Frequency of Germline Mutations in 25 Cancer Susceptibility Genes in a Sequential Series of Patients With Breast Cancer

Abstract

Purpose

Testing for germline mutations in BRCA1/2 is standard for select patients with breast cancer to guide clinical management. Next-generation sequencing (NGS) allows testing for mutations in additional breast cancer predisposition genes. The frequency of germline mutations detected by using NGS has been reported in patients with breast cancer who were referred for BRCA1/2 testing or with triple-negative breast cancer. We assessed the frequency and predictors of mutations in 25 cancer predisposition genes, including BRCA1/2, in a sequential series of patients with breast cancer at an academic institution to examine the utility of genetic testing in this population.

Methods

Patients with stages I to III breast cancer who were seen at a single cancer center between 2010 and 2012, and who agreed to participate in research DNA banking, were included (N = 488). Personal and family cancer histories were collected and germline DNA was sequenced with NGS to identify mutations.

Results

Deleterious mutations were identified in 10.7% of women, including 6.1% in BRCA1/2 (5.1% in non-Ashkenazi Jewish patients) and 4.6% in other breast/ovarian cancer predisposition genes including CHEK2 (n = 10), ATM (n = 4), BRIP1 (n = 4), and one each in PALB2, PTEN, NBN, RAD51C, RAD51D, MSH6, and PMS2. Whereas young age (P < .01), Ashkenazi Jewish ancestry (P < .01), triple-negative breast cancer (P = .01), and family history of breast/ovarian cancer (P = .01) predicted for BRCA1/2 mutations, no factors predicted for mutations in other breast cancer predisposition genes.

Conclusion

Among sequential patients with breast cancer, 10.7% were found to have a germline mutation in a gene that predisposes women to breast or ovarian cancer, using a panel of 25 predisposition genes. Factors that predict for BRCA1/2 mutations do not predict for mutations in other breast/ovarian cancer susceptibility genes when these genes are analyzed as a single group. Additional cohorts will be helpful to define individuals at higher risk of carrying mutations in genes other than BRCA1/2.

INTRODUCTION

Testing for mutations in high-penetrance breast cancer predisposition genes, particularly BRCA1 and BRCA2, has become standard practice for patients with breast cancer. Lifetime estimates of breast cancer risk in BRCA1 or BRCA2 (BRCA1/2) carriers range from 36% to 90% and of ovarian cancer risk range from 24% to 59% and 8% to 35% in BRCA1 and BRCA2 carriers, respectively.^1-5 Identification of BRCA1/2 mutations permits the implementation of prevention strategies, including magnetic resonance imaging screening or risk-reducing surgeries, which improves survival.^6,7 Genetic testing for other high-risk breast cancer susceptibility genes, such as TP53 (Li-Fraumeni syndrome), PTEN (Cowden’s syndrome), and CDH1 (hereditary diffuse gastric cancer), is also standard in appropriate patients.

More recently, next-generation sequencing (NGS) has enabled simultaneous testing for mutations in these high-penetrance genes and for other, more moderate-risk genes. Multigene panels are now commercially available and are increasingly being used in cancer risk assessment. Compared with high-penetrance genes, for which inherited mutations confer a five-fold or greater breast cancer risk, mutations in moderate-penetrance genes are associated with a two- to four-fold increase in risk. Cancer risks associated with mutations in these less potent predisposition genes are still being investigated. For example, mutations in PALB2, initially thought to confer a moderate risk of breast cancer, now seem to be associated with a five-fold or greater risk.⁸ NGS also allows simultaneous testing for other hereditary cancer risks, such as Lynch syndrome, in individuals with and without a suggestive family history. In addition, germline mutations in DNA repair genes such as BRIP1, RAD51C, and RAD51D are associated with an increased risk of ovarian cancer (Table 1).^9-15

Table 1.

Cancer Susceptibility Genes Other Than BRCA1/2

Cancer Susceptibility Gene	Breast Cancer RR (90% CI when available) or Inclusion Criteria
Breast
ATM	2.8 (2.2 to 3.7)35
BARD1	Breast cancer association reported; RR not yet determined17,46,47
BRIP1	2.0 (1.3 to 3.0)48; ovarian cancer RR 11.29
CDH1	6.6 (2.2 to 19.9)49
CHEK2	3.0 (2.6 to 3.5)35; most data for 1100delC
NBN	2.7 (1.9 to 3.7)35
PALB2	5.3 (3.0 to 9.4)35
PTEN	RR 2.0-5.050,51
STK11	RR 2.0-4.052,53
TP53	105 (62 to 165)35
Other
APC	Familial adenomatous polyposis
BMPR1A	Juvenile polyposis syndrome
CDK4	Melanoma syndrome
CDKN2A	Melanoma and pancreas cancer syndrome
EPCAM	Lynch syndrome
MLH1	Lynch syndrome
MSH2	Lynch syndrome
MSH6	Lynch syndrome
MUTYH*	MUTYH-associated polyposis
PMS2	Lynch syndrome
RAD51C	Ovarian cancer RR 5.2-6.311-13
RAD51D	Ovarian cancer RR 6.3-1212,15
SMAD4	Juvenile polyposis syndrome

Abbreviations: APC, adenomatous polyposis coli; ATM, ataxia telangiectasia mutated; BARD1, BRCA1-associated RING domain 1; BMPR1A, bone morphogenetic protein receptor, type 1A; BRCA1/2, early-onset breast cancer genes BRCA1 and BRCA2; BRIP1, BRCA1 interacting protein C-terminal helicase 1; CDH1, E-cadherin; CDK4, cyclin-dependent kinase 4; CDKN2A, cyclin-dependent kinase inhibitor 2A; CHEK2, checkpoint kinase 2; EPCAM, epithelial cell adhesion molecule; MLH1, mutL homolog 1; MSH2, mutS homolog 2; MSH6, mutS homolog 6; MUTYH, biallelic mutY homolog; NBN, nibrin; PALB2, partner and localizer of BRCA2; PMS2, PMS2 postmeiotic segregation increased 2; PTEN, phosphatase and tensin homolog; RAD51C, RAD51 paralog C; RAD51D, RAD51 paralog D; RR, relative risk; SMAD4, SMAD family member 4; STK11, serine/threonine kinase 11; TP53, tumor protein 53.

^*Only tumors with biallelic MUTYH mutations were considered for this analysis.

To date, studies evaluating the prevalence of mutations in moderate-penetrance breast cancer predisposition genes have been conducted in select breast cancer populations including African Americans,¹⁶ patients with triple-negative breast cancer (TNBC),¹⁷ and cases seen in high-risk genetic clinics.^18-21 The prevalence of mutations among patients with breast cancer, who are unselected for specific risk factors such as age at diagnosis, breast cancer subtype, or family cancer history, is unknown.

Evidence-based guidelines for BRCA1/2 testing in patients with breast cancer have been established. Criteria include young age at diagnosis, TNBC, Ashkenazi Jewish ancestry, or a significant family history of breast, ovarian, or other related cancers.^22,23 Existing recommendations for mutation detection in other high-penetrance genes are based on specific syndrome features.²² Criteria for testing of moderate-penetrance predisposition genes do not yet exist because predictive factors have not been identified and clinical utility is still being evaluated.

In this study, we assessed the frequency of deleterious germline mutations in 25 cancer susceptibility genes in a population of consecutive patients with breast cancer who presented to an academic cancer center. Our goals were to better understand the contribution of inherited mutations in moderate- and high-risk genes in a breast cancer cohort unselected for family history, breast cancer subtype, ethnicity, or age at diagnosis and to evaluate any clinical or pathologic factors that predict for mutations in moderate-risk genes.

