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Multi-Gene Panel Testing of 23,179 Individuals for Hereditary Cancer Risk Identifies Pathogenic Variant Carriers Missed by Current Genetic Testing Guidelines

Open AccessPublished:June 11, 2019DOI:https://doi.org/10.1016/j.jmoldx.2019.03.001
      Recent advancements in next-generation sequencing have greatly expanded the use of multi-gene panel testing for hereditary cancer risk. Although genetic testing helps guide clinical diagnosis and management, testing recommendations are based on personal and family history of cancer and ethnicity, and many carriers are being missed. Herein, we report the results from 23,179 individuals who were referred for 30-gene next-generation sequencing panel testing for hereditary cancer risk, independent of current testing guidelines—38.7% of individuals would not have met National Comprehensive Cancer Network criteria for genetic testing. We identified a total of 2811 pathogenic variants in 2698 individuals for an overall pathogenic frequency of 11.6% (9.1%, excluding common low-penetrance alleles). Among individuals of Ashkenazi Jewish descent, three-quarters of pathogenic variants were outside of the three common BRCA1 and BRCA2 founder alleles. Across all ethnic groups, pathogenic variants in BRCA1 and BRCA2 occurred most frequently, but the contribution of pathogenic variants in other genes on the panel varied. Finally, we found that 21.7% of individuals with pathogenic variants in genes with well-established genetic testing recommendations did not meet corresponding National Comprehensive Cancer Network criteria. Taken together, the results indicate that more individuals are at genetic risk for hereditary cancer than are identified by current testing guidelines and/or use of single-gene or single-site testing.
      Recent advancements in next-generation sequencing have greatly expanded the use of multi-gene testing panels in clinical diagnosis and management. Multi-gene panels are more sensitive and efficient than traditional testing paradigms and are increasingly more affordable. Furthermore, multi-gene panels increase the likelihood of detecting an underlying germline genetic component in diseases with genetic heterogeneity, such as cancer.
      • Rosenthal E.T.
      • Evans B.
      • Kidd J.
      • Brown K.
      • Gorringe H.
      • van Orman M.
      • Manley S.
      Increased identification of candidates for high-risk breast cancer screening through expanded genetic testing.
      Approximately 5% to 10% of all cancers are associated with hereditary cancer syndromes, most of which are inherited in an autosomal-dominant manner with high-to-moderate penetrance.
      • Nagy R.
      • Sweet K.
      • Eng C.
      Highly penetrant hereditary cancer syndromes.
      As such, the current approach to germline cancer testing is to recommend testing only for those individuals with a personal or family history that indicates an increased risk of disease or presence of a known familial pathogenic variant. However, because of phenotypic variability, age-related penetrance, and sex-specific cancer risks, this approach misses many carriers.
      • Grindedal E.M.
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      • Norum J.
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      Current guidelines for BRCA testing of breast cancer patients are insufficient to detect all mutation carriers.
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      Cost-effectiveness of population-based BRCA1, BRCA2, RAD51C, RAD51D, BRIP1, PALB2 mutation testing in unselected general population women.
      • Rosenthal E.T.
      • Bernhisel R.
      • Brown K.
      • Kidd J.
      • Manley S.
      Clinical testing with a panel of 25 genes associated with increased cancer risk results in a significant increase in clinically significant findings across a broad range of cancer histories.
      For example, recent studies have shown that up to half of women carrying BRCA1 and BRCA2 pathogenic variants had no family history of breast cancer.
      • King M.-C.
      • Levy-Lahad E.
      • Lahad A.
      Population-based screening for BRCA1 and BRCA2: 2014 Lasker Award.
      • Manchanda R.
      • Legood R.
      • Burnell M.
      • McGuire A.
      • Raikou M.
      • Loggenberg K.
      • Wardle J.
      • Sanderson S.
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      • Side L.
      • Balogun N.
      • Desai R.
      • Kumar A.
      • Dorkins H.
      • Wallis Y.
      • Chapman C.
      • Taylor R.
      • Jacobs C.
      • Tomlinson I.
      • Beller U.
      • Menon U.
      • Jacobs I.
      Cost-effectiveness of population screening for BRCA mutations in Ashkenazi Jewish women compared with family history-based testing.
      In another study of 360 ovarian cancer patients, three women with TP53 pathogenic variants had no family history of Li-Fraumeni syndrome, and two individuals with MSH6 pathogenic variants had no family history of Lynch syndrome.
      • Walsh T.
      • Casadei S.
      • Lee M.K.
      • Pennil C.C.
      • Nord A.S.
      • Thornton A.M.
      • Roeb W.
      • Agnew K.J.
      • Stray S.M.
      • Wickramanayake A.
      • Norquist B.
      • Pennington K.P.
      • Garcia R.L.
      • King M.-C.
      • Swisher E.M.
      Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing.
      These prior studies indicate a need in the field to perform a more systemic evaluation of the efficacy of personal- and family history–based screening as a prequalifier for genetic testing.
      This study analyzed the results of 23,179 individuals who received a 30-gene next-generation sequencing panel for risk of hereditary breast, ovarian, uterine/endometrial, colorectal, melanoma, pancreatic, prostate, and stomach cancer. Herein, we provide data on the frequency and spectrum of pathogenic or likely pathogenic variants by variant type, personal history of cancer, and ethnicity. More important, as these individuals were referred for genetic testing independent of testing guidelines, the results were also evaluated with respect to the genetic/familial high-risk assessments provided by the National Comprehensive Cancer Network (NCCN).
      • Daly M.B.
      • Pilarski R.
      • Berry M.
      • Buys S.S.
      • Farmer M.
      • Friedman S.
      • Garber J.E.
      • Kauff N.D.
      • Khan S.
      • Klein C.
      • Kohlmann W.
      • Kurian A.
      • Litton J.K.
      • Madlensky L.
      • Merajver S.D.
      • Offit K.
      • Pal T.
      • Reiser G.
      • Shannon K.M.
      • Swisher E.
      • Vinayak S.
      • Voian N.C.
      • Weitzel J.N.
      • Wick M.J.
      • Wiesner G.L.
      • Dwyer M.
      • Darlow S.
      NCCN Guidelines Insights: genetic/familial high-risk assessment: breast and ovarian, version 2.2017.
      • Provenzale D.
      • Gupta S.
      • Ahnen D.J.
      • Bray T.
      • Cannon J.A.
      • Cooper G.
      • David D.S.
      • Early D.S.
      • Erwin D.
      • Ford J.M.
      • Giardiello F.M.
      • Grady W.
      • Halverson A.L.
      • Hamilton S.R.
      • Hampel H.
      • Ismail M.K.
      • Klapman J.B.
      • Larson D.W.
      • Lazenby A.J.
      • Lynch P.M.
      • Mayer R.J.
      • Ness R.M.
      • Regenbogen S.E.
      • Samadder N.J.
      • Shike M.
      • Steinbach G.
      • Weinberg D.
      • Dwyer M.
      • Darlow S.
      Genetic/familial high-risk assessment: colorectal version 1.2016, NCCN Clinical Practice Guidelines in Oncology.
      National Comprehensive Cancer Network, Inc
      NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) Gastric Cancer Version 1.2017.

      Materials and Methods

       Participants

      The cohort in this retrospective study included 23,179 individuals who had Color Hereditary Cancer Test (Color Genomics, Inc., Burlingame, CA) results reported between May 2016 and September 2017. All individuals were ordered the test by a health care provider and gave informed consent to have their deidentified information used in anonymized studies. This population was not specifically selected for any particular metric, including sex, age, ethnicity, or history of cancer.

       Data Collection

      All phenotypic information was reported by the individual through an interactive, collaborative online health history tool; information not provided was noted as such. Individuals who reported more than one ethnicity were counted as multiple ethnicities, with the following exception: any individuals who reported Ashkenazi Jewish in addition to any other ancestry were counted as Ashkenazi Jewish.

