National Medical Policy

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1 National Medical Policy Subject: Policy Number: Genetic Testing for BRCA1 and BRCA2 NMP136 Effective Date*: April 2004 Updated: September 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 Medicaid Manuals for coverage 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)* Article (Local)* Molecular Diagnostic Testing: Genetic Testing: X Other CMS. Palmetto. MolDX: sf/docscat/moldx%20website~moldx~browse %20By%20Topic~Covered%20Tests~9BMLRK6 738?open&navmenu=%7C%7C MLN Matters. Number: MM7745. Related Change Request (CR). March 23, April Update to the Calendar Year (CY) 2012 Medicare Physician Fee Schedule Database (MPFSDB): Education/Medicare-Learning-Network- MLN/MLNMattersArticles/downloads/MM7745.pdf None Use Health Net Policy Genetic Testing for BRCA1 and BRCA2 Sep 15 1

2 Instructions 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 (Refer to NMP on Tumor Markers for Cancer) In general, Health Net, Inc. considers genetic testing medically necessary when all of the following are met: 1. The individual has personal or family history features suggestive of an inheritable condition 2. The test can be adequately interpreted; and 3. The results of the test will aid in diagnosis or directly impact the treatment being delivered to the patient and/or family members Important Note The responsibilities of the provider offering genetic testing includes risk assessment, pre-test education and post-test counseling. The clinician may choose to handle all aspects him or herself, or may work in concert with an expert in cancer genetic counseling and risk assessment. The requirement for submitting documentation for prior authorization varies among Health Net regional health plans. If documentation is required, the chart note should reflect a full and accurate history, including a history of familial cancer and a plan for pre and post-test counseling. In accordance with the guidelines set forth by the U.S. Preventive Services Task Force (USPFT), the American College of Medical Genetics, the American Society of Clinical Oncologists, the National Comprehensive Cancer Network (NCCN), and the American College of Obstetricans and Gyncologists (ACOG), Health Net Inc. considers genetic testing for BRCA1 or BRCA2 medically necessary for any of the following: 1. Individuals with a family member with a known deleterious BRCA1/BRCA2 mutation, or other cancer susceptibility gene; 2. Individuals with a personal history of breast cancer (including invasive and ductal carcinoma in situ), a known mutation in a cancer susceptibility gene within the family, and one or more of the following: Diagnosed < 45 years Genetic Testing for BRCA1 and BRCA2 Sep 15 2

3 Diagnosed < 50 y with any of the following: An additional primary [two breast primaries includes bilateral (contralateral) disease or two or more clearly separate ipsilateral primary tumors either synchronously or asynchronously] > 1 close blood relative 1 with breast cancer at any age An unknown or limited family history 2 > 1 close relative with pancreatic cancer > 1 relative with prostate cancer (Gleason score > 7) An unknown or limited family history Diagnosed < 60 y with a triple negative breast cancer (i.e., ER-, PR-negative, and HER2-negative) Diagnosed at any age with any of the following: >1 close blood relative 1 with breast cancer diagnosed < 50 y >2 close blood relative 1 with breast cancer >1 close blood relative 1 with invasive ovarian cancer (including fallopian tubes and primary peritoneal cancer 3 ) > 2 close blood relatives with pancreatic cancer or prostate cancer (Gleason score >7) A close male blood relative with breast cancer For an individual of ethnicity associated with higher mutation frequency (e.g. Ashkenazi Jewish) no additional family history may be required 4 3. Individuals with a personal history of ovarian cancer (including fallopian tubes and primary peritoneal cancer 3 ) 4. Individuals with a personal history of male breast cancer 5. Individuals with a personal history of pancreatic cancer or prostate cancer (Gleason score >7) at any age with > 2 1 close blood relative with breast (<50y) and/or invasive ovarian cancer and /or pancreatic or prostate cancer (Gleason score >7) at any age (NCCN, V2.2015) For an individual of ethnicity associated with higher mutation frequency (e.g. Ashkenazi Jewish) no additional family history may be required) 6. Individuals age 18 or older, who have not been diagnosed with either breast or ovarian cancer, with a family history of any of the following, with a known mutation in a cancer susceptibility gene within the family: (Significant limitations of interpreting test results for an unaffected individual should be discussed) First or second-degree blood relative with a history of breast cancer (including invasive and ductal carcinoma in situ) diagnosed < 45 years First or second-degree blood relative with a history of breast cancer (including invasive and ductal carcinoma in situ) diagnosed < 50 y with any of the following: An additional primary [two breast primaries includes bilateral (contralateral) disease or two or more clearly separate ipsilateral primary tumors either synchronously or asynchronously] > 1 close blood relative 1 with breast cancer at any age An unknown or limited family history 2 First or second-degree blood relative diagnosed <60 y with a triple negative breast cancer First or second-degree blood relative diagnosed at any age with any of the following: >1 close blood relative 1 with breast cancer diagnosed < 50 y >2 close blood relative 1 with breast cancer >2 close blood relative 1 with breast cancer primaries on the same side of family Genetic Testing for BRCA1 and BRCA2 Sep 15 3

4 >1 close blood relative 1 with invasive ovarian cancer (including fallopian tubes and primary peritoneal cancer3) > 2 close blood relatives 1 with pancreatic cancer or aggressive prostate cancer (Gleason score >7) at any age A close male blood relative with breast cancer For an individual of ethnicity associated with higher mutation frequency (e.g. Ashkenazi Jewish) no additional family history may be required 4 First or second-degree blood relative with a history of invasive ovarian cancer (including fallopian tubes and primary peritoneal cancer 3 ) First or second-degree blood relative with a history of male breast cancer First or second-degree blood relative with a history of pancreatic cancer or prostate cancer (Gleason score >7) at any age with > 1 close blood relatives with breast and/ovarian cancer and /or pancreatic or prostate cancer (Gleason score >7) For pancreatic cancer, if Ashkenazi Jewish ancestry, no additional affected relative is needed. Third-degree blood relative with breast cancer (including invasive and ductal carcinoma in situ) and/or invasive ovarian cancer (including fallopian tubes and primary peritoneal cancer 3 ) with > 2 close blood relatives 1 with breast cancer (at least one with breast cancer< 50y) and/or invasive ovarian cancer (including fallopian tubes and primary peritoneal cancer 3 ) 9. Personal and/or family history of three or more of the following (especially if early onset): Pancreatic cancer Prostate cancer (i.e., Gleason score >7) Sarcoma Adrenocortical carcinoma Brain tumors Endometrial cancer Thyroid cancer Kidney cancer Dermatologic manifestations and/or macrocephaly Hamartomatous polyps of GI tract; diffuse gastric cancer (can include multiple primaries in same individual) Note: Clinical judgment should be used to determine if the patient has reasonable likelihood of a mutation, considering the unaffected patient s current age and the age of female unaffected relatives who link the patient with the affected relatives. Testing of unaffected individuals should only be considered when an appropriate affected family member is unavailable for testing. Additional testing may be indicated if there is also a significant family history of cancer on the side of the family without the known mutation. Footnotes: 1. Close blood relatives include first-, second- and third degree relatives on same side of family. First-degree relatives include parents, siblings and children on both maternal and paternal sides. Second-degree relatives include grandparents, grandchildren, aunts and uncles, half-siblings, nieces and nephews on both maternal and paternal sides. Third-degree relatives are relatives with whom you share one-eighth of your genes, such as first cousins. Genetic Testing for BRCA1 and BRCA2 Sep 15 4

