National Medical Policy

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1 National Medical Policy Subject: Policy Number: Tumor Markers for Cancer NMP522 Effective Date*: July 2013 Updated: November 2015 This National Medical Policy is subject to the terms in the IMPORTANT NOTICE at the end of this document For Medicaid Plans: Please refer to the appropriate State's Medicaid manual(s), publication(s), citations(s) and documented guidance for coverage criteria and benefit guidelines prior to applying Health Net Medical Policies The Centers for Medicare & Medicaid Services (CMS) For Medicare Advantage members please refer to the following for coverage guidelines first: Use Source Reference/Website Link X National Coverage Determination (NCD) Carcinoembryonic Antigen: Tumor Antigen by Immunoassay - CA 125: Tumor Antigen by Immunoassay - CA 15-3/CA 27.29: Tumor Antigen by Immunoassay - CA 19-9: X National Coverage Manual Citation Local Coverage Determination (LCD)* Molecular Diagnostic Tests (MDT) ; Bladder Tumor Markers; MolDx:Prolaris Prostate Cancer Genomic Assay (L36340);MolDx Genomic Health Oncotype DX Prostate Cancer Assay (L36153): Article (Local)* x Other Palmetto GBA. MOLDX: sf/docscathome/moldx None Use Health Net Policy Instructions Tumor Markers for Cancer Nov 15 1

2 Medicare NCDs and National Coverage Manuals apply to ALL Medicare members in ALL regions. Medicare LCDs and Articles apply to members in specific regions. To access your specific region, select the link provided under Reference/Website and follow the search instructions. Enter the topic and your specific state to find the coverage determinations for your region. *Note: Health Net must follow local coverage determinations (LCDs) of Medicare Administration Contractors (MACs) located outside their service area when those MACs have exclusive coverage of an item or service. (CMS Manual Chapter 4 Section 90.2) If more than one source is checked, you need to access all sources as, on occasion, an LCD or article contains additional coverage information than contained in the NCD or National Coverage Manual. If there is no NCD, National Coverage Manual or region specific LCD/Article, follow the Health Net Hierarchy of Medical Resources for guidance. Current Policy Statement Health Net Inc., considers the following tumor markers medically necessary for the diagnosis and/or management of cancer (more information on each marker can be found following the table): Tumor Marker Afirma Thyroid FNA Analysis AFP (alpha-fetoprotein) AFP, lactate dehydrogenase (LDH), and beta-human chorionic gonadotropin (beta hcg) ALK Gene rearrangement B2M (beta-2 microglobulin) Bladder-tumor antigen stat test (BTA) Calcitonin CA (cancer antigen) 15-3, also known as CA or Truquant RIA CA 19.9 CA125 Carcinoembryonic antigen (CEA) CgA (chromogranin A) Cancer Thyroid Cancer Primary hepatocellular cancer Germ cell tumors Refer to policy, Molecular Tumor Markers for Non-Small lung Cancer Multiple myeloma; Chronic lymphocytic leukemia; Some lymphomas (see below) Bladder cancer Thyroid medullary carcinoma Metastatic breast cancer Pancreatic cancer; Gallbladder cancer; Cholangiocarcinoma; Carcinoma of the ampulla of Vater Epithelial ovarian cancer; Endometrial cancer Breast Cancer: Colorectal cancer; Medullary thyroid cancer Neuroendocrine tumors (e.g., carcinoid tumors, neuroblastoma, and small cell lung cancer) Tumor Markers for Cancer Nov 15 2

3 Tumor Marker CD- 117 (C-kit) (cluster of differentiation-117) Cyclin D1 EGFR (epidermal growth factor receptor) ER/PR (estrogen receptors and progesterone receptors) 5-HIAA (5-hydrocyindoleacetic acid) HE4 (human epididymis protein 4) HER2 (human epidermal growth factor receptor 2) IDH1, IDH2 Cancer Gastrointestinal stromal tumors Mantle cell lymphoma Refer to policy, Molecular Tumor Markers for Non-Small lung Cancer Breast cancer Carcinoid tumors Ovarian cancer Refer to policy, Her2/neu Gliomas ImmunoCyt/uCyte+ JAK2 KRAS gene sequencing MPO (myeloperoxidase) Nuclear-Matrix Protein (NMP22) NSE (neuron-specific enolase) Placental alkaline phosphatase (PLAP) PSA (prostate-specific antigen) Thyroglobulin UroVysion Bladder cancer Polycythemia vera, differential diagnosis of essential thrombocytosis, chronic myeloproliferative disorders (CMPD) and primary myelofibrosis (PMF) in adults Refer to policy, Molecular Tumor Markers for Non-Small lung Cancer and K-RAS Mutation Analysis of Colon Cancer Acute myeloid leukemia Bladder cancer Small cell lung cancer Germ cell seminoma and non-seminoma germ cell tumors in unknown primary cancers Prostate cancer Differentiated thyroid cancer Bladder cancer Afirma Thyroid FNA The Afirma Thyroid FNA Analysis is intended for adults with thyroid nodules at least 1 centimeter in size, that are indeterminate and who are being evaluated for the possibility of a thyroid malignancy. Tumor Markers for Cancer Nov 15 3

4 AFP (alpha-fetoprotein) Hepatocellular Cancer AFP is not frequently elevated in early stage hepatocellular carcinoma (HCC) thus its utility as a screening biomarker is limited. AFP testing can be useful in conjunction with other test results to guide management of patients for whom a diagnosis of HCC is suspected. AFP is also useful in monitoring individuals with HCC. AFP, lactate dehydrogenase (LDH), and beta-human chorionic gonadotropin (beta HCG) Germ Cell Tumors AFP, LDH and beta-hcg are critical in diagnosing germ cell tumors (GCTs), determining prognosis, and assessing treatment outcome. They are useful for monitoring all stages of nonseminomas. They are useful in monitoring metastatic seminomas because elevated marker levels are early signs of relapse. B2M (beta-2 microglobulin) Multiple Myeloma The level of beta-2 microglobulin reflects the tumor mass and is considered a standard measure of the tumor burden in multiple myeloma. Chronic Lymphocytic Leukemia Elevated levels of serum beta-2microglobulin have been shown to be a strong independent prognostic indicator for treatment-free intervals, response to treatment, and overall survival, in individuals with chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL) Lymphomas Beta-2 microglobulin may be useful prognostic indicator in anaplastic large cell lymphoma (ALCL) Beta-2 microglobulin is useful in the initial diagnostic work-up for Follicular lymphoma (FL), Diffuse Large B-Cell Lymphoma (DLBCL) Measurement of Beta-2 microglobulin may be useful in some circumstances in the initial work-up of mantle cell lymphoma, lymphoblastic lymphoma amnd Aids related B-Cell lymphoma. Bladder-tumor antigen stat test (BTA) Bladder Cancer Per the NCCN, urine molecular tests for urothelial tumor markers are now available. Most of these tests have a better sensitivity for detecting bladder cancer than urine cytology, but specificity is lower. However, it remains unclear whether these tests offer additional information that is useful for detection and management of non-muscle-invasive bladder tumors. NCCN considers this to be a 2B recommendation (i.e., Based on lower level of evidence, there is NCCN consensus that the intervention is appropriate) Calcitonin Medullary Thyroid Cancer Basal serum calcitonin and serum carcinoembryonic antigen (CEA) are generally accepted in the preoperative work-up for medullary thyroid cancer. Calcitonin is useful for post operative management and surveillance, to evaluate effectiveness of treatment and assess for recurrence of medullary thyroid cancer. CA 15-3; CA Tumor Markers for Cancer Nov 15 4

5 Metastatic breast cancer The NCCN states that monitoring of patients symptoms and cancer burden during treatment of metastatic breast disease is important to determine whether the treatment is providing benefit and that the patient does not have toxicity from an ineffective therapy. Increasing tumor markers (CEA, CA 15-3 and CA 27.29) are findings that are concerns for progression of disease, but may also be seen in the setting of responding disease. An isolated increase in tumor markers should rarely be used to declare progression of disease. Changes in bone lesions are often difficult to assess on plain or cross-sectional radiology or on bone scan. For these reasons, patient symptoms and serum tumor markers may be more helpful in patients with bone-dominant metastatic disease. CA 19.9 Pancreatic Cancer The NCCN reports that the best validated and most clinically useful biomarker in pancreatic cancer is CA CA 19-9 is commonly expressed and shed in pancreatic and hepatobiliary disease, and in many malignancies; thus it is not tumor specific. However, the degree of increase in CA 19-9 levels may be useful in differentiating pancreatic adenocarcinoma from inflammatory conditions of the pancreas. CA 19-9 is a good diagnostic marker in symptomatic individuals, but its low predictive value makes it a poor biomarker for screening. Preoperative CA 19-9 levels correlate with both AJCC staging and resectability and thus provide additional information for staging and determining resectability, along with information from imaging, laparoscopy and biopsy. CA 19-9 seems to have value as a prognostic marker. Low post operative levels or serial decrease following surgery have been found to correlate with survival for individuals undergoing resection for pancreatic cancer. Changes in CA 19-9 levels after chemotherapy in patients with advanced disease has been shown to be a reliable predictive marker for response to treatment. CA 19-9 may be undetectable in Lewis antigen-negative individuals. CA 19-9 may be falsely positive in cases of benign or malignant biliary obstruction and do not necessarily indicate cancer or advanced disease. NCCN recommends measurement of CA 19-9 levels prior to surgery (if bilirubin levels are normal), following surgery prior to administration of adjuvant therapy or for surveillance (category 2B, i.e., based on lower level evidence, there is consensus that intervention is appropriate) Biliary Tract Cancer CA 19-9 may be considered in initial workup of individuals with a suspicion of gallbladder cancer, although the markers are NOT specific for gallbladder cancer. CA 19-9 testing can be considered after biliary decompression. CA 19-9 may be considered in initial workup of individuals with a suspicion of cholangiocarcinoma, although the markers are NOT specific for cholangiocarcinoma. Ampullary Cancer Some ampullary cancers are associated with increased serum levels of CA 19-9 and/or carcinoembryonic antigen (CEA). Serial assay of these tumor markers may be useful for posttreatment follow-up. CA 125 Ovarian Cancer Per the NCCN, tumor markers (including CA-125, inhibin, AFP, and beta-hcg) can be measured if clinically indicated. Tumor Markers for Cancer Nov 15 5

6 Per the NCCN, CA 125 and other tumor markers may be done as clinically indicated prior to each cycle of chemotherapy. After completion of primary surgery and chemotherapy in individuals with all stages of ovarian cancer who have complete response, the recommendation is observation with follow-up. If the CA 125 level was initially elevated, the measurement of a CA 125 level or other tumor markers at each follow-up evaluation is recommended. The NCCN concurs with the Society of Gynecologic Oncology (SGO) opinion which states that individuals should discuss the pros and cons of CA 125 monitoring and that the use of CA 125 levels for surveillance is optional. The FDA has approved the use of HE4 and CA 125 for estimating the risk of ovarian cancer in women with a pelvic mass. Currently, the NCCN does not recommend the use of these biomarkers for determining the status of an undiagnosed pelvic mass. Endometrial Carcinoma Per the NCCN, in individuals with extrauterine disease, a serum CA 125 assay may be helpful in monitoring clinical response. However, serum CA 125 levels may be falsely increased in women who have peritoneal inflammation/infection or radiation injury, normal in women with isolated vaginal metastases and may not predict recurrence in the absence of other clinical findings. Both NCCN and SGO recommend that CA 125 and MRI/CT may be useful before surgery to assess if extrauterine disease is present. Hereditary Breast and Ovarian Cancer Syndrome (HBOC) For individuals with HBOC who have not elected salpingo-oophorectomy, the NCCN recommends considering concurrent transvaginal ultrasound (preferably day 1-10 of menstrual cycle in premenopausal women) and CA 125 (preferably after day 5 of menstrual cycle in premenopausal women) every 6 months starting at age 30y or 5-10 years before the earliest age of the first diagnosis of ovarian cancer in the family. Carcinoembryonic antigen (CEA) Breast Cancer Increasing tumor markers (CEA, CA 15-3 and CA 27.29) are findings that are concerns for progression of disease, but may also be seen in the setting of responding disease. An isolated increase in tumor markers should rarely be used to declare progression of disease. Changes in bone lesions are often difficult to assess on plain or cross-sectional radiology or on bone scan. For these reasons, patient symptoms and serum tumor markers may be more helpful in patients with bone-dominant metastatic disease. Colorectal Cancer Testing of CEA in the initial work-up is recommended for individuals who present with invasive colon cancer appropriate for resection. CEA determination should be included in the workup for individuals with metastatic synchronous adenocarcinoma. Colorectal Cancer Surveillance CEA testing is recommended at baseline and every 3-6 months for 2 years and then every 6 months for a total of 5 years, for patient with stage III disease and those with stage II disease if the clinician determines that the individual is a potential candidate for aggressive curative surgery. CEA testing is also recommended every 3-6 months for 2 years and then every 6 months for a total of 5 years, for individuals with stage IV disease with no NED after curative-intent surgery and subsequent adjuvant treatment. Tumor Markers for Cancer Nov 15 6

7 Surveillance of are similar to those recommendations for individuals with stage II/III disease, except that certain evaluations are performed more frequently. CEA testing is recommended every 3-6 months for the first 2 years. Routine CEA monitoring and routine CT scanning are not recommended beyond 5 years Medullary Thyroid Cancer Basal serum calcitonin and serum carcinoembryonic antigen (CEA) are generally accepted in the preoperative work-up for medullary thyroid cancer. A rapidly increasing CEA level, particularly in the setting of a stable calcitonin level may be important for predicting a worse prognosis. Measurement of CEA is useful for post operative management and surveillance, to evaluate effectiveness of treatment and assess for recurrence of medullary thyroid cancer. CgA (chromogranin A) Neuroendocrine tumors Chromogranin A may be used as a tumor marker; (NCCN category 3, i.e. based on any level of evidence, there is major NCCN disagreement that the intervention is appropriate) Although not diagnostic, elevated levels have been associated with recurrence. Analysis of a large prospective database showed that CgA levels elevated twice the normal limit or higher were associated with shorter survival times for individuals with metastatic neuroendocrine tumors. CgA levels can be elevated in other conditions (e.g., renal or hepatic insufficiency, hypertension, chronic gastritis) and are also commonly also elevated in the setting of concurrent proton pump inhibitors, thus caution should be used when considering initiating new therapy based on a rising CgA level in an asymptomatic individual with a tumor that appears stable. CD-117 (C-kit) (cluster of differentiation-117) Gastrointestinal Stromal Tumors Gastrointestinal stromal tumors (GISTs) that arise in adults are characterized by the near-universal expression of the CD117 antigen. The CD117 antigen is part of the KIT transmembrane receptor tyrosine kinase that is the product of the KIT (also denoted c-kit) protooncogene Cyclin D1 Non Hodgkins Lymphoma Per the NCCN, immunophenotyping/genetic testing is useful in the differential diagnosis of mature B-cell and NK/T-cell neoplasms in conjunction with clinical and morphologic correlation. In the differential diagnosis of small cell lymphomas [i.e., Chronic Lymphocytic Leukemia (CLL)/Small Lymphocytic Lymphoma (SLL), Mantle Cell Lymphoma (MCL), Hairy Cell Leukemia (HCL), Splenic Marginal Zone Lymphoma (MZL), extra-nodal MZL, nodal MZL, and Follicular Lymphoma] cyclin D1 is included in the panel for immunophenotyping. Distinguishing CLL/SLL from mantle cell lymphoma is essential and cyclin D1 expression is the most reliable marker for differentiating between CLL and MCL. In select cases, the use of cyclin D1 may be useful to differenciate PC-MZL from mantle cell lymphomas. Cyclin D1 is also include in the immunophenotyping panel for the diagnosis of medium-sized lymphomas [Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), blastoid variant of MCL.] Cyclin D1 positively confirms the diagnosis of blastoid MCL. Tumor Markers for Cancer Nov 15 7

8 ER/PR (estrogen receptors and progesterone receptors) Breast Cancer Per the NCCN, the work-up of individuals with invasive breast cancer should include determination of tumor estrogen/progesterone receptor (ER/PR) status and Her2 status. ER/PR tumor status is normally determined by immunohistochemistry (IHC) testing. Per NCCN, the revised AJCC cancer staging manual recommends the collection of prognostic factors, including tumor grade, estrogen receptor (ER) content, progesterone receptor (PR) content and human epidermal growth factor receptor 2 (HER2) status, although these characteristics do not specifically influence assigned stage of disease. 5-HIAA (5-hydrocyindoleacetic acid) Carcinoid Tumor 5-hydrocyindoleascetic acid (5-HIAA), a metabolite of serotonin may also be considered as a biochemical marker in some cases, particularly in individuals with small-intestinal carcinoid tumors. Baseline levels of 5-HIAA may be considered to monitor subsequent progression of disease. HE4 (human epididymis protein 4) Per NCCN, although human epididymis protein 4 (HE4) and CA125 appear to be useful in detecting ovarian cancer, recent data showthat several markers (including CA125, HE4, etc) do not increase early enough to be useful in detecting early-stage ovarian cancer. The FDA has approved the use of HE4 and CA 125 for estimating the risk of ovarian cancer in women with a pelvic mass. Currently, the NCCN does not recommend the use of these biomarkers for determining the status of an undiagnosed pelvic mass. IDH1, IDH2 (isocitrate dehydrogenase) Gliomas Per NCCN, mutations in the IDH genes are common in patients with low-grade gliomas and are reported to be a significant marker of positive prognosis. Routine IDH testing is not recommended because its impact on treatment is still unclear, however, testing may be useful for stratification of individuals with glioma, when the results from immunohistochemistry are negative or equivocal or when the results of the genetic test will direct treatment decisions. ImmunoCyt/uCyt+ Bladder cancer May be used for detecting primary bladder cancer in patients with signs/symptoms of bladder cancer or for detecting recurrent bladder cancer in patients with a history of bladder cancer, when used as an adjunct to cystoscopy and urinary cytology Per the NCCN, urine molecular tests for urothelial tumor markers are now available. Most of these tests have a better sensitivity for detecting bladder cancer than urine cytology, but specificity is lower. However, it remains unclear whether these tests offer additional information that is useful for detection and management of non-muscle-invasive bladder tumors. NCCN considers this to be a 2B recommendation (i.e., Based on lower level of evidence, there is NCCN consensus that the intervention is appropriate) MPO (myeloperoxidase) Tumor Markers for Cancer Nov 15 8

9 Acute Myeloid Leukemia Immunohistochemical staining for myeloperoxidase is the best method for determining which cells are committed to the myeloid lineage Nuclear-Matrix Protein (NMP22) Bladder Cancer Consideration may be given to the FDA approved urinary biomarker testing by fluorescence in situ hybridization or nuclear matrix protein 22 in the monitoring for recurrence. NSE (neuron-specific enolase) Small Cell Lung Cancer (SCLC) Neuroendocrine tumors account for approximately 20% of lung cancers; most (~15%) are small cell lung cancer. Most SCLCs stain positively for markers of neuroendocrine differentiation, including chromogranin A, neuron-specific enolase, neural cell adhesion molecule and synaptophysin. Placental alkaline phosphatase (PLAP) Occult Primary (Cancer of unknown Primary) Placental alkaline phosphatase (PLAP) is mainly found in seminomas but is also expressed in some nonseminoma germ cell tumors, and genitourinary, gastrointestinal, and pulmonary carcinomas. PSA (prostate-specific antigen) Prostate Cancer The decision to participate in an early detection program for prostate cancer is complex. Factors that should be considered include patient age, life expectancy, family history, race, and previous early detection test results. Initial suspicion of prostate cancer is based on abnormal digital rectal exam (DRE) or an elevated PSA level. Prostate cancers are characterized by clinical (TMN ) stage determined by DRE, Gleason score in the biopsy specimen, and serum PSA levels. NCCN guidelines incorporate a risk stratification scheme that uses a minimum of stage, grade and PSA to assign individuals to risk groups. These groups are used to select the appropriate options that should be considered for treatment and to predict the probability of biochemical failure after definitive local therapy. Biochemical progression-free survival can be reassessed post-operatively using age, serum PSA, and pathologic grade and stage. PSA levels are monitored in individuals choosing active surveillance and in individuals choosing active treatment. Many commercially available serum total PSA testing are currently available. They perform comparably, however, the levels are not interchangeable since they standardized against two different standards. An abnormal PSA result should be confirmed by retesting. Unbound or free PSA (fpsa) expressed as a ratio of total PSA has emerged as a clinically useful molecular form of PSA, with the potential to provide improvements in early detection, staging, and monitoring of prostate cancer. Studies have shown that the percentage of fpsa is significantly lower in men who have prostate cancer compared to men who have not. The US Food and Drug Administration (FDA) approved the use of percent-free PSA for the early detection of prostate cancer in men with PSA levels between 4-10 ng/ml. NCCN guidelines recommend the use of the percent-free PSA as an alternative in the management of patients with normal DREs and total PSA levels between 4-10ng/mL if there is a relative contraindication to biopsy. Physicians and patients Tumor Markers for Cancer Nov 15 9

10 electing to use percent-free PSA should be cautioned that this assay and the multi-institutional study performed to obtain its FDA approval were designed with the intention of avoiding unnecessary biopsies in men with a high likelihood of not having prostate cancer. Thyroglobulin Thyroid Cancer Thyroglobulin is most often measured in differentiated thyroid cancers (i.e., papillary, follicular, and Hurthle cell). Postoperative elevation of the thyroglobulin level above 10 nanograms per milliliter is suggestive of cancer recurrence UroVysion Bladder Cancer Per the NCCN, urine molecular tests for urothelial tumor markers are now available. Most of these tests have a better sensitivity for detecting bladder cancer than urine cytology, but specificity is lower. However, it remains unclear whether these tests offer additional information that is useful for detection and management of non-muscle-invasive bladder tumors. NCCN considers this to be a 2B recommendation (i.e., Based on lower level of evidence, there is NCCN consensus that the intervention is appropriate) Investigational Health Net Inc., considers the following tumor markers investigational, as their role in the management of various benign and cancerous conditions has not yet been demonstrated (may not be all inclusive): 1. BluePrint Molecular Subtyping Profile for Breast Cancer 2. Breast Cancer Index for Prognosis of Breast Cancer Recurrence (biotheranostics Inc.) 3. BreastNext Next-Gen Cancer Panel 4. BreastTrue High Risk Panel for Hereditary Breast Cancer (Pathway Genomics Corp). 5. BROCA Cancer Risk panel 6. CA 50 (Cancer antigen 50) 7. CA 72-4 (Cancer antigen 72-4) 8. CA 195 (Cancer antigen 195) 9. CA 549 (Cancer antigen 549) 10. CAM 17.1 (monoclonal antimucin antibody 17.1) 11. CancerNext Next-Gen Cancer Panel 12. CancerSelect (Personal Genome Diagnostics) 13. COLMOL: NGS Colon Cancer Panel 14. ColoNext 15. ColonSentry (GeneNews Ltd.) for Colorectal Cancer 16. ConfirmMDx for Prostate Cancer 17. Counsyl Universal Genetic Test 18. Decipher Prostate Cancer Classifier 19. Decision DX-GBM brain cancer assay 20. Decision DX-UM assay 21. Decision DX - Melanoma 22. Direct-to-consumer (DTC) Genetic tests (eg., 23andMe) 23. EarlyCDT-Lung Risk Assessment Test 24. FoundationOne Cancer Assay 25. FoundationOne Heme 26. HERmark breast cancer assay 27. High/Moderate Risk Panel Tumor Markers for Cancer Nov 15 10