       Multi-Gene Panel

      The Color Hereditary Cancer Test was used to analyze 30 genes in which pathogenic variants have been associated with an elevated risk of hereditary cancer, including breast, ovarian, uterine/endometrial, colorectal, melanoma, pancreatic, prostate, and stomach. This test is adapted from the multi-gene panel validated in Crawford et al.
      • Crawford B.
      • Adams S.B.
      • Sittler T.
      • van den Akker J.
      • Chan S.
      • Leitner O.
      • Ryan L.
      • Gil E.
      • van ’t Veer L.
      Multi-gene panel testing for hereditary cancer predisposition in unsolved high-risk breast and ovarian cancer patients.
      The 30 genes are APC, ATM, BAP1, BARD1, BMPR1A, BRCA1, BRCA2, BRIP1, CDH1, CDK4, CDKN2A (p14ARF and p16INK4a), CHEK2, EPCAM, GREM1, MITF, MLH1, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, POLD1, POLE, PTEN, RAD51C, RAD51D, SMAD4, STK11, and TP53. These genes were selected on the basis of published evidence of association with hereditary cancer and technical feasibility using the methods described below. Most of these genes were assessed for variants within all coding exons (±20 bp flanking each exon) and noncanonical splice regions. In PMS2, exons 12 to 15 could not be reliably assessed with the standard target enrichment protocol and, therefore, were not reported. In several genes, only specific positions known to impact cancer risk were analyzed (genomic coordinates in GRCh37): CDK4, only chromosome 12: g.58145429-58145431 (codon 24); MITF, only chromosome 3: g.70014091 (including c.952G>A); POLD1, only chromosome 19: g.50909713 (including c.1433G>A); POLE, only chromosome 12: g.133250250 (including c.1270C>G); EPCAM, only large deletions and duplications, including the 3′ end of the gene; and GREM1, only duplications in the upstream regulatory region.

       Laboratory Procedures

      Laboratory procedures were performed at the laboratory of Color Genomics, Inc. (Burlingame, CA) under Clinical Laboratory Improvement Amendments (number 05D2081492) and College of American Pathologists (number 8975161) compliance. DNA was extracted from blood or saliva samples and purified using the Perkin Elmer Chemagic DNA Extraction Kit (Perkin Elmer, Waltham, MA) automated on the Hamilton STAR (Hamilton, Reno, NV) and the Chemagic Liquid Handler (Perkin Elmer) instruments. The quality and quantity of the extracted DNA were assessed by UV spectroscopy (BioTek, Winooski, VT). High molecular weight genomic DNA was enzymatically fragmented and prepared using the Kapa HyperPlus Library Preparation Kit (Kapa Biosciences, Cape Town, South Africa) automated on the Hamilton STAR liquid handler. Target enrichment was performed with an automated (Hamilton STAR) hybrid capture procedure using SureSelect XT probes (Agilent, Santa Clara, CA) before being loaded onto the NextSeq 500/550 instrument (Illumina, San Diego, CA) for 150-bp paired-end sequencing.

       Bioinformatics Analysis

      Sequence reads were aligned against human genome reference GRCh37.p12 with the Burrows-Wheeler Aligner version 0.7.15,

      Li H: Aligning Sequence Reads, Clone Sequences and Assembly Contigs With BWA-MEM. arXiv 2013, arXiv:1303.3997v2 [q-bio.GN]

      and duplicate and low-quality reads were removed. Single-nucleotide variants and small insertions and deletions (2 to 50 bp) were called by the HaplotypeCaller module of GATK3.4.
      • DePristo M.A.
      • Banks E.
      • Poplin R.
      • Garimella K.V.
      • Maguire J.R.
      • Hartl C.
      • Philippakis A.A.
      • del Angel G.
      • Rivas M.A.
      • Hanna M.
      • McKenna A.
      • Fennell T.J.
      • Kernytsky A.M.
      • Sivachenko A.Y.
      • Cibulskis K.
      • Gabriel S.B.
      • Altshuler D.
      • Daly M.J.
      A framework for variation discovery and genotyping using next-generation DNA sequencing data.
      Variants in homopolymer regions were called by an internally developed algorithm using SAMtools version 1.8.
      • Li H.
      • Handsaker B.
      • Wysoker A.
      • Fennell T.
      • Ruan J.
      • Homer N.
      • Marth G.
      • Abecasis G.
      • Durbin R.
      1000 Genome Project Data Processing Subgroup
      The Sequence Alignment/Map format and SAMtools.
      Large structural variants (>50 bp) were detected using dedicated algorithms based on read depth (CNVkit version 0.8.5),
      • Talevich E.
      • Shain A.H.
      • Botton T.
      • Bastian B.C.
      CNVkit: genome-wide copy number detection and visualization from targeted DNA sequencing.
      paired reads, and split reads (LUMPY version 0.2.13,
      • Layer R.M.
      • Chiang C.
      • Quinlan A.R.
      • Hall I.M.
      LUMPY: a probabilistic framework for structural variant discovery.
      in-house developed algorithms). On pipeline completion, the sequencing run quality was checked. A no template control and two positive controls containing a set of known variants were concurrently run within every batch of samples. The coverage requirements for reporting were ≥20 unique reads (20×) for each base. Median coverage typically ranged between 200× and 300×.

       Variant Interpretation

      Variants were classified according to the American College of Medical Genetics and Genomics 2015 guidelines for sequence variant interpretation.
      • Richards S.
      • Aziz N.
      • Bale S.
      • Bick D.
      • Das S.
      • Gastier-Foster J.
      • Grody W.W.
      • Hegde M.
      • Lyon E.
      • Spector E.
      • Voelkerding K.
      • Rehm H.L.
      ACMG Laboratory Quality Assurance Committee
      Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.
      Every variant was reviewed by at least two variant scientists (R.O., R.C.C., E.C., Z.T., A.L., J.J., and A.Y.Z.), and all variant classifications were signed out by a board-certified medical geneticist or pathologist (Z.T., A.L., J.J., and S.T.). All pathogenic and likely pathogenic variants were confirmed on an orthogonal technology at an independent Clinical Laboratory Improvement Amendments–certified laboratory. Specifically, single-nucleotide variants and insertions and deletions were confirmed by Sanger sequencing, and structural variants were confirmed by variant-specific PCR, array comparative genomic hybridization, or multiplex ligation-dependent probe amplification.
      Results were reported as positive if one or more pathogenic or likely pathogenic variants (hereafter referred to as pathogenic variants) were detected and negative if no variant and/or only benign variants, likely benign variants, or variants of uncertain significance were detected at the time of data collection. Among the 30 genes tested, there are several alleles that are classified as pathogenic or likely pathogenic by multiple submitters in ClinVar but are known in the field to be commonly occurring and of low penetrance. Specifically, these include a single allele in APC, APC c.3920T>A (p.I1307K),
      • Prior T.W.
      • Chadwick R.B.
      • Papp A.C.
      • Arcot A.N.
      • Isa A.M.
      • Pearl D.K.
      • Stemmermann G.
      • Percesepe A.
      • Loukola A.
      • Aaltonen L.A.
      • De La Chapelle A.
      The I1307K polymorphism of the APC gene in colorectal cancer.
      and all monogenic pathogenic or likely pathogenic variants in MUTYH.
      • Cleary S.P.
      • Cotterchio M.
      • Jenkins M.A.
      • Kim H.
      • Bristow R.
      • Green R.
      • Haile R.
      • Hopper J.L.
      • LeMarchand L.
      • Lindor N.
      • Parfrey P.
      • Potter J.
      • Younghusband B.
      • Gallinger S.
      Germline MutY human homologue mutations and colorectal cancer: a multisite case-control study.
      This group of known high-frequency, low-penetrance alleles will be referred to as common low-penetrance alleles for brevity in the remainder of the article. Therefore, all pathogenic and likely pathogenic variant counts and frequency analyses have been reported as two values: one that includes all reported pathogenic variants and one that excludes the common low-penetrance alleles. To note, individuals with a monoallelic MUTYH pathogenic variant or APC p.I1307K were provided genetic testing reports that were distinct from genetic testing reports for biallelic MUTYH (ie, homozygous or compound heterozygous) pathogenic variants and other alleles in APC, respectively.