5 2. Individuals with unknown or limited family history/structure, such as fewer than 2 first or second degree female relatives having lived beyond age 45 in either, lineage, may have an underestimated probability of familial mutation detection. The likelihood of mutation detection may be very low in families with a large number of unaffected female relatives. Clinical judgment should be used to determine the appropriateness of genetic testing. The maternal and paternal sides should be considered independently. 3. Ovarian/fallopian tube/primary peritoneal cancers are components of Lynch syndrome/hereditary non-polyposis colorectal cancer. 4. Testing for Ashkenazi Jewish founder-specific mutation(s) should be performed first. Comprehensive genetic testing may be considered if ancestry also includes non- Ashkenazi Jewish relatives or if other HBOC criteria are met. Funder mutations exist in other populations. BRACAnalysis Large Rearrangement Test (BART) Health Net considers the BRACAnalysis Large Rearrangement Test (BART) medically necessary to detect large genomic rearrangements in individuals who are at risk for BRCA1/2 related cancers but have negative BRCA1/2 genetic sequence tests and meet the criteria for BRCA1/2 testing. Not Medically Necessary Health Net Inc. does not consider genetic testing for BRCA1 or BRCA2 medically necessary for any of the following indications: 1. Genetic testing in the absence of a personal or family history of breast or ovarian cancer 2. Unaffected family members with no known BRCA1 or BRCA2 mutation 3. Genetic testing for BRCA1 and BRCA2 mutations in minors under 18 years of age 4. BRCA testing to assess the risk of breast or prostate cancer in men without breast cancer Note: Health Net Inc. does not cover genetic testing when BRCA mutation analysis and sequencing is performed primarily for the medical management of other family members who are not covered under a Health Net plan. Health Net Inc. considers BRACAnalysis Rearrangement Test (BART) for screening of the general population or re-testing previously tested high-risk members for large genomic re-arrangements not medically necessary due to lack of information in the peer review literature validating this utility of this test or its effect on patient outcomes. Investigational Health Net, Inc. considers BRCAplus investigational since there is insufficient peerreviewed evidence on the analytical validity, clinical validity, and clinical utility. Health Net, Inc. also considers PALB2-Associated Hereditary Breast Cancer investigational in the following situations: Genetic Testing for BRCA1 and BRCA2 Sep 15 5

6 1. For PALB2 gene testing in breast cancer patients with a personal and/or family history consistent with hereditary breast cancer, including male breast cancer, who have not been tested for BRCA1/2 gene variants. 2. For PALB2 gene testing in breast cancer patients based on a personal or family history of pancreatic cancer, without additional features of hereditary breast cancer. 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 will be replaced by ICD-10 code sets. Health Net National Medical Policies will now include the preliminary ICD-10 codes in preparation for this transition. Please note that these may not be the final versions of the codes and that will not be accepted for billing or payment purposes until the October 1, 2015 implementation date. ICD-9 Codes 174 Malignant neoplasm of female breast Nipple and areola Central portion Upper-inner quadrant Lower-inner quadrant Upper-outer quadrant Lower-outer quadrant Axillary tail Other specified sites of female breast Breast (female) unspecified 175 Malignant neoplasm of male breast Nipple and areola Other and unspecified sites of male breast V10.30 Personal history of breast cancer V16.3 Family history of malignant neoplasm-breast V16.9 Unspecified malignant neoplasm ICD-10 Codes C C Malignant neoplasm of breast Z80.3 Family history of malignant neoplasm of breast Z80.9 Family history of malignant neoplasm, unspecified Z85.3 Personal history of malignant neoplasm of breast CPT Codes BRAF (v-raf murine sarcoma viral oncogene homolog B1) (eg. Colon cancer, gene analysis, V600E variant) BRCA1, BRCA2 (breast cancer 1 and 2) (eg, hereditary breast and ovarian cancer) gene analysis; full sequence analysis and common Genetic Testing for BRCA1 and BRCA2 Sep 15 6

7 duplication/deletion variants in BRCA1 (ie, exon 13 del 3.835kb, exon 13 dup 6kb, exon del 26kb, exon 22 del 510bp, exon 8-9 del 7.1kb) BRCA1, BRCA2 (breast cancer 1 and 2) (eg, hereditary breast and ovarian cancer) gene analysis; 185delAG, 5385insC, 6174delT variants BRCA1, BRCA2 (breast cancer 1 and 2) (eg, hereditary breast and ovarian cancer) gene analysis; uncommon duplication/deletion variants BRCA1 (breast cancer 1) (eg, hereditary breast and ovarian cancer) gene analysis; full sequence analysis and common duplication/deletion variants (ie, exon 13 del 3.835kb, exon 13 dup 6kb, exon del 26kb, exon 22 del 510bp, exon 8-9 del 7.1kb) BRCA1 (breast cancer 1) (eg, hereditary breast and ovarian cancer) gene analysis; known familial variant BRCA2 (breast cancer 2) (eg, hereditary breast and ovarian cancer) gene analysis; full sequence analysis BRCA2 (breast cancer 2) (eg, hereditary breast and ovarian cancer) gene analysis; known familial variant molecular diagnostics; molecular isolation or extraction, each nucleic acid type (ie, DNA or RNA) (Code have been deleted in To report, see 81161, ) isolation or extraction of highly purified nucleic acid, each nucleic acid type (ie, DNA or RNA) (Code have been deleted in To report, see 81161, ) enzymatic digestion, each enzyme treatment (Code have been deleted in To report, see 81161, ) dot/slot blot production, each nucleic acid preparation (Code have been deleted in To report, see 81161, ) separation by gel electrophoresis (eg. agarose, polyacrylamide), each nucleic acid preparation (Code have been deleted in To report, see 81161, ) nucleic acid probe, each (Code have been deleted in To report, see 81161, ) nucleic acid transfer (eg, Southern, Northern), each nucleic acid preparation (Code have been deleted in To report, see 81161, ) amplification of patient nucleic acid (eg PCR, LCR), single primer pair, each primer pair (Code have been deleted in To report, see 81161, ) amplification of patient nucleic acid, multiplex, first two nucleic acid sequences (Code have been deleted in To report, see 81161, ) amplification of patient nucleic acid, multiplex, each multiflex reaction (Code have been deleted in To report, see 81161, ) reverse transcription (Code have been deleted in To report, see 81161, ) mutation scanning, by physical properties (eg, single strand conformational polymorphisms (SSCP), heteroduplex, denaturing gradient gel electrophoresis (DGGE), RNA ase A), single segment, each (Code have been deleted in To report, see 81161, ) mutation identification by sequencing, single segment, each segment (Code have been deleted in To report, see 81161, ) mutation identification by allele specific transcription, single segment, each Genetic Testing for BRCA1 and BRCA2 Sep 15 7