11 28. igene Cancer Panel 29. MammaPrint 30. Mammastrat 31. MelanoSITE Fish Test 32. Molecular Intelligence (MI) (Caris Life Sciences) 33. Myriad MyRisk Hereditary Cancer Genetic Panel 34. Myeloma Prognostic Risk Signature (MyPRS Plus) Test for Myeloma 35. OncoType DX DCIS 36. Oncotype DX Prostate Cancer Assay 37. Ovanext 38. Ovasure 39. Panexia 40. PancNext Next-Gen Cancer Panel for Hereditary Pancreatic Cancer 41. Paradigm Cancer Diagnostic (PCDx) 42. Pathfinder TG 43. PreOvar KRAS-Variant Test for Ovarian Cancer 44. Progensa PCA3 Genetic Assay for Prostate Cancer 45. Prolaris Test for Prediction of Prostate Cancer Progression 46. ProOnc TumorSource 47. Prostate Core Mitomic Test for Prostate Cancer Diagnosis 48. ProstatePx 49. ProstRcision for prostate cancer 50. Proveri Prostate Cancer Assay (PPCA) 51. RenalNext Next-Generation Sequencing (NGS) Panel for Renal Cell Carcinoma 52. ResponseDX: Colon 53. Squamous Cell Carcinoma Antigen 54. Septin 9 (SEPT9) Methylation Analysis for Colorectal Cancer 55. SYMGENE68 NGS Cancer Panel 56. Target Now Molecular Profiling Test (Caris Life Sciences) 57. ThyGenX Thyroid Oncogene Panel/ThyraMIR [predecessor-mirinform Thyroid (Asuragen Inc)] 58. ThyroSeq v.2 Next Generation Sequencing Panel 59. Tissue of Origin Test (ResponseDX) 60. TreatmentMAP Definitions POC UC BC UCB AUC FNA HCC HCV HBV FFPE NPV AGEC HT TT IHC NSCLC FAP PFS GEP NGS Point of care Urothelial carcinoma Bladder cancer Urothelial carcinoma bladder Area under the curve Fine needle aspiration Hepatocellular carcinoma Hepatitis C virus Hepatitis B virus Formalin-fixed, paraffin-embedded Negative predictive value Afirma gene expression classifier Hemithyroidectomy Total thyroidectomy Immunohistochemistry Non small cell lung cancer Familial adenomatous polyposis Progression free survival Gene expression profiling Next generation sequencing Tumor Markers for Cancer Nov 15 11

12 GPS Genomic Prostate Score Codes Related To This Policy NOTE: The codes listed in this policy are for reference purposes only. Listing of a code in this policy does not imply that the service described by this code is a covered or noncovered health service. Coverage is determined by the benefit documents and medical necessity criteria. This list of codes may not be all inclusive. On October 1, 2015, the ICD-9 code sets used to report medical diagnoses and inpatient procedures have been replaced by ICD-10 code sets. ICD-9 Codes (May not be all inclusive) Malignant neoplasm of esophagus Malignant neoplasm of stomach Malignant neoplasm of small intestine, including duodenum Malignant neoplasm of colon Malignant neoplasm of rectum, rectosigmoid junction, and anus Maligant neoplasm of liver and intrahepatic bile ducts Malignant neoplasm of gallbladder and extrahepatic bile ducts Malignant neoplasm of pancreas Malignant neoplasm of the female breast 181 Malignant neoplasm of placenta (e.g., choriocarcinoma) Malignant neoplasm of ovary and other uterine adnexa 185 Malignant neoplasm of prostate Malignant neoplasm of testis Carcinoma in situ of stomach Carcinoma in situ of colon Carcinoma in situ of other and unspecified parts of intestine Carcinoma in situ of liver and biliary system Carcinoma in situ of other and unspecified digestive system Carcinoma in situ of breast Carcinoma in situ of bladder Carcinoma in situ of other and unspecified female genital organs Carcinoma in situ of prostate Carcinoma in situ of other and unspecified male genital organs Neoplasm of uncertain behavior of placenta Neoplasm of uncertain behavior of ovary Neoplasm of uncertain behavior of prostate Neoplasm of uncertain behavior of bladder Neoplasm of bladder Elevated prostate specific antigen (PSA) V10.3 Personal history of malignant neoplasm of breast V10.04 Personal history of malignant neoplasm of stomach V10.05 Personal history of malignant neoplasm of large intestine V10.06 Personal history of malignant neoplasm of rectum, rectosigmoid junction, and anus V10.07 Personal history of malignant neoplasm, liver V10.09 Personal history of malignant neoplasm of gastrointestinal tract, other V10.43 Personal history of malignant neoplasm of ovary V10.46 Personal history of malignant neoplasm of prostate Tumor Markers for Cancer Nov 15 12

13 V10.47 Personal history of malignant neoplasm of testis V76.44 Special screening for malignant neoplasm of prostate V76.46 Family history of malignant neoplasm of ovary ICD-10 Codes C15.3-C15.9 Malignant neoplasm of esophagus C16.0-C16.9 Malignant neoplasm of stomach C17.0-C17.9 Malignant neoplasm of small intestine C18.0-C18.9 Malignant neoplasm of colon C19 Malignant neoplasm of rectosigmoid junction C22.0-C22.9 Malignant neoplasm of liver and extrahepatic bile ducts C24.0-C24.9 Malignant neoplasm of other and unspecified parts of biliary tracts C25.0-C25.9 Malignant neoplasm of pancreas C C Malignant neoplasm of breast C56.1-C56.9 Malignant neoplasm of ovary C58 Malignant neoplasm of placenta C C Malignant neoplasm of breast C61 Malignant neoplasm of prostate C62.00-C62.92 Malignant neoplasm of testis D00.2 Carcinoma in situ of stomach D01.0 Carcinoma in situ of colon D01.40-D01.49 Carcinoma in situ of other and unspecified parts of the intestine D01.7 Carcinoma in situ of other specified digestive organs D01.9 Carcinoma in situ of digestive organ, unspecified D05.90-D05.92 Unspecified type of carcinoma in situ of breast D07.30-D07.39 Carcinoma in situ of other and unspecified female genital organs D07.5 Carcinoma in situ of prostate D07.60-D07.69 Carcinoma in situ of other and unspecified male genital organs D09.0 Carcinoma in situ of bladder D39.10 Neoplasm of uncertain behavior of unspecified ovary D39.2 Neoplasm of uncertain behavior of placenta D40.0 Neoplasm of uncertain behavior of prostate D41.1 Neoplasm of uncertain behavior of bladder D45 Polycythemia vera D47.1 Chronic myeloproliferative neoplasm D47.3 Essential (hemorrhagic) thrombocythemia D49.4 Neoplasm of unspecified behavior of bladder D75.81 Myelofibrosis R97.0-R97.8 Abnormal tumor markers Z12.5 Encounter for screening for malignant neoplasm of prostate Z12.73 Encounter for screening for malignant neoplasm of ovary Z Z Personal history of malignant neoplasm of digestive organs Z Z Personal history of malignant neoplasm of large intestine Z Z Personal history of malignant neoplasm of rectum, rectosigmoid junction, and anus Z85.05 Personal history of malignant neoplasm of liver Z Z Personal history of malignant neoplasm of small intestine Z85.07 Personal history of malignant neoplasm of pancreas Z85.09 Personal history of malignant neoplasm of other digestive organs Z85.3 Personal history of malignant neoplasm of breast Z85.43 Personal history of malignant neoplasm of ovary Z85.46 Personal history of malignant neoplasm of prostate Z85.47 Personal history of malignant neoplasm of testis CPT Codes Tumor Markers for Cancer Nov 15 13

14 81235 EFGR (epidermal growth factor receptor) (e.g., non-small cell lung cancer) gene analysis, common variants (e.g., exon 19 LREA deletion, L858R, T790M, G719A, G719S, L861Q KRAS (v-ki-ras2 Kirsten rat sarcoma viral oncogene) homolog (eg, carcinoma) gene analysis, variants in exon 2 (e.g. condons 12 and 13) (description revised in 2016) Unlisted multianalyte assay with algorithmic analysis Alpha-fetoprotein (AFP); serum Beta-2 microglobulin Calcitonin Carcinoembryonic antigen (CEA) Hydrocyindoleacetic acid, 5(HIAA) Lactate dehydrogenase (LD), LDH Lactate dehydrogenase (LD), (LDH); isoenzymes, separation and quantitation Myeloperoxidase (MPO) Serum assay, Oncoprotein (HER2/neu microtiter ELISA) Prosate specific antigen (PSA); complexed (direct measurement) Prosate specific antigen (PSA); total Prosate specific antigen (PSA); free Receptor assay; estrogen Receptor assay; progesterone Thyroglobulin Gonadotropin, chorionic (hcg); quantitative Gonadotropin, chorionic (hcg); qualitative Gonadotropin, chorionic (hcg); free beta chain Unlisted chemistry procedure Immunoassay for tumor antigen, qualitative or semiqualitative (e.g., bladder tumor antigen) Immunoassay for tumor antigen, quantitative; CA 15-3 (27.29) CA CA Human epididymis protein 4 (HE4) Immunoassay for tumor antigen, other antigen, quantitative (e.g. CA50, 72-4, 549); each Nuclear Matrix protein 22 (NMP22), qualitative Cytopathology, in situ hybridization (e.g. FISH), urinary tract specimen with morphometric analysis, 3-5 molecular probes, each specimen; manual Cytopathology, in situ hybridization (e.g. FISH), urinary tract specimen with morphometric analysis, 3-5 molecular probes each specimen; using computer-assisted technology Molecular cytogenetics: DNA probe, each (eg FISH) Interphase in situ hybridization, analyze cells Immunohistochemistry or immunocytochemistry, per specimen; initial single antibody stain procedure (Code revised in 2015) Tissue in situ hybridization, (eg. FISH) each probe (Code revised in 2015) Morphometric analysis; in situ hybridization (quantitative or semi-quantitative) each probe; using computer-assisted technology (Code revised in 2015) Tumor Markers for Cancer Nov 15 14

15 88365 In situ hybridization, (eg. FISH), per specimen; initial single probe stain procedure Morphometric analysis, in situ hybridization (quantitative or semi-quantitative), using computer-assisted technology, per specimen; initial single probe stain procedure HCPCS Codes G0103 Prostate cancer screening; prostate specific antigen test Scientific Rationale Update November 2015 Thyroid nodules are a common clinical problem, and differentiated thyroid cancer is becoming increasingly prevalent. The clinical importance of thyroid nodules rests with the need to exclude thyroid cancer, which occurs in 7 15% depending on age, sex, radiation exposure history, family history, and other factors. Differentiated thyroid cancer (DTC), which includes papillary and follicular cancer, comprises the vast majority (>90%) of all thyroid cancers. Nonpalpable nodules have the same risk of malignancy as do sonographicallyconfirmed palpable nodules of the same size. Generally, only nodules >1 cm should be evaluated, since they have a greater potential to be clinically significant cancers. Occasionally, there may be nodules <1 cm that require evaluation because of suspicious US findings, associated lymphadenopathy, or other high-risk clinical factors such as a history of childhood head and neck irradiation or a history of thyroid cancer in one or more first degree relatives. Occasionally, there may be nodules <1 cm that require further evaluation because of clinical symptoms or associated lymphadenopathy. Per the American Thyroid Association (ATA) Guidelines on Thyroid Nodules and Differentiated Thyroid Cancer (2015.), fine needle aspiration (FNA) is the most accurate and cost-effective method for evaluating thyroid nodules. US-guided FNA is preferred. The ATA states that thyroid nodule FNA cytology should be reported using diagnostic groups outlined in the Bethesda System for Reporting Thyroid Cytopathology. The Bethesda system recognizes six diagnostic categories and provides an estimation of cancer risk within each category based upon literature review and expert opinion. These categories include: nondiagnostic/unsatisfactory; benign; atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS); follicular neoplasm/suspicious for follicular neoplasm (FN), a category that also encompasses the diagnosis of Hürthle cell neoplasm/suspicious for Hürthle cell neoplasm; suspicious for malignancy (SUSP), and malignant. Molecular testing has emerged as a non-invasive way of managing FNA cytology indeterminate patients and for providing reliable diagnostic information. Two types of tests are currently available: mutation panel testing and classifiers. The largest studies of preoperative molecular markers in patients with indeterminate FNA cytology have respectively evaluated a 7-gene panel of genetic mutations and rearrangements (BRAF, RAS, RET/PTC, PAX8/PPARγ), a gene expression classifier (167 GEC; mrna expression of 167 genes), and galectin-3 immunohistochemistry (cell blocks). Commercially available classifiers include an mrna-based gene expression classifier (GEC) i.e., Afirma messenger RNA gene expression classifier (see Scientific Rationale Apr 2014) and, a more recently available microrna (mirna) expression classifier. Tumor Markers for Cancer Nov 15 15

16 ThyGenX and ThyraMIR Interpace Diagnostics has developed combination platform testing with ThyraMIR, and ThyGenX Thyroid Oncogene Panel. ThyGenX performs targeted next generation sequencing (NGS) analysis to identify >100 genetic alterations within 5 thyroid cancer-relevant genes, BRAF, HRAS, KRAS, NRAS, and PIK3CA, and 3 RNA fusion transcripts, PAX8/PPARγ, RET/PTC, and RET/PTC3. ThyGenX may be used for cytologically indeterminate thyroid nodules categorized as either AUS/FLUS or FN/SFN within the Bethesda classification scheme for FNA cytology. ThyGenX, along with other clinical information, may be used by physicians to help determine the need for surgery when patients are diagnosed with indeterminate thyroid nodules. The ThyGen X results are reported qualitatively as either mutation positive or mutation negative. ThyraMIR complements ThyGenX by providing increased sensitivity while maintaining the high specificity of ThyGenX. ThyraMIR thyroid mirna classifier is a PCR-based microrna (mirna) gene expression classifier which evaluates the expresson levels of 10 mirna genes within FNA biopsy: mir p, mir-31-5p, mir p, mir p, mir-146b-5p, mir-155. mir-204-5p, mir-222-3p, mir-375, and mir-551b- 3p. mirnas are endogenous non-coding RNAs that act as regulators of gene expression. The regulatory network is complex with a single mirna able to regulate several mrnas, and genes can be under the control of multiple mirnas. ThyraMIR may be used for cytologically indeterminate thyroid nodules categorized as either AUS/FLUS or FN/SFN within the Bethesda classification scheme for FNA cytology. It is performed following a negative ThyGenX result for all mutations or when mutations detected are not fully indicative of malignancy (i.e. ThyGenX results which favor a benign nodule but cancer could still be present). The test is used on the same FNA cytology sample. The ThyraMIR test reports a qualitative positive or negative result based on the gene expression levels. ThyGenX predecessor was the mirinform Thyroid test. The mirinform test was upgraded to the next-generation sequencing (NGS) platform used for ThyGenX. ThyGenX includes the same molecular targets as mirinform with the addition of PIK3CA and high concordance (99.7%) between MiRinform and ThyGenX has been demonsrated (per Interpace). ThyGenX requires 1 dedicated FNA biopsy containing a minimum of 50 ng of tissue. Per Interspace Diagnostics, Benefits of using ThyGenX include: One test for a broad panel of molecular markers Demonstrated specificity of 89% Helps inform clinical risk stratification and decision making Can aid in the selection of the appropriate surgery Uses your local cytopathology lab; no central cytopathology needed Per Interpace Diagnostics, An initial positive for ThyGenX alone has a risk of malignancy of 81% while a positive ThyraMIR result, after a negative ThyGenX test, has a risk of malignancy of 74%. This risk is similar to that of FNA cytology category suspicious for malignancy, which is normally managed with surgery. Interpace also notes that positive or negative test results should be interpreted in conjunction with all other available clinical data. Final diagnosis and optimal patient management are the responsibility of the referring physician or health care provider. ThyroSeq v.2 Next Generation Sequencing Panel ThyroSeq v.2 Next Generation Sequencing Panel (CBLPath) offers simultaneous sequencing and detection in >1000 hotspots of 14 thyroid cancer-related genes and for 42 types of gene fusions known to occur in thyroid cancer. Gene List for Mutations: AKT1, BRAF, CTNNB1, GNAS, HRAS, KRAS, NRAS, PIK3CA, PTEN, RET, Tumor Markers for Cancer Nov 15 16

17 TP53, TSHR, TERT, and EIF1AX. Gene List for Gene Fusions and Gene Expression: RET, PPARG, NTRK1, NTRK3, ALK, IGF2BP3, BRAF, MET, CALCA, PTH, SLC5A5, TG, TTF1, KRT7, and KRT20. The test is indicated for any of the following: Thyroid FNA diagnosed as indeterminate by cytology (Bethesda categories III, IV, V) Thyroid FNA diagnosed as malignant by cytology, when molecular testing is expected to affect the decision to perform surgery or determine the extent of surgery Thyroid FNA diagnosed as benign by cytology, when strong clinical suspicion for cancer exists based on ultrasonographic features or other imaging and clinical studies. Diagnosis of cancer is established in preoperative FNA or surgically excised thyroid tissue, when molecular profiling of cancer will affect clinical decision with regards to administration of radioactive iodine, intensity of follow up, or targeted therapies for advanced cancer American Thyroid Association Per the ATA guidelines, the principal proposed use of molecular markers in indeterminate thyroid FNA specimens is diagnostic (ruling out or in the presence of thyroid malignancy), with the implication of a companion use to inform decisionmaking on primary surgical treatment (i.e. the decision to perform surgery and if so, the extent of surgery). If molecular testing is being considered, patients should be counseled regarding the potential benefits and limitations of testing, and about the possible uncertainties in the therapeutic and long-term clinical implications of results. (Strong recommendation, low quality evidence). Per the guidelines, The largest studies of preoperative molecular markers in patients with indeterminate FNA cytology have respectively evaluated a 7-gene panel of genetic mutations and rearrangements (BRAF, RAS, RET/PTC, PAX8/PPARγ), a gene expression classifier (167 GEC; mrna expression of 167 genes), and galectin-3 immunohistochemistry (cell blocks). The guidelines state there is currently no single optimal molecular test that can definitively rule in or rule out malignancy in all cases of indeterminate cytology, and long-term outcome data proving clinical utility are needed. The ATA makes the following recommendations: If molecular testing is being considered, patients should be counseled regarding the potential benefits and limitations of testing, and about the possible uncertainties in the therapeutic and long-term clinical implications of results. (Strong recommendation, Low quality evidence) If intended for clinical use, molecular testing should be performed in CLIA/CAP (Clinical Laboratory Improvement Amendments/College of American Pathologists) certified molecular laboratories, or international equivalent, as reported quality assurance practices may be superior compared to other settings. (Strong recommendation, Low-quality evidence) For nodules with AUS/FLUS cytology, after consideration of worrisome clinical and sonographic features, investigations such as repeat FNA or molecular testing may be used to supplement malignancy risk assessment in lieu of proceeding directly with a strategy of either surveillance or diagnostic surgery. Informed patient preference and feasibility should be considered in clinical decisionmaking. (Weak recommendation, Moderate-quality evidence) Diagnostic surgical excision is the long-established standard of care for the management of follicular neoplasm/suspicious for follicular neoplasm (FN/SFN) cytology nodules. However, after consideration of clinical and sonographic features, molecular testing may be used to supplement malignancy risk Tumor Markers for Cancer Nov 15 17

18 assessment data, in lieu of proceeding directly with surgery. Informed patient preference and feasibility should be considered in clinical decision making. (Weak recommendation, Moderate-quality evidence) After consideration of clinical and sonographic features, mutational testing for BRAF or the 7-gene mutation marker panel (BRAF, RAS, RET/PTC, PAX8/PPARγ) may be considered in nodules with Suspicious for Malignancy (SUSP) cytology if such data would be expected to alter surgical decision-making. (Weak recommendation, Moderate-quality evidence) American Association of Clinical Endocrinologists 2010 Guidelines from the American Association of Clinical Endocrinologists (AACE), Associazione Medici Endocrinologi (AME), and European Thyroid Association (ETA) state that molecular and immunohistochemical markers may improve the accuracy of cytologic diagnosis, but they do not have consistent predictive value for malignancy and their use is still expensive and restricted to specialized centers. On the basis of current limited evidence, routine use of molecular and immunohistochemical markers in clinical practice is not recommended and should be reserved for selected cases. The Afirma Thyroid FNA Analysis. ThyGenX, ThyraMir and Thyroseq are not mentioned in the guidelines. National Comprehensive Care Network (NCCN) Per NCCN guidelines on Thyroid Carcinoma (2.2015), Molecular diagnostic testing to detect individual mutations [eg. BRAF V600E, RET/PTC, RAS, Pax8/PPAR (peroxisome proliferator-activated receptors) gamma] or pattern recognition approaches using molecular classifiers may be useful in the evaluation of FNA samples that are indeterminate to assist in management decisions. The NCCN Panel recommends (category 2B) molecular diagnostic testing for evaluating FNA results that are suspicious for follicular or Hurthle cell neoplasms or AUS/FLUS. The panel noted that the molecular testing (both the Gene Expression Classifier and individual mutational analysis) was available in the majority of NCCN Member Institutions (> 75%). The guideline notes that rather than proceeding to immediate surgical resection to obtain a definitive diagnosis for these indeterminate FNA cytology groups (follicular lesions), patients can be followed with observation if the application of a specific molecular diagnostic test (in conjunction with clinical and ultrasound features) results in a predicted risk of malignancy that is comparable to the rate seen in cytologically benign thyroid FNAs (approximately < 5%.) It is important to note that the predictive value of molecular diagnostics may be significantly influenced by the pretest probability of disease associated with the various FNA cytology groups. The guideline also notes that in the cytologically indeterminate groups, the risk of malignancy for FNA can vary widely among institutions. NCCN notes that because the published studies have focused primarily on adult patients with thyroid nodules, the diagnosis utility of molecular diagnostics in pediatric patients remains to be defined. Therefore, proper implementation of molecular diagnostics into clinincal care requires an understanding of both the performance characteristics of the specific molecular test and its clinical meaning across a range of pre-test disease probabilities. Studies Beaudenon-Huibregtse et al (2014) reported molecular testing for oncogenic gene mutations and chromosomal rearrangements plays a growing role in the optimal management of thyroid nodules, yet lacks standardized testing modalities and systematic validation data. The authors objective was to assess the performance of molecular cytology on preoperative thyroid nodule FNA s across a broad range of Tumor Markers for Cancer Nov 15 18

19 variables, including independent collection sites, clinical practices, and anatomic pathology interpretations. Single-pass FNAs were prospectively collected from 806 nodules 1cm or larger by ultrasonography at five independent sites across the United States. Specimens were shipped in a nucleic acid stabilization solution and tested at a centralized clinical laboratory. Seventeen genetic alterations (BRAF, KRAS, HRAS, and NRAS mutations, PAX8-PPARG and RET-PTC rearrangements) were evaluated by multiplex polymerase chain reaction and liquid bead array cytometry in 769 FNAs that met inclusion criteria. Cytology, histology, and clinical care followed local procedures and practices. All results were double-blinded. Thirty-two specimens (4.2%) failed to yield sufficient nucleic acid to generate molecular data. A single genetic alteration was detected in 80% of cytology malignant cases, 21% of indeterminate, 7.8% of nondiagnostic, and 3.5% of benign cases. Among 109 nodules with surgical histology reference standard, oncogenic mutations were present in 50% of malignant nodules missed by cytology. There were 14 cancers not identified by cytology or molecular tests, including 5 carcinomas with histologic sizes less than 1cm (3 multifocal) and 8 noninvasive follicular variants of papillary carcinoma (4 encapsulated). No mutations were detected in 89% of the nodules benign by histopathology with 6 false-positive molecular results in 5 adenomas (2-5.5cm) and 1 cystic nodule with an incidental papillary microcarcinoma (0.15cm). The posttest probability of thyroid cancer was 100% for nodules positive for BRAF or RET-PTC, 70% for RAS or PAX8-PPARG, and 88% for molecular cytology overall. The authors concluded centralized and standardized molecular testing for genetic alterations associated with a high risk of malignancy efficiently complements the local cytopathologic diagnosis of thyroid nodule aspirates in the clinical setting. Actionable molecular cytology can improve the personalized surgical and medical management of patients with thyroid cancers, facilitating one-stage total thyroidectomy and reducing the number of unnecessary diagnostic surgeries. Lastra et al (2014) reviewed their institutional experience with Afirma, used for the preoperative classification of thyroid nodules with indeterminate cytology. A cohort of 132 cases of thyroid FNA with Afirma testing was selected from the study files and relevant information was recorded and analyzed. At the study institution, Afirma is mainly performed on atypia of undetermined significance (AUS)/follicular lesion of undetermined significance (FLUS) cases when diagnosed as such on repeat FNA. The cohort included 98 female (74%) and 34 male (26%) patients. Cytology diagnosis was AUS/FLUS in 68 cases (51.5%), follicular neoplasm (FN) in 39 cases (29.5%), and FN with oncocytic features (FNOF) in 25 cases (19.0%). Of the FNOF cases with suspicious Afirma findings, 2 (15%) were malignant and 11 (85%) were benign. Of the FN cases with suspicious Afirma findings, 9 (53%) were malignant and 8 (47%) were benign. Of the AUS/FLUS cases with suspicious Afirma findings, 10 (63%) were malignant and 6 (37%) were benign. The authors concluded the Afirma classifier is a useful tool to aid in the distinction of cytologically indeterminate nodules. Performing Afirma in cases diagnosed as AUS/FLUS on repeat FNA would increase the positive predictive value, thereby minimizing the number of benign cases referred to surgery. Results of the Afirma test could be limited in cases diagnosed as FNOF. McIver et al (2014) assessed the performance of the Afirma GEC in a large academic medical center. Samples for the GEC were collected according to the manufacturer's recommended protocol from patients undergoing thyroid fine-needle aspiration. The authors requested GEC analysis on nodules reported cytologically as follicular neoplasm or atypia or follicular lesion of undetermined significance from patients willing to defer surgery. All patients undergoing thyroid fine-needle aspiration during the study period, whose cytology was reported as follicular neoplasm or atypia of undetermined significance/follicular lesion of undetermined significance, were offered access to the test and recruited to this study. PATIENTS whose GEC was "benign" Tumor Markers for Cancer Nov 15 19