       NCCN Consideration of Genetic Testing

      Health history was assessed to determine whether individuals met or did not meet NCCN consideration for genetic testing, as provided by the Genetic/Familial High-Risk Assessment: Breast and Ovarian Version 2.2017 (BRCA1, BRCA2, TP53, and PTEN),
      • Daly M.B.
      • Pilarski R.
      • Berry M.
      • Buys S.S.
      • Farmer M.
      • Friedman S.
      • Garber J.E.
      • Kauff N.D.
      • Khan S.
      • Klein C.
      • Kohlmann W.
      • Kurian A.
      • Litton J.K.
      • Madlensky L.
      • Merajver S.D.
      • Offit K.
      • Pal T.
      • Reiser G.
      • Shannon K.M.
      • Swisher E.
      • Vinayak S.
      • Voian N.C.
      • Weitzel J.N.
      • Wick M.J.
      • Wiesner G.L.
      • Dwyer M.
      • Darlow S.
      NCCN Guidelines Insights: genetic/familial high-risk assessment: breast and ovarian, version 2.2017.
      the Genetic/Familial High-Risk Assessment: Colorectal Version 2.2016 [MLH1, MSH2, MSH6, PMS2, EPCAM, APC (excluding APC p.I1307K), biallelic MUTYH, SMAD4, and BMPR1A],
      • Provenzale D.
      • Gupta S.
      • Ahnen D.J.
      • Bray T.
      • Cannon J.A.
      • Cooper G.
      • David D.S.
      • Early D.S.
      • Erwin D.
      • Ford J.M.
      • Giardiello F.M.
      • Grady W.
      • Halverson A.L.
      • Hamilton S.R.
      • Hampel H.
      • Ismail M.K.
      • Klapman J.B.
      • Larson D.W.
      • Lazenby A.J.
      • Lynch P.M.
      • Mayer R.J.
      • Ness R.M.
      • Regenbogen S.E.
      • Samadder N.J.
      • Shike M.
      • Steinbach G.
      • Weinberg D.
      • Dwyer M.
      • Darlow S.
      Genetic/familial high-risk assessment: colorectal version 1.2016, NCCN Clinical Practice Guidelines in Oncology.
      and the Gastric Cancer Version 1.2017 (CDH1).
      National Comprehensive Cancer Network, Inc
      NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) Gastric Cancer Version 1.2017.
      Phenotypic information, used to evaluate if an individual met or did not meet criteria, is outlined in Supplemental Table S1. Individuals who did not provide sufficient health history information were excluded from analyses or noted as such.

       Data Statement

      The data that support the findings in this study are available on request from the corresponding author (A.Y.Z.). The data are not publicly available as they contain information that could compromise research participant privacy or consent. All reported variants have been submitted to ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/submitters/505849).

      Results

       Population Study

      Our cohort of 23,179 individuals received a 30-gene next-generation sequencing panel for detection of pathogenic variants associated with elevated risk of hereditary cancer. The demographics of these individuals are described in Table 1. Most were women (83.1%) and older than 40 years (73.3%), and approximately half were Caucasian (52.1%). Two-fifths of individuals in the cohort (42.4%) reported no personal history of cancer. A total of 3845 individuals (16.6%) had a personal history of breast cancer, 341 (1.5%) had ovarian cancer, 438 (1.9%) had colorectal cancer, and 1476 (6.4%) had a personal history of another hereditary cancer associated with genes on the panel.
      Table 1Demographics of Individuals Tested with the 30-Gene Next-Generation Sequencing Hereditary Cancer Panel
      VariableIndividuals, nPopulation, %Individuals with PV, n (excluding common low-penetrance alleles, n)Pathogenic frequency, % (excluding common low-penetrance alleles, %)
      Total23,179100.02698 (2116)11.6 (9.1)
      Sex
       Female19,26383.12156 (1694)11.2 (8.8)
       Male391616.9542 (422)13.8 (10.8)
      Age, years
       18–3017477.5245 (208)14.0 (11.9)
       31–40444719.2517 (427)11.6 (9.6)
       41–50554423.9611 (489)11.0 (8.8)
       51–65725531.3825 (627)11.4 (8.6)
       >65418618.1500 (365)11.9 (8.7)
      Ethnicity
       Caucasian12,08352.11413 (1166)11.7 (9.6)
       Ashkenazi Jewish23019.9364 (218)15.8 (9.5)
       Hispanic14586.3201 (180)13.8 (12.3)
       Multiple ethnicities8523.761 (46)7.2 (5.4)
       Asian8243.696 (80)11.7 (9.7)
       African2341.029 (23)12.4 (9.8)
       Native American640.39 (8)14.1 (12.5)
       Unknown
      Unknown includes information not provided.
      536323.1525 (395)9.8 (7.4)
      Personal cancer history
      Number of individuals with personal history of cancer exceeds 23,179 because of multiple reported cancer types.
       Breast384516.6602 (471)15.7 (12.2)
       Ovarian
      Ovarian cancer includes fallopian cancer and primary peritoneal cancer.
      3411.568 (59)19.9 (17.3)
       Endometrial/uterine2040.924 (18)11.8 (8.8)
       Colorectal4381.9101 (75)23.1 (17.1)
       Melanoma4461.963 (48)14.1 (10.8)
       Pancreatic1070.518 (14)16.8 (13.1)
       Prostate6722.992 (73)13.7 (10.9)
       Stomach470.211 (9)23.4 (19.1)
       Other cancer
      Other cancer includes hematological malignancies, kidney cancer, thyroid cancer, and other cancers.
      13245.7172 (126)13.0 (9.5)
       No cancer982442.41005 (696)10.2 (7.1)
       Information not provided696330.0710 (492)10.2 (7.1)
      Number of individuals with a PV and pathogenic frequency when excluding common low-penetrance alleles (monoallelic MUTYH pathogenic variant and APC p.I1307K) are in parentheses.
      PV, pathogenic variant.
      Unknown includes information not provided.
      Number of individuals with personal history of cancer exceeds 23,179 because of multiple reported cancer types.
      Ovarian cancer includes fallopian cancer and primary peritoneal cancer.
      § Other cancer includes hematological malignancies, kidney cancer, thyroid cancer, and other cancers.

       Variants Detected

      In this cohort, 2811 pathogenic variants were identified in 2698 individuals, and an overall pathogenic frequency of 11.6% was reported (9.1%, excluding common low-penetrance alleles) (Table 1). The majority of individuals with a positive result had a pathogenic variant in genes with high-to-moderate penetrance (76.0%) (Figure 1A). BRCA1 and BRCA2 pathogenic variants accounted for 31.4% of positive results, which is not surprising given the number of individuals in the cohort with a personal history of breast cancer. Pathogenic variants in MLH1, MSH2, MSH6, and PMS2, which are associated with Lynch syndrome, accounted for 7.0% of positive results. A total of 647 positive results (24.0%) were monoallelic MUTYH pathogenic variants or APC p.I1307K. These alleles are classified as pathogenic or likely pathogenic by multiple submitters in ClinVar but are known in the field to be commonly occurring and of low penetrance. Because these alleles might confound the analysis, all subsequent calculations of pathogenic allele frequency and count will be explicitly reported with and without these common low-penetrance alleles. Finally, the frequency of individuals with a variant of uncertain significance in the cohort, irrespective of additional pathogenic variants, was 19.0% (n = 4414).
      Figure thumbnail gr1
      Figure 1Test outcomes by result type, pathogenic variant type, and effect. A: Number of negative and positive results in the cohort. Positive results were stratified into BRCA (BRCA1 and BRCA2), Lynch syndrome gene (MLH1, MSH2, MSH6, and PMS2), low-penetrance allele (a monoallelic MUTYH pathogenic variant or APC p.I1307K), and other (APC, ATM, BAP1, BARD1, BMPR1A, BRIP1, CDH1, CDK4, CDKN2A, CHEK2, GREM1, MITF, biallelic MUTYH pathogenic variants, NBN, PALB2, PTEN, RAD51C, RAD51D, SMAD4, STK11, and TP53). B: Effects of pathogenic variants in the cohort. Effect was predicted by SnpEff version 4.0d.
      • Cingolani P.
      • Platts A.
      • Wang L.L.
      • Coon M.
      • Nguyen T.
      • Wang L.
      • Land S.J.
      • Lu X.
      • Ruden D.M.
      A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3.
      A total of 251 missense variants were APC p.I1307K. There were 16 stop gained/start lost, 1 copy number variant (CNV), 4 splice donors, 27 splice acceptors, 8 in-frame deletions/insertions, 340 missense mutations, and 21 frameshift mutations that were monoallelic MUTYH variants. C: Number of pathogenic variants by gene, stratified by variant type: single-nucleotide variant (SNV; 1 bp), small insertions and deletion (indel; 2 to 50 bp), and large structural variant (SV; >50 bp). A total of 251 SNVs were APC p.I1307K. A total of 395 SNVs, 21 indels, and 2 SVs were monoallelic MUTYH variants. SVs include CNVs.
      A large majority [1730 (61.5%)] of the 2811 pathogenic variants identified were in CHEK2, BRCA2, MUTYH, and BRCA1, with the next most frequent in APC and ATM [268 (9.5%) and 174 (6.2%), respectively] (Figure 1C). No pathogenic variants were identified in EPCAM, POLD1, or POLE genes, which are primarily associated with colorectal cancer. Single-nucleotide variants accounted for 58.9% (n = 1657) of all pathogenic variants, whereas insertions and deletions and structural variants accounted for 35.4% (n = 995) and 5.7% (n = 159), respectively. Approximately half of all insertions and deletions were found in BRCA2 and BRCA1 [514 (51.7%)], and nearly one-third [51 (32.1%)] of structural variants were also in BRCA1. The functional consequences of the pathogenic variants identified were primarily missense and frameshift effects (38.7% and 33.7%, respectively) (Figure 1B). Of the 1088 missense variants, 612 (56.3%) were pathogenic and 476 (43.8%) were likely pathogenic. Copy number variants accounted for 5.7% (n = 159) of variants, of which 30.2% (n = 48) affected only a single exon.
      Most individuals with a positive result carried a single pathogenic variant [2576 (95.5%); 2630 (97.5%), excluding common low-penetrance alleles]. However, 119 individuals (4.4%) who carried two concurrent pathogenic variants (Figure 2) and 3 individuals (0.1%) who carried three concurrent pathogenic variants were identified. Not surprisingly, 45.3% (54/119) of the individuals who carried two concurrent pathogenic variants carried at least one common low-penetrance allele (Figure 2). More important, 23.5% of individuals (28/119) carried a pathogenic variant in BRCA1 or BRCA2 and another concurrent pathogenic variant outside of the common low-penetrance alleles (Figure 2).
      Figure thumbnail gr2
      Figure 2Heatmap of the number of individuals with two pathogenic variants by gene, including homozygotes. Genes with pathogenic variants (PVs) among concurrent carriers are listed on the x and y axes. The number of individuals with pathogenic variants in a set of two genes is specified by number and visualized by color gradient. BRCA includes BRCA1 and BRCA2. LS genes, Lynch syndrome genes (MLH1, MSH2, MSH6, and PMS2).

