Use of panel tests in place of single gene tests in the cancer genetics clinic

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1 Clin Genet 2015: 88: Printed in Singapore. All rights reserved CLINICAL GENETICS doi: /cge Short Report se of panel tests in place of single gene tests in the cancer genetics clinic Yorczyk A, Robinson LS, Ross TS. se of panel tests in place of single gene tests in the cancer genetics clinic. Clin Genet 2015: 88: The Authors. Clinical Genetics published by John Wiley & Sons A/S. Published by John Wiley & Sons Ltd., 2014 Improved technology has made it possible to test for mutations within multiple genes simultaneously. It is not clear when these gene panels should be used in the hereditary cancer setting. These analyses were intended to guide panel testing criteria. Offering hereditary panel testing as a first and final, single-tier, option was explored. A two-tiered approach, in which panel testing is offered reflexively following stricter criteria, was then applied to the same data. Within our cohort of 105 patients, the single-tier approach was associated with a higher mutation detection rate (6.7% vs 3.8%) and variant of uncertain significance (VS) rate (0.94 vs 0.23 average per person) compared to a two-tiered approach. Of the VSs also identified in other patients by another lab, 53% were classified differently between laboratories. Individuals reporting African American race had more VSs compared to other ancestry groups (p = 0.001). The test cost for a single-tier test was 21% more than a two-tiered approach. Single-tier panel testing was associated with higher mutation and VS rates, and there is inconsistent classification of the VS/low penetrant genes between laboratories. Conflict of interest The authors report no conflicts of interest. A. Yorczyk a,b, L.S. Robinson a,b and T.S. Ross a,b a Department of Cancer Genetics, niversity of Texas Southwestern Medical Center s Harold Simmons Comprehensive Cancer Center, Dallas, TX, SA and b Department of Cancer Genetics, niversity of Texas Southwestern Medical Center s Moncrief Cancer Institute, Fort Worth, TX, SA Key words: hereditary cancer next-generation sequencing panel testing Corresponding author: Theodora S. Ross, Department of Cancer Genetics, T Southwestern Medical Center, 5323 Harry Hines Blvd, MC 8852, Dallas, TX , SA. Tel.: ; fax: ; theo.ross@utsouthwestern.edu Received 17 May 2014, revised and accepted for publication 18 August 2014 Individuals with hereditary predispositions to cancer are at an increased risk to develop specific cancers compared to the general population. Next-generation sequencing (NGS) has allowed for the ability to analyze multiple genes simultaneously in panel tests at a reduced cost compared to Sanger sequencing (1, 2). Recently, we gained the ability to apply NGS technology to diagnose hereditary cancer syndromes accurately (3). Two-tiered strategies for incorporating hereditary cancer panels into a testing algorithm have been proposed (4, 5). This involves initial testing for genes with higher penetrance such as BRCA1/2 and reflexing to hereditary panels upon return of a negative results based on the level of risk and consideration of other syndromes (5). As part of the early phase for the panel test offered by Myriad Genetics, select providers were given the opportunity to offer a single, multi-cancer gene panel test at no extra cost to patients meeting specific criteria for hereditary breast and colon cancer testing. sing these data, we assessed the benefits and limitations of single-tier hereditary cancer panel testing, compared to the previously proposed two-tiered approach (5). The mutation rates, variant of undetermined significance (VS) rates, and test costs were compared between the two approaches. Materials and methods The panel test used, MyRisk, in the single-tier option included 25 genes; APC, ATM, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A, CHEK2, EPCAM, MLH1, MSH2, MSH6, MTYH, NBN, PALB2, PMS2, PTEN, RAD51C, RAD51D, SMAD4, STK11, and TP53. Patients were eligible for the panel test as a first-tier test at no extra cost if they The Authors. Clinical Genetics published by John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