20 were offered ultrasound follow-up in lieu of surgery. Those with a "suspicious" GEC were advised to undergo diagnostic lobectomy. The authors measured the rate of benign and suspicious calls from the Afirma GEC and histological diagnosis after surgery. A total of 72 nucleic acid samples were sent for GEC analysis. In 12 (17%) of these samples, there was insufficient mrna, leaving 60 Afirma results for analysis. Of these, 16 (27%) were benign, whereas 44 (73%) were suspicious. The rate of confirmed malignancy in GEC-suspicious nodules was only 17%. The authors concluded Afirma GEC demonstrated a lower than expected rate of benign reports in follicular or Hürthle cell neoplasm and a lower than anticipated malignancy rate within GEC-suspicious nodules. These data suggest that the positive predictive value of the GEC is lower than previously reported and call into question the performance of the test when applied in the context of specialized academic cytopathology. Nikiforov et al (2014) reported FNA cytology is a common approach to evaluating thyroid nodules, although 20% to 30% of FNAs have indeterminate cytology, which hampers the appropriate management of these patients. Follicular (or oncocytic) neoplasm/suspicious for a follicular (or oncocytic) neoplasm (FN/SFN) is a common indeterminate diagnosis with a cancer risk of approximately 15% to 30%. In this study, the authors tested whether the most complete next-generation sequencing (NGS) panel of genetic markers could significantly improve cancer diagnosis in these nodules. The evaluation of 143 consecutive FNA samples with a cytologic diagnosis of FN/SFN from patients with known surgical outcomes included 91 retrospective samples and 52 prospective samples. Analyses were performed on a proprietary sequencer using the targeted ThyroSeq v2 NGS panel, which simultaneously tests for point mutations in 13 genes and for 42 types of gene fusions that occur in thyroid cancer. The expression of 8 genes was used to assess the cellular composition of FNA samples. In the entire cohort, histologic analysis revealed 104 benign nodules and 39 malignant nodules. The most common point mutations involved the neuroblastoma RAS viral oncogene homolog (NRAS), followed by the Kirsten rat sarcoma viral oncogene homolog (KRAS), the telomerase reverse transcriptase (TERT) gene, and the thyroid-stimulating hormone receptor (TSHR) gene. The identified fusions involved the thyroid adenoma associated (THADA) gene; the peroxisome proliferator-activated receptor γ (PPARG) gene; and the neurotrophic tyrosine kinase, receptor, type 3 (NTRK3) gene. Performance characteristics were similar in the retrospective and prospective groups. Among all FN/SFN nodules, preoperative ThyroSeq v2 performed with 90% sensitivity (95% confidence interval [CI], 80%-99%), 93% specificity (95% CI, 88%-98%), a positive predictive value of 83% (95% CI, 72%-95%), a negative predictive value of 96% (95% CI, 92%- 100%), and 92% accuracy (95% CI, 88%-97%). The authors concluded the current results indicate that comprehensive genotyping of thyroid nodules using a broad NGS panel provides a highly accurate diagnosis for nodules with FN/SFN cytology and should facilitate the optimal management of these patients. Labourier et al (2015) aimed to evaluate a novel diagnostic algorithm combining mutation detection and mirna expression to improve the diagnostic yield of molecular cytology. Surgical specimens and preoperative FNAs (n = 638) were tested for 17 validated gene alterations using the mirinform Thyroid test and with a 10-miRNA gene expression classifier generating positive (malignant) or negative (benign) results. Cross-sectional sampling of thyroid nodules with atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) or follicular neoplasm/suspicious for a follicular neoplasm (FN/SFN) cytology (n = 109) was conducted at 12 endocrinology centers across the United States. Qualitative molecular results were compared with surgical histopathology to determine diagnostic performance and model clinical effect. Mutations were detected in 69% of nodules with malignant outcome. Among mutation-negative specimens, mirna testing correctly identified 64% of malignant cases and 98% of Tumor Markers for Cancer Nov 15 20

21 benign cases. The diagnostic sensitivity and specificity of the combined algorithm was 89% (95% confidence interval [CI], 73-97%) and 85% (95% CI, 75-92%), respectively. At 32% cancer prevalence, 61% of the molecular results were benign with a negative predictive value of 94% (95% CI, 85-98%). Independently of variations in cancer prevalence, the test increased the yield of true benign results by 65% relative to mrna-based gene expression classification and decreased the rate of avoidable diagnostic surgeries by 69%. The authors concluded multiplatform testing for DNA, mrna, and mirna can accurately classify benign and malignant thyroid nodules, increase the diagnostic yield of molecular cytology, and further improve the preoperative risk-based management of benign nodules with AUS/FLUS or FN/SFN cytology. Giordano et al (2014) reported molecular testing for oncogenic gene alterations provides clinically actionable information essential for the optimal management of follicular cell thyroid cancer. The authors aimed to establish the distribution and frequency of common oncogenic gene mutations and chromosomal rearrangements in a comprehensive set of benign and malignant thyroid lesions. A case-control study was conducted in 413 surgical cases comprising 17 distinct histopathologic categories, 244 malignant, 169 benign, and 304 double-blinded specimens. Seventeen alterations of BRAF, HRAS, KRAS, NRAS, PAX8, and RET genes were evaluated using a single validated technology platform. Following verification of analytical sensitivity, accuracy, and precision in model and surgical specimens, 152 molecular positive results were generated in lesions representing multiple stages of progression and epithelial differentiation as well as rare subtypes of primary, secondary, or recurring tumors. Single mutations were found in 58% of primary malignant lesions and 12% of benign (P <.001). In the blinded validation set, mutation distribution and frequency were distinct across variants of follicular and papillary carcinomas. BRAF or RET-PTC was detected exclusively in malignant lesions but not in follicular carcinomas (P <.001). RAS or PAX8-PPARG were present in 23% of adenomas, and NRAS was found in a single nonneoplastic lesion (P =.0014). These data substantiate the diagnostic utility of molecular testing for oncogenic mutations and validate its performance in a variety of surgical specimens. The authors concluded standardized and validated multianalyte molecular panels can complement the preoperative and postoperative assessment of thyroid nodules and support a growing number of clinical and translational applications with potential diagnostic, prognostic, or theranostic utility. Scientific Rationale Update October 2015 The Tissue of Origin Test (TOO) is a microarray-based RNA profiling test that compares the RNA expression of formalin-fixed paraffin-embedded (FFPE) tumor tissue from a patient with cancer of unknown primary (CUP) to the expression patterns of a panel of 15 known characterized tumor types (bladder, breast, colorectal, gastric, hepatocellular, kidney, melanoma, non-hodgkin s lymphoma, non-small cell lung, ovarian, pancreas, prostate, sarcoma, testicular germ cell, and thyroid) to identify the most likely primary tissue of origin. However, there is a paucity of peer-reviewed studies to the analytical validity of the TOO, and limited evidence to assess its clinical validity and utility in patients with CUP to identify the most likely primary tissue of origin. Therefore, at this time, it is considered investigational. Scientific Rationale Update September 2015 Multiple molecular prognostic tests are emerging, specifically with an aim to better risk stratify both untreated and treated men with localized prostate cancer. Some have become commercially available, while others are still being developed. Tumor Markers for Cancer Nov 15 21

22 The Prolaris test (Myriad) utilizes reverse transcription polymerase chain reaction (RT-PCR) methodology on RNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue from blocks or slides of prostatic adenocarcinoma biopsies. The test is available as Prolaris Biopsy and Prolaris Post Prostatectomy, depending on the sample used. Gene expression profiling is performed on 31 cell cycle progression (CCP) genes (representing multiple cancer proliferation pathways, the increased expression of which is associated with cellular proliferation) normalized to 15 reference genes by quantitative polymerase chain reaction (PCR) analysis; expression data are analyzed using a proprietary algorithm, and a Prolaris score is calculated that quantifies tumor aggressiveness, which can then be combined with clinical information to generate the 10-year biochemical recurrence risk. The Oncotype DX Prostate Cancer Assay (Genomic Health) is a gene expression test designed to predict the aggressiveness of a patient s cancer and thereby assist in treatment decisions. According to the laboratory website, the test is a reverse transcriptase polymerase chain reaction (RT-PCR) test that assays 12 genes in multiple biological pathways and compares their expression levels to that of 5 reference genes. The results are converted to a Genomic Prostate Score (GPS) of very low, low, or intermediate risk of cancer aggressiveness. NCCN guidelines on Prostate Cancer (1.2015) note that two tissue-based molecular assays appear further along in development and clinical use (i.e., Prolaris assay, Oncotype DX Prostate Cancer assay). The Prolaris assay produces a cell cycle progression (CCP) score from RNA expression levels of 31 genes involved in CCP. The Oncotype DX Prostate Cancer assay produces a Genomic Prostate Score (GPS) from RNA expression levels of 17 genes from 4 different molecular pathways (stromal response, cellular organization, androgen signaling and cell proliferation). The tissue based molecular assays can be performed on most formalin-fixed, paraffin-embedded prostate specimens. Per NCCN (1.2015), The Prolaris CCP score has been demonstrated predictive when applied in prospective-retrospective designs for biochemical recurrence or metastasis after radical prostatectomy, for survival when men were observed after diagnosis on transurethral resection of prostate or diagnostic needle biopsy, and for biochemical recurrence and survival after external beam radiation therapy. NCCN notes that Prolaris has changed treatment recommendations in 32-65% of cases and may enhance adherence to the treatment recommended. Per NCCN (1.2015), Both molecular biomarker tests have been developed with extensive industry support, guidance, and involvement, and have been marketed under less rigorous FDA regulatory pathway for biomarkers. Their clinical utility awaits evaluation by prospective, randomized clinical trials, which are unlikely to be done. The marketplace and comparative effectiveness research may be the only means for these tests and others like them to gain their proper place for better risk stratification for men with clinically localized prostate cancer. Prolaris Cuzick et al (2015) sought to validate the prognostic value of a CCP score independently and in a pre-specified linear combination with standard clinical variables, that is, a clinical-cell-cycle-risk (CCR) score. Paraffin sections from 761 men with clinically localized prostate cancer diagnosed by needle biopsy and managed conservatively in the United Kingdom, mostly between 2000 and 2003 were evaluated. The primary end point was prostate cancer death. Clinical variables consisted of centrally reviewed Gleason score, baseline PSA level, age, clinical stage, and extent of disease; these were combined into a single predefined risk assessment (CAPRA) score. Full data were available for 585 men who formed a fully independent Tumor Markers for Cancer Nov 15 22

23 validation cohort. In univariate analysis, the CCP score hazard ratio was 2.08 (95% CI (1.76, 2.46), P<10-13) for one unit change of the score. In multivariate analysis including CAPRA, the CCP score hazard ratio was 1.76 (95% CI (1.44, 2.14), P<10-6). The predefined CCR score was highly predictive, hazard ratio 2.17 (95% CI (1.83, 2.57), χ2=89.0, P<10-20) and captured virtually all available prognostic information. The authors concluded the CCP score provides significant pretreatment prognostic information that cannot be provided by clinical variables and is useful for determining which patients can be safely managed conservatively, avoiding radical treatment. Sommariva et al (2014) performed a systematic review to assess evidence on the value of the CCP in prostate cancer treatment by reviewing current publications and integrating the results via a meta-analysis. Sixteen publications were selected for inclusion. The authors reported the results show that use of the CCP score is better than existing assessments at elucidating the aggressive potential of prostate cancer in an individual. The pooled hazard ratio for biochemical recurrence per 1-unit increase in the CCP score was 1.88 in a univariate model and 1.63 in a multivariate model. Four studies showed that CCP testing can impact the decisions of physicians regarding treatment, and potentially lead to a decrease in surgical interventions for low-risk patients. Arsov et al (2014) reported initial inaccurate staging is a common problem associated with active surveillance (AS) for patients detected by transrectal ultrasound-guided prostate biopsy (TRUS-GB). Subsequently, repeated biopsies are necessary to monitor such patients. Thus, in addition to the already established clinicopathological criteria, there is a considerable demand for new, objective decision criteria to more accurately select AS candidates. Recently, a novel RNA expression signature derived from 31 cell-cycle progression (CCP) genes has been shown to be a strong predictor of outcome in patients after radical prostatectomy or radiotherapy. In a qualitative pilot study, the authors evaluated the prognostic value of the CCP-score (CCP-S) for the first time in men managed with AS after MRIguided prostate biopsy (MRI-GB). Nine patients previously diagnosed with prostate cancer during an ongoing, prospective trial assessing MRI-GB with additional TRUS- GB and were subsequently managed with AS were included in the study. CCP-S were retrospectively derived from biopsy specimens. The CCP-S is defined as the expression level of 31 CCP genes, normalized to 15 housekeeping genes, and is clinically validated in a range between -1.3 and 4.7. To assess the estimated 10-year mortality risk (without curative treatment), the CCP-S from each patient was combined with the individual CAPRA (Cancer of the Prostate Risk Assessment) score (CAPRA-S). Median patient age was 72 (range=58-77) years. Mean pre-biopsy PSA level was 6.33±1.94 (range ) ng/ml. Eight cases had Gleason score 6 (3+3) and one cancer had Gleason score 7 (3+4). Median CCP-S was -0.9 (range=- 1.5 to 0.0). Combining CCP-S with CAPRA-S [CAPRA-S: 1 (n=4), 2 (n=4), 3 (n=1)] the estimated 10-year mortality risk was not calculable for three patients because their CCP-S [CCP-S -1.4 (n=2) and -1.5 (n=1)] was outside the validated range. For the other 6 patients the estimated 10-year mortality ranged from %. The authors concluded the CCP-S confirms accurate staging of AS patients detected by MRI-based biopsy strategies and may significantly reduce inaccurate staging of AS patients and subsequent unnecessary re-biopsies. The CCP score may help to more accurately select for active surveillance candidates. Bishoff et al (2014) evaluated the CCP score in cohorts of patients from the Martini Clinic (283), Durham Veterans Affairs Medical Center (176) and Intermountain Healthcare (123). The score was derived from simulated biopsy (Martini Clinic) or diagnostic biopsy (Durham Veterans Affairs Medical Center and Intermountain Healthcare) and evaluated for an association with biochemical recurrence and Tumor Markers for Cancer Nov 15 23

24 metastatic disease. In all 3 cohorts the CCP score was associated with biochemical recurrence and metastatic disease. The association with biochemical recurrence remained significant after adjusting for other prognostic clinical variables. On combined analysis of all cohorts (total 582 patients) the score was a strong predictor of biochemical recurrence on univariate analysis (HR per score unit 1.60, 95% CI , p=2.4 10(-7)) and multivariate analysis (HR per score unit 1.47, 95% CI , p=4.7 10(-5)). Although there were few events (12), the CCP score was the strongest predictor of metastatic disease on univariate analysis (HR per score unit 5.35, 95% CI , p=2.1 10(-8)) and after adjusting for clinical variables (HR per score unit 4.19, 95% CI , p=8.2 10(-6)). The authors concluded the CCP score derived from a biopsy sample was associated with adverse outcomes after surgery. These results indicate that the score can be used at disease diagnosis to better define patient prognosis and enable more appropriate clinical care. Crawford et al (2014) evaluated the impact of the CCP report (Prolaris) on physician treatment recommendations for prostate cancer in a prospective study. Physicians ordering the CCP test in clinical practice completed surveys regarding treatment recommendations before and after they received and discussed the test results with patients. Clinicians also rated the influence of the test result on treatment decisions. Treatment selections were confirmed via third-party audit of patient charts following final survey responses. Overall, 65% of cases showed a change between intended treatment pre- and post-ccp test reporting. Pre-CCP testing, 164 of 305 cases received a recommendation for interventional treatment. Post-CCP test, interventional therapy was recommended for 103 of these cases, a reduction of 37.2%. Conversely, 141 of 305 cases were recommended pre-ccp testing for noninterventional treatment; 108 of these remained with non-interventional treatment while 33 shifted to interventional options, a 23.4% increase. There was a 49.5% reduction in surgical interventions and a 29.6% reduction in radiation treatment. A third-party audit identified 80.2% concordance between the post-ccp testing treatment recommendation and actual treatment. Re-assignment to intervention or non-intervention generally correlated with the result of the CCP report. Study limitations included physician selection of patients for testing, no evaluation of patient input in therapeutic choice, and other potential treatment decision factors not queried by the survey. The authors concluded based on responses of ordering physicians, the CCP report adds meaningful new information to risk assessment for localized prostate cancer patients. Test results led to changes in treatment with reductions and increases in interventional treatment that were directionally aligned with prostate cancer risk specified by the test. Shore et al (2014) evaluated the potential clinical utility of the CCP test (Prolaris) in a US-based clinical setting. Urologists who participated in a prospective clinical study were sent a retrospective questionnaire to assess the value of the CCP test result. Fifteen board-certified urologists participated in the study, representing 15 distinct community urology group practices. Questionnaires were received for 294 evaluable patients. All patients had localized prostate cancer (T1-T3b, N0, M0). Physicians found the CCP score valuable and indicated that 55% of tests generated a mortality risk that was either higher or lower than expected. Physicians also indicated that 32% of test results would lead to a definite or possible change in treatment. The data suggest that the test would have the net effect of shifting patients from more aggressive treatment to more conservative treatment. This was evidenced by the significant association between change in treatment and lower CCP scores (p<0.002) and by the fact that 62% of tests likely to lead to a definite or possible change in treatment had mortality risks lower than the physician expected versus only 10% with risks higher than expected. The study was limited as it measured the retrospectively assessed likelihood of change in treatment as estimated by the physician, not the actual change in treatment. The authors concluded the CCP score Tumor Markers for Cancer Nov 15 24

25 adds meaningful new information to risk assessment for localized prostate cancer patients. Real-world use of the test is likely to lead to a change in treatment in a significant portion of tested patients, particularly by shifting patients towards more conservative management. This could reduce overtreatment of patients with less aggressive disease, decreasing patient morbidity and costs for payers and the healthcare system. Freedland et al (2013) sought to evaluate the prognostic utility of the CCP score, a RNA signature based on the average expression level of 31 CCP genes, for predicting biochemical recurrence (BCR) in men with prostate cancer treated with external beam radiation therapy (EBRT) as their primary curative therapy. The CCP score was derived retrospectively from diagnostic biopsy specimens of men diagnosed with prostate cancer from 1991 to 2006 (n=141). All patients were treated with definitive EBRT; approximately half of the cohort was African American. Outcome was time from EBRT to BCR using the Phoenix definition. Median follow-up for patients without BCR was 4.8 years. Association with outcome was evaluated by Cox proportional hazards survival analysis and likelihood ratio tests. Of 141 patients, 19 (13%) had BCR. The median CCP score for patient samples was In univariable analysis, CCP score significantly predicted BCR (P=.0017). The hazard ratio for BCR was 2.55 for 1-unit increase in CCP score (equivalent to a doubling of gene expression). In a multivariable analysis that included Gleason score, prostate-specific antigen, percent positive cores, and androgen deprivation therapy, the hazard ratio for CCP changed only marginally and remained significant (P=.034), indicating that CCP provides prognostic information that is not provided by standard clinical parameters. With 10- year censoring, the CCP score was associated with prostate cancer-specific mortality (P=.013). There was no evidence for interaction between CCP and any clinical variable, including ethnicity. The authors concluded among men treated with EBRT, the CCP score significantly predicted outcome and provided greater prognostic information than was available with clinical parameters. If validated in a larger cohort, CCP score could identify high-risk men undergoing EBRT who may need more aggressive therapy. Cooperberg et al (2013) aimed to validate a previously described genetic risk score, denoted the CCP score, in predicting contemporary radical prostatectomy (RP) outcomes. RNA was quantified from paraffin-embedded RP specimens. The CCP score was calculated as average expression of 31 CCP genes, normalized to 15 housekeeper genes. Recurrence was defined as two prostate-specific antigen levels 0.2 ng/ml or any salvage treatment. Associations between CCP score and recurrence were examined, with adjustment for clinical and pathologic variables using Cox proportional hazards regression and partial likelihood ratio tests. The CCP score was assessed for independent prognostic utility beyond a standard postoperative risk assessment (Cancer of the Prostate Risk Assessment post-surgical [CAPRA-S] score), and a score combining CAPRA-S and CCP was validated. Eightytwo (19.9%) of 413 men experienced recurrence. The hazard ratio (HR) for each unit increase in CCP score (range, to 2.16) was 2.1 (95% CI, 1.6 to 2.9); with adjustment for CAPRA-S, the HR was 1.7 (95% CI, 1.3 to 2.4). The score was able to substratify patients with low clinical risk as defined by CAPRA-S 2 (HR, 2.3; 95% CI, 1.4 to 3.7). Combining the CCP and CAPRA-S improved the concordance index for both the overall cohort and low-risk subset; the combined CAPRA-S + CCP score consistently predicted outcomes across the range of clinical risk. This combined score outperformed both individual scores on decision curve analysis. The authors concluded the CCP score was validated to have significant prognostic accuracy after controlling for all available clinical and pathologic data. The score may improve accuracy of risk stratification for men with clinically localized prostate cancer, including those with low-risk disease. Tumor Markers for Cancer Nov 15 25

26 Oncotype DX Prostate Cancer Assay At the present time, there is currently insufficient evidence to evaluate the use of Oncotype DX Prostate Cancer Assay in the treatment of patients with prostate cancer. Knezevic et al (2013) sought to validate the analytical performance of the Oncotype DX Prostate Cancer Assay using predefined acceptance criteria. The lowest quartile of RNA yields from prostate needle biopsies (six 5 μm sections) was between 19 and 34 ng. Analytical validation of the process requiring as little as 5 ng of RNA met all pre-defined acceptance criteria. Amplification efficiencies, analytical sensitivity, and accuracy of gene assays were measured by serially diluting an RNA sample and analyzing features of the linear regression between RNA expression measured by the crossing point (Cp) versus the log2 of the RNA input per PCR assay well. Gene assays were shown to accurately measure expression over a wide range of inputs (from as low as ng to 320 ng). Analytical accuracy was excellent with average biases at qpcr inputs representative of patient samples <9.7% across all assays while amplification efficiencies were within ±6% of the median. Assessments of reproducibility and precision were performed by testing 10 prostate cancer RNA samples over multiple instruments, reagent lots, operators, days (precision), and RNA input levels (reproducibility) using appropriately parameterized linear mixed models. The standard deviations for analytical precision and reproducibility were 1.86 and 2.11 GPS units (100-unit scale) respectively. The authors concluded the Oncotype DX Prostate Cancer Assay, a clinical RT-PCR assay specifically designed for use with prostate needle biopsies, has been analytically validated using very limited RNA inputs. They noted the assay requirements and analytical performance will provide physicians with test results from a robust and reliable assay which will enable improved treatment decisions for men diagnosed with early-stage prostate cancer. Decipher Prostate Cancer Classifier The Decipher Prostate Cancer Classifier (GenomeDX) directly measures a patient's biological risk of developing metastatic prostate cancer. By assessing the activity of multiple genomic markers associated with metastatic disease, Decipher provides information about the aggressiveness of a patient's tumor information distinct from that provided by PSA and other clinical risk factors. Decipher is proposed to predict aggressive disease and help physicians make more informed treatment decisions for men with prostate cancer. Klein et al (2015) reported surgery is a standard first-line therapy for men with intermediate- or high-risk prostate cancer. Clinical factors such as tumor grade, stage, and prostate-specific antigen (PSA) are currently used to identify those who are at risk of recurrence and who may benefit from adjuvant therapy, but novel biomarkers that improve risk stratification and that distinguish local from systemic recurrence are needed. The authors sought to determine whether adding the Decipher genomic classifier, a validated metastasis risk-prediction model, to standard risk-stratification tools (CAPRA-S and Stephenson nomogram) improves accuracy in predicting metastatic disease within 5 yr after surgery (rapid metastasis [RM]) in an independent cohort of men with adverse pathologic features after radical prostatectomy (RP). The study population consisted of 169 patients selected from 2641 men who underwent RP at the Cleveland Clinic between 1987 and 2008 who met the following criteria: preoperative PSA>20 ng/ml, stage pt3 or margin positive, or Gleason score 8; pathologic node negative; undetectable post-rp PSA; no neoadjuvant or adjuvant therapy; and minimum of 5-yr follow-up for controls. The final study cohort consisted of 15 RM patients and 154 patients as non-rm controls. The performance of Decipher was evaluated individually and in combination with clinical risk factors using concordance index (c-index), decision curve analysis, and logistic regression for prediction of RM. RM patients developed metastasis at a median of 2.3 yr (interquartile range: ). In multivariable analysis, Decipher Tumor Markers for Cancer Nov 15 26

