National Medical Policy

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1 National Medical Policy Subject: Policy Number: MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer NMP520 Effective Date*: May 2013 Updated: June 2017 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), citation(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 National Coverage Determination (NCD) National Coverage Manual Citation X Local Coverage Determination (LCD)* Molecular Diagnostic Tests (MDT): MolDX: Breast Cancer Assay: Prosigna X Article (Local)* Molecular Diagnostic Tests (MDT). MolDX: MammaPrint Billing and Coding Guidelines: x Other Palmetto GBA. MOL DX. MammaPrint Billing and Coding Guidelines Update. 4/11/14: sf/docscat/moldx%20website~moldx~browse %20By%20Topic~Covered%20Tests~8SBH3U4 732?open&navmenu=Browse%5EBy%5ETopic% 7C%7C%7C%7C None Use Health Net Policy Instructions Medicare NCDs and National Coverage Manuals apply to ALL Medicare members in ALL regions. MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer Jun 17 1

2 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 use of the MammaPrint assay or Prosigna assay for the prognosis of breast cancer recurrence investigational. Although these assays may offer prognostic information beyond that offered by standard tools, the clinical utility has not been defined and there lacks evidence demonstrating that the use of these assays would influence clinical decision making regarding adjuvant therapy and/or improved outcomes. Other Assays of Genetic Expression Profiling Health Net, Inc. considers all of the following assays of genetic expression profiling as a technique of managing the treatment of breast cancer, including but not limited to the following as not medically necessary since there is insufficient evidence to determine whether they are better than conventional risk assessment tools in predicting breast cancer recurrence: 1. Breast Cancer Gene Expression Ratio (i.e. 2-gene ratio, H/I or HOXB13/IL17BR Aviara H/ISM) 2. Rotterdam Signature 76-Panel 3. The 41-gene signature assay 4. The Mammostrat 5. FoundationOne 6. OncoVue Definitions AOL Adjuvant! Online AST Adjuvant systemic treatment HR Hazard ratio DDFS Distant disease-free survival BCSS Breast cancer specific survival HER2+ Human epidermal growth factor receptor 2 ER Estrogen receptor MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer Jun 17 2

3 LN BC GLR GHL ACT DRFI DMFS DM IHC FISH PR CT ET Lymph node Breast cancer Genomic low risk Genomic high risk Adjuvant chemotherapy Distant-recurrence-free-interval Distant Metastasis Free Survival Distant metastasis Immunohistochemistry Fluorescence in situ hybridization Progesterone receptor Chemotherapy Endocrine therapy 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 Malignant neoplasm of female breast Malignant neoplasm of male breast V86.0 Estrogen receptor positive status [ER+] V86.1 Estrogen receptor negative status [ER-] ICD-10 Codes for use on and after October 2015 C C Malignant neoplasm of breast Z17.0 Estrogen receptor positive status [ER+] Z85.3 Personal history of malignant neoplasm of breast CPT Codes Oncology (breast), mrna, gene expression profiling by realtime RT-PCR of 21 genes, utilizing formalin-fixed paraffin embedded tissue, algorithm reported as recurrence score Unlisted chemistry procedure Unlisted cytogenetic study HCPCS Codes S3854 Gene expression profiling panel for use in the management breast cancer treatment Scientific Rationale (Alphabetical list) MammaPrint (Initial Review) MammaPrint (Agendia Inc.), also referred to as the Amsterdam signature and the 70-gene assay, is a qualitative in vitro diagnostic test service, performed in a single laboratory, using the gene expression profile of fresh breast cancer tissue samples to assess a patient's risk for distant metastasis. According to Agendia, MammaPrint MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer Jun 17 3

4 identifies which early-stage breast cancer patients are at risk of distant recurrence following surgery, independent of Estrogen Receptor status and any prior treatment. The MammaPrint assay uses a microarray technology platform, performed in a single laboratory, to analyze the expression of 70 genes from tumor samples that are fresh frozen or placed in an RNA molecular fixative solution provided in a kit from the manufacturer. The 70 genes included in the assay were selected using a combination of unsupervised and supervised classification of the correlations between tumor outcomes and genes that resulted in an optimal classification of the tumors into good and poor prognosis groups at 5 years of follow-up. A correlation classification threshold of 0.4 was chosen by limiting the false-negative rate to 10%. Patients with values > 0.4 are classified as having a good prognosis with a low risk of distant cancer recurrence, and those with correlation values 0.4 are considered at high risk with a poor prognosis. In the United States, the test is performed for breast cancer patients, with Stage I or Stage 1I disease, with tumor size < 5.0 cm and who are lymph node negative. When the test was first introduced, there was a requirement that patients are < 61 years of age; however, that requirement has now been removed from the Food and Drug Administration (FDA) labeling. Update: May 2015 Drucker et al. (2014) completed a study on 1,053 breast cancer patients primarily treated with breast-conserving treatment or mastectomy. Adjuvant treatment consisted of radiotherapy, chemotherapy, and/or endocrine therapy as indicated by guidelines used at the time. All patients were included in various 70-gene signature validation studies. After a median follow-up of 8.96 years with 87 LRRs, patients with a high-risk 70-gene signature (n = 492) had an LRR risk of 12.6 % (95 % CI ) at 10 years, compared to 6.1 % (95 % CI ) for low-risk patients (n = 561; P < 0.001). Adjusting the 70-gene signature in a competing risk model for the clinicopathological factors such as age, tumor