METHODS

Patient Selection

All women with stages I to III breast cancer seen at the Dana-Farber Cancer Institute (Boston, MA) between April 2010 and July 2012, who consented to DNA banking for clinical research, were eligible. Patients with a previous breast cancer were excluded. Cases were identified retrospectively and blood samples were obtained from the Dana-Farber/Harvard Cancer Center Specialized Program of Research Excellence (SPORE) in breast cancer biobank. Clinical and pathologic data abstracted from medical records as part of the Clinical Outcomes for Research Information Service program included personal and family cancer histories, cancer histology, stage and receptor status, ancestry, and history of genetic testing. All breast cancers were reviewed by breast pathologists in the Department of Pathology at Brigham and Women’s Hospital. ASCO/College of American Pathologists guidelines were used to define estrogen receptor (ER), progesterone receptor, and human epidermal growth factor receptor 2 (HER2) positivity. Women with bilateral breast cancer were eligible, provided their first breast cancer was diagnosed between April 2010 and July 2012; only features of the initial breast cancer were assessed. Genetic test results from this analysis were considered research and were not returned to study participants or used for clinical decision making. Specimens were collected for research purposes only and therefore did not comply with Clinical Laboratory Improvement Amendments chain of custody regulations for clinical testing. This study was approved by the institutional review board of the Dana-Farber/Harvard Cancer Center.

NGS Assay

Sample preparation for NGS was performed from frozen DNA using the RainDance microdroplet polymerase chain reaction (PCR) system (RainDance Technologies, Billerica, MA). Briefly, PCR products representing exons and proximal splicing elements of patient DNA were amplified in merged droplets consisting of fragmented patient DNA and select target enrichment primers. These PCR products were subsequently tagged with barcodes and sequencing adaptors for NGS on the Illumina HiSeq platform (Illumina, San Diego, CA). To circumvent highly homologous pseudogenes, modified sample preparation with long-range and nested PCR, followed by NGS on the Illumina MiSeq platform, was used for portions of the CHEK2 and PMS2 genes. All clinically actionable variants identified by NGS, as well as regions that did not meet our preset NGS quality metrics, were independently confirmed with orthogonal site-specific Sanger sequencing.

To detect exonic deletions and duplications, NGS dosage, microarray comparative genomic hybridization, multiplex ligation-dependent probe amplification, or a combination of these analyses was performed,²⁴ with all positive results confirmed by an orthogonal method. Gene variants deemed deleterious or suspected as deleterious were considered mutations. Analyzed genes were categorized into two groups (Table 1).

Variant Classification

Variants were classified using American College of Medical Genetics and Genomics recommendations, with supporting linkage, biochemical, clinical, functional, and statistical data used for specific missense and intronic alterations.^25-27

Statistics

Participant characteristics and sequencing results were summarized with descriptive statistics, including medians, means, and standard deviations for continuous data. For categorical data, proportions with 95% CIs were calculated by the Clopper-Pearson method. Demographic, clinical, and pathologic characteristics were compared using the χ² test (categorical variables) and the t test/analysis of variance (continuous variables). P values < .05 were considered significant.

RESULTS

Study Population

During the study period, 69.8% of patients with breast cancer seen at Dana-Farber Cancer Institute agreed to use of their clinical data and specimens for research. Sixty-one percent of blood samples were collected within 90 days of the initial breast cancer diagnosis and 94% within 1 year of diagnosis. The median time from diagnosis to blood sample collection was 77 days. Blood samples from 582 cases were analyzed and 87 failed due to insufficient DNA quantity or poor DNA quality. Six cases were excluded due to a prior breast cancer diagnosis and one was excluded for lack of clinical data, resulting in 488 cases which comprised the study cohort. Clinical and tumor pathologic features for study participants are provided in Table 2. The mean age at diagnosis was 50.3 years (range, 28 to 88 years); 7.8% of the study population were Ashkenazi Jewish and 81.4% were non-Ashkenazi white. Nearly 18% of women had TNBC, and 82.6% had stage I or II disease. Further, 49.0% of patients reported having a first- or second-degree relative with breast or ovarian cancer.

Table 2.

Clinical and Tumor Characteristics in Study Cohort (N = 488)

Study Characteristic	No.	%
Age at diagnosis, years
Mean ± SD	50.3 ± 11.3
Median	49
Range	28-88
≤ 45	180	36.9
46-60	199	40.8
> 60	109	22.3
Race/ethnicity
Ashkenazi Jewish	38	7.8
Non-Hispanic white (not Ashkenazi Jewish)	397	81.4
Hispanic	17	3.5
African American	12	2.5
Asian	10	2.0
Other	14	2.9
Ashkenazi Jewish ethnicity
Yes	38	7.8
No	450	92.2
Breast cancer subtypes, receptor status
TNBC	87	17.8
HR-positive/HER2-negative	301	61.7
HR-negative/HER2-positive	37	7.6
HR-positive/HER2-positive	63	12.9
Histology
Ductal	357	73.2
Lobular	36	7.4
Ductal and lobular	68	13.9
Other	27	5.5
Grade*
1	60	12.3
2	181	37.2
3	246	50.5
Stage
I	185	37.9
II	218	44.7
III	85	17.4
Bilateral disease
Yes	9	1.8
No	479	98.2
Patient history of prior cancer†
Yes	41	8.4
No	447	91.6
First-degree relative with any cancer†‡
Yes	271	56.7
No	207	43.3
First-/second-degree relative with any cancer†‡
Yes	403	84.3
No	75	15.7
First-/second-degree relative with breast or ovarian cancer†‡
Yes	234	49.0
No	244	51.0
First-/second-degree relative with breast cancer (< 50 years of age), male breast cancer, or ovarian cancer (any age)†‡
Yes	89	18.6
No	389	81.4

Abbreviations: HER2, human epidermal growth factor receptor 2; HR, hormone receptor; SD, standard deviation, TNBC, triple-negative breast cancer.

^*Tumor grade was missing for one patient.

^†Excludes in situ cancers and nonmelanoma skin cancers.

^‡Ten patients were missing family history information. These patients were excluded from analysis. If age at diagnosis was unavailable, it was conservatively considered to be older than 50 years.

Frequency of Deleterious Mutations

Among 488 patients with breast cancer, 55 deleterious mutations were identified in 52 (10.7%) women (Table 3; Appendix Table A1, online only). Thirty (6.1%) women had a germline BRCA1/2 mutation; 18 in BRCA1 and 12 in BRCA2. In addition, 20 (4.1%) women had a total of 21 deleterious mutations in non-BRCA1/2 breast cancer predisposition genes including CHEK2 (n = 10), ATM (n = 4), BRIP1 (n = 4), and one each in PALB2, PTEN, and NBN. One ATM mutation was identified in a woman with a BRCA2 mutation, and one patient had both an ATM and a CHEK2 mutation. Four (0.8%) women carried deleterious mutations in genes unrelated to breast cancer; two in Lynch-related genes (one each in MSH6 and PMS2), and one each in RAD51C and RAD51D. The patient with a PMS2 mutation also had a BRCA1 mutation. Thus, 49 (10.0%) women had an inherited mutation in a breast cancer predisposition gene and 52 (10.7%) in a gene associated with breast or ovarian cancer risk. Eight of the 10 CHEK2 mutations identified were 1100delC. No mutations in BARD1, CDH1, STK11, TP53, APC, BMPR1A, CDK4, CDKN2A p14, CDKN2A p16, EPCAM, MLH1, MSH2, MUTYH (biallelic), or SMAD4 were detected. Four patients with I1307K APC variants and nine women with monoallelic MUTYH mutations were identified but not included in this analysis due to lower associated cancer risk. Specific mutations identified and associated patient characteristics are provided in Appendix Table A1.

Table 3.