2 se of panel tests in the cancer genetics clinic 468 new patients seen by a genetic 370 underwent hereditary testing 98 did not meet criteria for testing or were unable to undergo testing for other reasons 318 qualified for BRCA1 or BRCA2 gene testing, or Lynch syndrome testing 52 patients underwent hereditary testing, but did not qualify for BRCA1or BRCA2 gene testing, or Lynch syndrome testing 301 underwent germline testing 17 underwent Lynch syndrome tumor testing that was either negative or guided gene-specific testing 132 underwent the hereditary cancer panel test offered by Myriad Genetics 169 excluded 143 patients did not undergo hereditary panel testing 26 patients underwent testing through a different laboratory 27 tests were either not resulted in time or were cancelled 105 tests results were returned for study participation Fig. 1. Details of patient accrual. All patients evaluated in our cancer genetics clinics during the fall of 2013 were offered panel testing if they qualified for BRCA1/2 or Lynch Syndrome testing. met their health insurance criteria for BRCA1/2 or Lynch syndrome gene testing. Patients were excluded from this multi-cancer syndrome panel if they underwent prior BRCA1/2 or Lynch syndrome gene testing, required a saliva sample, or needed a quick turn-around time. All patients meeting these criteria were offered the hereditary multi-cancer panel test and genetic counseling. Although the original goal was to analyze the first 100 results, 6 results were received on the final day, so 105 patients were included (Fig. 1). Following this single-tier approach, a review was performed to determine deleterious mutation rates and VSs rates. Genetics also provided their interpretations of the VSs identified in these patients. The significance of the average differences in the number of VSs between ancestry categories were evaluated using a one-way anova statistical test. sing the data from the same patient cohort, mutation and VS rates that would have resulted if the patient population had undergone a stricter, two-tiered approach were calculated based on the criteria originally proposed by Mauer et al. (Table S1, Supporting Information) (5). We also compared the costs of the single-tier method to a two-tiered method. The client prices for various NGS cancer panels, BRCA gene testing, and Lynch syndrome gene testing were averaged. The prices from nine clinical labs were used to determine average cost per person of the single-tier and two-tiered methods. Results From September through November 2013, 468 patients were seen for genetic counseling (Fig. 1). Of these, 370 elected to proceed with genetic testing, and

3 Yorczyk et al. Table 1. Deleterious mutations detected using single-tier method vs two-tier method Actionable mutations reported in our cohort by single-tier panel testing Mutation detected using single-tier (panel-only) method Mutation detected using two-tiered method Mutation detected in the first tier Mutation detected in the reflexive second tier APC c.3920t>a (I1307K) Yes No No BRCA1 c t>g Yes Yes n/a BRCA1 c t>g Yes Yes n/a BRCA2 c.9302del Yes Yes n/a BRCA2 del exons Yes Yes n/a CHEK2 c.1555c>t Yes No No EPCAM 3 term del Yes No No qualified for BRCA1/2 testing and/or Lynch syndrome testing. Of these, 17 had previous colon tumor testing that either obviated the need for germline testing or guided gene-specific testing. Of the remaining 301 patients, a total of 169 patients did not have the panel test because they met Myriad s exclusion criteria (see Materials and Methods) or they had panel testing through another lab (n = 26). A total of 132 patients (44%) underwent panel testing by Myriad Genetics and the first 105 test results were evaluated. Of the 105 study patients, 100 (95.2%) were female. Seventeen patients (16.2%) reported African American, two (1.9%) Ashkenazi Jewish, three (2.9%) Asian, thirteen (12.4%) Hispanic, Latin American, or Caribbean, six (5.7%) Native American, and 72 (68.6%) European descent. A total of 61 (58%) had a current or prior cancer diagnosis. sing the hereditary cancer panel as a single-tier test for individuals who met BRCA1/2 or Lynch syndrome testing criteria, seven individuals (6.7%) were reported to have a deleterious or suspected deleterious mutation. These were within APC, BRCA1/2, CHEK2, orepcam gene (Table 1). The seven mutation patients met NCCN guidelines for BRCA1/2 gene testing. One of these mutations (APC c.3920t>a) was classified as suspected deleterious. We next analyzed our data using a two-tiered method (Table S1) in which the most likely hereditary cancer syndrome is tested first then reflexing to panel if no mutation was detected. Four (3.8%) of the seven mutations would have been detected (all BRCA1/2) (Table 1). Of the 105 patients examined, 32 patients would have been recommended for a second-tier, reflex panel test following the criteria from Mauer et al. (Table S1) (5). No additional mutations would have been detected in the second tier in our cohort based on these criteria. The single-tier panel test approach increased our detection rate to 6.7% from 3.8% where three additional reported mutations (APC, CHEK2, and EPCAM), were detected. The family and medical history were not characteristic of the predicted phenotypes associated with these genes. sing the single-tier approach, 74 unique VSs were called by