size, grade, hormone receptor status, LVI, axillary lymph node involvement, surgical treatment, endocrine treatment, and chemotherapy resulted in a multivariable HR of 1.73 (95 % CI ; P = 0.042). Adding the signature to the model based on clinicopathological factors improved the discrimination, albeit non-significantly [C-index through 10 years changed from (95 % CI ) to (95 % CI )]. Calibration of the prognostic models was excellent. The 70-gene signature is an independent prognostic factor for LRR. A significantly lower local recurrence risk was seen in patients with a low-risk 70-gene signature compared to those with high-risk 70-gene signature. In 2014, Drukker et al. completed an additional study and reported additional comparisons between MammaPrint and clinical risk classifiers in RASTER patients. As measured by ROC analyses, MammaPrint improved prognostic performance of all 6 clinical classifiers studied. However, area under the ROC curve for the best performing combination, MammaPrint and PREDICT Plus, was only Among MammaPrint low-risk patients, 5-year survival estimates for patients classified as low or high risk by clinical risk classifiers were similar; only PREDICT Plus included the possibility of a less than 10% survival estimate for high risk patients. Survival estimates for untreated patients classified as high risk by MammaPrint were not reported, limiting full comparison of these risk stratifiers. Sapino et al. (2014) completed a study that described method optimization, validation, and performance of MammaPrint using analyte from FFPE tissue. Laboratory procedures for enabling the assay to be run on FFPE tissue were determined using 157 samples, and the assay was established using 125 matched FFPE and fresh tissues. Validation of MammaPrint-FFPE, compared with MammaPrintfresh, was performed on an independent series of matched tissue from five hospitals MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer Jun 17 4

5 (n = 211). Reproducibility, repeatability, and precision of the FFPE assay (n = 87) was established for duplicate analysis of the same tumor, interlaboratory performance, 20-day repeat experiments, and repeated analyses over 12 months. FFPE sample processing had a success rate of 97%. The MammaPrint assay using FFPE analyte demonstrated an overall equivalence of 91.5% (95% confidence interval, 86.9% to 94.5%) between the 211 independent matched FFPE and fresh tumor samples. Precision was 97.3%, and repeatability was 97.8%, with highly reproducible results between replicate samples of the same tumor and between two laboratories (concordance, 96%). Thus, with 580 tumor samples, MammaPrint was successfully translated to FFPE tissue. The assay has high precision and reproducibility, and FFPE results are substantially equivalent to results derived from fresh tissue. At this time, NCCN (Breast Cancer, Version ), ASCO and CTAF do not recommend MammaPrint as an option when evaluating breast cancer patients for risk of recurrence. On February 19, 2014, the BlueCross BlueShield Association Technology Evaluation Center Medical Advisory Panel voted that the use of MammaPrint to determine recurrence risk in women with unilateral, hormone receptor-positive, lymph nodenegative breast cancer who will receive hormonal therapy does not meet TEC criteria. Update: May 2014 Specimen requirements for the FDA-cleared MammaPrint assay, using a microarray technology platform to analyze the expression of 70 genes, are from a fresh tumor specimen that is placed in RNA molecular fixative solution, provided in a kit from Agendia Inc., or a fresh frozen tumor sample. The main limitation of the FDA cleared MammaPrint test is the requirement for fresh tumor samples. In the U.S., tumor samples are more commonly stored as FFPE samples, which do not have FDA clearance at this time. Agendia now accepts formalin-fixed paraffin-embedded (FFPE) specimens for analysis as well as fresh frozen samples. However, a detailed description of the MammaPrint assay methodology using FFPE specimens is not available on the manufacturer website or in published studies. Formalin-fixed paraffin-embedded (FFPE) tumor tissue cannot be used for the FDA-cleared version of the MammaPrint assay. The National Comprehensive Cancer Network (NCCN, Version3.2014) Guidelines on Breast Cancer notes: MammPrint is approved by the FDA to assist in assignment of women with ER-positive or ER-negative breast cancer into a high versus low risk for recurrence, but not for predicting benefit from adjuvant system therapy. Studies using MammaPrint as a prognostic and predictive tool are small and/or or retrospective in nature. Per the National Institute for Health and Care Excellence (NICE, September 2013): MammaPrint, IHC4 and Mammostrat are only recommended for use in research in people with ER+, LN and HER2 early breast cancer, to collect evidence about potentially important clinical outcomes and to determine the ability of the tests to predict the benefit of chemotherapy. The tests are not recommended for general use in these people because of MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer Jun 17 5