8 segment (Code have been deleted in To report, see 81161, ) mutation identification by allele specific translation, segment, each segment (Code have been deleted in To report, see 81161, ) interpretation and report (Code have been deleted in To report, see 81161, ) HCPCS Codes (N/A) Scientific Rationale Update September 2015 Rebbeck et al. (2015) completed an observational study of women who were found between 1937 and 2011, (i.e., median 1999), to carry disease-associated BRCA1 or BRCA2 mutations. The international sample comprised 19,581 carriers of BRCA1 mutations and 11,900 carriers of BRCA2 mutations from 55 centers in 33 countries on 6 continents. The authors estimated hazard ratios for breast and ovarian cancer based on mutation type, function, and nucleotide position. The RHR, the ratio of breast vs ovarian cancer hazard ratios, was also estimated. A value of RHR greater than 1 indicated elevated breast cancer risk; a value of RHR less than 1 indicated elevated ovarian cancer risk. Among BRCA1 mutation carriers, 9052 women (46%) were diagnosed with breast cancer, 2317 (12%) with ovarian cancer, 1041 (5%) with breast and ovarian cancer, and 7171 (37%) without cancer. Among BRCA2 mutation carriers, 6180 women (52%) were diagnosed with breast cancer, 682 (6%) with ovarian cancer, 272 (2%) with breast and ovarian cancer, and 4766 (40%) without cancer. In BRCA1, the authors identified 3 breast cancer cluster regions (BCCRs) located at c.179 to c.505 (BCCR1; RHR=1.46; 95% CI, ; P=2 10(-6)), c.4328 to c.4945 (BCCR2; RHR=1.34; 95% CI, ; P=.04), and c to c.5563 (BCCR2', RHR=1.38; 95% CI, ; P=6 10(-9)). We also identified an ovarian cancer cluster region (OCCR) from c.1380 to c.4062 (approximately exon 11) with RHR=0.62 (95% CI, ; P=9 10(-17)). In BRCA2, we observed multiple BCCRs spanning c.1 to c.596 (BCCR1; RHR=1.71; 95% CI, ; P=.03), c.772 to c.1806 (BCCR1'; RHR=1.63; 95% CI, ; P=.01), and c.7394 to c.8904 (BCCR2; RHR=2.31; 95% CI, ; P=.00002). We also identified 3 OCCRs: the first (OCCR1) spanned c.3249 to c.5681 that was adjacent to c.5946delt (6174delT; RHR=0.51; 95% CI, ; P=6X10(-17)). The second OCCR spanned c.6645 to c.7471 (OCCR2; RHR=0.57; 95% CI, ; P=.001). Mutations conferring nonsense-mediated decay were associated with differential breast or ovarian cancer risks and an earlier age of breast cancer diagnosis for both BRCA1 and BRCA2 mutation carriers. Breast and ovarian cancer risks varied by type and location of BRCA1/2 mutations. With appropriate validation, these data may have implications for risk assessment and cancer prevention decision making for carriers of BRCA1 and BRCA2 mutations. Per the NCCN guidelines, Genetic/Familial High-Risk Assessment: Breast and Ovarian (2.2015): BRCA-related ovarian cancers are associated with non-mucinous histology. Scientific Rationale Update October 2014 NCCN guidelines, Genetic/Familial High-Risk Assessment: Breast and Ovarian (2.2014), includes a section on multi-gene testing. Per NCCN: Genetic Testing for BRCA1 and BRCA2 Sep 15 8

9 The recent introduction of multi-gene testing for hereditary forms of cancer has rapidly altered the clinical approach to testing at-risk patients and their families. Based on next-generation sequencing technology, these tests simultaneously analyze a set of genes that are associated with a specific family cancer phenotype or multiple phenotypes. Multi-gene testing includes intermediate penetrant genes of unclear actionability, and may reveal missense variations of unknown significance. It is for these and other reasons that they are ideally offered in the context of professional genetic expertise for pre- and post- test counseling. The decision to use multi-gene testing for patient care should be no different than the rationale for testing a single gene known to be associated with the development of a specific type of cancer. Testing is focused on identifying a mutation known to be clinically actionable- that is, whether the management of an individual patient is altered based on the presence or absence of a mutation. However, as the genes on the multi-gene tests may differ in penetrance, cancer pathogenicity and the availability of specific prevention and screening guidelines, testing and post multi-gene testing management will require consultation with genetic professionals experienced in cancer susceptibility. The benefits, limitations, and management recommendations should all be reviewed by the provider and discussed with the patient when considering the use of multi-gene testing for cancer susceptibility. General Recommendations from NCCN: Provider: 1. Because of their complexity, hereditary cancer multigene tests should be ordered in consultation with a cancer genetics professional.(genetic counseling is highly recommended when genetic testing is offered and after results are disclosed. A genetic counselor, medical geneticist, oncologist, surgeon, oncology nurse, or other health professional with expertise and experience in cancer genetics should be involved early in counseling patients who potentially meet criteria for an inherited syndrome.) 2. As in other genetic testing, an affected family member should be tested first, whenever possible. 3. Multi-gene testing may be more cost and time effective in certain cases than sequentially testing more than 2-3 single genes associated with a phenotype 4. Since genes can be easily added or removed from multi-gene tests over time by a given lab, medical records must document which genes were included in the specific multi-gene test used for each patient, and in which labs they were performed. 5. Multi-gene tests vary in technical specifications (e.g., depth of gene coverage, extent of intron/exon boundary analysis, methodology of large deletion/duplication analysis). Tests should be chosen that maximize the likelihood of identifying mutations in the genes of interest and that will be likely alter patient management. 6. Under certain circumstances, technologies used in multi-gene testing may fail to identify mutations that might be identifiable through single-gene testing. If high clinical suspicion remains for a particular syndrome after negative multi-gene test results, consultation with the testing lab and/or additional targeted genetic testing may be warranted. Laboratories: Genetic Testing for BRCA1 and BRCA2 Sep 15 9

