sensitivity of 73.2% (95% CI, 65.9-79.4). Risk stratification using multivariate index assay would have resulted in referral of 429/770 (55.7%) patients, with sensitivity of 90.2% (95% CI, 84.7-93.9). MIA demonstrated statistically significant higher sensitivity (P .0001) and lower specificity (P .0001) for detecting malignancy compared with clinical assessment, CA125, and modified-ACOG guidelines. Goodrich et al (2014) investigated the relationship between imaging and the multivariate index assay (MIA) in the prediction of the likelihood of ovarian malignancy before surgery. Subjects were recruited in 2 related prospective, multiinstitutional trials that involved 44 sites across the United States. Women had ovarian imaging, biomarker analysis, and surgery for an adnexal mass. Ovarian tumors were classified as high risk for solid or papillary morphologic condition on imaging study. Biomarker and imaging results were correlated with surgical findings. Of the 1110 women who were enrolled with an adnexal mass on imaging, 1024 cases were evaluable. There were 255 malignant and 769 benign tumors. High-risk findings were present in 46% of 1232 imaging tests and 61% of 1024 MIA tests. The risk of malignancy increased with rising MIA scores; similarly, the likelihood of malignancy was higher for high-risk, compared with low-risk, imaging. Sensitivity and specificity for the prediction of malignancy were 98% (95% CI, 92-99) and 31% (95% CI, 27-34) for ultrasound or MIA; 68% (95% CI, 58-77) and 75% (95% CI, 72-78) for ultrasound and MIA, respectively. For computed tomography scan or MIA, sensitivity was 97% (95% CI, 92-99) and specificity was 22% (95% CI, 16-28); the sensitivity and specificity for computed tomography scan and MIA were 71% (95% CI, 62-79) and 70% (95% CI, 63-76). Only 1.6% of ovarian tumors were malignant when both tests indicated low risk. An assessment by the BlueCross BlueShield Association Technology Evaluation Center (BCBSA, 2013) stated: \"The evidence regarding the effect of OVA1 ...on health outcomes is indirect and based on studies of diagnostic performance of the tests in patients undergoing surgery for adnexal masses. Although the studies show improvements in sensitivity and worsening of specificity with the use of the tests in conjunction with clinical assessment, there are problems in concluding that this results in improved health outcomes. The clinical assessment performed in the studies is not well characterized. Although OVA1 improves sensitivity, specificity declines so much that most patients test positive.\" An technology assessment by the ECRI Institute (2015) concluded that the evidence on OVA1 consists of cross-sectional diagnostic accuracy studies. This evidence as reported in article abstracts is unclear as to whether use of OVA1 improves patient-oriented outcomes because none of the studies reported the direct impact of these tests on survival or quality of life. The primary rationale for using these tests is to select the type of surgeon to perform the primary surgery. Stewart et al (2016) reported on a survey of primary care physicians on how often they refer patients diagnosed with ovarian cancer to gynecological oncologists, finding thata total of 84% of primary care physicians (87% of family/general practitioners, 81% of internists and obstetrician/gynecologists) said they always referred patients to gynecologic oncologists for treatment. Common reasons for not always referring were patient preference or lack of gynecologic oncologists in the practice area. A total of 23% of primary care physicians had heard of the OVA1 test, which helps to determine whether gynecologic oncologist referral is needed. The authors noted that, although referral rates reported here are high, it is not clear whether ovarian cancer patients are actually seeing gynecologic oncologists for care. Eskander et al (2016) conducted a retrospective chart review of patients who received the OVA1.Twenty-two obstetricians/gynecologists were recruited from a variety of practices and hospitals throughoutthe United States. A total of 136 patients with elevated-risk assay results were assessed, of whom 122 underwent surgery to remove an adnexal mass. Prior to surgery, 98 (80%) of the patients were referred to a gynecologic oncologist with an additional 11 (9%) having a gynecologic oncologist available if required by intra-operative findings. Primary ovarian cancer was found in 65 (53%) patients, and gynecologic oncologists performed 61 (94%) of the initial surgeries these patients. Similar results were found in premenopausal and postmenopausal patients. Forde et al (2016) conducted an economic analysis modelto evaluate the clinical and cost implications of adoptingOVA1 in clinical practice versus the modified ACOG referral guidelines and CA-125 alone, over a lifetime horizon, from the perspective of the public payer. Clinical parameters used to characterize patients\' disease status, quality of life, and treatment decisions were estimated using the results of published studies; costs were approximated using reimbursement rates from CMS fee schedules. Model endpoints included overall survival (OS), costs, quality-adjusted life years (QALYs), and incremental cost-effectiveness ratio (ICER). The cost-effectiveness threshold was set to $50,000 per QALY. One-way sensitivity analysis was performed to assess uncertainty of individual parameters included in the analysis. All costs were reported in 2014 US dollars. Use of OVA1was cost-effective, resulting in fewer re-operations and pre-treatment CT scans. OverallOVA1 resulted in an ICER of $35,094/QALY gained.OVA1 was also cost-saving and QALY-increasing compared to use of CA-125 alone with an ICER of $12,189/QALY gained. One-way sensitivity analysis showed the ICER was most affected by the following parameters: sensitivity of OVA1; sensitivity of mACOG; and percentage of patients, not referred to a gynecologic oncologist, who were correctly diagnosed with advanced epithelial ovarian cancer (EOC). The authors concluded that OVA1is a more cost-effective triage strategy than mACOG or CA-125. It is expected to increase the percentage of women with ovarian cancer that are referred to gynecologic oncologists, which is shown to improve clinical outcomes. Limitations include the use of assumptions when published data was unavailable, and the use of multiple sources for