27 was a significant predictor of RM (odds ratio: 1.48; p=0.018) after adjusting for clinical risk factors. Decipher had the highest c-index, 0.77, compared with the Stephenson model (c-index: 0.75) and CAPRA-S (c-index: 0.72) as well as with a panel of previously reported prostate cancer biomarkers unrelated to Decipher. Integration of Decipher into the Stephenson nomogram increased the c-index from 0.75 (95% confidence interval [CI], ) to 0.79 (95% CI, ). The authors concluded Decipher was independently validated as a genomic metastasis signature for predicting metastatic disease within 5 yr after surgery in a cohort of high-risk men treated with RP and managed conservatively without any adjuvant therapy. Integration of Decipher into clinical nomograms increased prediction of RM. Decipher may allow identification of men most at risk for metastatic progression who should be considered for multimodal therapy or inclusion in clinical trials. Ross et al (2015) sought to evaluate the Decipher genomic classifier in a natural history cohort of men at risk who received no additional treatment until the time of metastatic progression in a retrospective case-cohort design. 356 men who underwent RP between 1992 and 2010 at intermediate or high risk and received no additional treatment until the time of metastasis were included in the study. Participants met the following criteria: Cancer of the Prostate Risk Assessment postsurgical (CAPRA-S) score 3; pathologic Gleason score 7; and post-rp prostate-specific antigen nadir <0.2 ng/ml. The primary endpoint was defined as regional or distant metastases. Time-dependent receiver operating characteristic (ROC) curves, extension of decision curve analysis to survival data, and univariable and multivariable Cox proportional-hazards models were used to measure the discrimination, net benefit, and prognostic potential of genomic and pathologic risk factors. Cumulative incidence curves were constructed using Fine-Gray competingrisks analysis with appropriate weighting of the controls to account for the casecohort study design. Ninety six patients had unavailable tumor blocks or failed microarray quality control. Decipher scores were then obtained for 260 patients, of whom 99 experienced metastasis. Decipher correlated with increased cumulative incidence of biochemical recurrence, metastasis, and prostate cancer-specific mortality (p<0.01). The cumulative incidence of metastasis was 12% and 47% for patients with low and high Decipher scores, respectively, at 10 yr after RP. Decipher was independently prognostic of metastasis in multivariable analysis (hazard ratio 1.26 per 10% increase; p<0.01). Decipher had a c-index of 0.76 and increased the c-index of Eggener and CAPRA-S risk models from 0.76 and 0.77 to 0.86 and 0.87, respectively, at 10 yr after RP. Although the cohort was large, the single-center retrospective design is an important limitation. The authors concluded in a patient population that received no adjuvant or salvage therapy after prostatectomy until metastatic progression, higher Decipher scores correlated with clinical events, and inclusion of Decipher scores improved the prognostic performance of validated clinicopathologic risk models. Cooperberg et al (2015) compared two previously validated post- after RP classifiersthe Cancer of the Prostate Risk Assessment Postsurgical (CAPRA-S) and the Decipher genomic classifier (GC)-to predict prostate cancer-specific mortality (CSM) in a contemporary cohort of RP patients. The authors sought to evaluate the combined prognostic ability of CAPRA-S and GC to predict CSM. A cohort of 1010 patients at high risk of recurrence after RP were treated at the Mayo Clinic between 2000 and High risk was defined by any of the following: preoperative prostate-specific antigen >20 ng/ml, pathologic Gleason score 8, or stage pt3b. A case-cohort random sample identified 225 patients (with cases defined as patients who experienced CSM), among whom CAPRA-S and GC could be determined for 185 patients. The scores were evaluated individually and in combination using concordance index (c-index), decision curve analysis, reclassification, cumulative incidence, and Cox regression for the prediction of CSM. Among 185 men, 28 experienced CSM. The c-indices for CAPRA-S and GC were 0.75 (95% confidence Tumor Markers for Cancer Nov 15 27

28 interval [CI], ) and 0.78 (95% CI, ), respectively. GC showed higher net benefit on decision curve analysis, but a score combining CAPRA-S and GC did not improve the area under the receiver-operating characteristic curve after optimism-adjusted bootstrapping. In 82 patients stratified to high risk based on CAPRA-S score 6, GC scores were likewise high risk for 33 patients, among whom 17 had CSM events. GC reclassified the remaining 49 men as low to intermediate risk; among these men, three CSM events were observed. In multivariable analysis, GC and CAPRA-S as continuous variables were independently prognostic of CSM, with hazard ratios (HRs) of 1.81 (p<0.001 per 0.1-unit change in score) and 1.36 (p=0.01 per 1-unit change in score). When categorized into risk groups, the multivariable HR for high CAPRA-S scores ( 6) was 2.36 (p=0.04) and was (p<0.001) for high GC scores ( 0.6). For patients with both high GC and high CAPRA-S scores, the cumulative incidence of CSM was 45% at 10 yr. The study is limited by its retrospective design. The authors concluded both GC and CAPRA-S were significant independent predictors of CSM. GC was shown to reclassify many men stratified to high risk based on CAPRA-S 6 alone. Patients with both high GC and high CAPRA-S risk scores were at markedly elevated post-rp risk for lethal prostate cancer. If validated prospectively, these findings suggest that integration of a genomic-clinical classifier may enable better identification of those post-rp patients who should be considered for more aggressive secondary therapies and clinical trials. Ross et al (2014) reported due to their varied outcomes, men with biochemical recurrence (BCR) following RP present a management dilemma. They sought to evaluate Decipher, a GC, for its ability to predict metastasis following BCR. The study population included 85 clinically high-risk patients who developed BCR after RP. Time-dependent ROC curves, weighted Cox proportional hazard models and decision curves were used to compare GC scores to Gleason score (GS), PSA doubling time (PSAdT), time to BCR (ttbcr), the Stephenson nomogram and CAPRA- S for predicting metastatic disease progression. All tests were two-sided with a type I error probability of 5%. GC scores stratified men with BCR into those who would or would not develop metastasis (8% of patients with low versus 40% with high scores developed metastasis, P<0.001). The area under the curve for predicting metastasis after BCR was 0.82 (95% CI, ) for GC, compared to GS 0.64 ( ), PSAdT 0.69 ( ) and ttbcr 0.52 ( ). Decision curve analysis showed that GC scores had a higher overall net benefit compared to models based solely on clinicopathologic features. In multivariable modeling with clinicopathologic variables, GC score was the only significant predictor of metastasis (P=0.003). The authors concluded when compared to clinicopathologic variables, GC better predicted metastatic progression among this cohort of men with BCR following RP. While confirmatory studies are needed, these results suggest that use of GC may allow for better selection of men requiring earlier initiation of treatment at the time of BCR. The American Urological Society guidelines on clinically localized prostate cancer (2011) do not address the use of these genomic tests. At this time, the impact of this testing on actual clinical outcomes not been validated in the peer review literature. In addition, it will be important to perform head-to-head analyses to determine if one test has greater prognostic ability, if their prognostic abilities are overlapping, or if they are synergistic. Scientific Rationale Update May 2015 The ResponseDX is a Colon test that is proposed to analyze gene expression of 5 genes, epidermal growth factor receptor (EGFR); excision repair crosscomplementation group 1 (ERCC1); thymidylate synthase (TYMS); kinase insert domain receptor (KDR; also known as vascular endothelial growth factor receptor 2 [VEFGR2]); and MET protooncogene, receptor tyrosine kinase (MET). This testing is done with specific sequence variants in 5 genes, Kirsten rat sarcoma viral oncogene Tumor Markers for Cancer Nov 15 28

29 homolog (KRAS); B-Raf proto-oncogene, serine/threonine kinase (BRAF); neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS); phosphatidylinositol- 4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA); and UDP glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1); as well as microsatellite instability (MSI) testing. However, it is not clear from the Response Genetics website how the information from these 11 tests is combined to predict chemotherapy response. No published studies regarding the ResponseDX Colon test were identified, and there were no studies that evaluated the combined predictive value of these 11 tests with respect to chemotherapy response. Therefore, there is currently insufficient peer-reviewed evidence to evaluate the impact of the ResponseDX: Colon test on patient outcomes. Scientific Rationale Update April 2015 The SYMGENE68 NGS Cancer Panel may be performed on solid tumor, blood, or bone marrow specimens, and uses next-generation sequencing technology to analyze 68 genes associated with a variety of malignancies and hereditary cancer syndromes. The panel includes genes associated with well-studied cancer predisposition syndromes (i.e., Li-Fraumeni syndrome, retinoblastoma, and von Hippel-Lindau syndrome) and genes associated with a variety of sporadic cancers, both solid tumors and hematologic malignancies. Upon review of the current literature, no study that specifically evaluated the SYMGENE68 NGS Cancer Panel was identified and no peer-reviewed study that included an analysis of all 68 genes included in the SYMGENE NGS Cancer Panel in cancer patients was identified. Therefore, there is currently insufficient published evidence to determine the impact of this test on the care of cancer patients or at-risk family members. There was no information in NCCN on SYMGENE68 NGS Cancer Panel. Scientific Rationale Update February 2015 The BreastTrue High Risk Panel is a next-generation sequencing test with deletion/duplication analysis to detect mutations in seven high-risk breast cancer susceptibility genes, including BRCA1, BRCA2 and PALB2. The test uses nextgeneration DNA sequencing (NGS) technology to sequence all coding regions, as well as direct Sanger sequencing to sequence areas with insufficient coverage by NGS and for confirmation of identified variants. Array comparative genomic hybridization (acgh) is used to detect large gene deletions and duplications. Samples for the BreastTrue test may consist of either saliva or whole blood collected using a manufacturer-provided test kit. No published studies were identified that evaluated the use of the BreastTrue test specifically, or the 7 genes included in the test, in the patients with, or at risk for, suspected familial breast cancer. There is currently insufficient published evidence to evaluate the clinical impact of this test on patient care. The National Comprehensive Cancer Network (NCCN) guideline titled Genetic/Familial High-Risk Assessment: Breast and Ovarian, version , indicates that the 7 genes included in the BreastTrue test panel contribute to familial forms of breast cancer, and the guideline promotes a gene-by-gene approach based on the personal and family history of the patient being evaluated. Caution is advocated in the use of multigene testing panels of these patients because of limitations, including an unknown percentage of variants of unknown clinical significance, uncertainty of the risk associated with most of the genes included in multigene panels, and a lack of clear guidelines regarding risk management in variant carriers The Breast Cancer Index (BCI, biotheranostics Inc.) is proposed as a predictive marker for the recurrence of breast cancer and a predictive model for efficacy of Tumor Markers for Cancer Nov 15 29

30 extended endocrine therapy. The BCI is intended to provide an estimate of the risk of patients with primary breast cancer developing metastases and to determine if there is a benefit to extending endocrine therapy past 5 years. The BCI was originally marketed as Theros Breast Cancer Index by biotheranostics Inc. in the U.S. but is now called the Breast Cancer Index. The BCI is intended for patients with ER+ breast cancer who are stage I or IIA with no lymph node involvement. Zhang et al. (2013) completed a study that assessed reproducibility of the BCI. BCI assays from 6 specimens were run 4 times, resulting in an intra-assay CV from 0.19% to 2.23% for the genes analyzed. In addition, BCI assays from 9 specimens were run by 3 operators, repeating at least 4 times over a 2-week period, resulting in an inter-assay CV from 0.91% to 1.77% for the genes analyzed. Lastly, using the universal reference RNA as a positive control, the inter-assay CV ranged from 0.86% to 1.40% for the genes analyzed from conducting the BCI assay in 42 independent batches. There was no assessment of how this variability affected the BCI score or any statistical analysis of the variability. The European Society for Medical Oncology (ESMO, 2013) published clinical practice guidelines for the diagnosis, treatment, and follow-up for primary breast cancer. Several commercially available molecular signatures (including the BCI) were mentioned as having no proven robust clinical utility so far, but in specific cases, with specific criteria, 2 of these tests (MammaPrint and Oncotype DX) may be used. There was no mention of the BCI in the NCCN Guidelines on Breast Cancer, version or the Genetic/Familial High-Risk Assessment: Breast and Ovarian, version The DX Prostate Cancer Assay for Prostate Cancer Prognosis (Genomic Health Inc.) is a gene expression test proposed to predict the aggressiveness of a patient s cancer and thereby assist in treatment decisions. The test is a reverse transcriptase polymerase chain reaction (RT-PCR) test that assays 12 genes in multiple biological pathways and compares their expression levels to that of 5 reference genes. The results are converted to a Genomic Prostate Score (GPS) of very low, low, or intermediate risk of cancer aggressiveness. Two studies directly related to the Oncotype DX Prostate Cancer Assay were identified, including 1 study describing analytical validation of the assay in formalin-fixed paraffin-embedded (FFPE) biopsy specimens and 1 study describing the derivation/validation of the test. At this time, there is currently insufficient evidence to evaluate the use of this test in the care of patients with prostate cancer, however based on the information from NCCN noted below this has not been added to the investigational list in this policy at this time. The NCCN Clinical Practice Guidelines in Oncology on Prostate Cancer, Version , note the following: The Oncotype DX Prostate Cancer assay produces a GPS from RNA expression levels of 17 genes from 4 different molecular pathways (stromal response, cellular organization, androgen signaling and cell proliferation). These tissue based molecular assays can be performed on most formalin-fixed, paraffin-embedded prostate specimens. The Oncotype DX GPS was developed from evaluation of a diagnostic prostate biopsy and radically prostatectomy series from Cleveland Clinic and validated in a diagnostic prostate biopsy and radical prostatectomy series from University of California, San Francisco. GPS performed in the diagnostic prostate biopsy has provided information beyond usual clinical information that predict the likelihood of Tumor Markers for Cancer Nov 15 30

31 Gleason sum 7 or extraprostatic disease on radical prostatectomy. Oncotype DX GPS improved upon NCCN risk group assignment, which may enhance rates of compliance with recommended active surveillance or diminish the number of surveillance prostate biopsies. The igene Cancer Panel is one of the 3 subpanels of the igene Personal Health and Disease Risk Panel. It is proposed to provide comprehensive hereditary information on 9 cancers: breast cancer, ovarian cancer, colorectal cancer, gastric cancer, pancreatic cancer, endometrial cancer, renal cell carcinoma, melanoma, and prostate cancer. This panel uses next-generation parallel sequencing technology to analyze 20 genes, including BRCA1 and BRCA2, etc. No studies were identified that specifically looked at the contribution of variants in the included genes to the hereditary forms of these cancers, or at the clinical impact of testing this panel of genes. Therefore, there is currently insufficient published evidence to determine the impact of this test on the care of patients with or at risk for a suspected familial cancer syndrome. The National Comprehensive Cancer Network (NCCN, Version ) guidelines titled 'Genetic/Familial High-Risk Assessment: Breast and Ovarian' note the following: At the present time, no consensus exists on recommendations for optimal management or surveillance approaches for carriers of lower or moderate penetrance genes, and no data are available to address cancer risk assessments in individuals who are found to carry multiple gene mutations with moderate penetrance. The Pancreatic Cancer Panel uses exon array comparative genomic hybridization (CGH)/next-generation sequencing technology to analyze 16 genes, including the breast cancer 1 (BRCA1) and 2 (BRCA2) genes, that have been reported to be associated with an increased risk of pancreatic and other hereditary cancers. Deletion/duplication analysis only is performed for the epithelial cellular adhesion molecule (EPCAM) gene. Functional and clinical interpretations are assigned to each variant identified, based upon the current state of scientific understanding, in the categories of Pathogenic, Expected Pathogenic, Variant of Unknown Clinical Significance, and Variant, Likely Benign. The preferred specimen for the Pancreatic Cancer Panel is a whole blood sample, or an oral rinse sample may also be submitted. No studies were identified that specifically looked at the variants in the 16 genes included in the Pancreatic Cancer Panel to the hereditary forms of pancreatic cancer, or at the clinical impact of testing this panel of genes. Therefore, there is currently insufficient published evidence to determine the impact of this test on the care of patients with or at risk for suspected familial pancreatic cancer. The NCCN Guidelines (Verion ) on Pancreatic Adenocarcinoma notes: The NCCN Pancreatic Cancer Panel currently supports pathology synoptic reports from the College of American Pathologists (CAP). The proposal is an abbreviated minimum analysis of pancreatic cancer specimens from the CAP recommendations. In addition to the standard TNM staging, other variables are included all of which have prognostic implications in the evolution of this disease. There was no other specific information about the pancreatic cancer panel noted. The High/Moderate Risk Panel uses next-generation sequencing technology to analyze 20 genes, (i.e BRCA1, BRCA2, etc.), that have been reported to be associated with an increased risk of breast, ovarian, colorectal, uterine, and other cancers. The preferred specimen for the High/Moderate Risk Panel is a whole blood sample, or an oral rinse sample. There was a paucity of peer-reviewed studies that identified the variants included in genes in the hereditary forms of these cancers, or at the clinical impact of testing this panel of genes. Therefore, there is currently insufficient published evidence to determine the impact of this test on the care of patients with or at risk for a suspected familial cancer syndrome. Tumor Markers for Cancer Nov 15 31

32 The NCCN Guidelines (Version ), Genetic/Familial High-Risk Assessment: Breast and Ovarian indicates that some of the genes included in the High/Moderate Risk Panel contribute to familial forms of breast cancer, and the guidelines promote a gene-by-gene approach based on the personal and family history of the patient being evaluated. Caution is advocated in the use of multigene testing panels of these patients because of limitations, including an unknown percentage of variants of unknown clinical significance, uncertainty of the risk associated with most of the genes included in multigene panels, and a lack of clear guidelines regarding risk management in variant carriers Scientific Rationale Update September 2014 Myeloma Prognostic Risk Signature (MyPRS Plus) test for myeloma includes a microarray-based GEP assay of a 70-gene signature of aggressive disease (GEP70). This test is done on plasma cells derived from bone marrow aspirate, a molecular subtyping analysis, and a virtual karyotype analysis. The MyPRS test is proposed as a method to risk stratify individuals with newly diagnosed and relapsed multiple myeloma. Clinical trials have shown that a high risk signature is present in approximately 15% of new cases of multiple myeloma (Shaughnessy, 2007). Kumar et al. (2011) completed a study examining the utility of two GEP-based risk stratification systems in a cohort of individuals undergoing initial therapy in the context of a phase III trial of lenalidomide in the treatment of multiple myeloma. Among 45 individuals studied at baseline, 7 (16%) and 10 (22%) respectively were high-risk using the GEP70 and GEP15 signatures. The median overall survival (OS) for the GEP70 high-risk group was 19 months versus not reached for the standard risk group (hazard ratio [HR]=14.1). While the medians were not reached, the GEP15 also predicted a poor outcome among the high-risk individuals. The estimated C-statistic (the test used to assess the predictive ability of the gene score) for GEP70 score was 0.74 (95% confidence interval [CI]: 0.61,0.88), a value conventionally considered as reflecting a prediction model with good discriminatory ability. In comparison, the C-statistic for FISH based risk stratification was 0.70 (95% CI: 0.55, 0.84). The median OS for the 10 participants considered to be high-risk by FISH was 39 months and did not reach median for the standard risk group (HR 5.8, 95% CI: 1.62, 20.5, p=0.007). The median time to progression (TTP) for the participants with GEP70 high-risk was 9 months compared to the standard risk group (23 months, p=0.3). In comparison the median TTP for the FISH high-risk group was 16 months versus the standard risk group (23 months, p=0.4). The authors concluded these results strengthen the rationale for a GEP based risk stratification approach. The objective of the study was not to determine whether one system was better than the other, due to the limited number of study participants; The results presented here confirms the value of the GEP scoring and highlights its value in the setting of initial therapy with lenalidomide and dexamethasone, one of the most commonly used treatments for myeloma, as well as its value in older patients. The small number of patients in this study, however, prevents a comparison of the GEP and FISH-based risk stratification, and an assessment of the incremental value of GEP over FISHbased risk stratification. While the information provided in this small populationbased study is encouraging, additional study with larger populations are required to confirm the potential of the GEP70 (MyPRS) to improve health outcomes for individuals with multiple myeloma. The NCCN (Version 1, 2015) Clinical Practice Guidelines in Oncology on Multiple Myeloma have no mention on the Myeloma Prognostic Risk Signature (MyPRS Plus) testing for myeloma. Tumor Markers for Cancer Nov 15 32

33 The current body of peer-reviewed evidence related to the commercially marketed test consists mostly of studies describing clinical validation of various GEP assays in patient populations with multiple myeloma. Myeloma Prognostic Risk Signature (MyPRS Plus) testing for myeloma is considered investigational since there is a low level of evidence of analytical validity and clinical validity and a paucity of peer reviewed published evidence of clinical utility. The DecisionDx-Melanoma gene expression profile (GEP) test was discovered and developed by Castle Biosciences. It is a multigene expression assay that is designed to predict distant metastasis in stage 1 and stage 2 melanoma patients. DecisionDx- Melanoma assesses the expression of 31 genes that have been associated with metastasis and classifies tumors as Class 1 (i.e., low risk of metastasis) or Class 2 (i.e., high risk of metastasis). The test is done using reverse transcription polymerase chain reaction (RT-PCR) on formalin-fixed paraffin-embedded (FFPE) melanoma specimens. According to the manufacturer s website, DecisionDx- Melanoma was developed using 107 samples from patients with stage 1 or stage 2 cutaneous melanoma, and was validated in a prospectively designed multicenter study, results of which were presented at the 2013 meeting of the American Society of Clinical Oncology. The validation study indicated that patients with Class 1 tumors had a 5-year metastasis-free survival rate of 97%, and patients with Class 2 tumors had a 5-year metastasis-free survival rate of 31%. However, no peer-reviewed published studies regarding the DecisionDx-Melanoma test were identified. Therefore, there is currently a paucity of evidence to evaluate the safety, efficacy, performance and clinical utility of this test in the care of patients with melanoma. There was no information on the DecisionDx-Melanoma test multigene expression assay in the NCCN Guidelines on Melanoma (Version 4,2014). Scientific Rationale Update August 2014 The Myriad MyRisk Hereditary Cancer Genetic Panel uses next-generation sequencing technology and large rearrangement testing to analyze 25 genes, including BRCA1 and BRCA2, that have been reported to be associated with an increased risk of breast, ovarian, colorectal, uterine, and other cancers. Functional and clinical interpretations are assigned to each variant identified, based upon the current state of scientific understanding, with a summary interpretative statement of either clinically actionable mutation identified or no clinically actionable mutation identified. When subsequently published evidence has the potential to alter the expected clinical impact of particular variants, an amended report will be provided by Myriad. The Myriad myrisk test may be performed using a whole blood sample. However, no studies were identified that specifically looked at the contribution of variants in the included genes to the hereditary forms of these cancers, or at the clinical impact of testing this panel of genes. Consequently, there is currently insufficient published peer-reviewed evidence to determine the impact of this test on the care of patients with, or at risk for, a suspected familial cancer syndrome. The Myriad myrisk Hereditary Cancer Panel is not yet commercially available, but may be ordered by genetic counselors for their patients. According to a laboratory representative from Myriad Genetics Inc. via telephone communication on April 29, 2014, the CPT code used in billing for Myriad myrisk will be that of the genetic test for which the patient qualifies. There was no listing for the MyRisk genetic test for Hereditary Cancer in the National Comprehensive Cancer Guidelines (NCCN, 2014). Tumor Markers for Cancer Nov 15 33