Germline Mutations Identified (N = 488)

Genes	No. of Patients With Mutation	Percent With Mutation	95% CI
Any deleterious mutation*	52 (55 total mutations)	10.7	8.1 to 13.7
Genes related to breast cancer*	49	10.0	7.5 to 13.1
BRCA1 or BRCA2	30	6.1	4.2 to 8.7
BRCA1*	18	3.7	2.2 to 5.8
BRCA2*	12	2.5	1.3 to 4.3
Other genes related to breast cancer*	20 (21 total mutations)	4.1	2.5 to 6.3
ATM*	4	0.8	0.2 to 2.1
BRIP1	4	0.8	0.2 to 2.1
CHEK2*†	10	2.1	1.0 to 3.7
NBN	1	0.2	0.01 to 1.1
PALB2	1	0.2	0.01 to 1.1
PTEN	1	0.2	0.01 to 1.1
Genes not clearly related to breast cancer*	4	0.8	0.2 to 2.1
MSH6	1	0.2	0.01 to 1.1
PMS2*	1	0.2	0.01 to 1.1
RAD51C	1	0.2	0.01 to 1.1
RAD51D	1	0.2	0.01 to 1.1

NOTE. No mutations were identified in the following genes: BARD1; CDH1; STK11; TP53; APC; BMPR1A; CDK4; CDKN2A p14; CDKN2A p16; EPCAM; MLH1; MSH2; MUTYH (biallellic); and SMAD4.

^*One patient had deleterious mutations in both BRCA2 and ATM. Another patient had deleterious mutations in both BRCA1 and PMS2. Another patient had deleterious mutations in both ATM and CHEK2.

^†Eight of 10 CHEK2 mutations were 1100delC (Appendix Table A1).

Of the 30 patients with a BRCA1/2 mutation, four (13.3%) had not been clinically identified after diagnosis but did meet National Comprehensive Cancer Network (NCCN) criteria for BRCA1/2 testing.

Variants of Uncertain Significance

At least one variant of uncertain significance (VUS) was identified in 162 (33.2%) women, with as many as three variants found per patient. Fifteen patients with a VUS also had a deleterious mutation. All VUSs identified are listed in Appendix Table A2 (online only).

Predictors of Deleterious Mutations

Age.

For BRCA1/2, the prevalence of deleterious mutations decreased with age at breast cancer diagnosis, with a frequency of 12.2%, 3.0%, and 1.8% for women diagnosed at age ≤ 45 years, 46 to 60 years, and older than 60 years, respectively (Table 4). In contrast, for these same age groups, the frequency of mutations in other genes related to breast cancer ranged from 3.7% to 4.4%, irrespective of age at diagnosis (Table 4).

Table 4.

Frequency of Deleterious Mutations by Age at Breast Cancer Diagnosis

	Patients ≤ 45 Years of Age With DM (n = 180)		Patients 46-60 Years of Age With DM (n = 199)		Patients > 60 Years of Age With DM (n = 109)
Genes	No.	% (95% CI)	No.	% (95% CI)	No.	% (95% CI)
Any deleterious mutation*	30	16.7 (11.5 to 22.9)	15	7.5 (4.3 to 12.1)	7	6.4 (2.6 to 12.8)
Genes related to breast cancer*	29	16.1 (11.1 to 22.3)	14	7.0 (3.9 to 11.5)	6	5.5 (2.1 to 11.6)
BRCA1 or BRCA2	22	12.2 (7.8 to 17.9)	6	3.0 (1.1 to 6.5)	2	1.8 (0.2 to 6.5)
BRCA1*	15	8.3 (4.7 to 13.4)	2	1.0 (0.1 to 3.6)	1	0.9 (0.02 to 5.0)
BRCA2*	7	3.9 (1.6 to 7.9)	4	2.0 (0.6 to 5.1)	1	0.9 (0.02 to 5.0)
Other genes related to breast cancer*	8	4.4 (1.9 to 8.6)	8	4.0 (1.8 to 7.8)	4	3.7 (1.0 to 9.1)
ATM*	3	1.7 (0.4 to 4.8)	1	0.5 (0.01 to 2.8)	0	0.0 (0.0 to 3.3)
BRIP1	1	0.6 (0.01 to 3.1)	2	1.0 (0.1 to 3.6)	1	0.9 (0.02 to 5.0)
CHEK2*	4	2.2 (0.6 to 5.6)	3	1.5 (0.3 to 4.3)	3	2.8 (0.6 to 7.8)
NBN	0	0.0 (0.0 to 2.0)	1	0.5 (0.01 to 2.8)	0	0.0 (0.0 to 3.3)
PALB2	1	0.6 (0.01 to 3.1)	0	0.0 (0.0 to 1.8)	0	0.0 (0.0 to 3.3)
PTEN	0	0.0 (0 to 2.0)	1	0.5 (0.01 to 2.8)	0	0.0 (0.0 to 3.3)
Genes not clearly related to breast cancer*	2	1.1 (0.1 to 4.0)	1	0.5 (0.01 to 2.8)	1	0.9 (0.02 to 5.0)
MSH6	0	0.0 (0.0 to 2.0)	1	0.5 (0.01 to 2.8)	0	0.0 (0.0 to 3.3)
PMS2*	1	0.6 (0.01 to 3.1)	0	0.0 (0.0 to 1.8)	0	0.0 (0.0 to 3.3)
RAD51C	0	0.0 (0.0 to 2.0)	0	0.0 (0.0 to 1.8)	1	0.9 (0.02 to 5.0)
RAD51D	1	0.6 (0.01 to 3.1)	0	0.0 (0.0 to 1.8)	0	0.0 (0.0 to 3.3)

NOTE. No mutations were identified in the following genes: BARD1; CDH1; STK11; TP53; APC; BMPR1A; CDK4; CDKN2A p14; CDKN2A p16; EPCAM; MLH1; MSH2; MUTYH (biallelic); and SMAD4.

Abbreviation: DM, deleterious mutation.

^*One patient diagnosed in the ≤ 45 years old group had deleterious mutations in both BRCA2 and ATM. Another patient diagnosed in the ≤ 45 years old group had deleterious mutations in both BRCA1 and PMS2. Another patient diagnosed in the ≤ 45 years old group had deleterious mutations in both ATM and CHEK2.

Breast cancer subtype.

Table 5 illustrates the prevalence of deleterious mutations according to breast cancer subtype. The highest prevalence of BRCA1/2 or of any mutations was in women with TNBC. Among 87 women with TNBC, 15 (17.2%) had a deleterious germline mutation, with 12 of these (13.8%) in BRCA1/2 (11 BRCA1, 1 BRCA2). Two (2.3%) women had a mutation in another breast cancer predisposition gene (one each in BRIP1 and NBN) and one (1.1%) in RAD51D. Among 301 women with ER-positive HER2-negative breast cancer, 26 (8.6%) had at least one mutation, with 15 (5.0%) in BRCA1/2 (5 BRCA1, 10 BRCA2) and 11 (3.7%) in another breast/ovarian cancer predisposition gene including CHEK2 (n = 4), ATM (n = 3), and one each in PALB2, BRIP1, PTEN, RAD51C, and MSH6. One woman had mutations in both ATM and CHEK2. Among 37 women with ER-negative HER2-positive disease, two (5.4%) had a BRCA1 mutation and two (5.4%) had a CHEK2 mutation. Eleven percent of 63 women with ER-positive HER2-positive breast cancer had a mutation, one (1.6%) in BRCA2 (also in ATM) and six (9.5%) in other breast cancer predisposition genes including CHEK2 (n = 4) and BRIP1 (n = 2).

Table 5.