       Frequency and Spectrum of Pathogenic Variants in Ethnic Populations

      After Caucasian, the second largest ethnic population in our cohort was individuals of Ashkenazi Jewish descent (9.9%), with a pathogenic frequency of 15.8% (9.5%, excluding common low-penetrance alleles) (Table 1 and Figure 3A). Three founder alleles in BRCA1 and BRCA2 are known to occur at a high population frequency (approximately 2.5%, collectively)
      • Friedman L.S.
      • Szabo C.I.
      • Ostermeyer E.A.
      • Dowd P.
      • Butler L.
      • Park T.
      • Lee M.K.
      • Goode E.L.
      • Rowell S.E.
      • King M.C.
      Novel inherited mutations and variable expressivity of BRCA1 alleles, including the founder mutation 185delAG in Ashkenazi Jewish families.
      • Oddoux C.
      • Struewing J.P.
      • Clayton C.M.
      • Neuhausen S.
      • Brody L.C.
      • Kaback M.
      • Haas B.
      • Norton L.
      • Borgen P.
      • Jhanwar S.
      • Goldgar D.
      • Ostrer H.
      • Offit K.
      The carrier frequency of the BRCA2 6174delT mutation among Ashkenazi Jewish individuals is approximately 1%.
      • Levy-Lahad E.
      • Catane R.
      • Eisenberg S.
      • Kaufman B.
      • Hornreich G.
      • Lishinsky E.
      • Shohat M.
      • Weber B.L.
      • Beller U.
      • Lahad A.
      • Halle D.
      Founder BRCA1 and BRCA2 mutations in Ashkenazi Jews in Israel: frequency and differential penetrance in ovarian cancer and in breast-ovarian cancer families.
      in Ashkenazi Jewish individuals: BRCA1 c.68_69delAG, BRCA1 c.5266dupC, and BRCA2 c.5946delT. In our cohort, these founder alleles were identified in 3.6% of Ashkenazi Jewish individuals (Figure 3A), and they accounted for 81.4% (n = 83) of the BRCA1 and BRCA2 pathogenic variants in Ashkenazi Jewish individuals—including one individual with two founder alleles (Figure 3B). More important, of individuals with pathogenic variants outside of common low-penetrance alleles, approximately 49.8% (n = 100) had pathogenic variants in genes other than BRCA1 and BRCA2 (Figure 3A).
      Figure thumbnail gr3
      Figure 3Pathogenic variants and frequency in ethnic subpopulations. A: Number of negative and positive results in the Ashkenazi Jewish (AJ) population. Positive results are BRCA (BRCA1 and BRCA2), low-penetrance allele (monoallelic MUTYH pathogenic variant or APC p.I1307K), and other (APC, ATM, BRIP1, CDH1, CHEK2, MITF, MSH2, MSH6, NBN, PALB2, RAD51C, and RAD51D). BRCA was stratified into Ashkenazi Jewish BRCA founder alleles (BRCA1 c.68_69delAG, BRCA1 c.5266dupC, and BRCA2 c.5946delT) and other BRCA. B: Percentage of AJ BRCA founder alleles among BRCA1 and BRCA2 pathogenic variants (PVs) by ethnicity. C: Number of pathogenic variants by gene in ethnicity minority groups, excluding common low-penetrance alleles (monoallelic MUTYH pathogenic variants and APC p.I1307K).
      A total of 14.8% of individuals reported non-Caucasian and non–Ashkenazi Jewish ethnicity (Table 1). The pathogenic frequency for Asians (11.7%; 9.7%, excluding common low-penetrance alleles) was similar to the overall pathogenic frequency (11.6%; 9.1%, excluding common low-penetrance alleles), whereas the pathogenic frequencies for Hispanics (13.8%; 12.3%, excluding common low-penetrance alleles), Africans (12.4%; 9.8%, excluding common low-penetrance alleles), and Native Americans (14.1%; 12.5%, excluding common low-penetrance alleles) were slightly higher (Table 1). As expected, the Ashkenazi Jewish BRCA founder alleles were largely absent in non–Ashkenazi Jewish individuals. They composed only 10.0% (n = 42) of BRCA1 and BRCA2 pathogenic variants for Caucasians and 5.0% (n = 5) for Hispanics, and they were absent in Asians, Africans, and Native Americans (Figure 3B). However, approximately half (54.1%) of the pathogenic variants outside of common low-penetrance alleles in these populations were in BRCA1 and BRCA2 (Figure 3C). Hispanic individuals also had a large number of Lynch syndrome gene pathogenic variants [32/181 (17.7%)] (Figure 3C), likely because of an enrichment of colorectal cancer in this subset of the cohort. Ashkenazi Jewish individuals also had a large number of pathogenic variants in CHEK2 [57/204 (27.9%)] (Figure 3C), most of which were the Ashkenazi Jewish CHEK2 founder alleles c.470T>C (p.I157T)
      • Kilpivaara O.
      • Vahteristo P.
      • Falck J.
      • Syrjäkoski K.
      • Eerola H.
      • Easton D.
      • Bartkova J.
      • Lukas J.
      • Heikkilä P.
      • Aittomäki K.
      • Holli K.
      • Blomqvist C.
      • Kallioniemi O.-P.
      • Bartek J.
      • Nevanlinna H.
      CHEK2 variant I157T may be associated with increased breast cancer risk.
      and c.1283C>T (p.S248F).
      • Shaag A.
      • Walsh T.
      • Renbaum P.
      • Kirchhoff T.
      • Nafa K.
      • Shiovitz S.
      • Mandell J.B.
      • Welcsh P.
      • Lee M.K.
      • Ellis N.
      • Offit K.
      • Levy-Lahad E.
      • King M.-C.
      Functional and genomic approaches reveal an ancient CHEK2 allele associated with breast cancer in the Ashkenazi Jewish population.