survival data. Urban et al (2017) reported that the addition of a patient-reported symptom index (SI), which captures subjective symptoms in an objective manner, improved the sensitivity of the OVA1 multivariate index assay (MIA). The investigators conducted aprospective study of patients seen at a tertiary care medical center. Following consent, patients completed an SI and preoperative serum was collected for an OVA1 multivariate index assay. Results for the SI andOVA1 were correlated with operative findings and surgical pathology. Of 218 patients enrolled, 124 (56.9%) had benign disease and 94 (43.1%) had borderline tumors or carcinomas. Sixty-six patients had a primary ovarian or fallopian tube cancer. The median age of patients enrolled in this study was 54 years (interquartile range, 44-63 years), of whom 148 (67.9%) were postmenopausal. More than a third (36.3%) of patients with benign masses was accurately identified as low risk by MIA and SI. The sensitivity, specificity, positive predictive value (PPV)and negative predictive value (NPV) of the SI relative to primary ovarian cancer was 87.9% (95% CI, 77.9%-93.7%), 70.2% (95% CI, 61.6%-77.5%), 61.1% (95% CI 51.0-70.2%)and 91.6% (95% CI, 84.3%-95.7%), respectively. The sensitivity, specificity, PPVand NPV of CA125 was 75.4% (95% CI, 63.7%-84.2%), 85.7% (95% CI 78.3%-90.9%), 74.2% (62.6%-82.3%)and 86.4% (95% CI, 79.1%-91.5%), respectively.The sensitivity, specificity, PPVand NPV of the MIA were 93.9% (95% CI, 85.4%-97.6%), 55.6 (95% CI 46.9%-64.1%), 53.0% (95% CI 44.0%-61.8%and 94.5% (95% CI, 94.5%-100%), respectively. The overall sensitivity for the combination of MIA plus SI was 100% (66/66; 95% CI, 94.5%-100%), and specificity was 36.3% (45/124; 95% CI, 28.4%-45.0%), with a PPV of 45.5% (37.6% to 53.6%) and a NPV of 100% (95% CI, 92.1%-100%). Limitations of this study noted by the authorsinclude the small sample size and thehigh prevalence of ovarian malignancies in thispopulationthat was largely from a tertiary care center.It should also be noted that the sensitivity and negative predictive value of SIplus CA 125 was 96.9% (95% CI 89.5%-99.2%) and 97.3% (95% CI 90.5%-99.2%), whichexceeded that of MIA alone but was somewhat less than MIA plus SI. Ovarian cancer guidelines from the National Comprehensive Cancer Network (2016) note that the Society of Gynecologic Oncology (SGO), the FDA, and the Mayo Clinic have stated that the OVA1 test should not be used as a screening tool to detect ovarian cancer. The NCCN explains that the OVA1 attempts to preoperatively classify adenixal masses as benign or malignant and suggests that patients can be assessed for who should undergo surgery by an experienced gynecologic oncologist and who can have surgery in the community. \"Based upon data documenting an increased survival, NCCN guidelines panel members recommended that all patients should undergo surgery by an experienced gynecologic oncologist (Category 1 recommendation).\" Guidelines on management of adnexal masses from the American College of Obstetricians and Gynecologists (ACOG, 2016)state that the OVA1 multivariate index assay has demonstrated higher sensitivity and negative predictive value comparedwith clinical impression and CA 125 alone.The guidelinesstate that serum biomarker panels [OVA1 and ROMA]may be used as an alternative to CA 125 alone in determining the need for referral to or consultation with a gynecological oncologist when an adenexal mass requires surgery. The guidelines state that trials that have evaluated the predictive value of these panelsshow potential for improved specificity; \"[h]owever, comparativeresearch has not yet definedthe best testing approach.\" ACOG guidelines (2016)state that, primarily based upon consensus and expert opinion (Level C), \"[s]erum biomarker panels may be used as an alternative to CA 125 level alone in determining the need for referral to or consultation with a gynecological oncologist when an adnexal mass requires surgery.\" The guidelines state that, based upon\"limited or inconsistent\" evidence (Level B),consultation or referral to a gynecological oncologist is recommended for women with an adnexal mass that meet one or more of the following criteria; postmenopausal with elevated CA 125 level, ultrasound findings suggestive of malignancy, ascites,a nodular or fixed pelvic mass, or evidence of abdominal or distant metastases; premenosausal with very elevated CA 125 level, ultrasound findings suggestive of malignancy, ascites, a nodular or fixed pelvic mass, or evidence of abdominal or distant metastases; premenopausal or postmenopausal with an elevated score on a formal risk assessment test such as the multivariate index assay, risk of malignancy index, or the Risk of Ovarian Malignancy algorithm or one of the ultrasound-based scoring systems from the International Ovarian Tumor Analysis group. The UK National Institute for Health Research Health Technology Assessment Programme has commissioned an assessment (Westwood, et al., 2016)comparing the Risk of Malignancy Index(RMI)to alternative risk scores for ovarian cancer, including Overa/OVA2 (Vermillion), as well as the ROMA score, simple rules ultrasound classification system (IOTA), Assessment of Different NEoplasias in the adnexa (ADNEX) model (IOTA group). The assessment is scheduled to be completed in 2017. ColonSentry The ColonSentry test (GeneNews, Toronto, Canada) measures the expression of seven genes, which serve as biomarkers to detect colorectal cancer. Interpretation of the status of these seven biomarkers is intended to assist physicians inidentifying patients who have an increased current risk. According to the manufacturer, individuals assessed as having an increased current risk of colorectal cancer should consider having a colonoscopy. Individuals assessed as having a decreased current risk of colorectal cancer should discuss with their doctor further screening, including repeating ColonSentry at regular intervals. There is a lack of evidence in the peer-reviewed published medical literature on the effectiveness of colorectal cancer screening with ColonSentry. No current evidence-based guidelines from medical professional organizations or public health agencies recommend ColonSentry for colorectal cancer screening. Prostate Px Prostate Px (Aureon) uses a prostate cancer patient\'s biopsy tissue to provide an assessment ofdisease severity