10 1. The classification of variants of uncertain significance remains an ongoing issue. The methods and data sources used to classify genetic variants can differ significantly by laboratory. Genetic testing laboratories should be transparent in the manner by which they classify variants 2. Since variant classification is greatly facilitated by public availability of data, laboratories should make their data available. 3. Laboratories have differing policies regarding whether or not to notify providers for interpretation changes of a previously identified variant. Because the notification of variant changes is an important aspect of care, there is an urgent need for standardized procedures across laboratories for handling such notifications. 4. Other next-generation sequencing methods, such as whole genome sequencing (WGS) and whole exome sequencing (WES), may become useful testing strategies in the future. At present, however, there is no evidence for the general use of these tests for cancer susceptibility testing. Scientific Rationale Update September 2014 PALB2 is a tumor suppressor gene that has been identified as a breast and pancreatic cancer susceptibility gene. It interacts with BRCA2 to repair damaged DNA and help maintain the rate of cell growth and division. PALB2 gene variants are inherited in an autosomal dominant manner, with carriers having a 50% recurrence chance of passing on the disease-causing variant to each of their offspring. However, PALB2 gene variants are an uncommon cause of hereditary breast cancer, although they are associated with a moderately increased risk for invasive breast cancer when present. In addition, it is possible that some cancer-affected family members of PALB2 carriers, especially those with later-onset disease, will test negative for the familial PALB2 gene variant. Antoniou et al. (2014) Germline loss-of-function mutations in PALB2 are known to confer a predisposition to breast cancer. However, the lifetime risk of breast cancer that is conferred by such mutations remains unknown. The authors analyzed the risk of breast cancer among 362 members of 154 families who had deleterious truncating, splice, or deletion mutations in PALB2. The age-specific breast-cancer risk for mutation carriers was estimated with the use of a modified segregation-analysis approach that allowed for the effects of PALB2 genotype and residual familial aggregation. The risk of breast cancer for female PALB2 mutation carriers, as compared with the general population, was eight to nine times as high among those younger than 40 years of age, six to eight times as high among those 40 to60 years of age, and five times as high among those older than 60 years of age. The estimated cumulative risk of breast cancer among female mutation carriers was 14% (95% confidence interval [CI], 9 to 20) by 50 years of age and 35% (95% CI, 26 to 46) by 70 years of age. Breast-cancer risk was also significantly influenced by birth cohort (P<0.001) and by other familial factors (P=0.04). The absolute breast-cancer risk for PALB2 female mutation carriers by 70 years of age ranged from 33% (95% CI, 25 to 44) for those with no family history of breast cancer to 58% (95% CI, 50 to 66) for those with two or more first-degree relatives with breast cancer at 50 years of age. Loss-of-function mutations in PALB2 are an important cause of hereditary breast cancer, with respect both to the frequency of cancer-predisposing mutations and to the risk associated with them. Our data suggest the breast-cancer risk for PALB2 mutation carriers may overlap with that for BRCA2 mutation carriers. Genetic Testing for BRCA1 and BRCA2 Sep 15 10

11 Additional analyses involving larger, peer reviewed studies are needed to establish the precise detection rate for PALB2 gene testing in the various patient populations and to identify the most appropriate candidates for PALB2 gene testing. NCCN (Version ) has guidelines on Genetic/Familial High-Risk Assessment: Breast and Ovarian. This includes the following information on gene panels: New genetic testing panels using next-generation sequencing for hereditary breast, ovarian, and other cancers have recently been introduced. These panels are intended for individuals who have tested negative for high penetrance genes (eg, BRCA1/2) and for those whose family history is suggestive of more than one syndrome. The genetic testing laboratories include somewhat different, but often overlapping, genes. Examples of currently available genes within these panels include: ATM, BARD1, BRIP, CDH1, CHEK2, MLH1, MSH2, MSH6, MUTYH, MRE11A, NBN, Palb2, PTEN, RAD50, RAD51B, RAD51C, RAD51D, STK11, and TP53. Limitations of these panels include an unknown percentage of variants of unknown significance, uncertainty of level of risk associated with most of these genes, and lack of clear guidelines on risk management of carriers of some of these mutations. Because of the complexity and limited data regarding their clinical utility, hereditary multigene cancer panels should only be ordered in consultation with a cancer genetics professional. Scientific Rationale Update July 2014 The BRCAplus is a multigene testing panel in which 6 breast cancer genes are analyzed. This includes BRCA1, BRCA2, CDH1, PTEN,TP53, and STK11. This genetic test looks for any mutations that would make the genes non-functional. Per the National Comprehensive Care Network (NCCN, Version1.2014) on Genetic/Familial High Risk Assessment: Breast and Ovarian, it indicates that the 6 genes included in this 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 risks 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 June 2013 Per the National Comprehensive Care Network (NCCN), An increased frequency of other malignancies has been reported in families with mutations in the BRCA1 or BRCA2 gene. Germline BRCA1 and BRCA2 mutations have been associated with an increased risk of prostate cancer in numerous reports. In particular, BRCA2 mutations have been associated with 2-5 fold increase in risk of prostate cancer, while increased risks were not observed for BRCA1 mutation carriers in some studes. Prostate cancer in patients with BRCA2 mutations has been associated with a higher histologic grade. Analysis of data obtained from cancer registries and treatment center databases showed that BRCA2 mutation carriers with prostate cancer had more aggressive or rapidly progressive disease, and significantly decreased survival compared with patients who were BRCA1 mutation carriers or non-carriers. In a study of patients with prostate cancer from a population-based cancer registry in Genetic Testing for BRCA1 and BRCA2 Sep 15 11

12 Iceland (n=596), patients with BRCA2 mutations had significantly decreased median survical compared to non-carriers (having wild type BRCA2) patients (2yrs vs 12 years; P<0.001). Moreover, in a study of patients with prostate cancer using data obtained from cancer center databases (n=301) 301 patients with BRCA2 mutations had significantly decreased median survival compared with patients with BRCA1 mutations (4 years vs 8 years; P<0.01). BRCA2 mutation carriers have also been reported to have a higher risk of pancreatic cancer and melanoma. Both BRCA1 and BRCA2 mutations have been associated with increased propensity for developing pancreatic cancer. In an analysis of samples taken from patients with familial pancreatic cancer (kindreds in which >3 family members had pancreatic cancer, at least 2 of which were first-degree relatives), BRCA2 mutations were detected in 17% of patient samples. Among the Ashkenazi Jewish population, BRCA2 mutations have been identified in about 4% of patients with pancreatic cancer. Mitra et al (2011) sought to evaluate the role of targeted prostate cancer screening in men with BRCA1 or BRCA2 mutations. A preliminary analysis of the data from the international study, IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in BRCA1/2 mutation carriers and controls), the first multicentre screening study targeting men with a known genetic predisposition to prostate cancer was reported. Men aged years from families with BRCA1 or BRCA2 mutations were offered annual prostate specific antigen (PSA) testing, and those with PSA > 3 ng/ml, were offered a prostate biopsy. Controls were men agematched (± 5 years) who were negative for the familial mutation. In total, 300 men were recruited (205 mutation carriers; 89 BRCA1, 116 BRCA2 and 95 controls) over 33 months. At the baseline screen (year 1), 7.0% (21/300) underwent a prostate biopsy. Prostate cancer was diagnosed in ten individuals, a prevalence of 3.3%. The positive predictive value of PSA screening in this cohort was 47 6% (10/21). One prostate cancer was diagnosed at year 2. Of the 11 prostate cancers diagnosed, nine were in mutation carriers, two in controls, and eight were clinically significant. Investigators concluded the present study shows that the positive predictive value of PSA screening in BRCA mutation carriers is high and that screening detects clinically significant prostate cancer. These results support the rationale for continued screening in such men Gallagher et al (2010) determined BRCA mutation prevalence in 832 Ashkenazi Jewish men diagnosed with localized prostate cancer between 1988 and 2007 and 454 Ashkenazi Jewish controls and compared clinical outcome measures among 26 BRCA mutation carriers and 806 noncarriers. Kruskal-Wallis tests were used to compare age of diagnosis and Gleason score, and logistic regression models were used to determine associations between carrier status, prostate cancer risk, and Gleason score. Hazard ratios (HR) for clinical end points were estimated using Cox proportional hazards models. BRCA2 mutations were associated with a 3-fold risk of prostate cancer [odds ratio, 3.18; 95% confidence interval (95% CI), ; P = 0.002] and presented with more poorly differentiated (Gleason score > or =7) tumors (85% versus 57%; P = ) compared with non-brca-associated prostate cancer. BRCA1 mutations conferred no increased risk. After 7,254 personyears of follow-up, and adjusting for clinical stage, prostate-specific antigen, Gleason score, and treatment, BRCA2 and BRCA1 mutation carriers had a higher risk of recurrence [HR (95% CI), 2.41 ( ) and 4.32 ( ), respectively] and prostate cancer-specific death [HR (95% CI), 5.48 ( ) and 5.16 ( ), respectively] than noncarriers. Investigators concluded BRCA2 mutation carriers had an increased risk of prostate cancer and a higher histologic grade, and BRCA1 or BRCA2 mutations were associated with a more aggressive clinical course. Genetic Testing for BRCA1 and BRCA2 Sep 15 12