       Individuals with a Personal History of Cancer

      A total of 5649 individuals (24.4%) in the cohort reported a personal history of cancer associated with genes on the panel (Table 1). A personal history of stomach or colorectal cancer correlated with the highest pathogenic frequencies at 23.4% and 23.1%, respectively (19.1% and 17.1%, excluding common low-penetrance alleles, respectively). The pathogenic variants most frequently identified in these individuals were in well-established stomach or colorectal cancer genes, including MLH1, MSH2, APC (excluding p.I1307K), CHEK2, MSH6, PMS2, and BMPR1A [61 (70.1%)] (Supplemental Table S2); five individuals were identified as biallelic MUTYH carriers. However, pathogenic variants were also identified in genes not typically associated with these cancers, such as BRCA1, BRCA2, MITF, CDKN2A, PALB2, ATM, NBN, and BARD1 [24 (27.6%)]. BRCA1 and BRCA2 pathogenic variants accounted for 13.8% (n = 12), indicating the utility of broader gene panel testing regardless of clinical phenotype. Individuals with a personal history of ovarian cancer had a similarly high pathogenic frequency at 19.9% (17.3%, excluding common low-penetrance alleles) (Table 1). A total of 50 (67.6%; 78.1%, excluding common low-penetrance alleles) pathogenic variants in individuals with a history of ovarian cancer were in BRCA1, BRCA2, BRIP, RAD51C, RAD51D, or Lynch syndrome genes. BRCA1 and BRCA2 pathogenic variants accounted for 51.4% (59.4%, excluding common low-penetrance alleles) (Supplemental Table S2).
      A total of 9824 individuals in our cohort (42.4%) reported that they did not have a personal history of cancer. This subpopulation had a pathogenic frequency of 10.2% (7.1%, excluding common low-penetrance alleles) (Table 1). A total of 766 pathogenic variants (73.4%) were in genes with high-to-moderate penetrance, including BRCA1 and BRCA2 [297 (28.5%)] and Lynch syndrome genes [57 (5.5%)] (Supplemental Table S2). These data suggest that personal history alone is a poor indicator of pathogenic variant carrier status. Monoallelic MUTYH pathogenic variants (n = 177) and APC p.I1307K (n = 100) accounted for 26.6% of pathogenic variants in individuals with no personal history of cancer, consistent with known population frequencies for these alleles.
      • Boursi B.
      • Sella T.
      • Liberman E.
      • Shapira S.
      • David M.
      • Kazanov D.
      • Arber N.
      • Kraus S.
      The APC p.I1307K polymorphism is a significant risk factor for CRC in average risk Ashkenazi Jews.
      • Theodoratou E.
      • Campbell H.
      • Tenesa A.
      • Houlston R.
      • Webb E.
      • Lubbe S.
      • Broderick P.
      • Gallinger S.
      • Croitoru E.M.
      • Jenkins M.A.
      • Win A.K.
      • Cleary S.P.
      • Koessler T.
      • Pharoah P.D.
      • Küry S.
      • Bézieau S.
      • Buecher B.
      • Ellis N.A.
      • Peterlongo P.
      • Offit K.
      • Aaltonen L.A.
      • Enholm S.
      • Lindblom A.
      • Zhou X.-L.
      • Tomlinson I.P.
      • Moreno V.
      • Blanco I.
      • Capellà G.
      • Barnetson R.
      • Porteous M.E.
      • Dunlop M.G.
      • Farrington S.M.
      A large-scale meta-analysis to refine colorectal cancer risk estimates associated with MUTYH variants.

       Genetic Testing Recommendations by NCCN

      NCCN guidelines provide recommendations for genetic testing and counseling for hereditary cancer syndromes and risk management recommendations for patients who are suspected to be at high risk for a genetic syndrome on the basis of personal and family history. To assess how many individuals in our cohort would or would not have met criteria for genetic testing, the number of individuals who provided insufficient health history information was determined (Supplemental Table S1), which excluded 5003 individuals (Table 2). Among the remaining 18,176 individuals, 11,147 (61.3%) would have met criteria for genetic testing for breast and ovarian,
      • Daly M.B.
      • Pilarski R.
      • Berry M.
      • Buys S.S.
      • Farmer M.
      • Friedman S.
      • Garber J.E.
      • Kauff N.D.
      • Khan S.
      • Klein C.
      • Kohlmann W.
      • Kurian A.
      • Litton J.K.
      • Madlensky L.
      • Merajver S.D.
      • Offit K.
      • Pal T.
      • Reiser G.
      • Shannon K.M.
      • Swisher E.
      • Vinayak S.
      • Voian N.C.
      • Weitzel J.N.
      • Wick M.J.
      • Wiesner G.L.
      • Dwyer M.
      • Darlow S.
      NCCN Guidelines Insights: genetic/familial high-risk assessment: breast and ovarian, version 2.2017.
      colorectal,
      • Provenzale D.
      • Gupta S.
      • Ahnen D.J.
      • Bray T.
      • Cannon J.A.
      • Cooper G.
      • David D.S.
      • Early D.S.
      • Erwin D.
      • Ford J.M.
      • Giardiello F.M.
      • Grady W.
      • Halverson A.L.
      • Hamilton S.R.
      • Hampel H.
      • Ismail M.K.
      • Klapman J.B.
      • Larson D.W.
      • Lazenby A.J.
      • Lynch P.M.
      • Mayer R.J.
      • Ness R.M.
      • Regenbogen S.E.
      • Samadder N.J.
      • Shike M.
      • Steinbach G.
      • Weinberg D.
      • Dwyer M.
      • Darlow S.
      Genetic/familial high-risk assessment: colorectal version 1.2016, NCCN Clinical Practice Guidelines in Oncology.
      or gastric cancer,
      National Comprehensive Cancer Network, Inc
      NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) Gastric Cancer Version 1.2017.
      and 7029 (38.7%) would not (Table 2). The pathogenic frequency of the NCCN-ineligible population was 8.2% (6.2%, excluding common low-penetrance alleles) (Table 2). Strikingly, 21.7% (n = 201) of individuals with pathogenic variants in genes with well-established genetic testing recommendations did not meet corresponding NCCN criteria for genetic testing, and this frequency varied greatly by hereditary cancer syndrome (Figure 4 and Supplemental Table S3). For example, of the 749 individuals who had a pathogenic variant in BRCA1, BRCA2, TP53, or PTEN, 138 (18.4%) would not have met criteria for genetic testing for breast and ovarian cancer (Figure 4 and Supplemental Table S3). Of the 144 individuals who had a pathogenic variant in MLH1, MSH2, PMS2, or MSH6, 52 (36.1%) would not have met criteria for genetic testing for Lynch syndrome (Figure 4 and Supplemental Table S3). An additional 57 individuals in the cohort, who would not have met corresponding NCCN criteria for the pathogenic variant that we identified, would have met criteria for genetic testing for another hereditary cancer indication (Supplemental Table S3). These data indicate that, given the phenotypic and genotypic heterogeneity that exists within various cancers, it is difficult to definitively categorize genes for specific indications without missing potential carriers.
      Table 2Individuals Who Would Have Met the Clinical Criteria for Genetic Testing for Breast and Ovarian, Colorectal, or Gastric Cancer, Those Who Would Not, and Those Who Did Not Provide Enough Information to Determine
      NCCNNegative result, n (excluding common low-penetrance alleles, n)Positive result, n (excluding common low-penetrance alleles, n)Total, nPathogenic frequency, % (excluding common low-penetrance alleles, %)
      Met criteria9502 (9824)1645 (1323)11,14714.8 (11.9)
      Did not meet criteria6453 (6594)576 (435)70298.2 (6.2)
      Not enough information4526 (4645)477 (358)50039.5 (7.2)
      Total20,481 (21,063)2698 (2116)23,17911.6 (9.1)
      Number of individuals with a pathogenic variant and pathogenic frequency when excluding common low-penetrance alleles (monoallelic MUTYH pathogenic variant and APC p.I1307K) are in parentheses. NCCN guidelines are outlined in Supplemental Table S1.
      NCCN, National Comprehensive Cancer Network.
      Figure thumbnail gr4
      Figure 4Individuals with a pathogenic variant in genes with National Comprehensive Cancer Network (NCCN) guidelines, by guideline. Percentage of individuals with a pathogenic variant in BRCA1, BRCA2, TP53, or PTEN who would not have met the clinical criteria for genetic testing of breast and ovarian cancer (means ± SD age, 51.1 ± 15.3 years) and those who would (means ± SD age, 46.9 ± 14.7 years). Percentage of individuals with a pathogenic variant in MLH1, MSH2, MSH6, or PMS2 who would not have met the clinical criteria for genetic testing for Lynch syndrome (means ± SD age, 50.3 ± 16.3 years) and those who would (means ± SD age, 47.3 ± 14.6 years). Percentage of individuals with a pathogenic variant in APC or biallelic MUTYH pathogenic variants who would not have met the clinical criteria for genetic testing for colorectal cancer (means ± SD age, 51.7 ± 16.5 years) and those who would (means ± SD age, 43.5 ± 12.4 years). APC does not include APC p.I1307K. Percentage of individuals with a pathogenic variant in SMAD4 or BMPR1A who would not have met the clinical criteria for genetic testing for juvenile polyposis syndrome (JPS; n = 0) and those who would (means ± SD age, 36.5 ± 18.2 years). NCCN guidelines are outlined in .