Deleterious Mutations by Breast Cancer Subtype (N = 488)

	Patients With TNBC Mutation (n = 87)		Patients With ER-Positive/HER2-Negative Mutation (n = 301)		Patients With ER-Negative/HER2-Positive Mutation (n = 37)		Patients With ER-Positive/HER2-Positive Mutation (n = 63)
Genes	No.	% (95% CI)	No.	% (95% CI)	No.	% (95% CI)	No.	% (95% CI)
Any deleterious mutation*	15	17.2 (10.0 to 26.8)	26	8.6 (5.7 to 12.4)	4	10.8 (3.0 to 25.4)	7	11.1 (4.6 to 21.6)
Genes related to breast cancer*	14	16.1 (9.1 to 25.5)	24	8.0 (5.2 to 11.6)	4	10.8 (3.0 to 25.4)	7	11.1 (4.6 to 21.6)
BRCA1 or BRCA2	12	13.8 (7.3 to 22.9)	15	5.0 (2.8 to 8.1)	2	5.4 (0.7 to 18.2)	1	1.6 (0.04 to 8.5)
BRCA1*	11	12.6 (6.5 to 21.5)	5	1.7 (0.5 to 3.8)	2	5.4 (0.7 to 18.2)	0	0.0 (0.0 to 5.7)
BRCA2*	1	1.1 (0.03 to 6.2)	10	3.3 (1.6 to 6.0)	0	0.0 (0.0 to 9.5)	1	1.6 (0.04 to 8.5)
Other genes related to breast cancer*	2	2.3 (0.3 to 8.1)	9	3.0 (1.4 to 5.6)	2	5.4 (0.7 to 18.2)	7	11.1 (4.6 to 21.6)
ATM*	0	0.0 (0.0 to 4.2)	3	1.0 (0.2 to 2.9)	0	0.0 (0.0 to 9.5)	1	1.6 (0.04 to 8.5)
BRIP1	1	1.1 (0.03 to 6.2)	1	0.3 (0.01 to 1.8)	0	0.0 (0.0 to 9.5)	2	3.2 (0.4 to 11.0)
CHEK2*	0	0.0 (0.0 to 4.2)	4	1.3 (0.4 to 3.4)	2	5.4 (0.7 to 18.2)	4	6.3 (1.8 to 15.5)
NBN	1	1.1 (0.03 to 6.2)	0	0.0 (0.0 to 1.2)	0	0.0 (0.0 to 9.5)	0	0.0 (0.0 to 5.7)
PALB2	0	0.0 (0.0 to 4.2)	1	0.3 (0.01 to 1.8)	0	0.0 (0.0 to 9.5)	0	0.0 (0.0 to 5.7)
PTEN	0	0.0 (0.0 to 4.2)	1	0.3 (0.01 to 1.8)	0	0.0 (0.0 to 9.5)	0	0.0 (0.0 to 5.7)
Genes not clearly related to breast cancer*	2	2.3 (0.3 to 8.1)	2	0.7 (0.1 to 2.4)	0	0.0 (0.0 to 9.5)	0	0.0 (0.0 to 5.7)
MSH6	0	0.0 (0.0 to 4.2)	1	0.3 (0.01 to 1.8)	0	0.0 (0.0 to 9.5)	0	0.0 (0.0 to 5.7)
PMS2*	1	1.1 (0.03 to 6.2)	0	0.0 (0.0 to 1.2)	0	0.0 (0.0 to 9.5)	0	0.0 (0.0 to 5.7)
RAD51C	0	0.0 (0.0 to 4.2)	1	0.3 (0.01 to 1.8)	0	0.0 (0.0 to 9.5)	0	0.0 (0.0 to 5.7)
RAD51D	1	1.1 (0.03 to 6.2)	0	0.0 (0.0 to 1.2)	0	0.0 (0.0 to 9.5)	0	0.0 (0.0 to 5.7)

NOTE. No mutations were identified in the following genes: BARD1; CDH1; STK11; TP53; APC; BMPR1A; CDK4; CDKN2A p14; CDKN2A p16; EPCAM; MLH1; MSH2; MUTYH (biallelic); and SMAD4.

Abbreviations: HER2, human epidermal growth factor receptor 2; HR, hormone receptor; TNBC, triple-negative breast cancer.

^*One HR-positive/HER2-positive patient had deleterious mutations in both BRCA2 and ATM. One TNBC patient had deleterious mutations in both BRCA1 and PMS2. One HR-positive/HER2-negative patient had deleterious mutations in both ATM and CHEK2.

All predictors.

Factors that significantly predicted for a BRCA1/2 mutation included younger age at breast cancer diagnosis (P < .01); Ashkenazi Jewish heritage (P < .01); TNBC (P = .01); tumor histologic grade 3 (P < .01); and family history of breast cancer diagnosed at age younger than 50 years, male breast cancer, or ovarian cancer (P < .01; Table 6). No factors predicted for a mutation in other breast cancer predisposition genes when these genes were analyzed as a single group.

Table 6.

Clinical and Pathologic Predictors of Germline Mutations in BRCA1/2 and Other Breast Cancer Predisposition Genes^*

Variable	No Mutation (n = 436)		BRCA1/2 Mutation (n = 30)†		Other BC Mutation (n = 19)†*		P
	No.	%	No.	%	No.	%	No Mutation v BRCA1/2 Mutation	No Mutation v Other BC Mutation
Patient characteristic
Age at BC diagnosis, years							< .01	.72
Mean ± SD	50.7 ± 11.2		42.6 ± 9.7		51.6 ± 10.9
Median	49		40		53
Range	28-88		31-66		34-68
≤ 45	150	34.4	22	73.3	7	36.8	< .01	.96
46-60	184	42.2	6	20.0	8	42.1
> 60	102	23.4	2	6.7	4	21.1
Ashkenazi Jewish heritage
Yes	29	6.7	7	23.3	2	10.5	< .01	.51
No	407	93.3	23	76.7	17	89.5
History of cancer‡
Yes	37	8.5	1	3.3	3	15.8	.32	.27
No	399	91.5	29	96.7	16	84.2
BC characteristic
Subtype
TNBC	72	16.5	12	40.0	2	10.5	.01	.11
HR-positive/HER2-negative	275	63.1	15	50.0	9	47.4
HR-negative/HER2-positive	33	7.6	2	6.7	2	10.5
HR-positive/HER2-positive	56	12.8	1	3.3	6	31.6
Histology
Ductal	325	74.5	22	73.3	10	52.6	.50	.08
Lobular	33	7.6	1	3.3	2	10.5
Ductal and lobular	58	13.3	4	13.3	4	21.1
Other	20	4.6	3	10.0	3	15.8
Histologic grade§
1	57	13.1	0	0.0	3	15.8	< .01	.94
2	167	38.4	4	13.3	7	36.8
3	211	48.5	26	86.7	9	47.4
Stage
I	169	38.8	12	40.0	4	21.1	.03	.12
II	198	45.4	8	26.7	9	47.4
III	69	15.8	10	33.3	6	31.6
Bilateral disease
Yes	8	1.8	0	0.0	1	5.3	.45	.29
No	428	98.2	30	100.0	18	94.7
Family history of cancer and prior genetic testing
First-degree relative with any cancer‡‖
Yes	242	56.8	15	50.0	12	63.2	.47	.58
No	184	43.2	15	50.0	7	36.8
First- or second-degree relative with any cancer‡‖
Yes	356	83.6	30	100.0	15	78.9	.02	.60
No	70	16.4	0	0.0	4	21.1
First- or second-degree relative with BC or ovarian cancer‡‖
Yes	202	47.4	22	73.3	9	47.4	.01	1.0
No	224	52.6	8	26.7	10	52.6
First- or second-degree relative < 50 years of age with BC, ovarian cancer, or male BC‡‖
Yes	71	16.7	12	40.0	5	26.3	< .01	.27
No	355	83.3	18	60.0	14	73.7

Abbreviations: BC, breast cancer; HER2, human epidermal growth factor receptor 2; HR, hormone receptor; SD, standard deviation; TNBC, triple-negative breast cancer.

^*Three patients with mutations not associated with breast cancer were not included in this analysis.