13 These results may have implications for tailoring clinical management of this subset of hereditary prostate cancer. Ferrone et al (2009) sought to define the prevalence of BRCA1 and BRCA2 in an unselected group of Jewish patients and to compare the clinical characteristics and overall survival (OS) of patients with resected BRCA mutation-associated pancreatic adenocarcinoma (PAC) to PAC patients without mutations. Jewish patients with PAC resected between January 1986 and January 2004 were identified. DNA was extracted from the archived material, anonymized, and genotyped for founder mutations in BRCA1 (185delAG, 5382insC) and BRCA2 (6174delT). Standard twosided statistical tests were utilized. Of the 187 Jewish patients who underwent resection for PAC, tissue was available for 145 patients. Eight subjects (5.5%) had a BRCA founder mutation (two with BRCA1 [1.3%], six with BRCA2 [4.1%]). The BRCA2 founder mutation was identified in 4.1% of patients with pancreatic adenocarcinoma compared with only 1.1% of cancer-free Washington, DC,-area controls (4.1% v 1.1%; P =.007; odds ratio, 3.85; 95% CI, 2.1 to 10.8). Patients with and without BRCA1 or BRCA2 mutations did not differ in age (mean, 66 v 73 years; P =.6) or other clinicopathologic features. OS was not significantly different (median, 6 v 16 months; P =.35). A previous cancer was reported by 24% (35 of 145) of patients with the most common sites being breast cancer (9 of 35; 74%) and prostate cancer (8 of 35; 23%). Investigators concluded founder mutations for BRCA1 and BRCA2 were identified in 5.5% of Ashkenazi patients operated on for PAC. BRCA2 mutations were more prevalent than documented by population studies. Consistent with previous reports, BRCA2 mutations are associated with an increased risk of PAC. Scientific Rationale Update December 2012 According to the National Cancer Comprehensive Network (NCCN, 2012), breast cancer is the second leading cause of cancer death in U.S. women. Up to 10% of breast cancers are due to specific mutations in single genes that are passed down in a family. Specific patterns of hereditary breast cancer/ovarian cancer are linked to mutations in the BRAC1 and BRAC2 genes. BRCA1/2 mutations confer an increased lifetime risk for the development of breast cancer (up to 80%), contralateral breast cancer (about 30% at 10 years), ovarian cancer (up to 40% for BRCA1 and 20% for BRCA2), and other cancers. Both BRAC1 and BRAC2 mutations have been associated with increased propensity for developing pancreatic cancer. There is a strong association between BRCA mutations and the diagnosis of triplenegative (hormone-receptor and human epidermal growth factor 2 [HER2] receptor negative) breast cancer. Up to 80 percent of BRCA mutation-associated breast cancers are triple-negative. Per the NCCN, Some histopathologic features have been reported to occur more frequently in breast cancer is characterized by a BRAC1/2 mutation. Several studies have shown that BRAC1 breast cancer is more likely characterized as ER-, PR negative, and HER2 negative. Studies have reported BRAC1 mutations in 11%-28% of patients with triple-negative breast cancer. In addition, it appears that among patients with triple-negative disease, BRAC mutation carriers were diagnosed at a younger age compared with non-carriers. An important limitation of genetic risk assessment with models and guideline criteria that are largely dependent on family history is that the probability of a mutation may be underestimated if there are too few female relatives (or too few surviving beyond Genetic Testing for BRCA1 and BRCA2 Sep 15 13

14 45 years of age). Per the 2012 NCCN guidelines on Breast and Ovarian Cancer Genetic Assessment, testing for BRCA1 or BRCA2 is appropriate In women with a personal history of breast cancer diagnosed age < 50y with a limited family history (i.e., fewer than 2 first, or second-degree female relatives or female relatives surviving beyond 45 years in either lineage, may have an underestimated probability of a familial mutation.) According to a revised position statement from the American Society of Breast Surgeons (Sept 2012), If a woman without a personal history of cancer seeks BRCA testing due to a high calculated personal risk, it is preferred to test one of her close relatives who has been diagnosed with breast cancer. This testing can establish whether the given familial pattern is actually associated with a BRCA mutation. If the affected family member has no BRCA mutation, the familial pattern in question can be assumed to be due to other genes and the family and the patient will continue to be managed as a high-risk group. On the other hand, for a woman whose affected family member carries a known BRCA mutation, a negative test means she has only ordinary risk for developing breast cancer and can thus avoid high-risk management. When an unaffected patient with no established familial BRCA mutation is tested, only a positive result can provide useful information. If no willing or living affected relative can be tested, a negative test does not provide information about an unaffected high-risk patient's true breast cancer risk; she will still be managed as high-risk based on her family history. The American Congress of Obstetricians and Gynecologists (ACOG) practice bulletin on Hereditary Breast and Ovarian Cancer Syndrome (reaffirmed 2011) states, Families with few female relatives may under represent female carrier despite the presence of a predisposing family mutation. Given these issues in families with few female relatives, it may be reasonable to consider genetic counseling in the setting of an isolated case of breast cancer at or before age 50 years. Weitzel et al (2007) sought to determine if BRCA gene mutations are more prevalent among single cases of early onset breast cancer in families with limited vs adequate family structure than would be predicted by currently available probability models. A total of 1543 women seen at US high-risk clinics for genetic cancer risk assessment and BRCA gene testing were enrolled in a prospective registry study between April 1997 and February Three hundred six of these women had breast cancer before age 50 years and no first- or second-degree relatives with breast or ovarian cancers. The main outcome measure was whether family structure, assessed from multigenerational pedigrees, predicts BRCA gene mutation status. Limited family structure was defined as fewer than 2 first- or second-degree female relatives surviving beyond age 45 years in either lineage. Family structure effect and mutation probability by the Couch, Myriad, and BRCAPRO models were assessed with stepwise multiple logistic regression. Model sensitivity and specificity were determined and receiver operating characteristic curves were generated. Family structure was limited in 153 cases (50%). BRCA gene mutations were detected in 13.7% of participants with limited vs 5.2% with adequate family structure. Family structure was a significant predictor of mutation status (odds ratio, 2.8; 95% confidence interval, ; P =.02). Although none of the models performed well, receiver operating characteristic analysis indicated that modification of BRCAPRO output by a corrective probability index accounting for family structure was the most accurate BRCA gene mutation status predictor (area under the curve, 0.72; 95% confidence interval, ; P<.001) for single cases of breast cancer. Investigators concluded family structure can affect the accuracy of mutation probability models. Genetic testing Genetic Testing for BRCA1 and BRCA2 Sep 15 14