and disease recurrence. Clinical data is integrated withan analysis of each patient’s cancer using tissue histology and molecular biomarkers, such as androgen receptor, associated with disease progression. Although the manufacturer states that the results of the Prostate Px can be used in decision-making, there is a lack of evidence of the clinical utility of this test in altering the management of patients such that clinical outcomes are improved. Post-Op Px Post-Op Px is a prognostic test that utilizes a patented systems pathology approach to analyze prostatectomy tissue by combining cellular, molecular and clinical information to provide a thorough and more accurate picture of each patient\'s individual risk of prostate cancer recurrence. (Aureon, 2010). Donovan et al (2011) evaluated the performance of a systems-based risk assessment tool with standard defined risk groups and the 10 year postoperative normogram for predicting disease progression. The systems model was found to be more accurate than standard risk groups both to predict significant disease progression (p 0.001) and for predicting prostate-specific antigen recurrence (p 0.001). However, this study has not been replicated in the peer-reviewed literature. An assessment by the BlueCross BlueShield Association Technology Evaluation Center (2010) concluded that tumor-cell epidermal growth factor receptor (EGFR) mutation analysis to predict response to erlotinib (Tarceva) in patients with advanced non-small cell lung cancer (NSCLC) meets the Blue Cross and Blue Shield Association Technology Evaluation Center (TEC) criteria. Furthermore, guidelines from the National Comprehensive Cancer Network (NCCN, 2010) recommend EGFR testing for the following histologic subtypes of NSCLC: adenocarcinoma, large cell, and NSCLC not otherwise specified. Epidermal growth factor receptor testing is not recommended for squamous cell carcinoma. The Alberta Provincial Thoracic Tumour Team’s clinical practice guideline on \"Non-small cell lung cancer stage IV\" (2011) stated that \"First-line monotherapy with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib is recommended for patients with EGFR mutation-positive NSCLC. Testing for EGFR mutations should take place for all eligible patients with advanced NSCLC and adenocarcinoma histology who are being considered for first-line therapy with gefitinib, irrespective of their gender, ethnicity, and smoking status\". NCCN non-small cell lung cancer guidelines (2015) state that EGFR and ALK testing should be conducted as part of a multiplex/next generation sequencing. TheNCCN NSCLC Guidelines Panel \"strongly endorses broader molecular profiling with the goal of identifying rare mutations for which effective drugs may already be available, or to appropriately counsel patients regarding the availability of clinical trials. Broad molecular profiling is a key component of the improvement of care of patients with NSCLC.\" Gao et al (2012) stated that gefiinib and erlotinib are 2 similar small molecules of selective and reversible epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), which have been approved for second-line or third-line indication in previously treated advanced NSCLC patients. The results of comparing the EGFR-TKI with standard platinum-based doublet chemotherapy as the first-line treatment in advanced NSCLC patients with activated EGFR mutation were still controversial. A meta-analysis was performed to derive a more precise estimation of these regimens. Finally, 6 eligible trials involved 1,021 patients were identified. The patients receiving EGFR-TKI as front-line therapy had a significantly longer PFS than patients treated with chemotherapy [median PFS was 9.5 versus 5.9 months; HR = 0.37; 95 % CI: 0.27 to 0.52; p 0.001]. The overall response rate (ORR) of EGFR-TKI was 66.60 %, whereas the ORR of chemotherapy regimen was 30.62 %, which was also a statistically significant favor for EGFR-TKI [relative risk (RR) = 5.68; 95 % CI: 3.17 to 10.18; p 0.001]. The OS was numerically longer in the patients received EGFR-TKI than patients treated by chemotherapy, although the difference did not reach a statistical significance (median OS was 30.5 versus 23.6 months; HR = 0.94; 95 % CI: 0.77 to 1.15; p = 0.57). Comparing with first-line chemotherapy, treatment of EGFR-TKI achieved a statistical significantly longer PFS, higher ORR and numerically longer OS in the advanced NSCLC patients harboring activated EGFR mutations, thus, it should be the first choice in the previously untreated NSCLC patients with activated EGFR mutation. Guidelines from the American Society for Clinical Oncology (2016) state: \"The clinician should not use HER1/epidermal growth factor receptor expression by IHC to guide adjuvant chemotherapy selection\" in breast cancer. CEACAM-7 Messick et al (2010) evaluated carcinoembryonic antigen cellular adhesion molecule-7 (CEACAM-7) expression in rectal cancer as a predictive recurrence factor. A single-institution colorectal cancer database and a frozen tissue biobank were queried for rectal cancer patients. CEACAM-7 messenger RNA (mRNA) expression from normal rectal mucosa and rectal cancers was analyzed using quantitative real-time polymerase chain reaction (PCR). Expression-level differences among normal tissue, disease-free survivors, and those that developed recurrence were analyzed. A total of 84 patients were included in the study, which consisted of 37 patients with non-recurrent disease (median follow-up of170 months), 29 patients with recurrent disease, and 18 patients with stage IV disease. CEACAM-7 expression was decreased 21-fold in rectal cancers compared with normal mucosa (p = 0.002). The expression levels of CEACAM-7 were relatively decreased in tumors that developed recurrence compared with non-recurrence, significantly for stage II patients (14-fold relative decrease,p = 0.002). For stages I-III, disease-free survival segregates were based on relative CEACAM-7 expression values (p = 0.036), specifically for stage II (p = 0.018). The authors concluded that CEACAM-7 expression is significantly decreased in rectal cancer. Expression differences between long-term survivors and those with recurrent disease introduce a potential tumor marker to define a subset of patients who benefit most from adjuvant therapy. Moreover, they stated that additional study and validationare