34 Scientific Rationale Update May 2014 The most common primary brain tumors are the gliomas. The World Health Organization (WHO) grades gliomas on a scale of I to IV, with higher grades corresponding to more malignant features. Pathologically, gliomas are divided into astrocytic or oligodendroglial tumors, depending on the primary histology, with rare cases of mixed histology. WHO grade II diffuse gliomas (also known as low-grade gliomas) and grade III (anaplastic) gliomas are less common than grade IV gliomas (glioblastomas). Glioblastoma is the most common malignant primary brain tumor in adults and accounts for > 30% of all gliomas. It is also the most lethal. Primary glioblastomas are defined as tumors that are discovered de novo, whereas secondary GBMs are tumors that arise after malignant progression via transformation of a lower-grade glioma. The best management strategy for infiltrative low-grade gliomas has yet to be defined. Surgery remains an important diagnostic and therapeutic modality. The primary surgical goal is to provide adequate tissue for a pathologic diagnosis and grading. Biopsy results can be misleading, because mitosis, or necrosis from one region to another, thus, small samples can provide a lower histologic grade. The role of maximal tumor resection in low-grade astrocytomas remains unresolved. The general recommendation for treating an astrocytoma is to first attempt as complete an excision of tumor as possible without compromising function. Lowgrade oligodendrogliomas are often amenable to total excision due to their location in the frontal lobes and distinct tumor margins. However, for tumors that involve eloquent areas, a total removal may not be feasible and an aggressive approach could result in neurological deficits. Major tumor removal should be performed whenever possible in individuals with Anaplastic astrocytoma (grade III) and glioblastoma (grade IV astrocytomas.) After surgical intervention, the choice of adjuvant therapy depends on the tumor pathology, status of the 1p/19q co-deleted loci, and performance status of the patient. Management of recurrent tumors depends on the extent of the disease and patient condition. Isocitrate dehydrogenase 1 (IDH1) mutations have recently been identified as early and frequent genetic alterations in astrocytomas, oligodendrogliomas, and oligoastrocytomas, (WHO grade II), as well as higher-grade gliomas (WHO grades III and IV), such as secondary glioblastomas. Primary glioblastomas very rarely contain IDH1 mutations. It is suggested that analyzing gliomas based on their genetic signatures allows for the stratification of these patients into distinct cohorts, with unique prognosis and survival. IDH1/2 mutations in glioma are associated with significantly prolonged progression-free and overall survival compared with comparable tumors without an IDH mutation. IDH mutations are strongly associated with other molecular markers of improved survival ( e.g., MGMT, CpG, TP53, TERT, 1p and 19q codeletion.) According to NCCN guidelines regarding low-grade infiltrative astrocytomas and oligodendrogliomas (Central Nervous System Cancers, V ): When possible, maximal safe resection is recommended for low-grade infiltrative astrocytomas and oligodendrogliomas, and the actual extent of the resection should be documented with a T2-weighted or FLAIR MRI scan with 72 hours after surgery. If the tumor is found to have components of oligodendroglioma, 1p/19q deletion testing should be considered, as it is a favorable prognostic factor. Managing the disease by serial observation alone is appropriate for selected patients. The NCCN Tumor Markers for Cancer Nov 15 34

35 panel also discussed the role of the isocitrate dehydrogenase 1 or 2 (IDH1, IDH2) genes in low-grade gliomas. Mutations in the IDH genes are common in patients and are reported to be a significant marker of positive prognosis. However, routine IDH testing as a recommendation is not included in the algorithm at this point because its impact on treatment is still unclear. Per the NCCN guidelines: The following are considered low-risk features for low grade glioma: age <40 years, Karnofsky Performance Status (KPS) > 70, minor or no neurological deficit, oligodendroglioma or mixed oligoastrocytoma, tumor dimension <6 cm, 1p and 19q codeleted, IDH1 or IDH2 mutated. Patients are categorized as having high risk if they have three or more of the following: Age > 40, KPS under 70, tumor larger than 6 cm, tumor crossing midline, or preoperative neurological deficit of more than minor degree. Other adverse factors to consider include increased perfusion on imaging and one or more deletions on 1p and 19q, wild type IDH 1 or 2. Beiko et al (2014) reported that IDH1 gene mutations identify gliomas with a distinct molecular evolutionary origin. The authors sought to determine the impact of surgical resection on survival after controlling for IDH1 status in malignant astrocytomas-world Health Organization grade III anaplastic astrocytomas and grade IV glioblastoma. Clinical parameters including volumetric assessment of preoperative and postoperative MRI were recorded prospectively on 335 malignant astrocytoma patients: n = 128 anaplastic astrocytomas and n = 207 glioblastoma. IDH1 status was assessed by sequencing and immunohistochemistry. IDH1 mutation was independently associated with complete resection of enhancing disease (93% complete resections among mutants vs 67% among wild-type, P <.001), indicating IDH1 mutant gliomas were more amenable to resection. The impact of residual tumor on survival differed between IDH1 wild-type and mutant tumors. Complete resection of enhancing disease among IDH1 wild-type tumors was associated with a median survival of 19.6 months versus 10.7 months for incomplete resection; however, no survival benefit was observed in association with further resection of nonenhancing disease (minimization of total tumor volume). In contrast, IDH1 mutants displayed an additional survival benefit associated with maximal resection of total tumor volume (median survival 9.75 y for >5 cc residual vs not reached for <5 cc, P =.025). The authors concluded the survival benefit associated with surgical resection differs based on IDH1 genotype in malignant astrocytic gliomas. Therapeutic benefit from maximal surgical resection, including both enhancing and nonenhancing tumor, may contribute to the better prognosis observed in the IDH1 mutant subgroup. Thus, individualized surgical strategies for malignant astrocytoma may be considered based on IDH1 status. Xie et al (2013) studied the correlation between IDH1 mutation and prognosis in supratentorial high-grade astrocytomas. There were 217 samples of supratentorial high-grade astrocytomas specimens collected for DNA extraction, IDH1 mutation of each patient was determined by PCR and direct sequencing. The differences of clinical features were compared between mutant group and wild type group. The relationship between IDH1 mutation and overall survival of the patients was studied with Kaplan-Meier survival curve, while multiple factors analysis was carried out by COX regression model. There were 43 (19.3%) IDH1 mutations in 217 specimens, of which 9 (24.3%) in WHO grade III, 34 (18.9%) in WHO grade IV. The mean age of primary glioblastoma multiforme (GBM) in mutant type group and wild type group were and years old respectively (P < 0.05). The median survival time was 64 weeks for the patients in IDH1 mutation group and 50 weeks for those in wild Tumor Markers for Cancer Nov 15 35

36 type group, and the difference was statistically significant (P < 0.001). The median survival time was 51 weeks for the wild type group of WHO grade III cases and 58 weeks for the mutant group of WHO grade IV cases (P < 0.001). COX multiple variable analysis showed that IDH1 mutation, surgical resection, preoperative Karnofsky performance, radiotherapy and chemotherapy were statistically significant in prognosis (P < 0.05). The authors concluded IDH1 mutation can be found in supratentorial high-grade astrocytomas, the patients with IDH1 mutation may have a better prognosis. Nobusawa et al (2009) sought to establish the frequency of IDH1 mutations in glioblastomas at a population level, and to assess whether they allow reliable discrimination between primary (de novo) glioblastomas and secondary glioblastomas that progressed from low-grade or anaplastic astrocytoma. The authors screened glioblastomas from a population-based study for IDH1 mutations and correlated them with clinical data and other genetic alterations. IDH1 mutations were detected in 36 of 407 glioblastomas (8.8%). Glioblastoma patients with IDH1 mutations were younger (mean, 47.9 years) than those with EGFR amplification (60.9 years) and were associated with significantly longer survival (mean, 27.1 versus 11.3 months; P < ). IDH1 mutations were frequent in glioblastomas diagnosed as secondary (22 of 30; 73%), but rare in primary glioblastomas (14 of 377; 3.7%: P < ). IDH1 mutations as genetic marker of secondary glioblastoma corresponded to the respective clinical diagnosis in 95% of cases. Glioblastomas with IDH1 mutation diagnosed as primary had clinical and genetic profiles similar to those of secondary glioblastomas, suggesting that they may have rapidly progressed from a less malignant precursor lesion that escaped clinical diagnosis and were thus misclassified as primary. Conversely, glioblastomas without IDH1 mutations clinically diagnosed as secondary typically developed from anaplastic rather than low-grade gliomas, suggesting that at least some were actually primary glioblastomas, that may have been misclassified, possibly due to histologic sampling error. The authors concluded IDH1 mutations are a strong predictor of a more favorable prognosis and a highly selective molecular marker of secondary glioblastomas that complements clinical criteria for distinguishing them from primary glioblastomas. Hartmann et al (2013) reported that the determinants of long-term survival in glioblastoma have remained largely obscure. IDH 1 or 2 mutations are common in World Health Organization (WHO) grades II and III gliomas, but rare in primary glioblastomas, and associated with longer survival. The authors compared clinical and molecular characteristics of 69 patients with centrally confirmed glioblastoma and survival >36 months (LTS-36), including 33 patients surviving >60 months (LTS-60), with 257 patients surviving <36 months. MGMT promoter methylation, 1p/19q codeletions, EGFR amplification, TP53 mutations, and IDH1/2 mutations were determined by standard techniques. The rate of IDH1/2 mutations in LTS-36 patients was 34% (23 of 67 patients) as opposed to 4.3% in controls (11 of 257 patients). Long-term survivors with IDH1/2-mutant glioblastomas were younger, had almost no EGFR amplifications, but exhibited more often 1p/19q codeletions and TP53 mutations than LTS patients with IDH1/2 wild-type glioblastomas. Long-term survivors with IDH1/2 wild-type showed no distinguishing features from other patients with IDH1/2 wild-type glioblastomas except for a higher rate of MGMT promoter methylation. Similarly, among 11 patients with IDH1/2-mutant glioblastomas without long-term survival, the only difference to IDH1/2-mutant longterm survivors was less-frequent MGMT promoter methylation. Compared with LTS- 36 patients, LTS-60 patients had less frequently TP53 mutations and radiotherapy alone as initial treatment. The authors concluded IDH1/2 mutations define a subgroup of tumors of LTS patients that exhibit molecular characteristics of WHO grade II/III gliomas and secondary glioblastomas. Determinants of LTS with IDH1/2 Tumor Markers for Cancer Nov 15 36

37 wild-type glioblastomas, which exhibit typical molecular features of primary glioblastomas, beyond MGMT promoter methylation, remain to be identified. Horbinski et al (2009) reported mutations in IDH1 and IDH2 have been identified in many adult astrocytomas and oligodendrogliomas. These mutations are targeted to specific codons, making assays to detect them in clinical specimens feasible. The authors describe a simple and accurate molecular assay for detection of IDH1/2 mutations on routine formalin-fixed paraffin-embedded tissues. Using this polymerase chain reaction-based assay, they tested 75 glial neoplasms and 57 nonneoplastic conditions that can mimic gliomas including radiation changes, viral infections, and infarcts. Of the gliomas, 37 (49%) were positive for IDH1 or IDH2 mutations; the most common mutation was IDH1 (97%). Two of 12 gangliogliomas were positive for IDH1 mutation, and both had unfavorable clinical outcomes (p < 0.03). None of the nonneoplastic cases were positive for IDH mutations. The assay detected IDH mutations in biopsy material containing mostly glioma and in concomitant near-miss stereotactic core biopsies that were otherwise equivocal for the presence of glioma by light microscopy. These results indicate that testing for IDH1/2 mutations can be effectively performed in a clinical setting and can enhance the accuracy of diagnosis of gliomas when traditional diagnostic methods are not definitive. Cheng et al (2013) performed a meta-analysis to determine if IDH1 mutation is associated with improved overall survival in patients with glioblastoma. Studies reporting overall survival by IDH1 mutation in patients with glioblastoma were considered potentially eligible for the meta-analysis. For the quantitative aggregation of the survival results, the IDH mutation effect was measured by the pooled hazard ratio (HR) with its 95% confidence interval (95%CI). Nine studies with a total of 1,669 patients with glioblastoma were finally included into this meta-analysis. Overall, the IDH1 mutation was associated with improved survival in patients with glioblastoma (random effects model HR = 0.45, 95%CI , P < 0.001). Sensitivity analysis further showed that the pooled estimates were stable in this meta-analysis. Therefore, the findings from this meta-analysis suggest that IDH1 mutation is associated with improved overall survival in patients with glioblastoma. Okita et al (2012) reported reliable prognostic biomarkers of grade II gliomas remain unclear. This study aimed to examine the role of mutations of IDH1/2, 1p/19q codeletion, and clinicopathological factors in patients with grade II glioma who were primarily treated with radiotherapy or chemoradiotherapy after surgery. Seventy-two consecutive patients, including 49 cases of diffuse astrocytomas (DA), 4 oligodendrogliomas (OL) and 19 oligoastrocytomas (OA), who underwent treatment from 1991 to 2010 at a single institution were examined. The overall survival (OS) of the DA patients (8.3 years) was significantly shorter than that of the OL and OA patients (11.7 years). IDH1/2 mutations were found in 46.9% of the DA patients and 82.6% of the OL and OA patients. The progression-free survival (PFS) and OS of the patients with IDH1/2 mutations (8.4 and 16.3 years) were significantly longer than those of the patients without IDH1/2 mutations (3.3 and 4.5 years). Among the patients with IDH1/2 mutations, those who were initially treated with chemoradiotherapy including nimustine hydrochloride (ACNU), had significantly longer PFS than those treated with radiotherapy alone, whereas no significant difference in PFS was observed between the chemoradiotherapy and radiotherapy groups in the patients without IDH1/2 mutations. Oligodendroglial tumors, age <40 years, initial Karnofsky performance status (KPS) 80, and IDH1/2 mutations were favorable prognostic factors regarding PFS and OS. IDH1/2 mutation was a predictive factor of response to chemoradiotherapy in grade II gliomas. Patients with IDH1/2 mutations may benefit more from chemoraiotherapy than those without IDH1/2 mutations. Tumor Markers for Cancer Nov 15 37

38 Ohno et al (2012) investigated the impact dehydrogenase (IDH1/2) mutations on the malignant progression of gliomas was investigated by comparing the histopathological features of 53 grade II and III gliomas after recurrence according to the IDH1/2 status. We identified IDH1/2 mutations in 44.4 % (16 of 36) of astrocytic tumors and 70.6 % (12 of 17) of oligodendroglial tumors. Histopathological malignant progression was observed in 68.8 % (11 in 16) and 55 % (11 in 20) of astrocytic tumors with and without IDH1/2 mutations, respectively. There were 8 secondary glioblastomas (GBM) that had progressed from 5 diffuse astrocytomas (DA) and 3 anaplastic astrocytomas (AA) with IDH1/2 mutations. Seven secondary GBMs were derived from 3 DAs and 4 AAs with wild-type IDH1/2. Malignant progression was observed in 47.1 % (8 of 17) of oligodendroglial tumors. All 12 oligodendroglial tumors with IDH1/2 mutations remained as such without progressing to GBM, whereas 3 of the 5 oligodendroglial tumors without IDH1/2 mutations progressed to GBM at recurrence. In conclusion, grade II and III gliomas developed to more malignant histological types, irrespective of the IDH1/2 mutation status, and the monitoring of the IDH1/2 status could be of value to predict the development of GBM in patients with oligodendroglial tumors. Houillier et al (2010) reports that IDH1 and IDH2 mutations occur frequently in gliomas, including low-grade gliomas. However, their impact on the prognosis and chemosensitivity of low-grade gliomas remains unclear. Search for IDH1 and IDH2 mutations, loss of heterozygosity on chromosomes 1p and 19q, MGMT promoter methylation, and p53 expression was performed in a series of 271 low-grade gliomas and correlated with overall survival. A subgroup of 84 patients treated up-front with temozolomide was individualized. Response to temozolomide was evaluated by progression-free survival, as well as by tumor size on successive MRI scans, and then correlated with molecular alterations. IDH (IDH1 or IDH2) mutations were found in 132/189 patients (70%). IDH mutation and 1p-19q codeletion were associated with prolonged overall survival in univariate (p = and p = ) and multivariate analysis (p = and p = 0.004). 1p-19q codeletion, MGMT promoter methylation, and IDH mutation (p = 0.01) were correlated with a higher rate of response to temozolomide. Further analysis of the course of the disease prior to any treatment except for surgery (untreated subgroup) showed that 1p-19q codeletion was associated with prolonged progression-free survival in univariate analysis, whereas IDH mutation was not. The authors concluded IDH mutation appears to be a significant marker of positive prognosis and chemosensitivity in lowgrade gliomas, independently of 1p-19q codeletion, whereas its impact on the course of untreated tumors seems to be limited. Ongoing and future prospective studies will further clarify the role of these markers in the clinical management of malignant gliomas. Scientific Rationale Update April 2014 Thyroid nodules are discrete masses present in the thyroid gland that are a common clinical finding among adults in the general population. Approximately 5% of women and 1% of men have a palpable thyroid nodule on physical examination. The evaluation of thyroid nodules, which may represent benign lesions or malignant thyroid tumors, typically includes a cytopathologic examination of a biopsy specimen obtained by fine-needle aspiration (FNA). Generally, only nodules >1 cm should be evaluated, since they have a greater potential to be clinically significant cancers. Occasionally, there may be nodules <1 cm that require evaluation because of suspicious US findings, associated lymphadenopathy, a history of head and neck irradiation, or a history of thyroid cancer in one or more first-degree relatives. The NCCN notes that FNA is a very sensitive test, however, false negative results are Tumor Markers for Cancer Nov 15 38

39 sometimes obtained, therefore, a reassuring FNA should not override worrisome clinical findings. Cytologic examination of an FNA specimen is typically categorized as: carcinoma; follicular or Hurthle cell neoplasm; follicular lesion of undetermined significance; thyroid lymphoma; benign (i.e., nodular goiter, colloid goiter, hyperplastic /adenomatoid nodule, Hashimoto s thyroiditis) or insufficient biopsy (nondiagnostic) (NCI 2007). Thyroid nodules are subsequently classified based on cytology, with 70% to 75% of nodules being benign and approximately 5% being malignant. Approximately 20% to 25% of thyroid nodules are classified as indeterminate and represent a significant diagnostic challenge for physicians. Genetic or genomic markers have been investigated to aid in evaluating indeterminate thyroid nodules. There are two approaches to the molecular characterization of FNA aspirates that are commercially available in the United States: identification of particular molecular markers of malignancy, such as BRAF and RAS mutational status, and use of high density genomic data for molecular classification (an FNA-trained mrna classifier) The mrna classifier measures the activity level of 167 genes within the nodule (using the FNA aspirate). The Afirma Gene Expression Classifier measures the expression of 142 genes to reclassify ambiguous thyroid FNA samples as either benign or suspicious for cancer. The molecular portion of the test, which involves an analysis of RNA extracted from thyroid nodule aspirates, utilizes the GEC to classify indeterminate nodules as either benign or suspicious. An analysis of an additional 25 genes is included in order to help classify rare tumor subtypes. FNA samples for both cytopathology and the Gene Expression Classifier are performed at the same visit. Patient samples submitted for the Afirma Thyroid FNA Analysis are read by specialized cytopathologists at Thyroid Cytopathology Partners (TCP), an independent group based in Austin, Texas. Veracyte believes TCP to be the largest thyroid-only cytopathology practice in the country. According to Veracyte Inc., The Afirma Thyroid FNA Analysis provides a novel solution for improved thyroid nodule assessment. This advanced offering combines specialized cytopathology with the Gene Expression Classifier (GEC) for the management of patients with thyroid nodules. The GEC reclassifies FNAs (fine needle aspirates) with indeterminate cytopathology results as either benign or suspicious for cancer. By helping physicians pre-operatively identify benign nodules (negative predictive value greater than 94%) in approximately half of patients with indeterminate cytopathology, our solution may enable clinical and sonographic follow-up for these patients in lieu of diagnostic surgery. Studies show that the risk of malignancy of a benign GEC result (less than 6%) is similar to that of a benign cytopathology diagnosis. Per NCCN (2013), Molecular diagnostics to detect individual mutations [eg. BRAF, RET/PTC, RAS, Pax8-PPAR (peroxisome proliferator-activated receptors) gamma] or pattern recognition approaches using molecular classifiers may be useful in the evaluation of FNA samples that are indeterminate. The NCCN recommends molecular diagnostics for evaluating FNA results that are suspicious for Follicular or Hurthle cell neoplasms; or follicular lesion of undetermined significance. They note, Rather than proceeding to immediate surgical resection to obtain a definitive diagnosis in these categories, patients can be followed with observation if the applications of a specific molecular diagnostic test results in a predicted risk of malignancy that is comparable to the rate seen in cytologically benign thyroid FNA. It is important to note that the predictive value of molecular diagnostics may be significantly influenced by the pre-test probability of disease associated with the various FNA cytology categories. Furthermore, in the cytologically indeterminate Tumor Markers for Cancer Nov 15 39

40 groups, the risk of malignancy for FNA can vary widely between institutions. Because the published studies have focused primarily on adult patients with thyroid nodules, the diagnostic utility of molecular diagnostics in pediatric patients remains to be defined. Therefore, proper implementation of molecular diagnostics into clinical care requires an understanding of both the performance characteristics of the specific molecular test and its clinical meaning across a range if pre-test probabilities. The Afirma Thyroid FNA Analysis is not specifically mentioned by name in the guidelines Guidelines from the American Association of Clinical Endocrinologists (AACE), Associazione Medici Endocrinologi (AME), and European Thyroid Association (ETA) state that molecular and immunohistochemical markers may improve the accuracy of cytologic diagnosis, but they do not have consistent predictive value for malignancy and their use is still expensive and restricted to specialized centers. On the basis of current limited evidence, routine use of molecular and immunohistochemical markers in clinical practice is not recommended and should be reserved for selected cases. The Afirma Thyroid FNA Analysis is not mentioned in the guidelines. The American Thyroid Association guidelines (updated 2009) reported that the use of molecular markers (e.g., BRAF, RAS, RET/PTC, Pax8-PPAR gamma, or galectin-3) may be considered for patients with indeterminate cytology on FNA to help guide management. (Recommendation rating: C - i.e., The recommendation is based on expert opinion.) The guidelines do not mention genomic tests, such as the Afirma Thyroid FNA Analysis. Alexander et al (2014) analyzed all patients who had received Afirma GEC testing at five academic medical centers between 2010 and Nodule and patient characteristics, fine needle aspiration cytology, Afirma GEC results, and subsequent clinical or surgical follow-up were obtained for 339 patients. Results were analyzed for pooled test performance, impact on clinical care, and site-to-site variation. Three hundred thirty-nine patients underwent Afirma gene expression classifier (GEC) testing of cytologically indeterminate nodules [165 atypical or follicular lesion of undetermined significance (AUS/FLUS); 161 follicular neoplasm (FN); 13 suspicious for malignancy] and 174 of 339 (51%) indeterminate nodules were GEC benign, whereas 148 GEC were suspicious (44%). GEC results significantly altered care recommendations, as 4 of 175 GEC benign were recommended for surgery in comparison to 141 of 149 GEC suspicious (P<.01). Of 121 Cyto Indeterminate/GEC Suspicious nodules surgically removed, 53 (44%) were malignant. Variability in siteto-site GEC performance was confirmed, as the proportion of GEC benign varied up to 29% (P=.58), whereas the malignancy rate in nodules cytologically indeterminate/gec suspicious varied up to 47% (P=.11). Seventy-one of 174 GEC benign nodules had documented clinical follow-up for an average of 8.5 months, in which 1 of 71 nodules proved cancerous. The investigators concluded these multicenter, clinical experience data confirm originally published Afirma GEC test performance and demonstrate its substantial impact on clinical care recommendations. Although nonsignificant site-to-site variation exists, such differences should be anticipated by the practicing clinician. Follow-up of GEC benign nodules thus far confirm the clinical utility of this diagnostic test. Dedhia et al (2014) reported increasing utilization of genetic expression profiling (GEP) for thyroid nodules with indeterminate FNA results will potentially decrease the number of patients requiring diagnostic thyroidectomy.a retrospective review of thyroidectomy procedures performed over 1 year at the University of Michigan in the endocrine surgery division evaluated the indications for thyroidectomy, FNA Bethesda classification, and final surgical pathology to determine how application of GEP on indeterminate FNA results would affect decision for surgery and subsequent Tumor Markers for Cancer Nov 15 40