6 uncertainty about their overall clinical benefit and consequently their cost effectiveness. Clinical Trials noted within the Initial Scientific Rationale, including PROMIS, and the MINDACT trial, are still ongoing. Although there are peer-reviewed studies on MammaPrint, there are low quality data to show it has clinical utility. An ongoing international study, the Microarray in Node- Negative Disease May Avoid Chemotherapy (MINDACT) trial, is noted in the Initial Scientific Rationale. Until the results of this study are available, definition of the clinical utility of the MammaPrint assay remains limited. It has not been shown in studies or clinical trials up to this point, that MammaPrint improves outcomes beyond what is currently available through clinical, histological and pathological means. Initial: May 2013 The use of the MammaPrint gene expression assay is not yet included in the 2013 Breast Cancer guidelines from the National Comprehensive Care Network (NCCN). Per the NCCN guidelines (version ): While many of the DNA microarray technologies are able to stratify patients into prognostic and/or predictive subsets on retrospective analysis, the gene subsets differ from study to study, and prospective clinical trials testing the utility of these techniques have yet to be reported. Currently, prospective randomized clinical trials are addressing the use of Oncotype DX and MammaPrint as predictive and/or prognostic tools in populations of women with early-stage, lymph node-negative breast cancer. Pending the results of the prospective trials, the Panel considers the 21 gene-gene RT-PCR assay as an option when evaluating patients with primary tumors characterized as 0.6 to 1.0 cm with unfavorable features or > 1 cm, and node-negative, hormonereceptor positive, and HER2-negative (category 2A; ie, based on lower-level evidence, there is uniform NCCN consensus that the intervention is appropriate). In this circumstance, the recurrence score may be determined to assist in estimating likelihood of recurrence and benefit from chemotherapy. The panel emphasizes that the recurrence score should be used for decision-making only in the context of other elements of risk stratification for an individual patient. Unplanned, retrospective subset analysis from a single randomized clinical trial in post-menopausal, ALNpositive, ER-positive breast cancer found that the 21-gene RT-PCR assay may provide predictive information for chemotherapy benefit in addition to tamoxifen. Patients with a high score in the study benefited from chemotherapy, whereas patients with a low score did not appear to benefit from the addition of chemotherapy regardless of the number of positive lymph nodes. Patient selection for assay use remains controversial. They noted further, The MINDACT trial is underway in Europe to compare the 70- gene signature with the commonly used clinical-pathologic criteria in selecting patients for adjuvant chemotherapy in breast cancer with 0-3 positive nodes. The findings from this trial will help determine the prognostic value of MammaPrint and the benefit of treating intermediate-risk patients with adjuvant chemotherapy. The California Technology Assessment Forum, The 70-Gene Signature (MammaPrint) As a Guide for the Management of Early Stage Breast Cancer (June 2010) concluded, MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer Jun 17 6

7 The majority of breast cancers in the United States are diagnosed at an early stage. Significant improvements in long-term outcomes for women with breast cancer have been achieved by targeting therapy based on the results of tests that predict response to therapy (hormone receptor status for tamoxifen and aromatase inhibitors; HER2 status for trastuzumab). Many women with early stage tumors receive no benefit from chemotherapy, but suffer all of its side effects. Current guidelines and risk assessment tools recommend that 80 to 90% of these women be offered chemotherapy, but fewer than 50% will benefit. The primary clinical goal of the current gene expression profile tools is to improve risk stratification of women with early stage breast cancers in order to more precisely individualize use of chemotherapy. Gene expression profiling describes several technologies that quantify the relative expression of mrna levels for many genes. Patterns of gene expression can be used to differentiate one tumor type from another and to separate tumors likely to be associated with a good prognosis from those with a poor prognosis. The MammaPrint test is the commercial version of the 70-gene prognostic signature developed at the NKI in Amsterdam. It was designed to predict five year rates of distant metastases in younger women with N0. The interpretation of the results from the initial validation study in 295 patients was clouded by the inclusion of patients used to develop the signature in the group of patients used to validate the signature, the inclusion of a large number of women with lymph node positive breast cancer (49%), and the use of a different array platform than the commercial test. A larger validation study of 307 patients with lymph node negative disease < 5 cm in diameter was the first to use the commercial microarray. Multiple retrospective cohorts have demonstrated that the 70-gene prognostic signature performed better than standard guidelines and risk assessment tools at predicting recurrent disease, but the magnitude of the association with the poor prognostic signature decreases with length of follow-up. Recent studies have confirmed that the 70-gene prognostic signature adds prognostic information in older women and in women with lymph node negative or up to three positive lymph nodes. However, it remains unclear if the 70-gene prognostic signature can predict response to chemotherapy. One study pooled retrospective data from multiple cohorts to evaluate this question. The study was underpowered to evaluate the response to chemotherapy in the low risk group and it was based on observational data rather than randomized data, so the results may not be accurate. That study reported that chemotherapy produced non-significant, but potentially clinically important 50% to 70% reductions in breast cancer recurrence in the low risk group that was approximately equivalent to the reductions observed in the high risk group. Giving chemotherapy to low risk women by the 70-gene prognostic signature lowered their risk of dying from breast cancer within five years from 3% to 1%. The difference in mortality was small, but many women would choose chemotherapy for this reduction in their risk of dying. These data are in contrast with that for the 21-gene recurrence score which has two randomized trials demonstrating a statistically significant interaction between the recurrence score and chemotherapy. Women with low recurrence scores experienced no improvements in outcomes when they received chemotherapy. In summary, a large number of patients have been evaluated with the commercial version of the 70-gene prognostic signature and the test clearly offers prognostic information beyond that offered by standard tools. However it remains unclear how the 70-gene prognostic signature should be used in managing patients with early stage breast cancer. A large, randomized clinical trial (MINDACT) enrolling 6000 patients in Europe will test the hypothesis that use of the 70-gene prognostic score can improve outcomes for women with early stage breast cancer. MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer Jun 17 7