needed before CEACAM-7 can be applied in clinical settings. Castro et al (2010) assessed the potential value of cofilin (CFL1) gene (main member of the invasion/metastasis pathway) as a prognostic and predictive NSCLC biomarker. Meta-analysis of tumor tissue microarray was applied to examine expression of CFL1 in archival lung cancer samples from 111 patients, and its clinicopathologic significance was investigated. The robustness of the finding was validated using another independent data set. Finally, the authors assayed in vitro the role of CFL1 levels in tumor invasiveness and drug resistance using 6 human NSCLC cell lines with different basal degrees of CFL1 gene expression. Cofilin levels in biopsies discriminated between good and bad prognosis at early tumor stages (IA, IB, and IIA/B), where high CFL1 levels are correlated with lower overall survival rate (p 0.0001). Biomarker performance was further analyzed by IHC,hazard ratio(p 0.001), and receiver-operating characteristic curve (area = 0.787; p 0.001). High CFL1 mRNA levels and protein content are positively correlated with cellular invasiveness (determined by Matrigel Invasion Chamber System) and resistance (2-fold increase in drug 50 % growth inhibition dose) against a list of 22 alkylating agents. Hierarchical clustering analysis of the CFL1 gene network had the same robustness for stratified NSCLC patients. The authors concluded that these findings indicated that the CFL1 gene and its functional gene network can be used as prognostic biomarkers for NSCLC and could also guide chemotherapeutic interventions. Moreover, prospective, large-scale, randomized clinical trials are needed to establish the role of CFL1 as a prognostic and drug resistance marker for NSCLC. EarlyCDT-Lung The EarlyCDT-Lung (Oncimmune, De Soto, KS)test measures antibodies to 6 tumor-associated antigens: p53, NY-ESO-1, CAGE, GBU4-5, Annexin 1, and SOX2. Elevation of any one of the panel of immuno-biomarkers above a predetermined cut-off value suggests that a tumor might be present. The test is designed to be used in conjunction with diagnostic imaging. High-risk individuals with a positive EarlyCDT-Lung would have additional testing such as a CT scan or the test would be used as a follow-up test for indeterminate lung nodules identified by CT. Boyle et al (2011) reported the sensitivity and specificity of an autoantibody panel of 6 tumor-related antigens (p53, NY-ESO-1, CAGE, GBU4-5, Annexin 1 and SOX2) in patients with lung cancer. Three cohorts of patients with newly diagnosed lung cancer were identified: group 1 (n = 145), group 2 (n = 241) and group 3 (n = 269). Patients were individually matched by gender, age and smoking history to a control individual with no history of malignant disease. Serum samples were obtained after diagnosis but before any anticancer treatment. Autoantibody levels were measured against the panel of 6 tumor-related antigens (p53, NY-ESO-1, CAGE, GBU4-5, Annexin 1 and SOX2). Assay sensitivity was tested in relation to demographic variables and cancer type/stage. The autoantibody panel demonstrated a sensitivity/specificity of 36 %/91 %, 39 %/89 % and 37 %/90 % in groups 1, 2 and 3, respectively, with good reproducibility. There was no significant difference between different lung cancer stages, indicating that the antigens included covered the different types of lung cancer well. The authors concluded that the assay confirms the value of an autoantibody panel as a diagnostic tool and offers a potential system for monitoring patients at high-risk of lung cancer. There is insufficient evidence of the effectiveness of the EarlyCDT-Lung as a screening test for the early detection of lung cancer. Systematic screening for lung cancer is not unequivocally recommended by any major professional organization. The USPSTF (2004) concluded that current evidence was insufficient to recommend for, or against, screening for lung cancer. Whether earlier detection of lung cancer will translate to a mortality benefit remains unclear. E-cad Deeb et al (2004) stated that E-cadherin (E-cad) and epidermal growth factor receptor (EGFR) are important cell adhesion and signaling pathway mediators. Theyr reported the results of a study which aimed to assess their expression in lung adenocarcinoma (AdC) and squamous cell carcinoma (SCC) and their association with clinicopathologic variables. Two to three cores from 130 resectable lung cancers (stages I-IIIA) were arrayed into three blocks using a Beecher system. Markers expression and coexpression were analyzed against clinicopathologic variables (age, gender, smoking status, performance status, weight loss, histology, grade, stage, and lymph node involvement) and patient survival. For E-cad, 65 cases (55%) were positive (+), 53 (45%) were negative (-); and for EGRF, 43 cases (34%) were (+), and 83 (66%) were (-). There was no significant association between E-cad or EGFR, and any of the clinicopathologic variables except for an association between EGFR(+) and SCC histologic type. Both negative and cytoplasmic staining of E-cad correlated with shorter patient survival with P=0.008 and 0.002, respectively. EGFR expression did not correlate with patient survival, but, patients with E-cad(-)/EGFR(+) phenotype had poorer survival than those with E-cad(+)/EGFR(-) (P=0.026). The authors concluded that lung AdC and SCC may be stratified based on expression of E-cad and EGFR with the E-cad(-)/EGFR(+) expression having a worse disease outcome. EML4-ALK Yoshida et al (2011) report that a subset of lung cancers harbors an EML4-ALK (echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase) gene fusion, and they examined 15 lung adenocarcinomas with reverse-transcriptase polymerase chain reaction-proven EML4-ALK fusion transcripts and 30 ALK-negative cases. Positive rearrangement signals (splits or isolated 3\' signals) were identified in 13 to 78% (mean ± SD, 41% ± 19%) of tumor cells in the ALK-positive cohort and in 0 to 15% (mean ± SD, 6% ± 4%) of cells in the ALK-negative cohort. Sensitivity was at 93% and specificity at 100%. The only false-negative tumor having only 13% CISH-positive cells displayed predominantly (76%) isolated 5\' signals unaccompanied by 3\' signals. FISH showed largely similar signal profiles, and the results were completely concordant with CISH.The authors stated that they have successfully introduced CISH