15 guidelines may need to be more inclusive for single cases of breast cancer when the family structure is limited and probability models need to be recreated using limited family history as an actual variable. Hartman et al (2012) assessed BRCA1 and BRCA2 mutation prevalence in an unselected cohort of patients with triple-negative breast cancer (BC). One hundred ninety-nine patients were enrolled. Triple negativity was defined as <1% estrogen and progesterone staining by immunohistochemistry and HER-2/neu not overexpressed by fluorescence in situ hybridization. Having given consent, patients had BRCA1 and BRCA2 full sequencing and large rearrangement analysis. Mutation prevalence was assessed among the triple-negative BC patients and the subset of patients without a family history of breast/ovarian cancer. Independent pathological review was completed on 50 patients. Twenty-one deleterious BRCA mutations were identified--13 in BRCA1 and 8 in BRCA2 (prevalence, 10.6%). In 153 patients (76.9%) without significant family history (first-degree or second-degree relatives with BC aged <50 years or ovarian cancer at any age), 8 (5.2%) mutations were found. By using prior National Comprehensive Cancer Network (NCCN) guidelines recommending testing for triple-negative BC patients aged <45 years, 4 of 21 mutations (19%) would have been missed. Two of 21 mutations (10%) would have been missed using updated NCCN guidelines recommending testing for triplenegative BC patients aged <60 years. Investigators concluded the observed mutation rate was significantly higher (P =.0005) than expected based on previously established prevalence tables among patients unselected for pathology. BRCA1 mutation prevalence was lower, and BRCA2 mutation prevalence was higher, than previously described. Additional mutation carriers would have met new NCCN testing guidelines, underscoring the value of the updated criteria. Study data suggest that by increasing the age limit to 65 years, all carriers would have been identified. Robertson et al (2012) evaluated the BRCA1 mutation frequency and the implications for clinical practice of undertaking genetic testing in women with TN breast cancer. They undertook BRCA1 mutation analysis in 308 individuals with TN breast cancer, 159 individuals from unselected series of breast cancer and 149 individuals from series ascertained on the basis of young age and/or family history. BRCA1 mutations were present in 45 out of 308 individuals. Individuals with TN cancer <50 years had >10% likelihood of carrying a BRCA1 mutation in both the unselected (11 out of 58, 19%) and selected (26 out of 111, 23%) series. However, over a third would not have been offered testing using existing criteria. We estimate that testing all individuals with TN breast cancer <50 years would generate an extra 1200 tests annually in England. Investigators concluded women with TN breast cancer diagnosed below 50 years have >10% likelihood of carrying a BRCA1 mutation and are therefore eligible for testing in most centres. However, implementation may place short-term logistical and financial burdens on genetic services. Xu et al (2012) investigated the mutations of BRCA1 and BRCA2 and sought to determine whether clinic-pathological factors related to BRCA gene mutation. Mastectomy specimens from 360 breast cancers were enrolled and examined in the study. The relationship between BRCA gene mutation and clinic-pathological factors was evaluated. Overall, 280 patients were BRCA negative and 80 got BRCA gene mutation. Triple-negative breast cancers, i.e., breast cancers that do not express estrogen receptors (ER), progesterone receptors (PR) or human epidermal growth factor receptor 2 (HER2/neu) were observed in 53.85% of the BRCA1 mutation patients, in 28.57% of the BRCA2 mutation cases, while 14.29% of BRCA negative patients. BRCA1 mutation patients got a heavy lymph node metastasis and higher Genetic Testing for BRCA1 and BRCA2 Sep 15 15

16 nuclear grade tumors than the others. Furthermore, BRCA mutation was also found to be significantly related to ER, PR and HER2/neu status. BRCA1 expression was not associated with breast cancer-specific survival in the triple-negative breast cancers. After Cox regression, BRCA1 mutation was not shown to be an independent prognostic factor for breast cancer. These findings substantiated the possibility of tumors associated with BRCA1 mutations divided into two distinct groups, triplenegative and non-triple-negative groups requires further investigation. Scientific Rationale Update June 2012 The National Cancer Comprehensive Network (NCCN, 2012) Guidelines for Genetic / Familial High-Risk Assessment for Breast and Ovarian Cancer notes the following: Certain mutations (i.e., large rearrangements) are not detectable by the primary sequencing assay and supplementary testing may be necessary. Comprehensive genetic testing includes full sequencing of BRCA1/BRCA2 and detection of large genomic rearrangements. These recommendations noted above by NCCN are for those who meet the testing criteria noted within the Health Net policy statement, which is in agreement with NCCN guidelines for BRCA testing. Judkins et al. (2012) completed a study in which the prevalence of BRCA1/2 large rearrangements (LRs) was investigated in 48,456 patients with diverse clinical histories and ancestries, referred for clinical molecular testing for suspicion of hereditary breast and ovarian cancer (HBOC). Current estimates of the contribution of large rearrangement (LR) mutations in the BRCA1 (breast cancer 1, early onset) and BRCA2 (breast cancer 2, early onset) genes responsible for hereditary breast and ovarian cancer are based on limited studies of relatively homogeneous patient populations. Sanger sequencing analysis was performed for BRCA1/2 and LR testing for deletions and duplications using a quantitative multiplex polymerase chain reaction assay. Prevalence data were analyzed for patients from different risk and ethnic groups from July 2007 through April Patients were designated as high-risk if their clinical history predicted a high prior probability, wherein LR testing was performed automatically in conjunction with sequencing. Elective patients did not meet the high-risk criteria, but underwent LR testing as ordered by the referring health care provider. Overall BRCA1/2 mutation prevalence among high-risk patients was 23.8% versus 8.2% for the elective group. The mutation profile for high-risk patients was 90.1% sequencing mutations versus 9.9% LRs, and for elective patients, 94.1% sequencing versus 5.9% LRs. This difference may reflect the bias in high-risk patients to carry mutations in BRCA1, which has a higher penetrance and frequency of LRs compared with BRCA2. There were significant differences in the prevalence and types of LRs in patients of different ancestries. LR mutations were significantly more common in Latin American/Caribbean patients. Comprehensive LR testing in conjunction with full gene sequencing is an appropriate strategy for clinical BRCA1/2 analysis. Scientific Rationale Update July 2011 Although a family history of breast and/or ovarian cancer is common in women diagnosed with breast or ovarian cancer, less than 10 percent of all breast cancers and less than 15 percent of ovarian cancers are associated with germline (inherited) genetic mutations. The majority of hereditary breast and ovarian cancers are Genetic Testing for BRCA1 and BRCA2 Sep 15 16