^†One patient with BRCA2 and ATM mutations is included with BRCA1/2 Mutation (not with Other BC Mutation).

^‡Excludes in situ cancers and nonmelanoma skin cancers.

^§Tumor grade was missing for one patient without a mutation.

^‖Ten patients were missing family history information. These patients were excluded from analysis. If age at diagnosis was unavailable, it was conservatively considered to be older than 50 years.

DISCUSSION

To our knowledge, this is the first study of the frequency of germline mutations in BRCA1/2 and other breast cancer predisposition genes retrospectively done in a prospectively collected, sequential series of patients with breast cancer who consented to DNA banking for clinical research. Among 488 patients, we found that 6.1% had a BRCA1/2 mutation (18.4% among Ashkenazi Jewish and 5.1% among non-Ashkenazi) and an additional 3.9% had a mutation in another breast cancer predisposition gene. In addition, four (0.8%) patients had a mutation in a gene linked to ovarian cancer, RAD51C, RAD51D, or a Lynch syndrome gene, one of whom also had a BRCA1 mutation. In total, 10.7% of patients had a deleterious mutation in at least one cancer predisposition gene. The only other report of germline mutations among unselected patients with breast cancer is from The Cancer Genome Atlas, which found that among 507 cases, 5.5% had a germline BRCA1/2 mutation and 4.3% had a mutation in another cancer predisposition gene. The mutation distribution was almost identical to that found in our study.²⁸ Likewise, the prevalence of mutations in non-BRCA1/2 predisposition genes was 3.7% in more than 1,800 TNBC cases¹⁷ and 4.5% in 289 African American women with breast cancer.¹⁶

Previous studies of multigene panel testing in patients with breast cancer, who were identified through high-risk clinics, have also consistently reported a prevalence of germline mutations in non-BRCA1/2 breast cancer predisposition genes of approximately 4%, with up to an additional 1% in other cancer susceptibility genes if heterozygous MUTYH mutations are excluded.^18-21 The prevalence of mutations in moderate-penetrance breast cancer genes seems to be similar in this sequential series of breast cancer cases and in cases from high-risk clinics. Predictive factors for BRCA1/2 and other high-penetrance genes that lead to referral to high-risk clinics do not seem to predict for mutations in moderate-penetrance genes. For example, as expected, the frequency of BRCA1/2 mutations decreased with increasing age at breast cancer diagnosis. However, the frequency of deleterious mutations in non-BRCA1/2 predisposition genes is independent of age at diagnosis (Table 4). As a result, among women diagnosed with breast cancer between ages 46 and 60 years in our cohort, more than half the mutations are in genes other than BRCA1/2. Among women diagnosed after 60 years of age, 6.4% had a deleterious mutation, with almost three-fourths in genes other than BRCA1/2.

As expected, factors known to enrich for BRCA1/2 mutations (such as Ashkenazi heritage, TNBC subtype, and strong family history of breast or ovarian cancer) predicted for BRCA1/2 mutations in our study. However, these factors were not significantly associated with mutations in other breast cancer predisposition genes when these genes were analyzed as a single group. We were unable to identify any factors that predicted for a mutation in non-BRCA1/2 breast cancer genes. Larger cohorts of women with mutations in each gene are required to identify gene-specific predictors. For example, the prevalence of ER-positive breast cancer is higher among germline CHEK2 1100delC mutation carriers than noncarriers.²⁹

We found that among non-BRCA1/2 genes, mutations in CHEK2 were most common. This is consistent with results from most studies that have evaluated multigene panels in breast cancer cohorts not enriched for TNBC.^19,20,30 The clinical significance of mutations in moderate-risk breast cancer genes such as CHEK2, ATM, and NBN is still being evaluated. An increased risk of contralateral breast cancer has been associated with germline mutations in PALB2 and CHEK2, suggesting that subsequent screening with breast magnetic resonance imaging may be indicated in these patients.^29,31-33 We identified one mutation in PTEN, a high-penetrance cancer predisposition gene associated with Cowden syndrome and an increased risk of breast, thyroid, endometrial, and other cancers. This result would allow initiation of appropriate surveillance and risk-reduction strategies.

We also identified six mutations in three genes (BRIP1, RAD51C, and RAD51D) that are associated with a six- to 12-fold increased risk of ovarian cancer.^10-13,15 Mutations were found even in women with breast cancer diagnosed after 60 years of age. Only two of the six mutation carriers had a personal or family history of ovarian cancer. It seems that identification of mutations in more moderate-penetrance ovarian cancer susceptibility genes will require testing of families with less notable cancer histories.³⁴ Finally, we found two mutations in genes for Lynch syndrome. Identification of such mutations can lead to increased surveillance for and identification of colorectal, endometrial, ovarian, and other cancers in these patients with breast cancer and their relatives.¹⁸

The use of multigene panels for assessment of cancer susceptibility has been increasing rapidly in clinical practice. In addition to including high-penetrance genes with established clinical utility, these panels contain genes for which clinical validity or significance is less certain at this time.³⁵ Proper interpretation of results is critical so that appropriate recommendations for risk management are offered. This presents challenges for clinicians, who often lack genetic training, and their patients, who face decisions about screening and prevention strategies.

Whereas the significance of mutations in several of the non-BRCA1/2 predisposition genes is still being studied, the benefit of identifying BRCA1/2 mutations is well established. In addition to cancer prevention strategies, BRCA-associated cancers have a greater response to therapies such as poly-(ADP) ribose polymerase inhibitors and platinum agents than sporadic cancers.^36-39 Studying a sequential series of patients with breast cancer allowed us to evaluate how often BRCA mutations might be missed in clinical practice, at least in an academic setting. Only four (13.3%) of the BRCA1/2 mutation carriers were first identified through this study, and all 30 carriers fulfilled NCCN 2015 genetic testing criteria. Thus, clinicians seem to be recognizing patients with breast cancer who are appropriate for BRCA1/2 testing, and the NCCN criteria seem to perform well. Increased clinician education about testing criteria might decrease the frequency of unidentified carriers even further.

Consistent with previous studies, we found the highest prevalence of BRCA1/2 mutations among cases with TNBC. For patients with ER-negative or ER-positive breast cancer, we found the frequency of BRCA mutations to be lower if the tumor also overexpressed HER2, consistent with previous reports (Table 5).^40-43

Approximately one-third of patients had at least one VUS, as has been reported in other series evaluating NGS panels.¹⁸ Most of these variants will eventually be reclassified, primarily as benign, but some will likely be deleterious.^27,44 VUSs should not be used to make clinical decisions.

Our study has limitations. Cases were ascertained from an academic center and may not reflect the breast cancer population or prevalence of mutations in the community. In our cohort, the median age at diagnosis was 50 years compared with 61 years in the United States.⁴⁵ Likewise, compared with the general population, a higher proportion of individuals (7.8%) were of Ashkenazi descent. However, although these factors may increase the prevalence of BRCA1/2 mutations, our results show that they do not seem to increase the frequency of mutations in moderate-penetrance breast cancer susceptibility genes. Indeed, one report found a lower frequency of non-BRCA1/2 mutations in the Ashkenazi population.¹⁹ Finally, only 11% of women in the study were nonwhite, limiting the generalizability of our findings in more diverse populations. Large population-based studies are needed to establish the true frequency of mutations in these genes.

In conclusion, this is the largest prospective study to date to assess the prevalence of mutations in cancer susceptibility genes among a sequential series of breast cancer cases seen at an academic institution and not otherwise selected for age, family history, ethnicity, or breast cancer subtype. We identified a mutation in 10.7% of patients, 6.1% in BRCA1/2 (5.1% in patients of non-Ashkenazi descent) and 4.6% in other breast/ovarian cancer predisposition genes. The prevalence of mutations in genes other than BRCA1/2 seems to be relatively consistent regardless of breast cancer population tested, when these genes are assessed as a single group. Although clinicians seem to be identifying the majority of patients with breast cancer who have BRCA1/2 mutations, the lack of predictive factors for mutations in other breast/ovarian cancer predisposition genes presents a challenge for identifying these carriers. Until better predictors emerge, it will be necessary to continue casting a wider net for identification of mutations in non-BRCA1/2 genes. This is despite the unclear clinical significance at present for several genes that are included in many commercially available, broad gene panels.