      Discussion

      The advancements in next-generation sequencing technologies and reductions in the cost of sequencing have greatly expanded the use of multi-gene panel testing for hereditary cancer risk in the clinic. Herein, we analyzed the results of 23,179 individuals who received physician-ordered genetic testing via a 30-gene panel for hereditary cancer risk. Compared with other studies to date on hereditary cancer panel testing, our cohort included a high proportion of men,
      • Rosenthal E.T.
      • Bernhisel R.
      • Brown K.
      • Kidd J.
      • Manley S.
      Clinical testing with a panel of 25 genes associated with increased cancer risk results in a significant increase in clinically significant findings across a broad range of cancer histories.
      • LaDuca H.
      • Stuenkel A.J.
      • Dolinsky J.S.
      • Keiles S.
      • Tandy S.
      • Pesaran T.
      • Chen E.
      • Gau C.-L.
      • Palmaer E.
      • Shoaepour K.
      • Shah D.
      • Speare V.
      • Gandomi S.
      • Chao E.
      Utilization of multigene panels in hereditary cancer predisposition testing: analysis of more than 2,000 patients.
      • Susswein L.R.
      • Marshall M.L.
      • Nusbaum R.
      • Vogel Postula K.J.
      • Weissman S.M.
      • Yackowski L.
      • Vaccari E.M.
      • Bissonnette J.
      • Booker J.K.
      • Cremona M.L.
      • Gibellini F.
      • Murphy P.D.
      • Pineda-Alvarez D.E.
      • Pollevick G.D.
      • Xu Z.
      • Richard G.
      • Bale S.
      • Klein R.T.
      • Hruska K.S.
      • Chung W.K.
      Pathogenic and likely pathogenic variant prevalence among the first 10,000 patients referred for next-generation cancer panel testing.
      • Kurian A.W.
      • Hare E.E.
      • Mills M.A.
      • Kingham K.E.
      • McPherson L.
      • Whittemore A.S.
      • McGuire V.
      • Ladabaum U.
      • Kobayashi Y.
      • Lincoln S.E.
      • Cargill M.
      • Ford J.M.
      Clinical evaluation of a multiple-gene sequencing panel for hereditary cancer risk assessment.
      unaffected individuals, and individuals of non-Caucasian ethnicity. Taken together, the data presented herein advance our understanding of the frequency and spectrum of pathogenic variants in these genes and highlight the utility of multi-gene panels.

       High-Frequency Alleles with Low Penetrance

      The rapid uptake of genetic testing has revealed that several less penetrant but more frequently occurring alleles are associated with hereditary cancer; however, the risks and/or screening recommendations for these alleles are different from those reported for high-to-moderate penetrance pathogenic variants.
      For example, in contrast to other germline pathogenic variants in APC, APC p.I1307K is not associated with familial adenomatous polyposis, in which hundreds to thousands of adenomatous colonic polyps develop during adolescence.
      • Jasperson K.W.
      • Patel S.G.
      • Ahnen D.J.
      APC-associated polyposis conditions.
      This nonsynonymous variant is primarily found in individuals of Ashkenazi Jewish descent, with an estimated prevalence of 5% to 10%.
      • Rozen P.
      • Shomrat R.
      • Strul H.
      • Naiman T.
      • Karminsky N.
      • Legum C.
      • Orr-Urtreger A.
      Prevalence of the I1307K APC gene variant in Israeli Jews of differing ethnic origin and risk for colorectal cancer.
      Although APC p.I1307K has been shown to increase risk of colorectal cancer in this subpopulation,
      • Boursi B.
      • Sella T.
      • Liberman E.
      • Shapira S.
      • David M.
      • Kazanov D.
      • Arber N.
      • Kraus S.
      The APC p.I1307K polymorphism is a significant risk factor for CRC in average risk Ashkenazi Jews.
      the frequency and penetrance in individuals who are not of Ashkenazi Jewish descent is unclear. In our cohort, 175 individuals of Ashkenazi Jewish descent (7.6%) had APC p.I1307K, and of those who were not concurrent carriers (n = 155), 16 individuals (10.3%) reported a personal history of colorectal cancer. However, only 1 non–Ashkenazi Jewish individual with APC p.I1307K reported a personal history of colorectal cancer, suggesting that this variant may have variable risk for colorectal cancer outside of the Ashkenazi Jewish subpopulation. Further longitudinal studies are warranted to determine the true associated risk in diverse populations.
      MUTYH-associated polyposis is an autosomal-recessive syndrome that is characterized by significantly increased lifetime risk of colorectal cancer, up to 100% in the absence of timely surveillance.
      • Cleary S.P.
      • Cotterchio M.
      • Jenkins M.A.
      • Kim H.
      • Bristow R.
      • Green R.
      • Haile R.
      • Hopper J.L.
      • LeMarchand L.
      • Lindor N.
      • Parfrey P.
      • Potter J.
      • Younghusband B.
      • Gallinger S.
      Germline MutY human homologue mutations and colorectal cancer: a multisite case-control study.
      The risk of developing colorectal cancer in individuals with a heterozygous germline MUTYH pathogenic variant is less clear, but several studies have demonstrated an increased cancer risk in monoallelic family members compared with the general population.
      • Jones N.
      • Vogt S.
      • Nielsen M.
      • Christian D.
      • Wark P.A.
      • Eccles D.
      • Edwards E.
      • Evans D.G.
      • Maher E.R.
      • Vasen H.F.
      • Hes F.J.
      • Aretz S.
      • Sampson J.R.
      Increased colorectal cancer incidence in obligate carriers of heterozygous mutations in MUTYH.
      • Win A.K.
      • Dowty J.G.
      • Cleary S.P.
      • Kim H.
      • Buchanan D.D.
      • Young J.P.
      • Clendenning M.
      • Rosty C.
      • MacInnis R.J.
      • Giles G.G.
      • Boussioutas A.
      • Macrae F.A.
      • Parry S.
      • Goldblatt J.
      • Baron J.A.
      • Burnett T.
      • Le Marchand L.
      • Newcomb P.A.
      • Haile R.W.
      • Hopper J.L.
      • Cotterchio M.
      • Gallinger S.
      • Lindor N.M.
      • Tucker K.M.
      • Winship I.M.
      • Jenkins M.A.
      Risk of colorectal cancer for carriers of mutations in MUTYH, with and without a family history of cancer.
      In our cohort, only 1.8% (n = 7) of individuals with a heterozygous MUTYH pathogenic variant reported a personal history of colorectal cancer, suggesting that monoallelic MUTYH carriers are not at significantly higher risk for colorectal cancer. This is consistent with the NCCN guidelines, which do not propose specific screening recommendations for individuals with a heterozygous MUTYH pathogenic variant.
      • Provenzale D.
      • Gupta S.
      • Ahnen D.J.
      • Bray T.
      • Cannon J.A.
      • Cooper G.
      • David D.S.
      • Early D.S.
      • Erwin D.
      • Ford J.M.
      • Giardiello F.M.
      • Grady W.
      • Halverson A.L.
      • Hamilton S.R.
      • Hampel H.
      • Ismail M.K.
      • Klapman J.B.
      • Larson D.W.
      • Lazenby A.J.
      • Lynch P.M.
      • Mayer R.J.
      • Ness R.M.
      • Regenbogen S.E.
      • Samadder N.J.
      • Shike M.
      • Steinbach G.
      • Weinberg D.
      • Dwyer M.
      • Darlow S.
      Genetic/familial high-risk assessment: colorectal version 1.2016, NCCN Clinical Practice Guidelines in Oncology.
      Regardless, returning positive results for these variants has important implications for carrier testing, as affected offspring are often missed because of lack of family history.