17 associated with mutations in two genes, breast cancer type 1 and 2 susceptibility genes (BRCA1 and BRCA2). Children of a parent with a BRCA1 or BRCA2 mutation have a 50 percent risk of having inherited the mutation. It is important that adult relatives are informed about this risk and the associated cancer risks, and should be made aware of the options for genetic counseling, testing, and management, as necessary. Narod (2010) Genetic testing for BRCA1 and BRCA2 mutations is gaining acceptance in clinical oncology worldwide and may help target unaffected high-risk women for prevention and for close surveillance. Annual screening with MRI seems to be an effective surveillance strategy, but the long-term follow-up of women with small MRI-detected breast cancers is necessary to establish its ultimate value. Women with cancer and a BRCA mutation may benefit from tailored treatments, such as cisplatin or olaparib. The treatment goals for a woman with a BRCA-associated breast cancer should be to prevent recurrence of the initial cancer and to prevent second primary breast and ovarian cancers. Rijnsburger et al. (2010) evaluated the long-term results including separate analyses of BRCA1 and BRCA2 mutation carriers and first results on survival. Women with higher than 15% cumulative lifetime risk (CLTR) of breast cancer were screened with biannual clinical breast examination and annual mammography and magnetic resonance imaging (MRI). Participants were divided into subgroups: carriers of a gene mutation (50% to 85% CLTR) and two familial groups with high (30% to 50% CLTR) or moderate risk (15% to 30% CLTR). The authors update contains 2,157 eligible women including 599 mutation carriers (median follow-up of 4.9 years from entry) with 97 primary breast cancers detected (median follow-up of 5.0 years from diagnosis). MRI sensitivity was superior to that of mammography for invasive cancer (77.4% v 35.5%; P<.00005), but not for ductal carcinoma in situ. Results in the BRCA1 group were worse compared to the BRCA2, the high, and the moderate-risk groups, respectively, for mammography sensitivity (25.0% v 61.5%, 45.5%, 46.7%), tumor size at diagnosis 1 cm (21.4% v 61.5%, 40.9%, 63.6%), proportion of DCIS (6.5% v 18.8%, 14.8%, 31.3%) and interval cancers (32.3% v 6.3%, 3.7%, 6.3%), and age at diagnosis younger than 30 years (9.7% v 0%). Cumulative distant metastasis-free and overall survival at 6 years in all 42 BRCA1/2 mutation carriers with invasive breast cancer were 83.9% (95% CI, 64.1% to 93.3%) and 92.7% (95% CI, 79.0% to 97.6%), respectively, and 100% in the familial groups (n=43). Screening results were somewhat worse in BRCA1 mutation carriers, but 6-year survival was high in all risk groups. Kurian et al. (2010) Women with BRCA1/2 mutations inherit high risks of breast and ovarian cancer; options to reduce cancer mortality include prophylactic surgery or breast screening, but their efficacy has never been empirically compared. The authors used decision analysis to simulate risk-reducing strategies in BRCA1/2 mutation carriers and to compare resulting survival probability and causes of death. A Monte Carlo model of breast screening was developed with annual mammography plus magnetic resonance imaging (MRI) from ages 25 to 69 years, prophylactic mastectomy (PM) at various ages, and/or prophylactic oophorectomy (PO) at ages 40 or 50 years in 25-year-old BRCA1/2 mutation carriers. With no intervention, survival probability by age 70 is 53% for BRCA1 and 71% for BRCA2 mutation carriers. The most effective single intervention for BRCA1 mutation carriers is PO at age 40, yielding a 15% absolute survival gain; for BRCA2 mutation carriers, the most effective single intervention is PM, yielding a 7% survival gain if performed at age 40 years. The combination of PM and PO at age 40 improves survival more than any Genetic Testing for BRCA1 and BRCA2 Sep 15 17

18 single intervention, yielding 24% survival gain for BRCA1 and 11% for BRCA2 mutation carriers. PM at age 25 instead of age 40 offers minimal incremental benefit (1% to 2%); substituting screening for PM yields a similarly minimal decrement in survival (2% to 3%). Although PM at age 25 plus PO at age 40 years maximizes survival probability, substituting mammography plus MRI screening for PM seems to offer comparable survival. These results may guide women with BRCA1/2 mutations in their choices between prophylactic surgery and breast screening. Malone et al. (2010) completed a nested case-control study that assessed the risk of subsequent contralateral breast cancer associated with carrying a BRCA1 or BRCA2 mutation. Patients with contralateral breast cancer diagnosed 1 year or more after a first primary breast cancer (n = 705) and controls with unilateral breast cancer (n = 1,398) were ascertained from an underlying population-based cohort of 52,536 women diagnosed with a first invasive breast cancer before age 55 years. Interviews and medical record reviews were used to collect risk factor and treatment histories. All women were tested for BRCA1/BRCA2 mutations. Relative (rate ratios) and absolute (5- and 10-year cumulative) risks of developing contralateral breast cancer following a first invasive breast cancer were computed. Compared with noncarriers, BRCA1 and BRCA2 mutation carriers had 4.5-fold (95% CI, 2.8- to 7.1-fold) and 3.4- fold (95% CI, 2.0- to 5.8-fold) increased risks of contralateral breast cancer, respectively. The relative risk of contralateral breast cancer for BRCA1 mutation carriers increased as age of first diagnosis decreased. Age-specific cumulative risks are provided for clinical guidance. The risks of subsequent contralateral breast cancer are substantial for women who carry a BRCA1/BRCA2 mutation. These findings have important clinical relevance regarding the assessment of BRCA1/BRCA2 status in patients with breast cancer and the counseling and clinical management of patients found to carry a mutation. Scientific Rationale Update Septemeber 2010 BRCA1 and BRCA2 are among several known susceptibility genes that have been associated with breast cancer and are thought to account for the majority of inherited breast cancers. The prevalence of BRCA mutations is highest among women of Ashkenazi Jewish descent. BRACAnalysis (Myriad Genetics Inc.) is a genetic test to identify mutations in the BRCA1 and BRCA2 genes. The test includes analysis for five common BRCA1 genomic rearrangements (large deletions and duplications). Large genomic rearrangements are estimated to account for approximately 5-10% of all disease-causing mutations in BRCA1 and BRCA2 genes in patients with hereditary breast and ovarian cancer syndrome (HBOC). According to the Myriad Genetics Inc, The BRAC Analysis Rearrangement Test, or BART, launched in August 2006, is designed to detect rare large rearrangements beyond the five tested in the BRAC Analysis test. The added test detects rare, large rearrangements of the DNA in the BRCA1 and BRCA2 genes and will be performed for women with exceptionally high risk who have tested negative for sequence mutations and the common large rearrangements already included in Myriad's test. According to Myriad, BART will automatically be performed concurrently with the sequence analysis of Comprehensive BRAC Analysis when any of the following criteria is met: Patient affected with: Breast cancer before age 50 Ovarian cancer at any age Additional Family History Required: 2 or more diagnoses of breast cancer before age 501 and/or ovarian cancer at any age2 2 or more diagnoses of breast cancer before age 501 and/or ovarian cancer at any age 2 Genetic Testing for BRCA1 and BRCA2 Sep 15 18