for diagnosing EML4-ALK-positive lung adenocarcinoma. This method allows simultaneous visualization of genetics and tumor cytomorphology and facilitates the molecular evaluation and could be applicable in clinical practice to detect lung cancer that may be responsive to ALK inhibitors. Ellis et al (2011) conducted a systematic review and a consensus meeting of Canadian lung cancer oncologists and pathologists to make recommendations on the use of biomarkers in NSCLC. The articles were reviewed by pairs of oncologists and pathologists to determine eligibility for inclusion. Ten oncologists and pathologists reviewed and summarized the literature at a meeting attended by 37 individuals. The findings included that there is some evidence that histology is prognostic for survival as well as evidence from multiple randomized clinical trials to recommend the following: histologic subtype is predictive of treatment efficacy and for some agents toxicity. Immunohistochemistry testing should be performed on NSCLC specimens that cannot be classified accurately with conventional H E staining. As EGFR mutations are predictive of benefit from tyrosine kinase inhibitors, diagnostic NSCLC samples should be routinely tested for EGFR-activating mutations. Clinical data on K-RAS mutations are inconsistent, therefore testing is not recommended. There is insufficient evidence to recommend other biomarker testing. No biomarkers to date reliably predict improved efficacy for anti-VEGF therapy. The authors concluded that outine assessment for EML4/ALK mutations is not recommended at present, although emerging data suggest that it may become valuable in the near future. Shanmugan and co-workers (2010) stated that mucin 4 (MUC4) is aberrantly expressed in colorectal adenocarcinomas (CRCs) but its prognostic value is unknown. Archival tissue specimens collected from 132 CRC patients who underwent surgical resection without pre-surgery or pos-tsurgery therapy were evaluated for expression of MUC4 by using a mouse monoclonal antibody and horseradish peroxidase. MUC4 expression levels were correlated with clinicopathologic features and patient survival. Survival was estimated by both uni-variate Kaplan-Meier and multi-variate Cox regression methods. In both normal colonic epithelium and CRCs, MUC4 staining was localized primarily in the cytoplasm. The optimal immunostaining cut-off value (greater than or equal to75 % positive cells and an immunostaining scoregreater than or equal to2.0), which was derived by using the bootstrap method, was used to categorize CRCs into groups of high expression (33 of 132 patients; 25 %) or low expression (99 of 132 patients; 75 %). Patients who had early stage tumors (stages I and II) with high MUC4 expression had a shorter disease-specific survival (log-rank; p = 0.007) than patients who had with low expression. Patients who had advanced-stage CRCs (stages III and IV) did not demonstrate such a difference (log-rank; p = 0.108). Multi-variate regression models that were generated separately for patients with early stage and advanced-stage CRC confirmed that increased expression of MUC4 was an independent indicator of a poor prognosis only for patients who had early stage CRCs (HR 3.77; 95 % CI: 1.46 to 9.73). The authors stated that aftr validating these findings in larger retrospective and prospective studies, a stage-based anayses could establish the utility of MUC4 as a prognostic molecular marker of early stage CRC. ProOnc TumorSourceDx ProOnc TumorSourceDx test is designed to identify tissue or origin for metastastic tumor. It identifies 25 possible classes of tissue origin corresponding to 17 distinct tissues and organs. It requires only 48 microRNAs to identify tissue of origin based on microRNA expression levels. However, there is insufficient evidence regarding its clinical value as tumor markers. Coccoand associates(2010) examined the expression of serum amyloid A (SAA) in endometrial endometrioid carcinoma and evaluated its potential as a serum biomarker. SAA gene and protein expression levels were evaluated in endometrial endometrioid carcinoma and normal endometrial tissues, by real-time PCR, IHC, and flow cytometry. SAA concentration in 194 serum samples from 50 healthy women, 42 women with benign diseases, and 102 patients including 49 grade 1, 38 grade 2, and 15 grade 3 endometrial endometrioid carcinoma was also studied by a sensitive bead-based immunoassay. SAA gene expression levels were significantly higher in endometrial endometrioid carcinoma when compared with normal endometrial tissues (mean copy number by real-time PCR = 182 versus 1.9;p = 0.001). IHC revealed diffuse cytoplasmic SAA protein staining in poorly differentiated endometrial endometrioid carcinoma tissues. High intra-cellular levels of SAA were identified in primary endometrial endometrioid carcinoma cell lines evaluated by flow cytometry, and SAA was found to be actively secreted in vitro. SAA concentrations (microg/ml) had medians of 6.0 in normal healthy women and 6.0 in patients with benign disease (p = 0.92). In contrast, SAA values in the serum of endometrial endometrioid carcinoma patients had a median of 23.7, significantly higher than those of the healthy group (p = 0.001) and benign group (p = 0.001). Patients harboring G3 endometrial endometrioid carcinoma were found to have SAA concentrations significantly higher than those of G1/G2 patients. The authors concluded that SAA is not only a liver-secreted protein, but is also an endometrial endometrioid carcinoma cell product. SAA is expressed and actively secreted by G3 endometrial endometrioid carcinoma, and it is present in high concentration in the serum of endometrial endometrioid carcinoma patients. SAA may represent a novel biomarker for endometrial endometrioid carcinoma to monitor disease recurrence and response to therapy. They stated that additional studies are needed to validate these findings. Caris Target Now / Caris Molecular Profiling Service Molecular Intelligence Services (formerly Target Now Molecular Profiling Test) uses a multi-platform profiling approach including gene sequencing (NGS and Sanger), protein expression analysis (immunohistochemistry) and gene copy number analysis (chromogenic or fluorescence in situ hybridization [FISH]).The test has been used toexamine tumor samples for underlying molecular alterations that may yield insights