8 The CTAF recommended that the use of the 70-gene prognostic signature (MammaPrint) does not meet technology assessment criterion 3 (i.e., technology must improve the net health outcomes); criterion 4 (i.e., technology must be as beneficial as any established alternatives) and criterion 5 (i.e., improvement must be attainable outside the investigational setting). The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group recommendations (2009) found insufficient evidence to make a recommendation for or against the use of tumor gene expression profiles to improve outcomes in defined populations of women with breast cancer. For one test, the EWG found preliminary evidence of potential benefit of testing results to some women who face decisions about treatment options (reduced adverse events due to low risk women avoiding chemotherapy), but could not rule out the potential for harm for others (breast cancer recurrence that might have been prevented). The evidence is insufficient to assess the balance of benefits and harms of the proposed uses of the tests. The EWG encourages further development and evaluation of these technologies. With regard to clinical utility, The EGAPP reported: No direct evidence links use of the MammaPrint test to clinical outcomes. No studies evaluated MammaPrint for ability to predict benefit from other treatments (e.g., chemotherapy). No studies determined whether use of MammaPrint in place of, or in addition to, current clinicopathologic markers (e.g., Adjuvant! Online, St. Gallen) changes management, and/or improves outcomes based on management with clinicopathologic markers alone. In the MicroarRAy PrognoSTics in Breast CancER (RASTER) study, Bueno-de- Mesquita et al (2008) sought to assess prospectively the feasibility of implementation of the 70-gene prognosis signature in community-based settings and its effect on adjuvant systemic treatment decisions when considered with treatment advice formulated from the Dutch Institute for Healthcare Improvement (CBO) and other guidelines. Between January, 2004 and December, 2006, 812 women aged less than 61 years with primary breast carcinoma (clinical T1-4N0M0) were enrolled. Fresh tumor samples were collected in 16 hospitals in the Netherlands within 1 h after surgery. Clinicopathological factors were collected and microarray analysis was done with a custom-designed array chip that assessed the mrna expression index of the 70 genes previously identified for the prognostic signature. Patients with a "good" signature were deemed to have a good prognosis and, therefore, could be spared adjuvant systemic treatment with its associated adverse effects, whereas patients with a "poor" signature were judged to have a poor prognosis and should be considered for adjuvant systemic treatment. Concordance between risk predicted by the prognosis signature and risk predicted by commonly used clinicopathological guidelines (ie, St Gallen guidelines, Nottingham Prognostic Index, and Adjuvant! Online) was assessed. Of 585 eligible patients, 158 patients were excluded because of sampling failure (n=128) and incorrect procedure (n=30). Prognosis signatures were assessed in 427 patients. The 70-gene prognosis signature identified 219 (51%) patients with good prognosis and 208 (49%) patients with poor prognosis. The Dutch CBO guidelines identified 184 patients (43%) with poor prognosis, which was discordant with those findings obtained with the prognosis signature in 128 (30%) patients. Oncologists recommended adjuvant treatment in 203 (48%) patients based on Dutch CBO guidelines, in 265 (62%) patients if the guidelines were used with the prognosis signature, and in 259 (61%) patients if Dutch CBO guidelines, prognosis signature, and patients' preferences for treatment were all taken into account. Adjuvant! Online guidelines identified more patients with poor prognosis than did the signature alone (294 [69%]), and discordance with the MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer Jun 17 8

9 signature occurred in 160 (37%) patients. St Gallen guidelines identified 353 (83%) patients with poor prognosis with the signature and discordance in 168 (39%) patients. Nottingham Prognostic Index recorded 179 (42%) patients with poor prognosis with the signature and discordance in 117 (27%) patients. The investigators concluded the use of the prognosis signature is feasible in Dutch community hospitals. Adjuvant systemic treatment was advised less often when the more restrictive Dutch CBO guidelines were used compared with that finally given after use of the prognosis signature. For the other guidelines assessed, less adjuvant chemotherapy would be given when the data based on prognosis signature alone are used, which might spare patients from adverse effects and confirms previous findings. The investigators noted that future studies should assess whether use of the prognosis signature could improve survival or equal survival while avoiding unnecessary adjuvant systemic treatment without affecting patients' survival, and further assess the factors that physicians use to recommend adjuvant systemic treatment. Drukker et al (2013) reported five year follow up results of the microarrayprognostics-in-breast-cancer (RASTER) study noted above, designed to prospectively evaluate the performance of the 70-gene signature, of which results were available for 427 patients (ct1-3n0m0). Adjuvant systemic treatment decisions were based on the Dutch CBO 2004 guidelines, the 70-gene signature and doctors and patients preferences. Five-year distant-recurrence-free-interval (DRFI) probabilities were compared between subgroups based on the 70-gene signature and Adjuvant! Online (AOL) (10-year survival probability <90% was defined as highrisk). Median follow-up was 61.6 months. Fifteen percent (33/219) of the 70-gene signature low-risk patients received adjuvant chemotherapy (ACT) versus 81% (169/208) of the 70-gene signature high-risk patients. The 5-year DRFI probabilities for 70-gene signature low-risk (n=219) and high-risk (n=208) patients were 97.0% and 91.7%. The 5-year DRFI probabilities for AOL low-risk (n=132) and high-risk (n=295) patients were 96.7% and 93.4%. For 70-gene signature low-risk-aol highrisk patients (n=124), of whom 76% (n=94) had not received ACT, 5-year DRFI was 98.4%. In the AOL high-risk group, 32% (94/295) less patients would be eligible to receive ACT if the 70-gene signature was used. The authors concluded, in this prospective community-based observational study, the 5-year follow up data confirmed the additional prognostic value of the 70-gene signature to clinicopathologic factors used in the AOL risk estimations. Omission of adjuvant chemotherapy as judged appropriate by doctors and patients and supported by a low-risk 70-gene signature result, appeared not to compromise outcome. The authors noted that limitations of this study included that treatment decisions in the study were based on restrictive Dutch guidelines of 2004 along with patient and doctor s preference; and reported 5 year outcome results whereas AOL risk estimations are based on 10 year outcomes. They noted the prognostic capacity of the 70 gene signature is best at a follow-up time of five years and has less discriminatory power in years They noted further, the data presented is immature regarding the effect of adjuvant endocrine therapy on long term outcomes and needs to be re-evaluated at 10 years of follow-up. Saghatchian et al (2013) reported the MammaPrint is a prognostic profile of distant recurrence and survival of primary breast cancer (BC). BC patients with 4-9 positive nodes (LN 4-9) are considered clinically at high-risk. The investigators sought to determine if MammaPrint added prognostic value in this group. MammaPrint profiles were generated from frozen tumors of patients operated from primary BC. Samples were classified as genomic Low Risk (GLR) or genomic High Risk (GHR). Among the 173 samples, 70 (40%) were classified as GLR and 103 (60%) as GHR. Tumors in the GHR group were significantly more often ductal carcinomas (93%), grade 3 (60%), estrogen and progesterone-negative, Her2 positive (25%). In the GLR MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer Jun 17 9

10 category, the 5-year overall survival was 97% vs. 76% for in the GHR group (p < 0.01); Distant Metastasis Free Survival (DMFS) at 5 years was 87% for GLR patients and 63% for GHR patients (p < 0.01). In the Luminal A subgroup, the genomic profile was the only independent risk factor for distant metastasis (DM) and BC specific death. Investigators concluded in the Luminal A subgroup, MammaPrint is an independent prognostic marker in BC patients with LN 4-9 and may be integrated in a selection strategy of patient candidate for more aggressive therapeutic approaches Azim et al (2013) critically evaluated the available evidence on genomic tests in breast cancer to define their prognostic ability and likelihood to determine treatment benefit. Independent evaluation of six genomic tests (Oncotype Dx, MammaPrint, Genomic Grade Index, PAM50 (ROR-S), Breast Cancer Index, and EndoPredict) was carried out by a panel of experts in three parameters: analytical validity, clinical validity, and clinical utility based on the principles of the EGAPP criteria. The majority of the working group members found the available evidence on the analytical and clinical validity of Oncotype Dx and MammaPrint to be convincing. None of the genomic tests demonstrated robust evidence of clinical utility: it was not clear from the current evidence that modifying treatment decisions based on the results of a given genomic test could result in improving clinical outcome. Investigators concluded the IMPAKT 2012 Working Group proposed the following recommendations: (i) a need to develop models that integrate clinicopathologic factors along with genomic tests; (ii) demonstration of clinical utility should be made in the context of a prospective randomized trial; and (iii) the creation of registries for patients who are subjected to genomic testing in the daily practice. Nguyen et al (2012) compared breast cancer subtyping with the three centrally assessed microarray-based assays BluePrint, MammaPrint, and TargetPrint with locally assessed clinical subtyping using immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). BluePrint, MammaPrint, and TargetPrint were all performed on fresh tumor samples. Microarray analysis was performed at Agendia Laboratories, blinded for clinical and pathological data. IHC/FISH assessments were performed according to local practice at each institution; estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) assessments were performed on 132 samples, and Ki-67 on 79 samples. The concordance between BluePrint and IHC/FISH subtyping was 94 % for the Luminal-type, 95% for the HER2-type, and 94% for the Basal-type subgroups. The concordance of BluePrint with subtyping using mrna single gene readout (TargetPrint) was 96% for the Luminal-type, 97% for the HER2-type, and 98% for the Basal-type subgroups. The concordance for substratification into Luminal A and B using MammaPrint and Ki-67 was 68%. The concordance between TargetPrint and IHC/FISH was 97% for ER, 80% for PR, and 95% for HER2. Investigators concluded the implementation of multigene assays such as TargetPrint, BluePrint, and MammaPrint may improve the clinical management of breast