Myriad where each panel gene had a VSs at least once in this cohort (Table S2). We compared the interpretation of these variants with another laboratory, Genetics. Of the 74 VSs found in the study, observed and interpreted 32 of them. Of these 32 VSs, 17 (53%) were classified differently by (Table 2). One of the six deleterious mutations, the well-studied APC alteration (p.i1307k) would have been classified as a pathogenic mutation with reduced penetrance by Genetics. This difference reflects the considerable debate about the clinical significance of this mutation. Thirteen of the Myriad VSs would have been reported as polymorphisms by Genetics, and three of the VSs would have been classified as likely benign. One variant reported by Myriad (CHEK2 c.470>t) would have been reported as a moderate penetrance mutation with a comment regarding variant specific risks. Thirty-three VSs reported by Myriad were not yet classified by at the time of data collection and nine intronic variants would not have been reported by. sing the single-tier approach, 99 VSs (0.94 per person) were detected overall (Table S3). If a stricter, two-tiered approach had been applied, only 24 VSs (0.23 per person) would have been detected. As expected, those who reported African American ethnicity had statistically higher rate of VSs compared to other ethnicities (p = 0.001). sing the average cost of hereditary panels from various labs, the cost of a single-tier, panel-only test would have been $4099 per person, or an estimated $430,425 for all 105 patients. Had a two-tiered approach been used instead, $2190 would have been spent per person on the first-tier test, and $3392, would have been spent in test charges per person upon completion of the second tier ($356,244 in total) (Table S3). The cost of the single-tier hereditary panel test was therefore 21% higher compared to the stricter two-tiered approach. Discussion As the cancer genetic community is moving to NGS panel testing, there are still many unanswered questions. By analyzing our cohort of patients that had initial panel testing against published two-tiered testing criteria, we found that more mutations would be found, but at a higher test cost. In addition, the types of mutations found were not always expected based on the family 280

4 se of panel tests in the cancer genetics clinic Table 2. The VSs and mutations reported by Myriad Genetics were not all interpreted the same by Genetics at the time of test report a Myriad VS APC c.1145g>a APC c.4336g>a APC c.6670a>g APC c.721g>a APC c.8161c>t ATM c.1132a>g ATM c.1430a>g ATM c.1464g>t ATM c.1516g>t ATM c.1810c>t ATM c.186-7c>t ATM c.2289t>a ATM c.2362a>c ATM c.2442c>a ATM c.320g>a ATM c.334g>a ATM c.4388t>g ATM c a>g ATM c.6088a>g ATM c.6919c>t BARD1 c.1216c>g BARD1 c.1738g>a BARD1 c.1793c>a BARD1 c.776a>g BARD1 c.899c>t BMPR1A c A>G BRCA1 c.2346t>a BRIP1 c.151g>a BRIP1 c.205g>a BRIP1 c.258_269del BRIP1 c.2873t>a BRIP1 c.890a>g CDH1 c c>g CDH1 c.688-4t>c CDK4 c.776c>t CDKN2A c.-25c>t CDKN2A c.-2g>a CDKN2A c.316g>a CDKN2A c.369t>a CDKN2A c.430c>t CHEK2 c.1510g>c CHEK2 c.382c>a CHEK2 c.410g>a CHEK2 c.442a>g CHEK2 c.470t>c CHEK2 c.707t>c CHEK2 c.715g>a MLH1 c.1245t>g MLH1 c.2217t>g MLH1 c.2239c>t MLH1 c.-25t>c MSH2 c.1004c>g MSH2 c c>a MSH6 c.1028c>t MSH6 c G>A MTYH c.1450g>a MTYH c.821g>a VS VS VS VS VS VS VS VS b VS VS VS VS Table 2. Continued Myriad VS MTYH c.925c>t NBN c.1489a>g NBN c.1882g>a NBN c G>A NBN c.680t>c PALB2 c.2816t>g PMS2 c.1438g>c PTEN c _802-14del RAD51C c.146-8a>g RAD51C c.431t>c RAD51C c.790g>a RAD51D c.362a>g RAD51D c.698a>g SMAD4 c.1573a>g STK11 c.835g>a TP53 c.1079g>c TP53 c.97-6c>t Myriad mutations APC c.3920t>a b BRCA1 c t>g BRCA2 c.9302del BRCA2 del exons CHEK2 c.1555c>t EPCAM 3 term del VS VS VS c, deleterious/suspected deleterious mutation;, not reported; = polymorphism; = unknown; = variant likely benign. a Intronic alterations beyond ± 5 are only reported by Genetics if they are pathogenic or likely pathogenic mutations. b reported as suspected deleterious ; test report describes fold increased risk for colon cancer. c Reduced penetrance. history and are not well-defined in terms of their clinical significance. In our population, we found an APC mutation (I1307K) in a male non-jewish breast cancer patient, with a mother and grandfather with late onset colon cancer. Thus, the APC gene mutation could help to explain the colon cancer in his family and would not have been detected with two-tiered testing. Another woman with breast cancer at 39 was found to have a CHEK2 c.1555c>t mutation. Her father reportedly had colon cancer at age 46 and a maternal uncle had colon cancer at age 65. The patient did not meet previously proposed criteria (5) for two-tiered testing following negative BRCA1/2 gene testing, nor did she meet criteria previously outlined for CHEK2 gene testing (5). The third mutation, EPCAM 3 term del, was found in a 54-year-old unaffected female who had a family history of breast and ovarian cancer, but no colon cancer. This mutation was not expected based on the family history. It is possible that these patients had incorrectly documented their family histories (6). Offering first-tier panel testing, which includes genes tertiary on the differential, could prevent inaccurate, or even limited, family histories from restricting testing options. 281