into potentially overlapping and different therapeutic options for individuals with these tumor types. According to the manufacturer, the Caris Life Sciences molecular profiling test, Caris Target Now, examines the genetic and molecular changes unique to a patient\'s tumor so that treatment options may be matched to the tumor\'s molecular profile. The manufacturer states that theCaris Target Now test is performed after a cancer diagnosis has been established and the patient has exhausted standard of care therapies or if questions in therapeutic management exist. Using tumor samples obtained from a biopsy, the tumor is examined to identify biomarkers that may have an influence on therapy. Using this information, Caris Target Now is intended to provide information on the drugs that will be more likely to produce a positive response. The manufacturer states that Caris Target Now can be used with any solid cancer such as lung cancer, breast cancer, and prostate cancer. There is insufficient evidence to support the use of Caris Target Now molecular profiling.A study (Von Hoff et al, 2010) compared the progression-free survival (PFS) of patients with refractory metastatic cancers using a treatment regimen selected by Caris Target Now molecular profiling of the patient\'s tumorwith the PFS for the most recent regimen on which the patient had experienced progression.The investigators prespecified that amolecular profilingapproachwould bedeemed of clinical benefit for the individual patient who had a PFS ratio (defined as a ratio of PFS on molecular profiling-selected therapy to PFS on prior therapy) ofgreater than or equal to1.3.In 86 patients who hadmolecular profilingattempted, there was a molecular target detected in 84 (98 %). Sixty-six of the 84 patients were treated according tomolecular profilingresults. Eighteen (27 %) of 66 patients had a PFS ratio ofgreater than orequal1.3 (95 % CI:17 % to 38 %). Therefore, the null hypothesis (thatlessthan or equal to15 % of this patient population would have a PFS ratio ofgreater than or equal to1.3) was rejected. The authors concluded that, in 27 % of patients, themolecular profilingapproach resulted in a longer PFS on an molecular profiling-suggested regimen than on the regimen on which the patient had just experienced progression. An accompanying editorial (Doroshow, 2010) noted that the trial had a number of significant limitations, including uncertainty surrounding the achievement of time to progression (the study’s primary endpoint), and a lack of a randomized design for this trial. A report by the National Horizon Scanning Centre (2013) stated that the company stated that the tumor profiling service provided by Caris Life Sciences has been extensively altered with the addition of several new technologies. The new service is named Caris Life Sciences Molecular Intelligence Services. The NHSC stated that randomized controlled trials comparing clinical outcomes for patients using Carismolecular profiling to those receiving standardspecialist care are needed to determine whether this testing service is effective and cost-effective. CoA racemase (P504S) and HMWCK (34betaE12) Kumaresan et al (2010) reviewed 1034 cases of morphologically difficult prostate cancer, which were divided into benign (585), malignant (399) and suspicious (50) and evaluated using CoA racemase (P504S) and HMWCK (34betaE12). Forty nine suspicious cases were resolved by using both markers whereas 1 case was resolved by further support with CD68. The original diagnosis was changed in 15 of 50 suspicious cases from benign to malignant, one case from benign to high grade PIN, and in one case from malignant to benign. The authors concluded that a combination of HMWCK and AMACR is of value in combating morphologically suspicious cases and that although the sensitivity and specificity of HMWCK and AMACR in this study were high, \"it should be used with caution, keeping in mind all their pitfalls and limitations.\" P504S Murray et al (2010) studied P504S expressing circulating prostate cells as a marker for prostate cancer. The authors stated that PSA is the only biomarker routinely used in screening. This study aimed to develop a system to test the presence of circulating prostate cells in men without a diagnosis of prostate cancer in realation with age, serum PSA levels and prostate biopsy by determining the co-expression of several markers such as CD82, HER-2 and matrix metalloproteinase 2 MMP-20. The results indicated that among 409 men screened for prostate cancer 16.6% were positive for circulating prostate cells. The authors concluded that the study of circulating prostate cells with various markers could be a useful complementary screening test for prostate cancer in men with increased PSA level. FLT3 has been used to predict prognosis in acute myelogenous leukemia (Chin, et al, 2006). Mutations in FLT3 are common in AML and have been associated with poorer survival in children and in younger adults with normal cytogenetics receiving intensive chemotherapy. The NCCN Task Force issued a report in November of 2011 which updated their position regarding molecular markers for diagnosis, prognosis, prediction, and companion diagnostic markers (Febbo et. al., 2011). As a result of these recommendations, use of MGMT, IDH mutation and 1p/19qcodeletionare now established for glioma. Also, use of ALK gene fusion has been established for non-small cell lung cancer. The updated NCCN guidelines have not yet established the efficacy of ColoPrint, CIMP, LINE-1 hypomethylation, or Immune cells for colon cancer. Similarly, the efficacy of FLT3-TKD mutation, WT1 mutation, RUNX1 mutation, MLL-PTD, IDH1 mutation, IDH2 R172, and IDH2 codon 140 mutation has not been established for use in acute myeloid leukemia. ColoPrint ColoPrint (Agendia) is an 18-gene profile that classifies colon cancer into Low Risk or High Risk of relapse, by measuring genes representative of the metastatic pathways of colon cancer metastases which were selected for their predictive relationship to 5-year distant metastases probability (Raman, et al., 2013). ColoPrint is indicated for stage II colon cancer, and provides relapse risk stratification independent of clinical and pathologic factors such as T4-stage and MSI status. ColoPrint determines if the patient is a candidate for chemotherapy. An NCCN Task Force report (NCCN, 2011) concluded