41 thyroidectomy volume. During the study period, 358 thyroidectomies were performed. The indication for procedure included: FNA findings, n = 122; symptomatic multinodular goiter, n = 85; nodule >4 cm, n = 30; Graves', n = 26; other, n = 95. FNA was performed in 231 patients. Bethesda classification included: benign, n = 69; malignant, n = 55; follicular lesion of undetermined significance, n = 59; follicular neoplasm, n = 20; suspicious for malignancy, n = 16; nondiagnostic, n = 12. If standard GEP was performed for all indeterminate FNA results, it would have influenced the decision for surgery in 68 (19 %) patients. Assuming 38 % of indeterminate FNA specimens will have benign results on genetic profiling, 27 patients would not have undergone thyroidectomy, translating into a 7.2 % decrease in overall thyroidectomy volume over a year. Reviewers concluded that in an academic endocrine surgery program, the most common indication for thyroidectomy is an FNA result; however, standard application of GEP for all indeterminate thyroid FNAs would result in a minimal reduction in overall thyroidectomy volume. Harrell and Bimston (2013) analyzed the performance of the GEC over 27 months in a community hospital-based thyroid surgery practice. Authors began using GEC and Thyroid Cytopathology Partners (TCP) exclusively for thyroid FNA analysis in 1/2011, shortly after the Afirma GEC first became commercially available. They focused on patients with indeterminate FNA results and the outcomes of GEC analysis, with particular attention to the calculation of the negative predictive value of the Afirma test. They performed 645 FNA's in 519 patients over 27 months. 58 of the FNA's (9%) were read as indeterminate, with 36 of these classified as suspicious by GEC (62%), 20 characterized as GEC benign (34%) and 2 determined to be inadequate due to low mrna content. Of the 36 suspicious GEC patients, 30 underwent thyroidectomy and 21 of the 30 had malignant final pathology. Of the 20 benign GEC patients, 5 underwent thyroid surgery and 2 were discovered to have malignancies. Negative predictive value for the Afirma GEC in their practice environment was 89.6%. The authors concluded in a practice with a high incidence of thyroid cancer in patients with indeterminate FNA's (33% for their practice), the negative predictive value of the Afirma GEC test may not be as robust as suggested in the literature to date. Gomberawalla and Elaraj (2014) reported multiple genetic mutations have been found to be associated with thyroid cancer, and molecular testing of thyroid nodule FNA specimens has been proposed as an adjunct to the cytologic diagnosis. The reviewers examined how molecular testing of FNAs could be used to guide surgical decision-making. B-type RAF kinase mutations in papillary thyroid cancer have been found to be associated with extrathyroidal extension, lymph node metastases, and advanced stage in two meta-analyses that are based largely on retrospective data. Testing for a panel of gene mutations has been found to have high specificity and positive predictive value, whereas microarray testing using a commercially available gene-expression classifier has been found to have high sensitivity and negative predictive value for the diagnosis of malignancy in cytologically indeterminate FNAs. Although there is no consensus regarding the use of such tests, they have already started to change clinical practice. Reviewers concluded molecular testing of FNA specimens may help to avoid diagnostic thyroidectomy or may help in deciding the extent of surgery in a patient with an indeterminate FNA biopsy. The use of these tests is currently undergoing review by a task force within the American Thyroid Association. The evidence supporting the validity and utility of the Afirma Thyroid FNA Analysis is somewhat limited, although it consistently supports the assertion that the assay may be useful as a rule out test, used to help exclude malignancy in thyroid nodules with indeterminate FNA cytology. Given its relatively high NPV (i.e., approximately 90% to 96%), the likelihood of a thyroid malignancy may be decreased significantly Tumor Markers for Cancer Nov 15 41

42 in many patients with indeterminate FNA biopsies. In addition, evidence supporting the clinical utility indicates that GEC results may influence medical management decisions in patients with indeterminate thyroid nodules and can lead to a significant decrease in diagnostic thyroid surgeries Scientific Rationale Update February 2014 Molecular Intelligence (MI) is a group of molecular biomarker tests provided by Caris Life Sciences, based upon a previous test, Target Now Molecular Profiling. Molecular Intelligence differs from Target Now in its methodology, which incorporates nextgeneration sequencing (NGS), and in the biomarkers analyzed. The MI profile includes immunohistochemistry (IHC), fluorescence/chromogenic in situ hybridization (FISH/CISH), next-generation sequencing (NGS), and quantitative polymerase chain reaction (qpcr) methodologies performed on formalin-fixed, paraffin-embedded (FFPE) tissues or biopsies preserved in neutral buffered formalin. Molecular Intelligence is proposed to provide information based upon literature review that associates a tumor s biomarker status with therapeutic agents that may have potential or lack of potential clinical benefit. It is proposed to identify open clinical trials relevant to particular biomarkers, with information provided in the form of a MI Profile Report. The MI Profile is for use in solid tumors, intended for therapeutic decision support and qualification for clinical trials in the treatment of aggressive, rare, or refractory cancers. Although Caris Molecular Intelligence is available for all solid tumors, including cancers of unknown primary, it is especially helpful when treating rare tumors or cancers with undefined standard of care, (e.g. sarcoma or glioma), aggressive cancers with few standard treatment options, (e.g. melanoma, pancreatic cancer), and difficult-to-treat cancers, such as certain metastatic and refractory diseases, e.g.(ovarian or triple-negative breast cancer). Although there are studies on molecular biomarkers, there are currently no Clinical Trials and a paucity of published peer reviewed data specifically on the Molecular Intelligence assay for cancer, therefore, it is considered investigational at this time. There is no information in NCCN Guidelines on (2014) Colon Cancer, (2014) Occult Primary Cancer, (2013) Soft Tissue Sarcoma, (2014) Melanoma, (2014) Pancreatic Cancer, (2013) Ovarian Cancer. or (2014) Breast Cancer, that specifically addresses the Molecular Intelligence assay. Scientific Rationale Initial Tumor markers are indicators of cellular, biochemical, molecular, or genetic alterations whereby neoplasia can be recognized. Tumor markers fall into three broad categories proteins, genetic mutations, and epigenetic changes (e.g., differential promoter-region methylation). The utility of tumor markers is adjunctive to medical and surgical management of malignancies, serving to help detect recurrences as well as predict prognosis. The clinical value of any given tumor marker will depend on its specificity and sensitivity as well as its intended clinical use. Serum tumor markers are currently used for screening, diagnosing, and predicting prognosis and treatment response. Use of tests for screening of disease, even those with high sensitivity and specificity, should be confined as much as possible to populations at risk for the disease. Bladder Cancer According to the National Comprehensive Cancer Network (NCCN), bladder cancer is the fourth most common cancer, occurring three times more often in men than women in the U.S. Bladder cancer is rarely diagnosed in individuals younger than 40 years. Bladder cancer is divided into three categories: non-muscle-invasive tumors, Tumor Markers for Cancer Nov 15 42

43 muscle invasive lesions and metastatic lesions. Prognosis, management and therapeutic aims differ among the categories. The diagnosis of bladder cancer commonly is suggested by the presence of hematuria, which may be either gross or microscopic. However, hematuria is frequently seen in a wide range of benign conditions. The diagnosis of bladder cancer ultimately requires a histologic diagnosis, which usually comes from a biopsy that is obtained at cystoscopy. Cytology may provide strong evidence for the presence of malignancy, but it has a low sensitivity and occasional false positives are reported. Urine biomarkers have potential applications in individuals in whom bladder cancer is suspected based upon the presence of hematuria, symptoms, or in whom there is an unusually high risk of tumor. Urine biomarkers may also have a role in detecting recurrences in patients who have been treated for non-muscle-invasive disease. Although cystoscopy is the gold standard for surveillance in patients with a history of bladder cancer, it does not detect all recurrences nor does it visualize the upper urinary tract. Performing a biomarker test in addition to cystoscopy may minimize the risk of missing a high grade tumor. A number of urine biomarkers and techniques are being evaluated. Many of these are based upon immunologic detection of soluble molecules in the urine [e.g., Bladder tumor antigen (BTA-STAT, Nuclear matrix protein 22 (NMP 22)]. Other techniques analyze exfoliated cells that are isolated from urine or bladder washings by centrifugation (e.g., UroVysion, ImmunoCyt). Per 2013 NCCN guidelines on Bladder cancer, Management of bladder cancer is based on pathologic findings of biopsy specimen, with attention to histology, grade and depth of lesion. These factors are used to estimate the probability of recurrence and progression to a more advanced stage. Consideration may be given to the FDA approved urinary biomarker testing by fluorescence in situ hybridization or nuclear matrix protein 22 in the monitoring for recurrence. NCCN guidelines on bladder cancer also state that urine molecular tests for urothelial tumor markers are now available. Most of these tests have a better sensitivity for detecting bladder cancer than urine cytology, but specificity is lower. However, it remains unclear whether these tests offer additional information that is useful for detection and management of non-muscle-invasive bladder tumors. NCCN considers this to be a 2B recommendation (i.e., Based on lower level of evidence, there is NCCN consensus that the intervention is appropriate). Miyake et al (2012) investigated the influence of hematuria on the performance of the bladder tumor antigen (BTA) tests in a clinical cohort and in an experimental model. Urine samples from a cohort of 126 subjects (64 with BCa and 62 controls) were analyzed by ELISA for hemoglobin and BTA. The experimental model involved the spiking of urine with blood from the same subject, and hemoglobin, red blood cell count, and BTA levels (BTA stat and BTA-TRAK). BTA-TRAK analyses were also performed on serum samples obtained from 40 subjects (20 with confirmed with BCa). In the 126 subject cohort, correlation between hemoglobin and BTA was Of the 64 BCa samples, 72 % had a positive BTA assay, but 47 % of controls were also positive. The sensitivity and specificity of BTA to detect BCa was 72 and 53 %, respectively. Hematuria, measured by urinary hemoglobin, was a better indicator of BCa with 75 % sensitivity and 90 % specificity. Spiking of BTA-negative urine samples with as little as 1 μl/10 ml was enough to produce a positive BTA test. High levels of BTA were found equally in the serum of subjects with or without BCa (mean BTA levels 355,159 vs. 332,329 U/ml, respectively). Investigators concluded rather than detecting a bladder tumor antigen, urinary BTA assays may be measuring serum cfh introduced by bleeding, a common presenting factor in BCa subjects. The Tumor Markers for Cancer Nov 15 43

44 presence of hematuria in subjects without malignant disease can result in falsepositive BTA assays. Lüdecke et al (2012) tested the suitability of three point of care (POC) test systems, UBC rapid, NMP22 BladderChek and BTA stat, available on the market, with respect to interference due to blood contamination in urine samples. Urine samples were obtained from voluntary asymptomatic individuals without a history of bladder cancer. A specimen negative in all test systems was selected for further study. This sample was treated with fresh heparinized blood in a 1:10 ratio and then titrated in a dilution series. All the urine samples and their consecutive test results were photographed and a urinalysis was performed on each sample. In none of the samples of the dilution series did UBC rapid or NMP22 BladderChek show a falsepositive result due to blood contamination. In contrast, with the BTA stat testing system, false-positive results were obtained from all samples with macrohaematuria and with densities up to 150 erythrocytes/μl, indicating a suspected tumor, whereas the sample was actually proven to be tumor free. Investigators concluded for the primary diagnosis of bladder carcinoma, neither the UBC rapid nor the NMP22 BladderChek POC test systems are sensitive to the presence of blood in the urine, whereas BTA stat consistently yields false-positive results due to cross-reactivity to macrohaematuria and microhaematuria up to a density of 150 erythrocytes/μl, thus this system should not be employed for this examination. Todenhöfer et al (2013) investigated whether combinations of 4 of the most broadly available tests [cytology, FISH, ucyt+, and nuclear matrix protein 22 (NMP22- ELISA)] may improve their diagnostic performance. The study was comprised of 808 patients who were suspected of having urothelial carcinoma (UC). All patients underwent urethrocystoscopy and upper urinary tract imaging and, in the case of positive findings, transurethral resection/biopsy. FISH, ucyt+, cytology, and NMP22- ELISA were performed in all patients. UC was diagnosed in 115 patients (14.2%). Cytology and FISH were found to be the single tests with the best overall performance (area under the curve [AUC], 0.78/0.79). Combinations of 2, 3, and 4 markers were found to increase the AUC to various extents compared with the use of single markers. Combining cytology and FISH improved the sensitivity and performance (AUC, 0.83) compared with the single tests and identified 12 tumors that were not detected by cytology alone. The percentage of WHO grade 3/carcinoma in situ tumors not detected by cytology was reduced by 62.5% when FISH was performed in cytology-negative patients. The addition of ucyt+ as a third test further improved performance (AUC, 0.86), whereas the addition of NMP22- ELISA was not found to have any additional influence on the performance of the test combination. Investigators concluded the results of the current study support the combined use of urine markers and may form the basis of further studies investigating whether risk stratification based on urine marker combinations may individualize diagnostic algorithms and the surveillance of patients suspected of having UC. Hatzichristodoulou et al (2012) compared nuclear matrix protein 22 expression by BladderChek and ELISA, as urine-based assays for bladder cancer (BC) detection. Urine samples of 100 BC patients and 100 controls were analyzed. Comparative statistical evaluations were based on sensitivity and specificity. Seventy-one patients had primary and 29 recurrent BC. The sensitivity of BladderChek was significantly higher compared to ELISA in the overall cancer cohort and in patients with primary BC (p< and p=0.0001, respectively). Both tests demonstrated significant correlation of sensitivities and tumor stage/grade for the overall cancer cohort and for patients with primary BC. Both tests had specificity values of 100% in healthy individuals. Specificity was 93% for BladderChek and 99% for ELISA in patients with benign diseases (p=0.048). Investigators concluded BladderChek may Tumor Markers for Cancer Nov 15 44

45 be clinically more useful for BC detection. Due to high specificity, BladderChek could be used for high-risk screening. However, due to its low sensitivity, BladderChek cannot replace but only complement cystoscopy for BC detection. Schlake et al (2012) compared the performance of NMP-22, urinary cytology and office cystoscopy when utilized in a urology practice for 1 year. A total of 391 consecutive office cystoscopy procedures performed over 1 year were included in the study. NMP-22 and cytology were performed on the urine specimens of patients presenting for cystoscopy. Tumor resection/bladder biopsy was performed when cystoscopy, NMP-22 or urinary cytology were abnormal. Cystoscopy, NMP-22, and urinary cytology data were available in 351 encounters and 69 tumor resections were performed. Urothelial carcinoma bladder (UCB) was identified in 37 bladder specimens. NMP-22, urinary cytology and cystoscopy demonstrated sensitivity and specificity of (51%/96%), (35%/97%), and (92%/88%), respectively. Investigators concluded the study demonstrates cystoscopy was the most sensitive test in the diagnosis of UCB. NMP-22 had a higher sensitivity than urinary cytology and similar specificity to cytology. The authors concluded additional urinary marker testing has a limited role in the management of bladder cancer in the office setting. When adjunct testing is desired in the diagnosis and surveillance of bladder cancer, NMP-22 is a cost effective alternative to urinary cytology. Dimashkieh et al (2013) evaluated the effectiveness of multiprobe FISH and urine cytology in detecting urothelial cell carcinoma (UC) in the same urine sample. In total, 1835 cases with the following criteria were selected: valid results from both the multiprobe FISH assay and urine cytology in the same urine sample, histologic and/or cystoscopic follow-up within 4 months of the original tests, or at least 3 years of clinical follow-up information. The results of FISH and cytology were correlated with clinical outcomes derived from a combination of histologic, cystoscopic, and clinical follow-up information. Of 1835 cases, 1045 cases were from patients undergoing surveillance of recurrent UC, and 790 were for hematuria. The overall sensitivity, specificity, positive predictive value, and negative predictive value in detecting UCC were 61.9%, 89.7%, 53.9%, and 92.4%, respectively, for FISH and 29.1%, 96.9%, 64.4%, and 87.5%, respectively, for cytology. The performance of both FISH and cytology generally was better in the surveillance population and in samples with high-grade UC. In 95 of 296 cases with atypical cytology that were proven to have UCC, 61 cases, mostly high-grade UC, were positive using the multiprobe FISH assay. Investigators concluded the UroVysion multiprobe FISH assay was more sensitive than urine cytology in detecting UC, but it produced more false-positive results. The current data suggest that the use of FISH as a reflex test after an equivocal cytologic diagnosis may play an effective role in detecting UC. Comploj et al (2013) reported the results of 7422 ucyt+/immunocyt and cytology analyses that were performed over the course of 9 years at the study institution for the evaluation and follow-up of patients with urothelial carcinoma. Between January 2002 and March 2011, 2217 patients with a mean age of 69.5 years (range, 15 years-99 years) were enrolled in the study. All patients seen for the follow-up of bladder and/or upper tract urothelial cancer as well as those with a history that was suspicious for bladder cancer were recruited. In all patients, a voided urinary cytology and ucyt+/immunocyt test was performed. Patients underwent routine cystoscopy as well as cystoscopy when cytology and/or the ucyt+/immunocyt test yielded positive results. Lesions that were detected cystoscopically were biopsied and removed transurethrally. A total of 7422 ucyt+/immunocyt and cytology analyses were performed. Of the 7422 ucyt+/immunocyt tests and cytologies that were performed, 7075 (95.3%) were considered adequate. A total of 578 patients (with 1156 analyses) underwent biopsy and 728 (63%) samples had a histologically proven urothelial carcinoma. Overall sensitivity was 34.5% for cytology, 68.1% for Tumor Markers for Cancer Nov 15 45

46 ucyt+/immunocyt, and 72.8% for the 2 tests combined. Overall specificity was 97.9% for cytology, 72.3% for ucyt+/immunocyt, and 71.9% for the 2 tests combined. Cytology and the ucyt+/immunocyt test together had an overall sensitivity of 72.8%, with 59% for grade 1, 77% for grade 2, and 90% for grade 3 tumors (according to the 1973 World Health Organization grading classification system). Investigators concluded on the basis of their 9-year experience, the value of ucyt+/immunocyt and cytology analyses in the follow-up of patients with nonmuscle-invasive urothelial cancer is confirmed. This could potentially reduce the number and cost of routine cystoscopic examinations in patients who are followed for bladder carcinoma. Breast Cancer Per the NCCN, breast cancer is the most common malignancy in women in the U.S. and is second only to lung cancer as a cause of cancer death. Routine pathologic evaluation remains the most critical element in determining the prognosis of patients with breast cancer. Among the most potent prognostic factors available are lymph node status, tumor size and histologic grade, histologic tumor type, and lymphatic vascular invasion. Assay of hormone receptors has become a routine part of the evaluation of breast cancers, since the results predict the clinical response to hormone therapy, both in the adjuvant setting and for those with metastatic disease. Along with ER and PR, the determination of HER2 tumor status for all newly diagnosed invasive breast cancers and for first recurrences of breast cancer whenever possible if previously unknown or negative. Hormone receptor expression should be used to guide endocrine therapy decisions. HER2 expression should be used to select patients for whom HER2-directed therapy use is appropriate in the metastatic and adjuvant setting. (Refer to Medical Policy HER2neu ) Gene expression profiling has identified molecular signatures, such as the 21-gene recurrence score [Oncotype Dx, the Amsterdam 70-gene prognostic profile (Mammaprint), and the Rotterdam/Veridex 76-gene signature] are used in addition to conventional prognostic indicators in their ability to predict breast cancer outcome and response to treatment. (Refer to Medical Policy, Oncotype DX for Cancer and MammaPrint for additional information.) Monitoring of patients symptoms and cancer burden during treatment of metastatic breast disease is important to determine whether the treatment is providing benefit and that the patient does not have toxicity from an ineffective therapy. Increasing tumor markers (CEA, CA 15-3 and CA 27.29) are findings that are concerns for progression of disease, but may also be seen in the setting of responding disease. An isolated increase in tumor markers should rarely be used to declare progression of disease. Changes in bone lesions are often difficult to assess on plain or crosssectional radiology or on bone scan. For these reasons, patient symptoms and serum tumor markers may be more helpful in patients with bone-dominant metastatic disease. Hepatocellular Carcinoma (HCC) Hepatocellular carcinoma (HCC) is a highly lethal carcinoma. Risk factors for developing HCC include viral infections caused by Hepatitis B virus (HBV) and/or hepatitis C virus (HCV), alcoholic cirrhosis, and rarely inherited errors of metabolism. Diagnostic testing used to diagnose HCC include imaging, biopsy and AFP serology. Serum AFP has long been used as a marker for HCC, however, it is not a sensitive or specific diagnostic test for HCC, as values > 400ng/ml which are considered diagnostic of HCC, are observed only in a small percentage of individuals. AFP can be Tumor Markers for Cancer Nov 15 46

47 useful in conjunction with other testing to guide management of individuals for whom a diagnosis of HCC is suspected. Prostate Cancer NCCN reports that a fine balance exists between maximizing early detection of potentially curable cancers and minimizing the cost, anxiety and treatment burden associated with diagnosis of indolent tumors. NCCN states their recommendations on Prostate cancer are not population screening guidelines but rather a set of recommendations to aid men who have chosen to engage in early detection of prostate cancer. When the first recommendations for early detection programs for prostate cancer were made, serum total PSA was the only PSA-based test available. Subsequent years have seen the development of a series of PSA derivatives that are possible useful in increasing specificity and decreasing unnecessary biopsies. Many commercially available serum total PSA testing are currently available. They perform comparably, however, the levels are not interchangeable since they standardized against two different standards. An abnormal PSA result should be confirmed by retesting. Studies have shown that a PSA level above 4ng/ml increases the chance of detecting prostate cancer at prostate biopsy to nearly 30%-35%. Large programs for the early detection of prostate cancer have shown that nearly 70% of cancer cases can be detected using a PSA cutoff level of 4ng/ml in the first four years. Overall, appropriate use of PSA alone can provide a diagnostic lead time of nearly 5-10 years compared with digital rectal exam (DRE). Unbound or free PSA (fpsa) expressed as a ratio of total PSA has emerged as a clinically useful molecular form of PSA, with the potential to provide improvements in early detection, staging, and monitoring of prostate cancer. Most clinical work investigating the use of molecular forms of PSA for early detection of prostate cancer has focused on the percentage of PSA found circulating in the free or unbound form. Numerous studies have shown that the percentage of fpsa is significantly lower in men who have prostate cancer compared to men who have not. The U.S. Food and Drug Administration (FDA) approved the use of percent-free PSA for the early detection of prostate cancer in men with PSA levels between 4-10 ng/ml. Since the FDA approval, testing for percent free PSA has gained widespread clinical acceptance in the U.S., specifically for patients with normal DRE s who have previously undergone prostate biopsy because they had a total PSA level within the diagnostic grey zone. NCCN guidelines recommend the use of the percent-free PSA as an alternative in the management of patients with normal DREs and total PSA levels between 4-10ng/mL if there is a relative contraindication to biopsy. Physicians and patients electing to use percent-free PSA should be cautioned that this assay and the multi-institutional study performed to obtain its FDA approval were designed with the intention of avoiding unnecessary biopsies in men with a high likelihood of not having prostate cancer. Afirma Thyroid FNA Analysis The evaluation of thyroid nodules, typically includes a cytopathological examination of a biopsy specimen obtained by fine-needle aspiration (FNA), however, approximately 20% to 25% are classified as indeterminate. Evaluation of patients with indeterminate results on FNA often includes surgical resection of the nodule or the entire thyroid gland. Most patients will prove to be benign after evaluation of the Tumor Markers for Cancer Nov 15 47