       Concurrent Pathogenic Variant Carriers

      One advantage of multi-gene panel testing compared with single-gene testing is the detection of additional pathogenic variants in other genes that may also contribute to cancer risk. Recent reports have estimated that up to 3.1% of individuals who test positive on a multi-gene hereditary cancer test have more than one pathogenic variant.
      • Rosenthal E.T.
      • Bernhisel R.
      • Brown K.
      • Kidd J.
      • Manley S.
      Clinical testing with a panel of 25 genes associated with increased cancer risk results in a significant increase in clinically significant findings across a broad range of cancer histories.
      • LaDuca H.
      • Stuenkel A.J.
      • Dolinsky J.S.
      • Keiles S.
      • Tandy S.
      • Pesaran T.
      • Chen E.
      • Gau C.-L.
      • Palmaer E.
      • Shoaepour K.
      • Shah D.
      • Speare V.
      • Gandomi S.
      • Chao E.
      Utilization of multigene panels in hereditary cancer predisposition testing: analysis of more than 2,000 patients.
      • Susswein L.R.
      • Marshall M.L.
      • Nusbaum R.
      • Vogel Postula K.J.
      • Weissman S.M.
      • Yackowski L.
      • Vaccari E.M.
      • Bissonnette J.
      • Booker J.K.
      • Cremona M.L.
      • Gibellini F.
      • Murphy P.D.
      • Pineda-Alvarez D.E.
      • Pollevick G.D.
      • Xu Z.
      • Richard G.
      • Bale S.
      • Klein R.T.
      • Hruska K.S.
      • Chung W.K.
      Pathogenic and likely pathogenic variant prevalence among the first 10,000 patients referred for next-generation cancer panel testing.
      In our cohort, 122 individuals with a positive result (4.5%) had two or more pathogenic variants (n = 68; 2.5%, excluding common low-penetrance alleles). More important, a second concurrent pathogenic variant in a different gene with high-to-moderate penetrance would have been missed in 55.4% (n = 36) of individuals if they had been tested for only BRCA1, BRCA2, or Lynch syndrome genes. The knowledge of a second pathogenic variant could lead to additional preventions and more accurate genetic counseling. Identifying individuals with multiple pathogenic variants also has important implications for family members. Family members who had previously undergone single-gene or single-gene testing may have been erroneously informed they were true negatives. Further detailed studies are warranted to determine the clinical implications of carrying more than one pathogenic variant, especially with regard to variation in expressivity.

       Pathogenic Variant Spectrum in the Ashkenazi Jewish Subpopulation

      One ethnicity in which single-site, single-gene, and BRCA1- and BRCA2-only testing has been commonly used is the Ashkenazi Jewish subpopulation. Initial reports suggested that three founder alleles in BRCA1 and BRCA2 accounted for 98% to 99% of BRCA1 and BRCA2 pathogenic variants in individuals of Ashkenazi Jewish descent, with a pathogenic frequency of 1 in 40.
      • Roa B.B.
      • Boyd A.A.
      • Volcik K.
      • Richards C.S.
      Ashkenazi Jewish population frequencies for common mutations in BRCA1 and BRCA2.
      • Phelan C.M.
      • Kwan E.
      • Jack E.
      • Li S.
      • Morgan C.
      • Aubé J.
      • Hanna D.
      • Narod S.A.
      A low frequency of non-founder BRCA1 mutations in Ashkenazi Jewish breast-ovarian cancer families.
      • Frank T.S.
      • Deffenbaugh A.M.
      • Reid J.E.
      • Hulick M.
      • Ward B.E.
      • Lingenfelter B.
      • Gumpper K.L.
      • Scholl T.
      • Tavtigian S.V.
      • Pruss D.R.
      • Critchfield G.C.
      Clinical characteristics of individuals with germline mutations in BRCA1 and BRCA2: analysis of 10,000 individuals.
      Furthermore, these founder alleles were estimated to account for up to 30% of early-onset breast cancer and 60% of ovarian cancer in this subpopulation.
      • Abeliovich D.
      • Kaduri L.
      • Lerer I.
      • Weinberg N.
      • Amir G.
      • Sagi M.
      • Zlotogora J.
      • Heching N.
      • Peretz T.
      The founder mutations 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2 appear in 60% of ovarian cancer and 30% of early-onset breast cancer patients among Ashkenazi women.
      As a result, there were suggestions that genetic testing for the BRCA founder alleles within the Ashkenazi Jewish population may be sufficient. However, the data presented herein indicate that this approach would be insufficient, as 18.6% of BRCA1 and BRCA2 pathogenic variants in individuals of Ashkenazi Jewish descent were non-BRCA founder alleles (Figure 3B). Furthermore, 59.2% of pathogenic variants outside of common low-penetrance alleles were non-BRCA founder alleles (Figure 3A). The most prevalent pathogenic variant in this subpopulation other than the three BRCA1 and BRCA2 founder alleles (when excluding low-penetrance alleles) was CHEK2 c.1283C>T (p.S428F), which has previously been reported to increase breast cancer risk in Ashkenazi Jewish women by approximately twofold.
      • Shaag A.
      • Walsh T.
      • Renbaum P.
      • Kirchhoff T.
      • Nafa K.
      • Shiovitz S.
      • Mandell J.B.
      • Welcsh P.
      • Lee M.K.
      • Ellis N.
      • Offit K.
      • Levy-Lahad E.
      • King M.-C.
      Functional and genomic approaches reveal an ancient CHEK2 allele associated with breast cancer in the Ashkenazi Jewish population.
      Taken together, these data support emerging research that other gene pathogenic variants contribute to the high incidence of breast and ovarian cancer within this subpopulation
      • Crawford B.
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      Multi-gene panel testing for hereditary cancer predisposition in unsolved high-risk breast and ovarian cancer patients.
      • Walsh T.
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      • Casadei S.
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      • Lee M.K.
      • King M.-C.
      Genetic predisposition to breast cancer due to mutations other than BRCA1 and BRCA2 founder alleles among Ashkenazi Jewish women.
      • Rosenthal E.
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      • Wenstrup R.J.
      Incidence of BRCA1 and BRCA2 non-founder mutations in patients of Ashkenazi Jewish ancestry.
      and suggest that multi-gene panels are a more efficient testing paradigm.

       Limited Genetic Testing and Ethnicity

      Much of the literature on multi-gene panel testing is composed of cohorts that are predominantly Caucasian and of non-Hispanic ancestry. Recent efforts have been made to correct this ascertainment bias and have demonstrated that multi-gene panel testing is relevant across racial and ethnic groups.
      • Ricker C.
      • Culver J.O.
      • Lowstuter K.
      • Sturgeon D.
      • Sturgeon J.D.
      • Chanock C.R.
      • Gauderman W.J.
      • McDonnell K.J.
      • Idos G.E.
      • Gruber S.B.
      Increased yield of actionable mutations using multi-gene panels to assess hereditary cancer susceptibility in an ethnically diverse clinical cohort.
      This study adds support to these claims as the pathogenic frequency from our multi-gene panel was elevated among all reported ethnicities in the cohort. The pathogenic frequencies for Caucasians and Asians were similar, whereas the pathogenic frequencies for other ethnicities were slightly higher, perhaps revealing a selection bias for high-risk individuals who have, to date, had limited genetic testing for hereditary cancer risk. The combined frequency of pathogenic variants in BRCA1 and BRCA2 was remarkably similar across all ethnicities, despite large discrepancies in the number of individuals who underwent genetic testing. In contrast, the other pathogenic variants were distributed nonuniformly across other genes. This suggests that the association of true high-penetrance genes, such as BRCA1 and BRCA2, with cancer risk is conserved across ethnic groups, whereas low- or moderate-penetrance genes show selected variability. As genetic testing volume expands and more data become available about genetic variants in non-European ethnicities, multi-gene panel testing will likely continue to show improved utility in all ethnicities and reveal if the prevalence and penetrance that had been previously established in individuals of Caucasian and of non-Hispanic ancestry stand true.