that the efficacy of ColoPrint has not been established. DecisionDx-UM The DecisionDx test is a gene expression profile that determines the molecular signature of a patient\'s melanoma. The results of the test provide knowledge regarding the risk of near term metastasis (5 years). Tumors with a Class 1 signature are associated with a good prognosis and a low potential to spread (or metastasize), while tumors with a Class 2 signature have a high potential to spread. Aaberg et al (2014) conducted a chart review and cross-sectional survey of ophthalmologists who treat uveal melanomato assesscurrent clinical practices for uveal melanoma (UM) and the impact of molecular prognostic testing on treatment decisions. This study involved a chart reviewof all Medicare beneficiaries tested by UM gene expression profile in 2012, conducted under an institutional review board-approved protocol.In addition, 109 ophthalmologists specializing in the treatment of UM were invited to participate in 24-question survey in 2012; 72 were invited to participate in a 23-question survey in 2014. The review of Medicare medical records included 191 evaluable patients, 88 (46%) with documented medical treatment actions or institutional policies related to surveillance plans. Of these 88, all gene expression profiling (GEP) Class 1 UM patients were treated with low-intensity surveillance. All GEP Class 2 UM patients were treated with high-intensity surveillance (P 0.0001 versus Class 1). There were 36 (19%) with information concerning referrals after initial diagnosis. Of these 36, all 23 Class 2 patients were referred to medical oncology; however, none of the 13 Class 1 patients were referred (P 0.0001 versus Class 1). Only Class 2 patients were recommended for adjunctive treatment regimens. 2012 survey: 50 respondents with an annual median of 35 new UM patients. The majority of respondents (82%) performed molecular analysis of UM tumors after fine needle biopsy (FNAB); median: 15 FNAB per year; 2014 survey: 35 respondents with an annual median of 30 new UM patients. The majority offered molecular analyses of UM tumor samples to most patients. Patients with lowmetastatic risk (disomy 3 or GEP Class 1) were generally assigned to less frequent (every 6 or 12 months) and less intensive clinical visits. Patients with high metastatic risk (monosomy 3 or GEP Class 2) were assigned to more frequent surveillance with hepatic imaging and liver function testing every 3-6 months. High-risk patients were considered more suitable for adjuvant treatment protocols.Chappell et al (2012) reported on a retrospective case series of uveal melanoma patients gene expression profiles to characterize the clinical spectrum of class 1 and class 2 uveal melanomas and their relationship with intraocular proton radiation response. A total of 197 uveal melanoma patients from a single institution were analyzed for pathology, clinical characteristics, and response to radiation therapy. A total of 126 patients (64%) had class 1 tumors and 71 (36%) had class 2 tumors. Patients with class 2 tumors had more advanced age (mean: 64 years vs 57 years; P = .001), had thicker initial mean ultrasound measurements (7.4 mm vs 5.9 mm; P = .0007), and were more likely to have epithelioid or mixed cells on cytopathology (66% vs 38%; P = .0004). Although mean pretreatment and posttreatment ultrasound thicknesses were significantly different between class 1 and class 2 tumors, there was no difference in the mean change in thickness 24 months after radiation therapy (mean difference: class 1 = -1.64 mm, class 2 = -1.47; P = .47) or in the overall rate of thickness change (slope: P = .64). Class 2 tumors were more likely to metastasize and cause death than class 1 tumors (disease-specific surviva [DSS]: P .0001).Worley et al (2007)compared a gene expression-based classifier versus the standard genetic prognostic marker, monosomy 3, for predicting metastasis in uveal melanoma. Gene expression profiling, fluorescence in situ hybridization (FISH), and array comparative genomic hybridization (aCGH) were done on 67 primary uveal melanomas. Clinical and pathologic prognostic factors were also assessed. The investigators found that thegene expression-based molecular classifier assigned 27 tumors to class 1 (low risk) and 25 tumors to class 2 (high risk). By Cox univariate proportional hazards, class 2 signature (P = 0.0001), advanced patient age (P =0.01), and scleral invasion (P = 0.007) were the only variables significantly associated with metastasis. Only the class 2 signature was needed to optimize predictive accuracy in a Cox multivariate model. A less significant association with metastasis was observed for monosomy 3 detected by aCGH (P = 0.076) and FISH (P = 0.127). The sensitivity and specificity for the molecular classifier (84.6% and 92.9%, respectively) were superior to monosomy 3 detected by aCGH (58.3% and 85.7%, respectively) and FISH (50.0% and 72.7%, respectively). Positive and negative predictive values (91.7% and 86.7%, respectively) and positive and negative likelihood ratios (11.9 and 0.2, respectively) for the molecular classifier were also superior to those for monosomy 3. In a prospective case series study, Corrêaand Augsburger (2016) sought todetermine whether any conventional clinical prognostic factors for metastasis from uveal melanoma retain prognostic significance in multivariate models incorporating gene expression profile (GEP) class of the tumor cells. The investigators conducted asingle-institution study of GEP testing and other conventional prognostic factors for metastasis and metastatic death in 299 patients with posterior uveal melanoma evaluated by fine-needle aspiration biopsy (FNAB) at the time of or shortly prior to initial treatment. Univariate prognostic significance of all evaluated potential prognostic variables (patient age, largest linear basal diameter of tumor [LBD], tumor thickness, intraocular location of tumor, melanoma cytomorphologic subtype, and GEP class) was performed by comparison of Kaplan-Meier event rate curves and univariate Cox proportional hazards modeling. Multivariate prognostic significance of combinations of significant prognostic factors identified by univariate analysis was performed using step-up and step-down Cox proportional hazards modeling. GEP class was the strongest prognostic factor for metastatic death in this