19 Male breast cancer at any age Breast cancer at or after age 50 and ovarian cancer at any age Breast cancer before age 50 and ovarian cancer at any age 2 or more diagnoses of breast cancer before age 501 and/or ovarian cancer at any age2 1 or more diagnoses of breast cancer before age 501 and/or ovarian cancer at any age2 No additional relatives required 1. Male breast cancer qualifies at any age 2. At least one relative must be a first or second degree relative and qualifying cancers must be on the same side of the family Note: For the above criteria, breast cancer includes ductal carcinoma in situ (DCIS) and invasive breast cancers. According to Myriad, if the clinical criteria are met, BART will automatically be performed concurrently with Comprehensive BRACAnalysis. According to the National Society of Genetic Counselors: "When a mutation has not been previously identified in the family, a "no mutation detected" result in an affected patient means that the current technology did not find a mutation in BRCA1 or BRCA2. The cause of the pattern of cancer in the client and the family is still undetermined, and the risk assessment must be based on the clinical history. There are three possible explanations for a "no mutation detected" result; for an individual patient, it is necessary to consider which is most likely by reviewing the patient's personal and family histories of cancer, and considering the pre-test probability of detecting a mutation in BRCA1 or BRCA2: The cancer history may be due to the combined effects of chance, environmental factors, and lifestyle factors, as opposed to a mutation in a single gene. A BRCA1 or BRCA2 mutation is present, but current technology is not able to detect such a change. When interpreting a "no mutation detected" test result, it is important for the healthcare provider to have an understanding of the testing methodology used by the laboratory and its estimated sensitivity. For clients that meet defined clinical criteria, it may be appropriate to request additional analysis to detect large genomic rearrangements in both BRCA1 and BRCA2 genes. Research opportunities may be available for families with a significant history of breast and ovarian cancer, in whom no mutation was found through clinical testing. Such families should be encouraged to contact their genetic counselor on a regular basis, to determine if additional technology has been added to the clinically available testing methods that may find a previously undetectable mutation. The cancer history in the client or family may be due to a mutation in a different set of genes. Mutations in BRCA1 or BRCA2 are responsible for the majority of strong family histories of early-onset breast cancer and ovarian cancer. Among for some individuals that meet defined clinical criteria, it may be appropriate to request additional analysis to detect large genomic rearrangements in both BRCA1 and BRCA2 genes." Genetic Testing for BRCA1 and BRCA2 Sep 15 19

20 Per the National Comprehensive Care Network (NCCN) Clinical Practice Guidelines in Oncology, Genetic/Familial High-Risk Assessment: Breast and Ovarian: It is important to mention that certain large genomic rearrangements are not detectable by a primary sequencing assay thereby necessitating supplementary testing, in some cases. For example, there are tests that detect rare, large cancer-associated rearrangements of DNA in the BRCA1 and BRCA2 genes that not detected by sequencing the BRCA 1/2 genes. The National Cancer Institute notes that, "Mutation-screening methods vary in their sensitivity. Methods widely used in research laboratories, such as single-stranded conformational polymorphism (SSCP) analysis and conformation-sensitive gel electrophoresis (CSGE), miss nearly a third of the mutations that are detected by DNA sequencing. In addition, large genomic alterations such as translocations, inversions, or large deletions or insertions are missed by most of the techniques, including direct DNA sequencing, but testing for these are commercially available. Such rearrangements are believed to be responsible for 12% to 18% of BRCA1 inactivating mutations but are less frequently seen in BRCA2 and in individuals of Ashkenazi Jewish descent." Walsh et al (2006) investigated the frequency and types of undetected cancerpredisposing mutations in BRCA1, BRCA2, CHEK2, TP53, and PTEN among patients with breast cancer from high-risk families with negative (wild-type) genetic test results for BRCA1 and BRCA2. Probands from 300 US families with 4 or more cases of breast or ovarian cancer but with negative (wild-type) commercial genetic test results for BRCA1 and BRCA2 were screened by multiple DNA-based and RNA-based methods to detect genomic rearrangements in BRCA1 and BRCA2 and germline mutations of all classes in CHEK2, TP53, and PTEN. The main outcome measure was the frequency of mutations in previously undetected germline mutations in BRCA1, BRCA2, CHEK2, TP53, and PTEN that predispose to breast cancer, among families with negative genetic test results. The investigators reported of the 300 probands, 52 (17%) carried previously undetected mutations, including 35 (12%) with genomic rearrangements of BRCA1 or BRCA2, 14 (5%) with CHEK2 mutations, and 3 (1%) with TP53 mutations. At BRCA1 and BRCA2, 22 different genomic rearrangements were found, of sizes less than 1 kb to greater than 170 kb; of these, 14 were not previously described and all were individually rare. At CHEK2, a novel 5.6-kb genomic deletion was discovered in 2 families of Czechoslovakian ancestry. This deletion was found in 8 of 631 (1.3%) patients with breast cancer and in none of 367 healthy controls in the Czech and Slovak Republics. For all rearrangements, exact genomic breakpoints were determined and diagnostic primers validated. The 3 families with TP53 mutations included cases of childhood sarcoma or brain tumors in addition to multiple cases of breast cancer. The investigators concluded the mutational spectra of BRCA1 and BRCA2 include many high-penetrance, individually rare genomic rearrangements. Among patients with breast cancer and severe family histories of cancer who test negative (wild type) for BRCA1 and BRCA2, approximately 12% can be expected to carry a large genomic deletion or duplication in one of these genes, and approximately 5% can be expected to carry a mutation in CHEK2 or TP53. Effective methods for identifying these mutations should be made available to women at high risk. Wenstrup et al (2007) reported on the clinical assay for large rearrangements referred to as BART (BRCA1/2 Rearrangement Test) developed by Myriad Lab. The performance of BART was validated using a completely anonymous large number of Genetic Testing for BRCA1 and BRCA2 Sep 15 20