5 Yorczyk et al. One of the unexpected findings in our study was the differences between labs classifications of the 74 unique VSs reported by Myriad. Of the 32 variants interpreted by both and Myriad, 17 (53%) were classified differently between the labs. Each lab has a different process for determining the significance of variants, and clinicians need to be aware of these inconsistencies. Sharing information on variant classifications will improve patient care by speeding up the VSs reclassification process. Another challenge of panel testing is the high VSs rate. Individuals of African American ancestry comprised 16.2% of our cohort and had a greater VS frequency compared to those of other ancestries (p = 0.001). Panel testing will certainly result in the detection of moderate penetrance genes, such as ATM and CHEK2. No consensus on recommendations for management or surveillance exists for lower-penetrant genes (3, 7), nor for individuals found to have multiple mutations of moderate penetrance (7). There is also an issue of finding unexpected mutations, such as the EPCAM mutation in our cohort. Clinicians need to counsel patients about unexpected genetic findings or findings that may not affect the management. Of the seven initially reported deleterious mutation or suspected deleterious mutation carriers, six (5.7%) were clinically actionable. The remaining variant, APC c.3920t>a (I1307K) was reported as suspected deleterious by Myriad Genetics with special interpretation that indicated a lower penetrance than standard loss of function APC mutations. Literature review indicates controversy over whether this mutation is associated with a lower penetrance (1.5 to 1.9-fold increased non-polyposis colon cancer risk) (8) or no increased risk (9), thus leading to different interpretations by different laboratories. Single-tier panel test cost was on average $4099 per person, compared to $3392 per person for a two-tiered test. Thus, it was $707 more per person, or $74,235 altogether, to increase the detection rate by 2.9%, and to detect 75% more mutations in our cohort of 105 individuals. This is approximately the cost of treatment for one cancer. Although this is not a complete cost analysis, it appears that offering panel testing as a single-tier test would be cost-effective given the higher detection rate and the elimination of the costs associated with a return to clinic for further testing. Conclusion This is the first analysis that compares the outcomes of a single-tier, panel-only approach to the outcomes that would have resulted using a two-tiered approach. se of a single-tier approach led to clinical reports of three deleterious mutations that would have otherwise gone undetected. There was also a higher VSs rate in the single-tier approach. Similarly, both provider and patient should be aware of the potential to find lower-penetrant mutations for which management recommendations may or may not exist. There was also variability in the interpretation of VSs and lower-penetrant genes between laboratories; data-sharing would help reduce these inconsistencies. The increased diagnosis rate with first-tier hereditary cancer panel testing suggests that this strategy should be discussed with all patients, as the benefits may outweigh the risks and costs for different patients. Supporting Information Additional supporting information may be found in the online version of this article at the publisher s web-site. Acknowledgements We thank Myriad Genetics for the opportunity to perform this study with their MyRisk test. We also thank Genetics, especially Laura Panos and Jill Dolinsky and their colleagues. Finally, we are grateful to the staff of the T Southwestern Genetics group and Marion-Stewart Thomas for her administrative assistance. References 1. Walsh T, Lee MK, Casadei S et al. Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing. Proc Natl Acad Sci S A 2010: 107: Ku CS, Cooper DN, Iacopetta B et al. Integrating next-generation sequencing into the diagnostic testing of inherited cancer predisposition. Clin Genet 2013: 83: Domchek SM, Bradbury A, Garber JE et al. Multiplex genetic testing for cancer susceptibility: out on the high wire without a net? 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