GLOSSARY TERMS

germline mutation:

an inherited variation in the lineage of germ cells. Germline mutations can be passed on to offspring.

NextGen Sequencing:

a non-Sanger rapid DNA sequencing method that can be done with greater speed, developed after the first methodologic articles describing relatively rapid DNA sequencing produced by Sanger et al (1977).

penetrance:

the likelihood that a given gene mutation will produce disease. This likelihood is calculated by examining the proportion of people with the particular genetic mutation that show symptoms of disease.

triple-negative breast cancer (TNBC):

breast tumors that are negative for estrogen and progesterone receptor expression and that also underexpress HER-neu.

Appendix

Table A1.

Mutations Identified in Study Cohort

Study ID	Ashkenazi Decent	Bilateral Breast Cancer	Gene	Mutation	Mutation Effect	Age at Dx (Years)	Breast Cancer Subtype	Family History
BOB16452	N	N	ATM	c.5623C>T (p.Arg1875*)	Truncation	60	HR+/HER-	None
BOB17213	N	N	BRCA1	c.5467+3A>C	Splice site	34	TNBC	CO, LK, Ovary
BOB17227	Y	N	BRCA1	c.68_69del (p.Glu23Valfs*17)	Frameshift	45	HR-/HER+	Breast, ES, UNP
BOB17377	Y	N	BRCA2	c.1754del (p.Lys585Argfs*29)	Frameshift	62	HR+/HER-	BL, LG, Ovary, LYM, CO
BOB17390	N	N	BRCA1	c.4327C>T (p.Arg1443*)	truncation	32	TNBC	Breast, Breast, Ovary, PAN
BOB17443	Y	N	BRCA1	c.68_69del (p.Glu23Valfs*17)	Frameshift	66	TNBC	Other, Ovary
BOB17447	Y	N	BRCA1	c.68_69del (p.Glu23Valfs*17)	Frameshift	34	HR-/HER+	Breast, CNS, LK
BOB17521	N	N	CHEK2	c.1368dupA (p.Glu457Argfs*33)	Frameshift	42	HR+/HER+	None
BOB17625	N	N	PALB2	c.599del (p.Leu200*)	Frameshift	34	HR+/HER-	Breast, UNP, UNP, LG
BOB17832	N	N	RAD51D	del exon 1	Large rearrangement	40	TNBC	STO, PR, Other, PR, Breast
BOB17843	N	N	CHEK2	c.1100del (p.Thr367Metfs*15)	Frameshift	36	HR-/HER+	Breast, HD, Breast, LK
BOB17913	Y	N	BRIP1	c.2392C>T (p.Arg798*)	Truncation	40	HR+/HER+	PR
BOB18197	N	N	BRCA2	c.5682C>A (p.Tyr1894*)	Truncation	37	HR+/HER-	Breast, STO, CO
BOB18292	N	N	BRCA1	c.4964_4982del (p.Ser1655Tyrfs*16)	Frameshift	37	HR-/HER+	Ovary
BOB18356	N	N	BRCA1	c.220C>T (p.Gln74*)	Truncation	59	TNBC	LG, LG, LG
BOB18732	N	N	CHEK2	c.1100del (p.Thr367Metfs*15)	Frameshift	68	HR+/HER+	PR, ES, Breast, FG, Breast
BOB18736	N	N	BRCA1	del exons 1-2	Large rearrangement	43	TNBC	Breast, Breast
BOB18885	N	N	BRIP1	c.2392C>T (p.Arg798*)	Truncation	50	HR+/HER+	PR, Other, Ovary, STO, Breast, ENDO
BOB200145	N	N	CHEK2	c.1100del (p.Thr367Metfs*15)	Frameshift	58	HR-/HER+	None
BOB20037	N	N	BRCA1	c.4675+1G>A	Splice site	39	HR+/HER-	UNP
BOB20040	N	N	CHEK2	c.1100del (p.Thr367Metfs*15)	Frameshift	64	HR+/HER+	CO, CO, LG, PAN
BOB20049	N	N	NBN	c.127C>T (p.Arg43*)	Truncation	56	TNBC	LG, LG, LG, CNS, PAN
BOB20068	N	N	BRCA1	c.5266dupC (p.Gln1756Profs*74)	Frameshift	43	HR+/HER-	Breast, CO, Breast, LYM
BOB20216	N	N	BRCA2	c.6644dupA (p.Tyr2215*)	Frameshift	34	HR+/HER+	Breast
BOB20297	N	N	CHEK2	c.1100del (p.Thr367Metfs*15)	Frameshift	56	HR+/HER-	MM, CO, LG, WT
BOB20304	Y	N	BRCA2	c.5946del (p.Ser1982Argfs*22)	Frameshift	58	HR+/HER-	PR
BOB20356WB	Y	N	CHEK2	c.1100del (p.Thr367Metfs*15)	Frameshift	46	HR+/HER-	Breast, ENDO, CLL, Breast, STO, PR
BOB20421WB	N	N	ATM	c.7705del (p.Asp2569Metfs*4)	Frameshift	39	HR+/HER-	Other
BOB20467	N	N	ATM	c.3381_3384del (p.Gln1128Lysfs*3)	Frameshift	33	HR+/HER+	Breast, ES, THY, PAN, Breast
BOB20467	N	N	BRCA2	c.6267_6269delinsC (p.Glu2089Aspfs*2)	Frameshift	33	HR+/HER+	Breast, ES, THY, PAN, Breast
BOB20605WB	N	N	BRCA2	c.658_659del (p.Val220Ilefs*4)	Frameshift	47	TNBC	OCMEL
BOB20702WB	N	N	BRIP1	c.2392C>T (p.Arg798*)	Truncation	66	HR+/HER-	CO
BOB20756	N	N	BRCA1	c.5137del (p.Val1713*)	Frameshift	31	HR+/HER-	Breast, Breast, BL, PR, LG, BL
BOB20822WB	N	N	CHEK2	c.1100del (p.Thr367Metfs*15)	Frameshift	45	HR+/HER+	KID, Breast, Breast, KID, LYM, PR
BOB20858WB	N	N	BRCA1	c.5266dupC (p.Gln1756Profs*74)	Frameshift	38	TNBC	STO, BL, Breast, CO, PR
BOB20980WB	N	N	BRCA2	c.5946del (p.Ser1982Argfs*22)	Frameshift	53	HR+/HER-	PR, BL
BOB20988WB	N	N	BRCA2	c.7618-1G>A	Splice site	48	HR+/HER-	Breast, LG
BOB21280WB	N	N	BRCA2	c.8585dupT (p.Glu2863Argfs*6)	Frameshift	38	HR+/HER+	FT, Breast, Ovary, PR
BOB21299WB	N	N	BRCA1	c.5503C>T (p.Arg1835*)	Truncation	33	HR-/HER+	LG, Breast
BOB21299WB	N	N	PMS2	c.137G>T (p.Ser46Ile)	Missense	33	HR-/HER+	LG, Breast
BOB21399WB	N	N	BRCA1	c.415C>T (p.Gln139*)	Truncation	45	TNBC	Breast, CO, BL, CO, Other, CO
BOB21568WB	N	N	CHEK2	c.1100del (p.Thr367Metfs*15)	Frameshift	68	HR+/HER+	Ovary, PAN
BOB21578WB	N	N	BRCA2	c.8537_8538del (p.Glu2846Glyfs*22)	Frameshift	41	HR+/HER-	Breast, Breast, Breast, PR
BOB21663WB	N	N	BRCA1	c.5266dupC (p.Gln1756Profs*74)	Frameshift	33	TNBC	Breast, Breast
BOB21668WB	N	N	BRCA1	del exons 1-23	Large rearrangement	42	HR+/HER+	HN, Breast, Ovary, Cx
BOB21887WB	N	N	BRIP1	c.2255_2256del (p.Lys752Argfs*12)	Frameshift	53	TNBC	BL, Breast, CNS, MEL
BOB21984WB	Y	N	BRCA2	c.5946del (p.Ser1982Argfs*22)	Frameshift	39	HR+/HER-	LG
BOB22130WB	N	Y	ATM	c.3993+1G>A	Splice site	44	HR+/HER-	CO, Breast, Breast
BOB22130WB	N	Y	CHEK2	c.444+1G>A	Splice site	44	HR+/HER-	CO, Breast, Breast
BOB22416WB	N	N	RAD51C	c.577C>T (p.Arg193*)	Truncation	77	HR+/HER-	None
BOB23112WB	N	N	BRCA1	c.962G>A (p.Trp321*)	Truncation	56	HR-/HER+	Breast, Breast
BOB23117WB	N	N	PTEN	c.388C>T (p.Arg130*)	Truncation	56	HR+/HER-	PR, THY, ENDO, LG, PAN
BOB23508WB	N	N	MSH6	del exons 3-9	Large rearrangement	50	HR+/HER+	ENDO, ENDO, LG
BOB24634	Y	N	BRCA2	c.2808_2811del (p.Ala938Profs*21)	Frameshift	45	HR+/HER+	ES, PR
BOB24943	N	N	BRCA1	c.5266dupC (p.Gln1756Profs*74)	Frameshift	34	HR+/HER-	LARYNX, Breast, LK, Breast, CO