cancer patients. High discordance between Luminal A and B substratification based on MammaPrint versus locally assessed Ki-67 or grade indicates that chemotherapy decisions should not be based on the basis of Ki-67 readout or tumor grade alone. TargetPrint serves as a second opinion for those local pathology settings where high-quality standardization is harder to maintain. Kok et al (2012) reported breast cancer patients with node positive disease can have an excellent outcome with tamoxifen only. It is unclear whether analyzing both the 70-gene signature and hormone receptors provides superior prediction of outcome in tamoxifen-treated patients than either alone. Three series were evaluated: 121 patients (81% node positive) received adjuvant tamoxifen, 151 patients did not receive tamoxifen (10% node positive) and 92 patients received tamoxifen for MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer Jun 17 10

11 metastatic disease. The 70-gene signature was analyzed using MammaPrint. estrogen receptor (ER) and progesterone receptor (PR) immunohistochemistry was evaluated following St. Gallen Consensus (Highly Endocrine Responsive: ER and PR 50%, Incompletely Endocrine Responsive: ER and/or PR low or either one absent). In patients treated with adjuvant tamoxifen, both the 70-gene signature (adjusted for Endocrine Response Categories HR 2.17, 95%CI ) as well as the Endocrine Response Categories (adjusted for 70-gene signature HR 6.35, 95%CI ) were associated with breast-cancer-specific-survival (BCSS). Also in patients treated with tamoxifen for metastatic disease, combined analysis of the 70- gene signature and ER/PR revealed additional value (multivariate Cox regression, p = 0.013). In patients who did not receive tamoxifen, only the 70-gene signature was associated with outcome. Investigators concluded in the series analyzed, the 70- gene signature was mainly a prognostic factor, while ER and PR levels were mainly associated with outcome after tamoxifen. Combination of these three factors may improve outcome prediction in tamoxifen-treated patients. Hartmann et al (2012) evaluated the prognostic value of the MammaPrint signature in women 60 years with invasive breast cancer. 60 female patients were included in this prospective study. Eligibility criteria included: pt1c-3, pn0-1a, grade 2/3, hormone receptor-positive and HER2-negative tumor. The clinical risk was determined by Adjuvant! Online (AOL), 38 patients (63%) were considered to be low-risk patients by the 70-gene signature, while 22 (37%) were considered to be high-risk patients. No statistically significant differences between low- and high-risk groups could be detected for conventional prognostic parameters, particularly not for Ki-67. By AOL, 33 patients (55%) were considered to be at high risk, of which 20 had a discordant MammaPrint result. The discordance rate between the profile and AOL was 48%, which is higher than in previous publications. When the 70-gene signature was used in combination with the clinical risk assessment, the recommendation for adjuvant systemic treatment differed in 11 patients (18%). Investigators concluded in the intermediate-risk subgroup, the 70-gene signature could be useful to decide in elderly patients whether they may benefit from adjuvant chemotherapy or not. Conventional clinicopathological factors were not suitable for a prediction of the 70-gene signature results in these patients. Prat et al (2012) evaluated the ability of six clinically relevant genomic signatures to predict relapse in patients with ER-positive (ER+) tumors treated with adjuvant tamoxifen only. ER-positive (ER+) breast cancer includes all of the intrinsic molecular subtypes, although the luminal A and B subtypes predominate. Four microarray datasets were combined and research-based versions of PAM50 intrinsic subtyping and risk of relapse (PAM50-ROR) score, 21-gene recurrence score (Oncotype DX), MammaPrint, Rotterdam 76 gene, index of sensitivity to endocrine therapy (SET) and an estrogen-induced gene set were evaluated. Distant relapsefree survival (DRFS) was estimated by Kaplan-Meier and log-rank tests, and multivariable analyses were done using Cox regression analysis. Harrell's C-index was also used to estimate performance. All signatures were prognostic in patients with ER+ node-negative tumors, whereas most were prognostic in ER+ node-positive disease. Among the signatures evaluated, PAM50-ROR, Oncotype DX, MammaPrint and SET were consistently found to be independent predictors of relapse. A combination of all signatures significantly increased the performance prediction. Importantly, low-risk tumors (>90% DRFS at 8.5 years) were identified by the majority of signatures only within node-negative disease, and these tumors were mostly luminal A (78%-100%). Investigators concluded most established genomic signatures were successful in outcome predictions in ER+ breast cancer and provided statistically independent information. From a clinical perspective, multiple signatures combined together most accurately predicted outcome, but a common finding was that each signature identified a subset of luminal A patients with node-negative MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer Jun 17 11