       Broadening Access of Genetic Testing

      Current recommendations and reimbursements for genetic testing for hereditary cancer risk are based on personal and family clinical history or presence of a known family pathogenic variant. However, several groups have recently proposed broadening access of genetic testing beyond these high-risk populations for highly penetrant conditions that have well-defined genetic causes and well-established clinical interventions. Indeed, several studies focused on the US Centers for Disease Control and Prevention Tier 1 genomic cancer conditions (hereditary breast and ovarian cancer and Lynch syndrome) have demonstrated that population testing leads to early detection and intervention, improved survival rates, and reduced cost.
      • Manchanda R.
      • Patel S.
      • Gordeev V.S.
      • Antoniou A.C.
      • Smith S.
      • Lee A.
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      • Turnbull C.
      • Ramus S.J.
      • Gayther S.A.
      • Pharoah P.D.P.
      • Menon U.
      • Jacobs I.
      • Legood R.
      Cost-effectiveness of population-based BRCA1, BRCA2, RAD51C, RAD51D, BRIP1, PALB2 mutation testing in unselected general population women.
      • King M.-C.
      • Levy-Lahad E.
      • Lahad A.
      Population-based screening for BRCA1 and BRCA2: 2014 Lasker Award.
      • Manchanda R.
      • Legood R.
      • Burnell M.
      • McGuire A.
      • Raikou M.
      • Loggenberg K.
      • Wardle J.
      • Sanderson S.
      • Gessler S.
      • Side L.
      • Balogun N.
      • Desai R.
      • Kumar A.
      • Dorkins H.
      • Wallis Y.
      • Chapman C.
      • Taylor R.
      • Jacobs C.
      • Tomlinson I.
      • Beller U.
      • Menon U.
      • Jacobs I.
      Cost-effectiveness of population screening for BRCA mutations in Ashkenazi Jewish women compared with family history-based testing.
      • Long E.F.
      • Ganz P.A.
      Cost-effectiveness of universal BRCA1/2 screening: evidence-based decision making.
      Furthermore, it has been reported that approximately 50% of individuals with pathogenic variants in BRCA1, BRCA2, and Lynch syndrome genes would not have met clinical criteria for genetic testing.
      • Grindedal E.M.
      • Heramb C.
      • Karsrud I.
      • Ariansen S.L.
      • Mæhle L.
      • Undlien D.E.
      • Norum J.
      • Schlichting E.
      Current guidelines for BRCA testing of breast cancer patients are insufficient to detect all mutation carriers.
      • Buchanan A.H.
      • Manickam K.
      • Meyer M.N.
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      • Servano 3rd, P.O.
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      • Pearlman R.
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      In our cohort, among those who provided sufficient health history information, 21.3% (n = 190) of individuals with a pathogenic variant in one of these genes would not have met NCCN criteria for genetic testing for hereditary breast and ovarian cancer or Lynch syndrome. One common concern with broadening access to population-level genetic testing is the possibility of a negative psychological impact. However, recent studies have shown that screening does not adversely affect short-term psychological or quality-of-life outcomes and that study participation was associated with decreased anxiety and uncertainty linked to genetic testing.
      • Lieberman S.
      • Tomer A.
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      The lack of detrimental psychological outcomes coupled with clinically actionable findings supports population-based genetic testing for these hereditary cancers.

       Study Limitations

      This study may be limited by self-reporting of demographics and health history by the individual as opposed to a health care provider. Health care provider reports are considered the gold standard for collection of patient medical history data. The data collected in our study are entirely self-reported, which we recognize may be a limitation of this study. However, several studies have shown that self-reported data on personal and family history of cancer have high concordance with data reported by a health care provider or electronic medical records. Concordance varied by cancer site and was highest (>85.1%) in breast, prostate, and colorectal cancer and lowest in melanoma.
      • Gentry-Maharaj A.
      • Fourkala E.-O.
      • Burnell M.
      • Ryan A.
      • Apostolidou S.
      • Habib M.
      • Sharma A.
      • Parmar M.
      • Jacobs I.
      • Menon U.
      Concordance of National Cancer Registration with self-reported breast, bowel and lung cancer in England and Wales: a prospective cohort study within the UK Collaborative Trial of Ovarian Cancer Screening.
      • D'Aloisio A.A.
      • Nichols H.B.
      • Hodgson M.E.
      • Deming-Halverson S.L.
      • Sandler D.P.
      Validity of self-reported breast cancer characteristics in a nationwide cohort of women with a family history of breast cancer.
      • Bergmann M.M.
      • Calle E.E.
      • Mervis C.A.
      • Miracle-McMahill H.L.
      • Thun M.J.
      • Heath C.W.
      Validity of self-reported cancers in a prospective cohort study in comparison with data from state cancer registries.
      Reliability and accuracy of self-reported family history were also high (up to 95.4%) and dependent on type of cancer and relationship to the individual.
      • Ziogas A.
      • Anton-Culver H.
      Validation of family history data in cancer family registries.
      • Ferrante J.M.
      • Ohman-Strickland P.
      • Hahn K.A.
      • Hudson S.V.
      • Shaw E.K.
      • Crosson J.C.
      • Crabtree B.F.
      Self-report versus medical records for assessing cancer-preventive services delivery.
      • Chang E.T.
      • Smedby K.E.
      • Hjalgrim H.
      • Glimelius B.
      • Adami H.-O.
      Reliability of self-reported family history of cancer in a large case-control study of lymphoma.
      Similarly, several recent studies have reported discrepancies in how individuals self-report their race/ethnicity and the subjective assignments made by health care providers or administrators.
      • Witzig R.S.
      • Dery M.
      Subjectively-assigned versus self-reported race and ethnicity in US healthcare.
      • Mersha T.B.
      • Abebe T.
      Self-reported race/ethnicity in the age of genomic research: its potential impact on understanding health disparities.
      • Magaña López M.
      • Bevans M.
      • Wehrlen L.
      • Yang L.
      • Wallen G.R.
      Discrepancies in race and ethnicity documentation: a potential barrier in identifying racial and ethnic disparities.
      Misclassifications were highest among those individuals who are racial/ethnic minority or multiple ethnicities, suggesting that the way in which information on race and ethnicity is collected may need to be reevaluated. Finally, our analyses also assumed that individuals represent unrelated probands; however, it is possible that two or more family members may have been referred for genetic testing for hereditary cancer risk and were present in the cohort.

       Future Directions

      Most individuals in this cohort self-reported as female and Caucasian, indicating a need in the broader community for better outreach and education of genetic testing in males and minorities. The paucity of younger test takers (aged <40 years) in our cohort highlights the importance of increasing awareness and uptake of genetic testing within this population when preventative care is more relevant. Finally, as the cost of sequencing continues to decrease and genetic testing becomes more accessible to a broader population, we will gain a better understanding of the genes associated with elevated risk for hereditary cancer. With this additional knowledge, it will be imperative to reevaluate the genes included on multi-gene panels. The genes on this panel were selected on the basis of studies in high-risk cohorts and, thus, the population-based prevalence and penetrance of many of these genes are unknown. The data presented herein advance our understanding of the true frequency and spectrum of pathogenic variants in these genes and highlight the clinical actionability and utility of multi-gene panels for hereditary cancer risk.

      Acknowledgments

      We thank Justin Lock, Danny DeSloover, Valerie Ngo, Hau-Ling Poon, and the entire Color Genomics, Inc., laboratory team for sample processing; Ziga Mahkovec, Gilad Mishne, and the entire Color Genomics, Inc., bioinformatics team for bioinformatics analysis; Serra Kim and the entire Color Genomics, Inc., genetics team for variant interpretation; and Lauren Ryan, Lily Servais, and the entire Color Genomics, Inc., genetic counseling team for genetic counseling expertise.
      A.Y.Z. designed the study; C.L.N., A.D.Z., W.S., J.v.d.A., and S.T. acquired and analyzed data; R.O., R.C.C., E.C., Z.T., A.L., J.J., and A.Y.Z. performed variant classification; C.L.N., A.D.Z., J.v.d.A., S.T., and A.Y.Z. drafted or critically revised the manuscript for important intellectual content; A.Y.Z. is the guarantor of this work and, as such, has full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

      Supplemental Data

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