48 surgical thyroid tissue specimen. In an effort to help resolve the diagnostic ambiguity of indeterminate thyroid nodules, recent studies have sought to identify genetic or genomic markers that may aid in differentiating benign and malignant thyroid tumors. The Afirma Thyroid FNA Analysis (Veracyte Inc.) is a recently developed test that examines the expression of genes known to be involved in cell growth, in order to identify indeterminate thyroid nodules with expression patterns characteristic of benign thyroid lesions. In the analysis, a proprietary gene expression classifier (GEC) is used to categorize nodules as either benign or suspicious. According to Veracyte Inc., the primary goal of this assay is to identify patients who likely have benign thyroid masses so that they may avoid unnecessary thyroid surgery. The Afirma Thyroid FNA Analysis (Veracyte Inc.) includes a standard cytopathological evaluation of FNA specimens and an expression analysis of 142 genes in nodules with indeterminate FNA cytopathology. The molecular portion of the test, which involves an analysis of RNA extracted from thyroid nodule aspirates, utilizes the GEC to classify indeterminate nodules as either benign or suspicious. An analysis of an additional 25 genes is included in order to help classify rare tumor subtypes. The Afirma Thyroid FNA Analysis is intended for adults (21 years of age or older) with thyroid nodules at least 1 centimeter (cm) in size, who are being evaluated for the possibility of a thyroid malignancy. At this time, the data regarding the Afirma Thyroid FNA Analysis is too limited to draw meaningful conclusions about the performance and value of this assay in patients with thyroid nodules. While the available evidence suggests that the test has a high negative predictive value (NPV) and may lead to a reduction in diagnostic surgeries for benign thyroid nodules, additional evidence supporting its analytical validity, clinical validity, and clinical utility is needed. Duick et al (2012) evaluated how the Afirma gene expression classifier (AGEC) impacted the joint decision of the endocrinologist and patient to operate when FNA cytology was indeterminate, but the AGEC reading of the nodule was benign. In this cross-sectional cohort study, data were contributed retrospectively by 51 endocrinologists at 21 practice sites that had previously obtained 3 benign AGEC readings in 1cm nodules with indeterminate FNA cytology readings. Information regarding demographic data, nodule size and location, decision to operate, surgery type (hemithyroidectomy [HT] or total thyroidectomy [TT]), and reason for recommending surgery was retrospectively collected. Compared to a 74% previous historical rate of surgery for cytologically indeterminate nodules, the operative rate fell to 7.6% during the period that AGEC were obtained in the clinical practices, a highly significant reduction in the decision to operate (p<0.001). The rate of surgery on cytologically indeterminate nodules that were benign by the AGEC reading did not differ from the historically reported rate of operation on cytologically benign nodules (p=0.41). The four primary reasons reported by the physicians for operating on nodules with a benign AGEC reading, in descending order: large nodule size (46.4%), symptomatic nodules (25.0%), rapidly growing nodules (10.7%), or a second suspicious or malignant nodule in the same patient (10.7%). These reasons are concordant with those typically given for operation on cytologically benign nodules. Investigators concluded in a substantial group of medical practices, obtaining an AGEC test in patients with cytologically indeterminate nodules was associated with a striking reduction in the rate of diagnostic thyroidectomy. Approximately, one surgery was avoided for every two AGEC tests run on thyroid FNAs with indeterminate cytology. In a study funded by Veracyte, Alexander et al (2012) performed a 19-month, prospective, multicenter validation study involving 49 clinical sites, 3789 patients, and 4812 fine-needle aspirates from thyroid nodules 1 cm or larger that required Tumor Markers for Cancer Nov 15 48

49 evaluation. The investigators obtained 577 cytologically indeterminate aspirates, 413 of which had corresponding histopathological specimens from excised lesions. Results of a central, blinded histopathological review served as the reference standard. After inclusion criteria were met, a gene-expression classifier was used to test 265 indeterminate nodules in this analysis, and its performance was assessed. Of the 265 indeterminate nodules, 85 were malignant. The gene-expression classifier correctly identified 78 of the 85 nodules as suspicious (92% sensitivity; 95% confidence interval [CI], 84 to 97), with a specificity of 52% (95% CI, 44 to 59). The negative predictive values for "atypia (or follicular lesion) of undetermined clinical significance," "follicular neoplasm or lesion suspicious for follicular neoplasm," or "suspicious cytologic findings" were 95%, 94%, and 85%, respectively. Analysis of 7 aspirates with false negative results revealed that 6 had a paucity of thyroid follicular cells, suggesting insufficient sampling of the nodule. Investigators concluded the data suggest consideration of a more conservative approach for most patients with thyroid nodules that are cytologically indeterminate on fine-needle aspiration and benign according to gene-expression classifier results. Chudova et al (2010) set out to develop a molecular test that distinguishes benign and malignant thyroid nodules using fine-needle aspirates (FNA). Investigators used mrna expression analysis to measure more than 247,186 transcripts in 315 thyroid nodules, comprising multiple subtypes. The data set consisted of 178 retrospective surgical tissues and 137 prospectively collected FNA samples. Two classifiers were trained separately on surgical tissues and FNAs. The performance was evaluated using an independent set of 48 prospective FNA samples, which included 50% with indeterminate cytopathology. Performance of the tissue-trained classifier was markedly lower in FNAs than in tissue. Exploratory analysis pointed to differences in cellular heterogeneity between tissues and FNAs as the likely cause. The classifier trained on FNA samples resulted in increased performance, estimated using both 30- fold cross-validation and an independent test set. On the test set, negative predictive value and specificity were estimated to be 96 and 84%, respectively, suggesting clinical utility in the management of patients considering surgery. Using in silico and in vitro mixing experiments, investigators demonstrated that even in the presence of 80% dilution with benign background, the classifier can correctly recognize malignancy in the majority of FNA samples. Investigators concluded the FNA-trained classifier was able to classify an independent set of FNAs in which substantial RNA degradation had occurred and in the presence of blood. High tolerance to dilution makes the classifier useful in routine clinical settings where sampling error may be a concern. An ongoing multicenter clinical trial will allow us to validate molecular test performance on a larger independent test set of prospectively collected thyroid FNAs. Target Now Molecular Profiling Test According to manufacturer s website, (Caris life Sciences) the Target Now Molecular Profiling Test is designed to evaluate the tumors of patients who have exhausted standard-of care-therapies in order to determine potential treatment options based on biomarkers identified within the tumor. Target Now uses formalin-fixed, paraffinembedded (FFPE) and/or frozen tumor specimens and a combination of immunohistochemistry (IHC), gene expression microarray, and targeted gene variant analysis, to establish a molecular profile of the tumor. This information is then used as the basis of a literature review to recommend potential therapies. The Target Now test is available as either Target Now Select, which focuses the analysis on five cancer types [non-small cell lung cancer (NSCLC), breast, colorectal, melanoma, and ovarian surface epithelial] or Target Now Comprehensive, which is a broader molecular profile. Von Hoff et al (2010) describing the use of the test describes molecular profiling in 86 patients with refractory metastatic cancer. Both FFPE and fresh frozen tissue specimens were required for the analysis. A molecular target was detected in 84 (98%) patients. Of these patients, 66 (78.6%) were treated according Tumor Markers for Cancer Nov 15 49

50 to the molecular profile results, with 18 (27%) having a progression-free survival (PFS) ratio (defined as PFS on molecular profile selected therapy divided by PFS on prior therapy) of 1.3 (95% confidence interval, 17% to 38%; P=0.007). The authors interpreted these results to suggest that using a molecular profile to guide cancer therapy resulted in a longer PFS than prior therapy in a significant proportion of patients. However, given the small study population and the fact that these results have not been replicated in an independent dataset, there is currently insufficient evidence to evaluate the use of the Target Now Molecular Profiling Test in guiding treatment decisions in cancer patients. ColoNext The ColoNext test (Ambry Genetics) is a next-generation sequencing panel that tests for variants in 14 genes that have been associated with hereditary colorectal cancer (CRC), including the genes that cause Lynch syndrome (MLH1, MSH2, MSH6, and PMS2) and the gene that causes familial adenomatous polyposis (FAP) (APC). While testing these genes may be appropriate in individuals with clinical or family histories that suggest a specific syndrome, there is no evidence that mass screening of all 14 genes in individuals suspected of having or being at risk for a hereditary CRC syndrome has an impact on clinical outcomes. Therefore, it is currently not possible to assess the use of the ColoNext test in the care of such individuals. DecisionDx-GBM The DecisionDx-GBM test (Castle Biosciences Inc.) is a multigene expression assay that is designed to predict which patients are likely to experience long-term (> 2 years) progression-free survival (PFS). According to the manufacturer s website, GBM specimens from four datasets were used to identify an initial set of 200 genes that were differentially expressed in long-term survivors. From these, 38 genes that were robustly associated with survival were validated in an independent dataset and a final group of 9 genes was identified. These 9 genes are: aquaporin 1 (AQP1), chitinase 3-like 1 (CHI3L1), epithelial membrane protein 3 (EMP-3), glycoprotein NMB (GPNMB), insulin-like growth factor binding protein 2 (IGFBP2), galectin 3 (LGALS3), oligodendrocyte lineage transcription factor 2 (OLIG2), podoplanin (PDPN), and reticulon 1 (RTN1). Three control genes were also identified: eukaryotic translation elongation factor 1, alpha-1 (EEF1A1); beta-glucoronidase (GUSB); and ribosomal protein S27 (RPS27). The 9 genes and 3 control genes were then validated in a third dataset. Expression of the 9 genes was found through multivariate analysis to be an independent predictor of PFS (Cox hazard ratio [HR], 2.7; P=0.0003) and overall survival (Cox HR, 2.7; P=0.0003) compared with age, performance score, and methylation status of the methylguanine methyltransferase gene (MGMT). MGMT methylation has been previously reported to be an independent predictor of response of GBM to radiation and chemotherapy. MGMT methylation was not found to be an independent predictor of survival in the DecisionDx-GBM validation studies. There is currently insufficient evidence to evaluate the performance and clinical utility of this test in the care of patients with GBM. The NCCN does not address DecisionDx-GBM test in their guidelines on Central Nervous System Cancers. The DecisionDx-UM The DecisionDx-UM (Castle Biosciences Inc.) test is a gene expression profiling test intended for use in patients with uveal melanoma (UM). It is a 15-gene polymerase chain reaction (PCR)-based assay that stratifies UM patients into two classes based on the molecular signature of tumor tissue. There is very limited published literature investigating the DecisionDx-UM test. Long term studies, preferably prospective in nature, with large study populations are needed to clearly define molecular classes of UM and adequately assess the relationship between tumor class and metastasis. There is lack of direct evidence of clinical utility and very low evidence of analytical validity and clinical validity of the test. Tumor Markers for Cancer Nov 15 50

51 In a prospective, multicenter study, Onken et al (2012) evaluated the prognostic performance of a 15 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. Patients were managed for their primary tumor and monitored for metastasis. The 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 = ). 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. Investigators concluded the GEP assay had a high technical success rate and was the most accurate prognostic marker among all of the factors analyzed. The GEP provided a highly significant improvement in prognostic accuracy over clinical TNM classification and chromosome 3 status. Chromosome 3 status did not provide prognostic information that was independent of GEP. Direct-to-consumer (DTC) Genetic tests Direct-to-consumer (DTC) genetic tests, also known as at-home genetic tests, are marketed and sold directly to the customer without a doctor's order or a consultation with a genetic counselor. In recent years, the tests have become more accessible as many new genetic testing companies have emerged. Although direct-to-consumer genetic testing may promote awareness of genetic diseases, allow consumers to take a more proactive role in their health care, and offer a means for people to learn about their ancestral origins, these tests have significant risks and limitations. Consumers are vulnerable to being misled by the results of unproven or invalid tests. Without guidance from a healthcare provider, they may make important decisions about treatment or prevention based on inaccurate, incomplete, or misunderstood information about their health. More research is needed to fully understand the benefits and limitations of direct-toconsumer genetic testing. According to the National Cancer Institute, individuals are considered to be candidates for cancer risk assessment if they have a personal and/or family history (maternal or paternal lineage) with features suggestive of hereditary cancer. These features vary by type of cancer and specific hereditary syndrome. Genetic testing may be considered when the following factors are present: An individual's personal history (including ethnicity) and/or family history is suspicious for a genetic predisposition to cancer. The genetic test has sufficient sensitivity and specificity to be interpreted. Tumor Markers for Cancer Nov 15 51

52 The test will impact the individual's diagnosis, cancer management or cancer risk management, and/or help clarify risk in family members. A candidate for genetic testing receives genetic education and counseling before testing to facilitate informed decision making and adaptation to the risk or condition. Genetic education and counseling gives an individual time to consider the various medical uncertainties, diagnosis, or medical management based on varied test results, and the risks, benefits, and limitations of genetic testing. Review History July 2013 February 2014 April 2014 May 2014 August 2014 September 2014 February 2015 April 2015 May 2015 August 2015 September 2015 October 2015 November 2015 January 2016 Medical Advisory Council, initial approval Update. Added Molecular Intelligence as investigational. Updated Codes. Added Afirma Thyroid FNA Analysis as medically necessary for adults with thyroid nodules at least 1 centimeter in size, that are indeterminate and who are being evaluated for the possibility of a thyroid malignancy. Code updates. Update- Added IDH testing medically necessary for glioma in limited scenarios. Update Added MyRisk Hereditary Cancer genetic test as investigational due to a paucity of published peer-reviewed studies. Added Myeloma Prognostic Risk Signature (MyPRS Plus) Test for Myeloma and DecisionDx-Melanoma as investigational since there is a paucity of peer-reviewed evidence of analytical validity and clinical validity. Added Breast Cancer Index, BreastTrue High Risk Panel, the High/Moderate Risk Panel, and the igene Cancer Panel, as investigational since there is a paucity of peer reviewed studies to support them. Codes reviewed and updated. Added SYMGENE68 NGS Cancer Panel as investigational since there is a paucity of peer reviewed studies to support this. Added ResponseDX:Colon as investigational since there is a paucity of peer-reviewed literature to support this. Added TreatmentMAP, Paradigm Cancer Diagnostic (PCDx) Test and COLMOL:NGS Colon Cancer Panel as investigational. Update Added Oncotype DX Prostate Cancer Assay and Decipher Prostate Cancer Classifier to investigational section of the policy. Added Tissue of Origin (ResponseDX) to the investigational section of the policy, since there is a paucity of peerreviewed literature to support it. Added ThyGenX Thyroid Oncogene Panel/ThyraMIR ([predecessor -mirinform Thyroid) and ThyroSeq v.2 Next Generation Sequencing Panel as investigational. Added JAK2 as medically necessary for specific diagnoses Added FoundationOne Heme as investigational since there is a paucity of peer reviewed literature to support this testing. This policy is based on the following evidence-based guidelines: 1. American College of Medical Genetics. ACMG Statement on Direct-to-Consumer Genetic Testing. April Tumor Markers for Cancer Nov 15 52

53 2. Basch E, Oliver TK, Vickers A, et al. Screening for prostate cancer with prostatespecific antigen testing: American Society of Clinical Oncology Provisional Clinical Opinion. J Clin Oncol Aug 20;30(24): Available at: 3. Gilligan TD, Hayes DF, Seidenfeld J, Temin S. ASCO Clinical Practice Guideline on Uses of Serum Tumor Markers in Adult Males With Germ Cell Tumors. J Oncol Pract Jul;6(4): Available at: 4. Harris L, Fritsche H, Mennel R, et al. American Society of Clinical Oncology 2007 update of recommendations for the use of tumor markers in breast cancer. J Clin Oncol Nov 20;25(33): Available at: update-recommendations-use-tumor-markers 5. Hayes Genetic Test Overview. GTE Report. Afirma Thyroid FNA Analysis. Aug Update Feb Hayes Genetic Test Overview. GTE Synopsis. Target Now Molecular Profiling Test (Caris Life Sciences). Sept Hayes Genetic Test Overview. GTE Synopsis. ColoNext. June Hayes Genetic Test Overview. GTE Synopsis. DecisionDx-GBM. Oct Hayes Genetic Test Overview. GTE Report. DecisionDx-UM Gene Expression Assay for Risk Stratification of Patients with Uveal Melanoma. Dec Update Apr Hayes Medical Technology Directory. Ancillary ImmunoCyt/uCyt+ Testing for Bladder Cancer Screening and Detection. Sept Update Sept Hayes Medical Technology Directory. Ancillary UroVysion Fluorescence In Situ Hybridization (FISH) Testing for Bladder Cancer Screening and Detection. Jul Update Jul Locker GY, Hamilton S, Harris J, et al. ASCO 2006 update of recommendations for the use of tumor markers in gastrointestinal cancer. J Clin Oncol Nov 20;24(33): Available at: update-recommendations-use-tumor-markers-gastrointestinal-cancer 13. National Cancer Institute. Cancer Genetics Risk Assessment and Counseling. (PDQ)last modified June Available at: National Comprehensive Care Network. NCCN Clinical Practice Guidelines in Oncology. Bladder Cancer. Version National Comprehensive Care Network. NCCN Clinical Practice Guidelines in Oncology. Breast Cancer. Version Updated Version Updated Version National Comprehensive Care Network. NCCN Clinical Practice Guidelines in Oncology. Central Nervous System cancers. Version update Version National Comprehensive Care Network. NCCN Clinical Practice Guidelines in Oncology. Genetic/Familial High-Risk Assessment: Breast and Ovarian. Version Updated version National Comprehensive Care Network. NCCN Clinical Practice Guidelines in Oncology. Non-Hodgkin s Lymphomas. Version National Comprehensive Care Network. NCCN Clinical Practice Guidelines in Oncology. Neuroendocrine Tumors. Version National Comprehensive Care Network. NCCN Clinical Practice Guidelines in Oncology. Occult Primary. Version I Updated Version 1, National Comprehensive Care Network. NCCN Clinical Practice Guidelines in Oncology. Ovarian Cancer. Version Tumor Markers for Cancer Nov 15 53

54 22. National Comprehensive Care Network. NCCN Clinical Practice Guidelines in Oncology. Pancreatic Adenocarcinoma. Version Updated Version 1, Updated Version National Comprehensive Care Network. NCCN Clinical Practice Guidelines in Oncology. Prostate Cancer. Version Updated Version Updated Version Update Version National Comprehensive Care Network. NCCN Clinical Practice Guidelines in Oncology. Testicular Cancer. Version I National Comprehensive Care Network. NCCN Clinical Practice Guidelines in Oncology. Thyroid Carcinoma. Version Update National Comprehensive Care Network. NCCN Clinical Practice Guidelines in Oncology. Uterine Neoplasms. Version I Society of Gynecologic Oncology. Use of CA125 for Monitoring Ovarian Cancer. June Available at: 2/use-of-ca125-for-monitoring-ovarian-cancer/ 28. Hayes. Genetic Test Evaluation (GTE). Molecular Intelligence for Colorectal Cancer (CRC) (Caris Life Sciences). January 15, National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology. Colon Cancer. Version 2, National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology. Soft Tissue Sarcoma. Version National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology. Melanoma. Version Updated Version Updated Version Version Gharib H, Papini E, Paschke R, Duick DS, Valcavi R, Hegedus L, Vitti P, AACE/AME/ETA Task Force on Thyroid Nodules. American Association of Clinical Endocrinologists, Associazione Medici Endocrinologi, and European Thyroid Association medical guidelines for clinical practice for the diagnosis and management of thyroid nodules. Endocr Pract May-Jun;16(Suppl 1):1-43. Available at: American Thyroid Association (ATA) Guidelines Taskforce on Thyroid Nodules, Cooper DS, Doherty GM, Haugen BR, Kloos RT, Lee SL, Mandel SJ, Mazzaferri EL, McIver B, Pacini F, Schlumberger M, Sherman SI, Steward DL, Tuttle RM. Revised American Thyroid Association management guidelines for patients with thyroid nodules and differentiated thyroid cancer. Thyroid Nov;19(11): Available at: Febbo, PG, Ladanyi, M, Aldape, KD, et al. NCCN Task Force Report: Evaluating the Clinical Utility of Tumor Markers in Oncology. J Natl Compre Canc Netw. 2011;9:S1-S Hayes. GTE Overview. Myriad myrisk Hereditary Cancer, May 5, Hayes. GTE Overview. Myeloma Prognostic Risk Signature (MyPRS Plus) Test for Myeloma. July 17, Hayes. GTE Overview. DecisionDx-Melanoma. September 4, Hayes. GTE Overview. BreastTrue High Risk Panel for Hereditary Breast Cancer (Pathway Genomics Corp.) January 8, Hayes. GTE Overview. Breast Cancer Index. Breast Cancer Index for Prognosis of Breast Cancer Recurrence (biotheranostics Inc.) December 11, Hayes. GTE Overview. Oncotype DX Prostate Cancer Assay for Prostate Cancer Prognosis (Genomic Health Inc.) December 9, National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology. Prostate Cancer. Version Hayes. GTE Overview. igene Cancer Panel. June 15, Hayes. GTE Overview. High/Moderate Risk Panel for Hereditary Cancer Risk (GeneDx Inc.). January 29, Tumor Markers for Cancer Nov 15 54

55 44. Hayes. GTE Overview. Pancreatic Cancer Panel for Hereditary Pancreatic Cancer (GeneDx Inc.). January 29, Hayes. GTE Synopsis. SYMGENE68 NGS Cancer Panel. March 12, Hayes. GTE Overview. TreatmentMAP. July 30, Hayes. GTE Overview. COLMOL: NGS Colon Cancer Panel. August 6, Hayes. GTE Overview. Paradigm Cancer Diagnostic (PCDX) Test. August 27, Hayes. GTE Report. Tissue of Origin Test (ResponseDX). October 1, Hayes GTE Report. mirinform Thyroid. Apr Update Mar Hagen BR, Alexander EK, Bible KC, et al American Thyroid Association Management Guidelines for Adult Patients with Thyroid Nodules and Differentiated Thyroid Cancer. Thyroid Oct 14. Available at: Hayes GTE Synopsis. ThyroSeq. Apr Hayes. GTE Overview. FoundationOne Heme. December References Update November Bhatia P, Deniwar A, Friedlander P, et al. Diagnostic potential of ancillary molecular testing in differentiation of benign and malignant thyroid nodules. Anticancer Res Mar;35(3): Beaudenon-Huibregtse S1, Alexander EK, Guttler RB, et al. Centralized molecular testing for oncogenic gene mutations complements the local cytopathologic diagnosis of thyroid nodules. Thyroid Oct;24(10): Brauner E, Holmes BJ, Krane JF, et al. Performance of the Afirma Gene Expression Classifier in Hürthle Cell Thyroid Nodules Differs from Other Indeterminate Thyroid Nodules. Thyroid Jul;25(7): CBLPath. ThyroSeq v.2 Next Generation Sequencing. Available at: d=gtesyn.thyroseq Ferris RL, Baloch Z, Bernet V, et al. American Thyroid Association Statement on Surgical Application of Molecular Profiling for Thyroid Nodules: Current Impact on Perioperative Decision Making. Thyroid Jul;25(7): Giordano TJ, Beaudenon-Huibregtse S, Shinde R., et al. Molecular testing for oncogenic gene mutations in thyroid lesions: a case-control validation study in 413 postsurgical specimens. Human Patholology, 45(7): Labourier E, Shifrin A, Busseniers AE, et al. Molecular Testing for mirna, mrna, and DNA on Fine-Needle Aspiration Improves the Preoperative Diagnosis of Thyroid Nodules With Indeterminate Cytology. J Clin Endocrinol Metab Jul;100(7): Lastra RR, Pramick MR, Crammer CJ, et al. Implications of a suspicious afirma test result in thyroid fine-needle aspiration cytology: an institutional experience. Cancer Cytopathol Oct;122(10): Liu S, Gao A, Zhang B, et al. Assessment of molecular testing in fine-needle aspiration biopsy samples: an experience in a Chinese population. Exp Mol Pathol Oct;97(2): Marti JL, Avadhani V, Donatelli LAet al. Wide Inter-institutional Variation in Performance of a Molecular Classifier for Indeterminate Thyroid Nodules. Ann Surg Oncol Nov;22(12): McIver B, Castro MR, Morris JC, et al. An independent study of a gene expression classifier (Afirma) in the evaluation of cytologically indeterminate thyroid nodules. J Clin Endocrinol Metab Nov;99(11): Nikiforov YE, Carty SE, Chiosea SI, et al. Impact of the Multi-Gene ThyroSeq Next-Generation Sequencing Assay on Cancer Diagnosis in Thyroid Nodules with Atypia of Undetermined Significance/Follicular Lesion of Undetermined Significance Cytology. Thyroid Sep 10. Tumor Markers for Cancer Nov 15 55