12 disease who might be considered suitable candidates for adjuvant endocrine therapy alone. The European Organisation for Research and Treatment of Cancer, is sponsoring the MINDACT (Microarray In Node negative Disease may Avoid ChemoTherapy) trial, a multicentre, prospective, phase III randomized study comparing the 70-gene expression signature with a common clinical-pathological prognostic tool (Adjuvant! Online) in selecting patients for adjuvant chemotherapy in node-negative breast cancer. The study is ongoing, but not recruiting participants. The estimated primary completion date is March Rutgers et al (2011) presented the results of the pilot phase consisting of first 800 patients included. MINDACT has enrolled 6600 patients, classified into high or low risk by MammaPrint and clinicopathological risk through Adjuvant! Online. Patients with both clinical (C) and genomic (G) high risks are offered adjuvant CT; those with both C and G low risks do not receive CT; patients with discordant risk are randomized for the decision of adjuvant CT based on C or G risk. CT randomization of anthracycline-based versus docetaxel/capecitabine and endocrine therapy randomization between letrozole and tamoxifen letrozole are offered. During the pilot phase 46% of screened patients were enrolled. Main reasons for non-enrolment were node positivity before trial amendment, sample quality problems and failure to meet logistic settings. Among the 800 patients, 386 (48%) were C-low/G-low, 198 (24.8%) as C-high/G-high, 75 (9.4%) as C-low/G-high and 141 (17.6%) as C-high/G-low. In total 216 (27%) cases were discordant. The difference between patients with C-high (42%) and G-high risk (34%) is 8.25% (95% confidence interval (CI), %; P<.0001). Compliance with the treatment decision was high (>92%). The authors concluded the logistically complex MINDACT trial is feasible in a multinational setting. The proportion of discordant patients, the potential reduction in CT by using the genomic signature and compliance to treatment assignment are in accordance with the trial hypotheses. Paik et al (2011) reviewed evidence for the clinical utility of five multi-gene expression based prognostic tests for breast cancer offered as reference lab tests - MammaPrint, MapQuant Dx, Oncotype Dx, PAM50 Breast Cancer Intrinsic Subtype Classifier, and Theros Breast Cancer Index, to determine whether any of them should be considered as routine clinical test. Peer reviewed publications, meeting abstracts, and information provided by company web sites have been reviewed to compile information regarding their clinical utility according to the following criteria; (1) Analytical validity and regulatory approval of the reference lab test. (2) Level of evidence for clinical utility. (3) Whether published evidences support prognostic and/or predictive claim. While published evidences for clinical claims for Oncotype Dx and MammaPrint used reference lab tests, and the supporting evidences for other tests come from academic assays before being converted to reference lab tests, results from two large randomized clinical trials testing the clinical utility of Oncotype Dx and MammaPrint are still several years away and until that time none of the markers would reach level I evidence by Marker Utility Grading System. However, Oncotype Dx has reached a level IB evidence according to Simon modification to Marker Utility Grading System. Therefore, Oncotype Dx may be considered for routine clinical use as an adjunct to clinical and pathological information and has been incorporated into clinical guidelines in USA. While MammaPrint, MapQuantDx, and PAM50 have been repeatedly demonstrated to provide robust prognostic information, evidence for its worth as a predictive marker for chemotherapy benefit is yet to come from randomize clinical trials and therefore its utility is limited to prognostication. Meta-analysis of publicly available microarray based gene expression studies demonstrated that gene expression assays provide similar information and the most important information they provide is the proliferation activity. In untreated population, the prognostic impact of proliferation genes is limited to ER+HER2- subset since HER2+ or ER-HER2- subsets are associated with MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer Jun 17 12

13 high proliferation activity. Therefore the clinical utility of these gene expression based tests is mainly for ER+HER2- subset. Since they are usually treated with adjuvant anti-estrogen therapies, for their clinical utility, demonstration of the interaction between the gene expression markers and chemotherapy in anti-estrogen treated cohort in a randomized clinical trial would be required. While Oncotype is the only test supported by studies in a randomized clinical trial for adjuvant chemotherapy, other gene expression based tests are expected to provide similar information. Gene expression profiling assays as more reproducible and precise surrogates for tumor grade (MapQauntDx and Theros Breast Cancer Index) are very promising assays. However, absence of definitive predefined cut-off for defining the subset that benefit from chemotherapy validated in cohorts from randomized trials limit their clinical application. Knauer et al (2010) reported overexpression of HER-2 is observed in 15-25% of breast cancers, and is associated with increased risk of recurrence. Current guidelines recommend trastuzumab and chemotherapy for most HER-2-positive patients. However, the majority of patients does not recur and might thus be overtreated with adjuvant systemic therapy. The authors investigated whether the 70-gene MammaPrint signature identifies HER-2-positive patients with favorable outcome. 168 T1-3, N0-1, HER-2-positive patients were identified from a pooled database, classified by the 70-gene signature as good or poor prognosis, and correlated with long-term outcome. A total of 89 of these patients did not receive adjuvant chemotherapy. In the group of 89 chemotherapy-naive patients, after a median follow-up of 7.4 years, 35 (39%) distant recurrences and 29 (33%) breast cancer-specific deaths occurred. The 70-gene signature classified 20 (22%) patients as good prognosis, with 10-year distant disease-free survival (DDFS) of 84%, compared with 69 (78%) poor prognosis patients with 10-year DDFS of 55%. The estimated hazard ratios (HRs) were 4.5 (95% confidence interval (CI) , P=0.04) and 3.8 (95% CI , P=0.07) for DDFS and breast cancer-specific survival (BCSS), respectively. In multivariate analysis adjusted for known prognostic factors and hormonal therapy, HRs were 5.8 (95% CI , P=0.03) and 4.7 (95% CI , P=0.05) for DDFS and BCSS, respectively. Investigators concluded the 70-gene prognosis signature is an independent prognostic indicator