Abbreviations: BL, bladder cancer; Breast, breast cancer; CLL, chronic lymphocytic leukemia; CNS, central nervous system cancer; CO, colorectal cancer; Cx, cervical cancer; Dx, diagnosis; ENDO, endometrial/uterine cancer; ES, esophageal cancer; FG, female genital cancer, unspecified; FT, fallopian tube cancer; HD, Hodgkins lymphoma; HER, human epidermal growth factor receptor; HN, head and neck cancer; HR, hormone receptor; KID, kidney cancer unspecified; larynx, laryngeal cancer; LG, lung cancer; LK, leukemia; LYM, lymphoma; MEL, melanoma; MM, multiple myeloma; N, no; OCMEL, ocular melanoma; Other, cancer unspecified; Ovary, ovarian cancer; PAN, pancreatic cancer-exocrine; PR, prostate cancer; STO, stomach cancer; THY, thyroid cancer; TNBC, triple-negative breast cancer; UNP, cancer of unknown primary; WT, wilms tumor; Y, yes.

APC NM_000038.5 ATM NM_000051.3 BARD1 NM_000465.3 BMPR1A NM_004329.2 BRCA1 NM_007294.3 BRCA2 NM_000059.3 BRIP1 NM_032043.2 CDH1 NM_004360.3 CDK4 NM_000075.3 CHEK2 NM_007194.3

EPCAM NM_002354.2 MLH1 NM_000249.3 MSH2 NM_000251.2 MSH6 NM_000179.2 MUTYH NM_001128425.1 NBN NM_002485.4 P14ARF NM_058195.3

P16 NM_000077.4 PALB2 NM_024675.3 PMS2 NM_000535.5 PTEN NM_000314.4 RAD51C NM_058216.2 RAD51D NM_002878.3 SMAD4 NM_005359.5 STK11 NM_000455.4 TP53 NM_000546.5

Table A2.