56 13. Nikiforov YE, Carty SE, Chiosea S et al. Highly accurate diagnosis of cancer in thyroid nodules with follicular neoplasm/suspicious for a follicular neoplasm cytology by ThyroSeq v2 next-generation sequencing assay. Cancer Dec 1;120(23): Nishino M. Molecular cytopathology for thyroid nodules: A review of methodology and test performance. Cancer Cytopathol Sep Paskaš S, Janković J, Živaljević V Cancer. Malignant risk stratification of thyroid FNA specimens with indeterminate cytology based on molecular testing. Cancer Cytopathol Aug;123(8): Smith DL, Lamy A, Beaudenon-Huibregtse S, et al. A multiplex technology platform for the rapid analysis of clinically actionable genetic alterations and validation for BRAF p.v600e detection in 1549 cytologic and histologic specimens. Arch Pathol Lab Med Mar;138(3): Stokowy T, Wojtaś B, Krajewska J, et al. A two mirna classifier differentiates follicular thyroid carcinomas from follicular thyroid adenomas. Mol Cell Endocrinol Jan 5;399: Wei WJ, Shen CT, Song HJ, et al. MicroRNAs as a potential tool in the differential diagnosis of thyroid cancer: a systematic review and meta-analysis. Clin Endocrinol (Oxf) Dec Witt RL. Outcome of thyroid gene expression classifier testing in clinical practice. Laryngoscope Sep Wu JX, Lam R, Levin M, et al. Effect of malignancy rates on cost-effectiveness of routine gene expression classifier testing for indeterminate thyroid nodules. Surgery Oct Yang SE, Sullivan PS, Zhang J, et al. Has Afirma gene expression classifier testing refined the indeterminate thyroid category in cytology? Cancer Cytopathol Sep 30. References Update September Arsov C, Jankowiak F, Hiester A, et al. Prognostic value of a cell-cycle progression score in men with prostate cancer managed with active surveillance after MRI-guided prostate biopsy--a pilot study. Anticancer Res May;34(5): Bishoff JT, Freedland SJ, Gerber L, et al. Prognostic utility of the cell cycle progression score generated from biopsy in men treated with prostatectomy. J Urol Aug;192(2): Boström PJ, Bjartell AS, Catto JW, et al. Genomic Predictors of Outcome in Prostate Cancer. Eur Urol Apr Cooperberg MR, Davicioni E, Crisan A, et al. Combined value of validated clinical and genomic risk stratification tools for predicting prostate cancer mortality in a high-risk prostatectomy cohort. Eur Urol Feb;67(2): Cooperberg MR, Simko JP, Cowan JE, et al. Validation of a cell-cycle progression gene panel to improve risk stratification in a contemporary prostatectomy cohort. J Clin Oncol Apr 10;31(11): Crawford ED, Scholz MC, Kar AJ, et al. Cell cycle progression score and treatment decisions in prostate cancer: results from an ongoing registry. Curr Med Res Opin Jun;30(6): Cuzick J, Stone S, Fisher G, et al. Validation of an RNA cell cycle progression score for predicting death from prostate cancer in a conservatively managed needle biopsy cohort. Br J Cancer Jun Falzarano SM, Ferro M, Bollito E, et al. Novel biomarkers and genomic tests in prostate cancer: a critical analysis. Minerva Urol Nefrol Jun Freedland SJ, Gerber L, Reid J, et al. Prognostic utility of cell cycle progression score in men with prostate cancer after primary external beam radiation therapy. Int J Radiat Oncol Biol Phys Aug 1;86(5): Tumor Markers for Cancer Nov 15 56

57 10. Klein EA, Yousefi K, Haddad Z, et al. A genomic classifier improves prediction of metastatic disease within 5 years after surgery in node-negative high-risk prostate cancer patients managed by radical prostatectomy without adjuvant therapy. Eur Urol Apr;67(4): Knezevic D, Goddard AD, Natraj N, et al. Analytical validation of the Oncotype DX prostate cancer assay - a clinical RT-PCR assay optimized for prostate needle biopsies. BMC Genomics. 2013;14: Nguyen HG, Welty CJ, Cooperberg MR. Diagnostic associations of gene expression signatures in prostate cancer tissue. Curr Opin Urol Jan;25(1): Ross AE, D'Amico AV, Freedland SJ. Which, when and why? Rational use of tissue-based molecular testing in localized prostate cancer. Prostate Cancer Prostatic Dis Jun Ross AE, Johnson MH, Yousefi K, et al Tissue-based Genomics Augments Postprostatectomy Risk Stratification in a Natural History Cohort of Intermediateand High-Risk Men. Eur Urol Jun Ross AE, Feng FY, Ghadessi M, et al. A genomic classifier predicting metastatic disease progression in men with biochemical recurrence after prostatectomy. Prostate Cancer Prostatic Dis Mar;17(1): Sartori DA, Chan DW. Biomarkers in prostate cancer: what's new? Curr Opin Oncol May;26(3): Shore N, Concepcion R, Saltzstein D, et al. Clinical utility of a biopsy-based cell cycle gene expression assay in localized prostate cancer. Curr Med Res Opin Apr;30(4): Sommariva S, Tarricone R, Lazzeri M, et al. Prognostic Value of the Cell Cycle Progression Score in Patients with Prostate Cancer: A Systematic Review and Meta-analysis. Eur Urol Dec 4. References Update February Klein EA, Cooperberg MR, Magi-Galluzzi C, et al. A 17-gene Assay to Predict Prostate Cancer Aggressiveness in the Context of Gleason Grade Heterogeneity, Tumor Multifocality, and Biopsy Undersampling. Eur. Urol. 2014;66(3): Knezevic D, Goddard AD, Natraj N, et al. Analytical validation of the Oncotype DX prostate cancer assay - a clinical RT-PCR assay optimized for prostate needle biopsies. BMC Genomics. 2013;14:690. References Update September Dhillon, et al. Gene expression profile signature (DecisionDx-Melanoma) to predict visceral metastatic risk in patients with Stage I and Stage II cutaneous melanoma. J Clin Oncol 2012; Kumar SK, Uno H, Jacobus SJ, et al. Impact of gene expression profiling-based risk stratification in patients with myeloma receiving initial therapy with lenalidomide and dexamethasone. Blood. 2011; 118(16): Nair B, Shaughnessy JD Jr, Zhou Y, et al. Gene expression profiling of plasma cells at myeloma relapse from tandem transplantation trial Total Therapy 2 predicts subsequent survival. Blood. 2009; 113(26): Shaughnessy JD Jr, Zhan F, Burington BE, et al. A validated gene expression model of high-risk multiple myeloma is defined by deregulated expression of genes mapping to chromosome 1. Blood. 2007b; 109(6): References Update August Myriad. MyRisk Hereditary Cancer Available at: References Update May 2014 Tumor Markers for Cancer Nov 15 57

58 1. Ahmadi R, Stockhammer F, Becker N, et al. No prognostic value of IDH1 mutations in a series of 100 WHO grade II astrocytomas. J Neurooncol Aug;109(1): Beiko J, Suki D, Hess KR, et al. IDH1 mutant malignant astrocytomas are more amenable to surgical resection and have a survival benefit associated with maximal surgical resection. Neuro Oncol Jan;16(1): Cheng HB, Yue W, Xie C, et al. IDH1 mutation is associated with improved overall survival in patients with glioblastoma: a meta-analysis. Tumour Biol Dec;34(6): Hartmann C, Hentschel B, Wick W, et al. Patients with IDH1 wild type anaplastic astrocytomas exhibit worse prognosis than IDH1-mutated glioblastomas, and IDH1 mutation status accounts for the unfavorable prognostic effect of higher age: implications for classification of gliomas. Acta Neuropathol Dec;120(6): Hartmann C, Hentschel B, Simon M, et al. Long-term survival in primary glioblastoma with versus without isocitrate dehydrogenase mutations. Clin Cancer Res Sep 15;19(18): Hartmann C, Meyer J, Balss J, et al. Type and frequency of IDH1 and IDH2 mutations are related to astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse gliomas. Acta Neuropathol Oct;118(4): Horbinski C, Kofler J, Kelly LM, et al. Diagnostic use of IDH1/2 mutation analysis in routine clinical testing of formalin-fixed, paraffin-embedded glioma tissues. J. Neuropathol Exp Neurol Dec;68(12): Houillier C, Wang X, Kaloshi G, et al..idh1 or IDH2 mutations predict longer survival and response to temozolomide in low-grade gliomas. Neurology Oct 26;75(17): Killela PJ, Pirozzi CJ, Healy P, et al. Mutations in IDH1, IDH2, and in the TERT promoter define clinically distinct subgroups of adult malignant gliomas. Oncotarget Jan Nobusawa S, Watanabe T, Kleihues P, Ohgaki H..IDH1 mutations as molecular signature and predictive factor of secondary glioblastomas. Clin Cancer Res Oct 1;15(19): Ohno M, Narita Y, Miyakita Y, et al. Histopathological malignant progression of grade II and III gliomas correlated with IDH1/2 mutation status. Brain Tumor Pathol Oct;29(4): Okita Y, Narita Y, Miyakita Y, et al. IDH1/2 mutation is a prognostic marker for survival and predicts response to chemotherapy for grade II gliomas concomitantly treated with radiation therapy. Int J Oncol Oct;41(4): Preusser M, Bent Mv. Clinical neuropathology practice news : immunohistochemistry pins IDH in glioma - molecular testing procedures under scrutiny. Clin Neuropathol Mar-Apr;32(2):82-3. Clin Neuropathol Sep-Oct;30(5): Preusser M, Capper D, Hartmann C; Euro-CNS Research Committee. IDH testing in diagnostic neuropathology: review and practical guideline article invited by the Euro-CNS research committee. Clin Neuropathol Sep-Oct;30(5): Taylor JW, Chi AS, Cahill DP. Tailored therapy in diffuse gliomas: using molecular classifiers to optimize clinical management. Oncology (Williston Park) Jun;27(6): Takano S, Tian W, Matsuda M, et al. Detection of IDH1 mutation in human gliomas: comparison of immunohistochemistry and sequencing. Brain Tumor Pathol Apr;28(2): Xie F, Tang JJ, Wang X, et al. Correlation between IDH1 mutation and prognosis in supratentorial high-grade astrocytomas. Sichuan Da Xue Xue Bao Yi Xue Ban Mar;44(2):184-7, 192. Tumor Markers for Cancer Nov 15 58

59 18. Yan H, Parsons DW, Jin G, et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med Feb 19;360(8): References Update April Alexander EK, Schorr M, Klopper J, et al. Multicenter clinical experience with the Afirma gene expression classifier. J Clin Endocrinol Metab Jan;99(1): Dedhia PH, Rubio GA, Cohen MS, et al. Potential effects of molecular testing of indeterminate thyroid nodule fine needle aspiration biopsy on thyroidectomy volume. World J Surg Mar;38(3): Gomberawalla A, Elaraj DM. How to use molecular testing results to guide surgery: a surgeon's perspective. Curr Opin Oncol Jan;26(1): Harrell RM, Bimston DN. Surgical Utility of Afirma: Effects of High Cancer Prevalence and Oncocytic Cell Types in Patients with Indeterminate Thyroid Cytology. Endocr Pract Nov 18:1-16. References Updated February Caris Molecular Intelligence. Frequently asked questions for Physicians. Available at: 2. Kim, et al. The BATTLE trial: personalizing therapy for lung cancer. Cancer Discov Jun;1(1): Epub 2011 Jun Tsimberidou AM, et al. Clin Cancer Res Sep Von Hoff, et al. Pilot study using molecular profiling of patients tumors to find potential targets and select treatments for their refractory cancers. J Clin Oncol Nov 20;28(33): Epub 2010 Oct 4. References Initial 1. Ahn JS, Kim HS, Chang SG, Jeon SH. The clinical usefulness of nuclear matrix protein-22 in patients with atypical urine cytology. Korean J Urol Sep;52(9): Alexander EK, Kennedy GC, Baloch ZW, et al. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. N Engl J Med. 2012;367(8): Ali SZ Md, Fish SA Md, Lanman R Md, et al. Use of the Afirma Gene Expression Classifier for Preoperative Identification of Benign Thyroid Nodules with Indeterminate Fine Needle Aspiration Cytopathology. PLoS Curr Feb 11;5. 4. Allingham-Hawkins D, Lea A, Levine S. DecisionDx-GBM Gene Expression Assay for Prognostic Testing in Glioblastoma Multiform. PLoS Curr Oct 12 [revised 2010 Nov 26];2:RRN Barbieri CE, Cha EK, Chromecki TF, et al. Decision curve analysis assessing the clinical benefit of NMP22 in the detection of bladder cancer: secondary analysis of a prospective trial. BJU Int Mar;109(5): Bonberg N, Taeger D, Gawrych K, et al. Chromosomal instability and bladder cancer: the UroVysion(TM) test in the UroScreen study. BJU Int Jan Budman LI, Kassouf W, Steinberg JR. Biomarkers for detection and surveillance of bladder cancer. Can Urol Assoc J Jun;2(3): Chudova D, Wilde JI, Wang ET, et al. Molecular classification of thyroid nodules using high-dimensionality genomic data. J Clin Endocrinol Metab. 2010;95(12): Cook SA, Damato B, Marshall E, Salmon P. Psychological aspects of cytogenetic testing of uveal melanoma: preliminary findings and directions for future research. Eye (Lond). 2009;23(3): Comploj E, Mian C, Ambrosini-Spaltro A, Dechet C, et al. ucyt+/immunocyt and cytology in the detection of urothelial carcinoma: An update on 7422 analyses. Cancer Cytopathol Mar 12. Tumor Markers for Cancer Nov 15 59

60 11. Couturier J, Saule S. Genetic determinants of uveal melanoma. Dev Ophthalmol. 2012;49: Dimashkieh H, Wolff DJ, Smith TM, et al. Evaluation of urovysion and cytology for bladder cancer detection: A study of 1835 paired urine samples with clinical and histologic correlation. Cancer Cytopathol Jun Duick DS, Klopper JP, Diggans JC, et al. The impact of benign gene expression classifier test results on the endocrinologist-patient decision to operate on patients with thyroid nodules with indeterminate fine-needle aspiration cytopathology. Thyroid Oct;22(10): Feil G, Stenzl A. Tumor marker tests in bladder cancer. Actas Urol Esp Jan;30(1): Gill HS, Char DH. Uveal melanoma prognostication: from lesion size and cell type to molecular class. Can J Ophthalmol Jun;47(3): Harbour JW. Molecular prognostic testing and individualized patient care in uveal melanoma. Am J Ophthalmol. 2009;148(6): Harbour JW. Genomic, prognostic, and cell-signaling advances in uveal melanoma. Am Soc Clin Oncol Educ Book. 2013;2013: Harbour JW, Chen R. The DecisionDx-UM Gene Expression Profile Test Provides Risk Stratification and Individualized Patient Care in Uveal Melanoma. PLoS Curr Apr 9;5. pii: ecurrents.eogt.af8ba80fc776c8f1ce8f5dc485d4a Hatzichristodoulou G, Kübler H, Schwaibold H, et al. Nuclear matrix protein 22 for bladder cancer detection: comparative analysis of the BladderChek and ELISA. Anticancer Res Nov;32(11): Hosseini J, Golshan AR, Mazloomfard MM, et al. Detection of recurrent bladder cancer: NMP22 test or urine cytology? Urol J Winter;9(1): Hwang EC, Choi HS, Jung SI, Kwon, et al. Use of the NMP22 BladderChek test in the diagnosis and follow-up of urothelial cancer: a cross-sectional study. Urology Jan;77(1): Huber S, Schwentner C, Taeger D, et al. Nuclear matrix protein-22: a prospective evaluation in a population at risk for bladder cancer. Results from the UroScreen study. BJU Int Sep;110(5): Kehinde EO, Al-Mulla F, Kapila K, Anim JT. Comparison of the sensitivity and specificity of urine cytology, urinary nuclear matrix protein-22 and multitarget fluorescence in situ hybridization assay in the detection of bladder cancer. Scand J Urol Nephrol Mar;45(2): Kloos RT, Reynolds JD, Walsh PS, et al. Does addition of BRAF V600E mutation testing modify sensitivity or specificity of the Afirma Gene Expression Classifier in cytologically indeterminate thyroid nodules? J Clin Endocrinol Metab Apr;98(4):E Lotan Y, Shariat SF, Schmitz-Dräger BJ, et al. Considerations on implementing diagnostic markers into clinical decision making in bladder cancer. Urol Oncol Jul-Aug;28(4): Lüdecke G, Pilatz A, Hauptmann A, et al. Comparative analysis of sensitivity to blood in the urine for urine-based point-of-care assays (UBC rapid, NMP22 BladderChek and BTA-stat) in primary diagnosis of bladder carcinoma. Interference of blood on the results of urine-based POC tests. Anticancer Res May;32(5): Malkhasian KA, Petrov SV, Ul'ianin MIu, et al. Use of fluorescent in situ hybridization in the cytogenetic diagnosis of urinary bladder urothelial carcinoma. Vopr Onkol. 2011;57(4): Miyake M, Goodison S, Rizwani W, et al. Urinary BTA: indicator of bladder cancer or of hematuria. World J Urol Dec;30(6): Onken MD, Worley LA, Char DH, et al. Collaborative Ocular Oncology Group report number 1: prospective validation of a multi-gene prognostic assay in uveal melanoma. Ophthalmology Aug;119(8): Tumor Markers for Cancer Nov 15 60

61 30. Onken MD, Worley LA, Dávila RM, et al. Prognostic testing in uveal melanoma by transcriptomic profiling of fine needle biopsy specimens. J Mol Diagn. 2006a;8(5): Onken MD, Worley LA, Ehlers JP, Harbour JW. Gene expression profiling in uveal melanoma reveals two molecular classes and predicts metastatic death. Cancer Res. 2004;64(20): Onken MD, Worley LA, Harbour JW. Association between gene expression profile, proliferation and metastasis in uveal melanoma. Curr Eye Res. 2010a;35(9): Onken MD, Worley LA, Tuscan MD, Harbour JW. An accurate, clinically feasible multi-gene expression assay for predicting metastasis in uveal melanoma. J Mol Diagn. 2010b;12(4): O'Sullivan P, Sharples K, Dalphin M, et al. A multigene urine test for the detection and stratification of bladder cancer in patients presenting with hematuria. J Urol Sep;188(3): Raitanen MP; FinnBladder Group. The role of BTA stat Test in follow-up of patients with bladder cancer: results from FinnBladder studies. World J Urol Feb;26(1): Schlake A, Crispen PL, Cap AP, et al. NMP-22, urinary cytology, and cystoscopy: a 1 year comparison study. Can J Urol Aug;19(4): Seo S, Cho S, Hong K, Shim B, Kwon S. Usefulness of NMP22 BladderChek for the diagnosis and monitoring of bladder cancer. Korean J Lab Med Feb;27(1): Srivastava R, Arora VK, Aggarwal S, et al. Cytokeratin-20 immunocytochemistry in voided urine cytology and its comparison with nuclear matrix protein-22 and urine cytology in the detection of urothelial carcinoma. Diagn Cytopathol Sep;40(9): Shariat SF, Savage C, Chromecki TF, et al. Assessing the clinical benefit of nuclear matrix protein 22 in the surveillance of patients with nonmuscle-invasive bladder cancer and negative cytology: a decision-curve analysis. Cancer Jul 1;117(13): Todenhöfer T, Hennenlotter J, Esser M, et al. Combined application of cytology and molecular urine markers to improve the detection of urothelial carcinoma. Cancer Cytopathol May;121(5): Von Hoff DD, Stephenson JJ Jr, Rosen P, et al. Pilot study using molecular profiling of patients' tumors to find potential targets and select treatments for their refractory cancers. J Clin Oncol Nov 20;28(33): Ward LS, Kloos RT. Molecular markers in the diagnosis of thyroid nodules. Arq Bras Endocrinol Metabol Mar;57(2): Important Notice General Purpose. Health Net's National Medical Policies (the "Policies") are developed to assist Health Net in administering plan benefits and determining whether a particular procedure, drug, service or supply is medically necessary. The Policies are based upon a review of the available clinical information including clinical outcome studies in the peer-reviewed published medical literature, regulatory status of the drug or device, evidence-based guidelines of governmental bodies, and evidence-based guidelines and positions of select national health professional organizations. Coverage determinations are made on a case-by-case basis and are subject to all of the terms, conditions, limitations, and exclusions of the member's contract, including medical necessity requirements. Health Net may use the Policies to determine whether under the facts and circumstances of a particular case, the proposed procedure, drug, service or supply is medically necessary. The conclusion that a procedure, drug, service or supply is medically necessary does not constitute coverage. The member's contract defines which procedure, drug, service or supply is covered, excluded, limited, or subject to dollar caps. The policy provides for clearly written, reasonable and current criteria that have been approved by Health Net s National Medical Advisory Council (MAC). The clinical criteria and medical policies provide guidelines for determining the medical necessity criteria for specific procedures, equipment, and services. In order to be eligible, all services must be medically necessary and otherwise defined in the member's benefits contract as described this "Important Notice" disclaimer. In all cases, final benefit determinations are based on the applicable contract language. To the extent there are Tumor Markers for Cancer Nov 15 61

62 any conflicts between medical policy guidelines and applicable contract language, the contract language prevails. Medical policy is not intended to override the policy that defines the member s benefits, nor is it intended to dictate to providers how to practice medicine. Policy Effective Date and Defined Terms. The date of posting is not the effective date of the Policy. The Policy is effective as of the date determined by Health Net. All policies are subject to applicable legal and regulatory mandates and requirements for prior notification. If there is a discrepancy between the policy effective date and legal mandates and regulatory requirements, the requirements of law and regulation shall govern. * In some states, prior notice or posting on the website is required before a policy is deemed effective. For information regarding the effective dates of Policies, contact your provider representative. The Policies do not include definitions. All terms are defined by Health Net. For information regarding the definitions of terms used in the Policies, contact your provider representative. Policy Amendment without Notice. Health Net reserves the right to amend the Policies without notice to providers or Members. In some states, prior notice or website posting is required before an amendment is deemed effective. No Medical Advice. The Policies do not constitute medical advice. Health Net does not provide or recommend treatment to members. Members should consult with their treating physician in connection with diagnosis and treatment decisions. No Authorization or Guarantee of Coverage. The Policies do not constitute authorization or guarantee of coverage of particular procedure, drug, service or supply. Members and providers should refer to the Member contract to determine if exclusions, limitations, and dollar caps apply to a particular procedure, drug, service or supply. Policy Limitation: Member s Contract Controls Coverage Determinations. Statutory Notice to Members: The materials provided to you are guidelines used by this plan to authorize, modify, or deny care for persons with similar illnesses or conditions. Specific care and treatment may vary depending on individual need and the benefits covered under your contract. The determination of coverage for a particular procedure, drug, service or supply is not based upon the Policies, but rather is subject to the facts of the individual clinical case, terms and conditions of the member s contract, and requirements of applicable laws and regulations. The contract language contains specific terms and conditions, including pre-existing conditions, limitations, exclusions, benefit maximums, eligibility, and other relevant terms and conditions of coverage. In the event the Member s contract (also known as the benefit contract, coverage document, or evidence of coverage) conflicts with the Policies, the Member s contract shall govern. The Policies do not replace or amend the Member s contract. Policy Limitation: Legal and Regulatory Mandates and Requirements The determinations of coverage for a particular procedure, drug, service or supply is subject to applicable legal and regulatory mandates and requirements. If there is a discrepancy between the Policies and legal mandates and regulatory requirements, the requirements of law and regulation shall govern. Reconstructive Surgery CA Health and Safety Code requires health care service plans to cover reconstructive surgery. Reconstructive surgery means surgery performed to correct or repair abnormal structures of the body caused by congenital defects, developmental abnormalities, trauma, infection, tumors, or disease to do either of the following: (1) To improve function or (2) To create a normal appearance, to the extent possible. Reconstructive surgery does not mean cosmetic surgery," which is surgery performed to alter or reshape normal structures of the body in order to improve appearance. Requests for reconstructive surgery may be denied, if the proposed procedure offers only a minimal improvement in the appearance of the enrollee, in accordance with the standard of care as practiced by physicians specializing in reconstructive surgery. Reconstructive Surgery after Mastectomy California Health and Safety Code requires treatment for breast cancer to cover prosthetic devices or reconstructive surgery to restore and achieve symmetry for the patient incident to a mastectomy. Coverage for prosthetic devices and reconstructive surgery shall be subject to the co-payment, or deductible and coinsurance conditions, that are applicable to the mastectomy and all other terms and conditions applicable to other benefits. "Mastectomy" means the removal of all or part of the breast for medically necessary reasons, as determined by a licensed physician and surgeon. Tumor Markers for Cancer Nov 15 62

63 Policy Limitations: Medicare and Medicaid Policies specifically developed to assist Health Net in administering Medicare or Medicaid plan benefits and determining coverage for a particular procedure, drug, service or supply for Medicare or Medicaid members shall not be construed to apply to any other Health Net plans and members. The Policies shall not be interpreted to limit the benefits afforded Medicare and Medicaid members by law and regulation. Tumor Markers for Cancer Nov 15 63