that identifies a subgroup of HER-2-positive early breast cancer with a favorable long-term outcome. They noted however, the study was limited by analyses of retrospective data, small sample size, and limited follow-up time for some patients and a somewhat heterogeneous group of patients with or without endocrine therapy. The author notes that further validation is ongoing in the MINDACT Trial. Knauer et al (2010) assessed the additional predictive value of the 70-gene MammaPrint signature for chemotherapy (CT) benefit in addition to endocrine therapy (ET) from pooled study series. For 541 patients who received either ET (n = 315) or ET + CT (n = 226), breast cancer-specific survival (BCSS) and distant disease-free survival (DDFS) at 5 years were assessed separately for the 70-gene high and low risk groups. The 70-gene signature classified 252 patients (47%) as low risk and 289 (53%) as high risk. Within the 70-gene low risk group, BCSS was 97% for the ET group and 99% for the ET + CT group at 5 years with a non-significant univariate hazard ratio (HR) of 0.58 (95% CI ; P = 0.62). In the 70-gene high risk group, BCSS was 81% (ET group) and 94% (ET + CT group) at 5 years with a significant HR of 0.21 (95% CI ; P < 0.01). DDFS was 93% (ET) versus 99% (ET + CT), respectively, in the 70-gene low risk group, HR 0.26 (95% CI ; P = 0.20). In the high risk group DDFS was 76 versus 88%, HR of 0.35 (95% CI ; P < 0.01). Results were similar in multivariate analysis, showing significant survival benefit by adding CT in the 70-gene high risk group. The authors concluded, a significant and clinically meaningful benefit was observed by adding chemotherapy to endocrine treatment in 70-gene high risk patients. This benefit was MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer Jun 17 13

14 not significant in low risk patients, who were at such low risk for recurrence and cancer-related death, that adding CT does not appear to be clinically meaningful. They concluded further, it seems reasonable to use the multigene assays whenever indicated in hormone receptor-positive patients for improved decision-making regarding the role of adding adjuvant CT to hormonal treatment. Gevensleben et al (2010) performed a validation study of MammaPrint and TargetPrint in an unselected German breast cancer population and was designed to determine the degree of concordance with currently applied clinical parameters. One hundred and forty cases of breast cancer stage I and II were classified as being low or high risk for distant metastasis using MammaPrint. Results were compared to current clinical risk classifications and adjuvant treatment management. Immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH)/chromogenic in situ hybridization (CISH) assessments of ER, PR and HER2 were further compared with gene expression read-outs using TargetPrint. Thirty-two percent of patients (19/59) with a poor prognosis-signature identified via MammaPrint did not receive adjuvant systemic treatment apart from endocrine therapy and were potentially undertreated; whereas 42% (35/77) of patients with a good prognosis-signature received chemotherapy and were potentially overtreated. Comparison of microarray receptor results with IHC and FISH/CISH were concordant in 97% for ER; 86% for PR; and 94% for HER2. The investigators concluded in this German study population, MammaPrint would have resulted in altered treatment advice for adjuvant systemic therapy in 40% of patients. Kunz (2011) assessed the accuracy of MammaPrint in this younger patient group, performing a meta-analysis on a subgroup from previously described retrospective studies (n = 689). 44 patients had MammaPrint results and were eligible for the analysis. The results revealed a low risk result in 29 patients and a high risk result in 15 patients. Using St. Gallen 2007 guidelines for risk assessment, the numbers were 4 for low risk and 6 for high risk, respectively, while 34 patients were classified as intermediate risk. In the group of women with intermediate risk, MammaPrint assigned 23 patients to low risk and 11 to the high risk group. In the meta-analysis of retrospective studies, 10 year overall survival for the low and high MammaPrint groups was 90.2 and 65.2%, respectively. The authors concluded using gene expression analysis as additional tool, patients with an intermediate clinical risk can be accurately separated into low and high risk groups. The gene expression analysis provides more accurate information on recurrence risk compared to conventional clinicopathological criteria and thus may provide additional guidance in daily clinical practice in future. The authors noted results of further prospectively designed studies like the MINDACT trial will confirm the retrospective analysis and will determine how both low and high risk patients can best be treated. Knauer et al (2010) assessed the additional predictive value of the 70-gene MammaPrint signature for chemotherapy (CT) benefit in addition to endocrine therapy (ET) from pooled study series. For 541 patients who received either ET (n = 315) or ET + CT (n = 226), breast cancer-specific survival (BCSS) and distant disease-free survival (DDFS) at 5 years were assessed separately for the 70-gene high and low risk groups. The 70-gene signature classified 252 patients (47%) as low risk and 289 (53%) as high risk. Within the 70-gene low risk group, BCSS was 97% for the ET group and 99% for the ET + CT group at 5 years with a non-significant univariate hazard ratio (HR) of 0.58 (95% CI ; P = 0.62). In the 70-gene high risk group, BCSS was 81% (ET group) and 94% (ET + CT group) at 5 years with a significant HR of 0.21 (95% CI ; P < 0.01). DDFS was 93% (ET) versus 99% (ET + CT), respectively, in the 70-gene low risk group, HR 0.26 (95% CI ; P = 0.20). In the high risk group DDFS was 76 versus 88%, HR of 0.35 (95% CI ; P < 0.01). Results were similar in multivariate analysis, showing MammaPrint, Prosigna and Other Gene Expression Tests for Breast Cancer Jun 17 14