series. However, tumor LBD, tumor thickness, and intraocular tumor location also proved to be significant individual prognostic factors in this study. On multivariate analysis, a 2-term model that incorporated GEP class and largest basal diameter was associated with strong independent significance of each of the factors.Correaand Augsburger (2014) sought to determine the relative sufficiency of paired aspirates of posterior uveal melanomas obtained by FNAB for cytopathology and GEP, and their prognostic significance for predicting death from metastasis The investigators conducted aprospective non-randomized single-centerstudy of 159 patients with posterior uveal melanoma sampled by FNAB in at least two tumor sites betweenSeptember 2007and December 2010. Cases were analyzed with regard to sufficiency of the obtained aspirates for cytopathologic classification and GEP classification. Statistical strength of associations between variables and GEP class was computed using Chi-square test. Cumulative actuarial survival curves of subgroups of these patients based on their cytopathologic versus GEP-assigned categories were computed by the Kaplan-Meier method. The endpoint for this survival analysis was death from metastatic uveal melanoma. FNAB aspirates were insufficient for cytopathologic classification in 34 of 159 cases (21.9%). In contrast, FNAB aspirates were insufficient for GEP classification in only one of 159 cases (0.6%). This difference is statistically significant (P   0.001). Six of 34 tumors (17.6%) that yielded an insufficient aspirate for cytopathologic diagnosis were categorized as GEP class 2, while 43 of 125 tumors (34.7%) that yielded a sufficient aspirate for cytopathologic diagnosis were categorized as GEP class 2. To date, 14 of the 49 patients with a GEP class 2 tumor (28.6%) but only five of the 109 patients with a GEP class 1 tumor (5.6%) have developed metastasis. Fifteen of 125 patients (12%) whose tumors yielded sufficient aspirates for cytopathologic classification but only four of 34 patients (11.8%) whose tumors yielded insufficient aspirates for cytopathologic classification developed metastasis. The median post-biopsy follow-up time for surviving patients in this series was 32.5months. Cumulative actuarial 5-year probability of death from metastasis 14.1% for those with an insufficient aspirate for cytopathologic classification versus 22.4% for those with a sufficient aspirate for cytopathologic classification (log rank P = 0.68). In contrast, the cumulative actuarial 5-year probability of metastatic death was 8.0% for those with an insufficient/unsatisfactory aspirate for GEP classification or GEP class 1 tumor, versus 45.0% for those with a GEP class 2 tumor (log rank P = 0.005).In a prospective study, Oniken et al (2012)evaluated the prognostic performance ofthe DecisionDx15 gene expression profiling (GEP) assay that assigns primary posterior uveal melanomas to prognostic subgroups: class 1 (low metastatic risk) and class 2 (high metastatic risk). A total of 459 patients with posterior uveal melanoma were enrolled from 12 independent centers. Tumors were classified by GEP as class 1 or class 2. The first 260 samples were also analyzed for chromosome 3 status using a single nucleotide polymorphism assay. Net reclassification improvement analysis was performed to compare the prognostic accuracy of GEP with the 7th edition clinical Tumor-Node-Metastasis (TNM) classification and chromosome 3 status. The investigators found thatthe GEP assay successfully classified 446 of 459 cases (97.2%). The GEP was class 1 in 276 cases (61.9%) and class 2 in 170 cases (38.1%). Median follow-up was 17.4 months (mean, 18.0 months). Metastasis was detected in 3 class 1 cases (1.1%) and 44 class 2 cases (25.9%) (log-rank test, P 10(-14)). Although there was an association between GEP class 2 and monosomy 3 (Fisher exact test, P 0.0001), 54 of 260 tumors (20.8%) were discordant for GEP and chromosome 3 status, among which GEP demonstrated superior prognostic accuracy (log-rank test, P = 0.0001). By using multivariate Cox modeling, GEP class had a stronger independent association with metastasis than any other prognostic factor (P 0.0001). Chromosome 3 status did not contribute additional prognostic information that was independent of GEP (P = 0.2). At 3 years follow-up, the net reclassification improvement of GEP over TNM classification was 0.43 (P = 0.001) and 0.38 (P = 0.004) over chromosome 3 status. Klufas et al (2015) reported their experience with uveal melanoma (UM)-specific GEP testing on a series of choroidal metastatic tumors confirmed by cytopathology so that clinicians may be aware that receiving a class 1 or class 2 test result in non-melanoma is possible. These investigators performed a retrospective review of all cytopathology and DecisionDx-UM GEP reports between January 2012 to December 2014 from intra-operative FNA biopsy of choroidal tumors undergoing brachytherapy. A total of 4 patients were identified to have cytopathology consistent with a non-melanoma primary. All 4 patients presented with a unilateral, single choroidal tumor, which was treated with iodine-125 brachytherapy and underwent intra-operative FNA biopsy for cytopathology and UM-specific GEP testing for molecular prognostication. Gene expression profile testing of the choroidal tumor in each patient revealed class 1A in 3 patients and class 2 in 1 patient. The authors concluded that DecisionDx-UM GEP may be a helpful test for molecular prognostication in patients with UM; however, class 1 and class 2 test results are indeed possible in the setting of a non-melanoma malignancy. They recommended that cytopathology and/or other melanoma-specific testing be performed in all cases of suspected choroidal melanoma because GEP with this assay is unable to rule out the diagnosis of a choroidal melanoma. Plasseuard et al (2016)sought to evaluate the clinical validity and utility of DecisionDx-UM.Beginning March 2010, 70 patients were enrolled in a prospective,multicenter, IRB-approved study to document patient management differences and clinical outcomes associated with low-risk Class 1 and high-risk Class 2 results indicated by DecisionDx-UM testing. Thirty-seven patients in the prospective study were Class 1 and 33 were Class 2. Class 1 patients had 100% 3-year metastasis-free survival compared to 63% for Class 2 (log rank test