Variants of Unknown Significance Identified in Study Cohort

Study ID	Gene	Variant of Uncertain Significance
BOB21387WB	APC	c.5424_5426del (p.Asn1808del)
BOB20704WB	APC	c.437C>T (p.Ala146Val)
BOB20752WB	APC	c.1276G>T (p.Ala426Ser)
BOB20406WB	APC	c.2204C>T (p.Ala735Val)
BOB19922	APC	c.8462A>G (p.Asp2821Gly)
BOB21973WB	APC	c.420G>C (p.Glu140Asp)
BOB20898WB	APC	c.95A>G (p.Asn32Ser)
BOB17786	APC	c.7399C>A (p.Pro2467Thr)
BOB21159WB	APC	c.3511C>T (p.Arg1171Cys)
BOB21291WB	APC	c.4766G>A (p.Arg1589His)
BOB17786	APC	c.5026A>G (p.Arg1676Gly)
BOB20049	APC	c.5357G>A (p.Arg1786Lys)
BOB21384WB	APC	c.5357G>C (p.Arg1786Thr)
BOB24011	APC	c.5503A>G (p.Arg1835Gly)
BOB16413	APC	c.7589G>A (p.Arg2530Gln)
BOB17440	APC	c.2717C>T (p.Ser906Phe)
BOB20365WB	APC	c.2725A>G (p.Thr909Ala)
BOB20054	APC	c.3374T>C (p.Val1125Ala)
BOB18356	ATM	c.1960C>A (p.Gln654Lys)
BOB21660WB	ATM	c.2096A>G (p.Glu699Gly)
BOB20412	ATM	c.2275A>G (p.Ser759Gly)
BOB21410WB	ATM	c.2494C>T (p.Arg832Cys)
BOB22428WB	ATM	c.2494C>T (p.Arg832Cys)
BOB20047	ATM	c.2552A>G (p.Asp851Gly)
BOB16447	ATM	c.2699T>C (p.Met900Thr)
BOB17825	ATM	c.3014A>G (p.Asn1005Ser)
BOB20403WB	ATM	c.3240C>A (p.Asp1080Glu)
BOB200138	ATM	c.3467C>T (p.Thr1156Met)
BOB17688	ATM	c.3590T>C (p.Val1197Ala)
BOB20212	ATM	c.3925G>A (p.Ala1309Thr)
BOB21585WB	ATM	c.3925G>A (p.Ala1309Thr)
BOB21877WB	ATM	c.3993+5G>T
BOB21139WB	ATM	c.4148C>T (p.Ser1383Leu)
BOB21979WB	ATM	c.4324T>C (p.Tyr1442His)
BOB200137	ATM	c.4375G>A (p.Gly1459Arg)
BOB22337WB	ATM	c.4388T>G (p.Phe1463Cys)
BOB24634	ATM	c.4388T>G (p.Phe1463Cys)
BOB20412	ATM	c.4420C>G (p.His1474Asp)
BOB20920WB	ATM	c.4424A>G (p.Tyr1475Cys)
BOB18118	ATM	c.4709T>C (p.Val1570Ala)
BOB17701	ATM	c.4949A>G (p.Asn1650Ser)
BOB20521WB	ATM	c.5693G>A (p.Arg1898Gln)
BOB17446	ATM	c.6067G>A (p.Gly2023Arg)
BOB23405	ATM	c.6067G>A (p.Gly2023Arg)
BOB18191	ATM	c.6332A>G (p.His2111Arg)
BOB20888WB	ATM	c.6543G>T (p.Glu2181Asp)
BOB20885WB	ATM	c.6860G>C (p.Gly2287Ala)
BOB17443	ATM	c.6919C>T (p.Leu2307Phe)
BOB21067WB	ATM	c.6919C>T (p.Leu2307Phe)
BOB21289WB	ATM	c.6919C>T (p.Leu2307Phe)
BOB21984WB	ATM	c.6919C>T (p.Leu2307Phe)
BOB17646	ATM	c.7618G>A (p.Val2540Ile)
BOB20767WB	ATM	c.7740A>C (p.Arg2580Ser)
BOB17523	ATM	c.7919C>T (p.Thr2640Ile)
BOB18813	ATM	c.7988T>C (p.Val2663Ala)
BOB18356	ATM	c.8147T>C (p.Val2716Ala)
BOB21385WB	ATM	c.8734A>G (p.Arg2912Gly)
BOB20044	ATM	c.977T>C (p.Ile326Thr)
BOB21282WB	BARD1	c.1042G>A (p.Val348Ile)
BOB20970WB	BARD1	c.2183C>T (p.Ser728Phe)
BOB16447	BARD1	c.2282G>A (p.Ser761Asn)
BOB20049	BARD1	c.2282G>A (p.Ser761Asn)
BOB21068WB	BARD1	c.2282G>A (p.Ser761Asn)
BOB21742WB	BARD1	c.581G>A (p.Arg194Lys)
BOB20751WB	BARD1	c.620A>G (p.Lys207Arg)
BOB20606WB	BARD1	c.632T>C (p.Leu211Ser)
BOB22413WB	BARD1	c.668A>G (p.Glu223Gly)
BOB200140	BARD1	c.716T>A (p.Leu239Gln)
BOB21660WB	BARD1	c.841C>T (p.Pro281Ser)
BOB20049	BARD1	c.928T>G (p.Ser310Ala)
BOB17708	BMPR1A	c.1327C>T (p.Arg443Cys)
BOB21384WB	BMPR1A	c.1327C>T (p.Arg443Cys)
BOB213892WB	BMPR1A	c.1327C>T (p.Arg443Cys)
BOB20235	BMPR1A	c.560G>A (p.Arg187His)
BOB21048WB	BMPR1A	c.676-3A>C
BOB18108	BRCA1	c.1263A>C (p.Glu421Asp)
BOB21874WB	BRCA1	c.5513T>A (p.Val1838Glu)
BOB18622	BRCA2	c.4901T>C (p.Phe1634Ser)
BOB23117WB	BRCA2	c.7925T>G (p.Phe2642Cys)
BOB18803	BRCA2	c.7434A>C (p.Leu2478Phe)
BOB21143WB	BRCA2	c.8902A>G (p.Thr2968Ala)
BOB20819WB	BRIP1	c.1616G>A (p.Arg539Lys)
BOB17429	BRIP1	c.1899C>G (p.Ile633Met)
BOB17713	BRIP1	c.1899C>G (p.Ile633Met)
BOB20231	BRIP1	c.205+5G>A
BOB18605	BRIP1	c.2071A>C (p.Ile691Leu)
BOB22122WB	BRIP1	c.2120G>A (p.Arg707His)
BOB20986WB	BRIP1	c.262_264del (p.Cys88del)
BOB21880WB	BRIP1	c.2830C>G (p.Gln944Glu)
BOB17212	BRIP1	c.337A>C (p.Thr113Pro)
BOB17786	BRIP1	c.3464G>A (p.Gly1155Glu)
BOB20223	BRIP1	c.3651G>T (p.Trp1217Cys)
BOB17776	BRIP1	c.380-17T>A
BOB20592WB	BRIP1	c.728T>C (p.Ile243Thr)
BOB21143WB	BRIP1	c.728T>C (p.Ile243Thr)
BOB21573WB	BRIP1	c.778A>G (p.Thr260Ala)
BOB17377	BRIP1	c.790C>T (p.Arg264Trp)
BOB20756	BRIP1	c.790C>T (p.Arg264Trp)
BOB20988WB	BRIP1	c.820A>G (p.Thr274Ala)
BOB18605	BRIP1	del exon 7
BOB21139WB	CDH1	c.1090A>T (p.Thr364Ser)
BOB18734	CDH1	c.1297G>A (p.Asp433Asn)
BOB21048WB	CDH1	c.2329G>A (p.Asp777Asn)
BOB18354	CDH1	c.499G>A (p.Glu167Lys)
BOB18287	CDH1	c.88C>A (p.Pro30Thr)
BOB21284WB	CDK4	c.209A>G (p.Asn70Ser)
BOB18539	CDK4	c.820-15T>G
BOB21587WB	CHEK2	c.1217G>A (p.Arg406His)
BOB17443	CHEK2	c.1283C>T (p.Ser428Phe)
BOB17433	CHEK2	c.1343T>G (p.Ile448Ser)
BOB20594WB	CHEK2	c.1343T>G (p.Ile448Ser)
BOB200150	CHEK2	c.1558_1559insC (p.Lys520Thrfs*5)
BOB17184	CHEK2	c.190G>A (p.Glu64Lys)
BOB22413WB	CHEK2	c.190G>A (p.Glu64Lys)
BOB18002	CHEK2	c.275C>T (p.Pro92Leu)
BOB18810	CHEK2	c.410G>A (p.Arg137Gln)
BOB17992	CHEK2	c.422A>C (p.Lys141Thr)
BOB18283	CHEK2	c.432T>G (p.Phe144Leu)
BOB21805WB	CHEK2	c.470T>C (p.Ile157Thr)
BOB20237	CHEK2	c.499G>A (p.Gly167Arg)
BOB21046WB	CHEK2	c.598G>A (p.Val200Ile)
BOB17828	CHEK2	c.715G>A (p.Glu239Lys)
BOB21787WB	CHEK2	c.787G>C (p.Glu263Gln)
BOB21923WB	CHEK2	c.931G>A (p.Asp311Asn)
BOB21384WB	MLH1	dup entire MLH1 gene
BOB17691	MLH1	c.226G>A (p.Val76Ile)
BOB18664	MSH2	c.982G>C (p.Ala328Pro)
BOB17227	MSH2	c.944G>T (p.Gly315Val)
BOB20421WB	MSH2	c.2458+6T>C
BOB18197	MSH2	c.835C>G (p.Leu279Val)
BOB17446	MSH2	c.775C>T (p.Pro259Ser)
BOB20874WB	MSH2	c.2728C>A (p.Gln910Lys)
BOB17994	MSH2	c.440T>G (p.Val147Gly)
BOB17828	MSH6	c.3974_3976del (p.Lys1325del)
BOB20594WB	MSH6	c.3294C>G (p.Cys1098Trp)
BOB18110	MSH6	c.1599G>C (p.Glu533Asp)
BOB16469	MSH6	c.3173-10C>A
BOB18399	MSH6	c.3801+5G>A
BOB21391WB	MSH6	c.1793A>G (p.Lys598Arg)
BOB18268	MSH6	c.2225A>G (p.Asn742Ser)
BOB23707WB	MSH6	c.41C>T (p.Ser14Phe)
BOB17777	MYH	c.1013_1014delinsGC (p.Gln338delinsArg)
BOB20856WB	MYH	c.1013_1014delinsGC (p.Gln338delinsArg)
BOB20894WB	MYH	c.1013_1014delinsGC (p.Gln338delinsArg)
BOB18198	MYH	c.821G>A (p.Arg274Gln)
BOB17696	MYH	c.820C>T (p.Arg274Trp)
BOB17212	MYH	c.1276C>T (p.Arg426Cys)
BOB18489	MYH	c.1276C>T (p.Arg426Cys)
BOB200139	MYH	c.305G>A (p.Ser102Asn)
BOB21157WB	NBN	c.1036G>A (p.Val346Met)
BOB20888WB	NBN	c.1354A>C (p.Thr452Pro)
BOB21973WB	NBN	c.1444A>G (p.Arg482Gly)
BOB24387	NBN	c.1690G>A (p.Glu564Lys)
BOB17995	NBN	c.1720T>A (p.Leu574Ile)
BOB18279	NBN	c.1952C>T (p.Pro651Leu)
BOB18481	NBN	c.643C>T (p.Arg215Trp)
BOB20974WB	NBN	c.643C>T (p.Arg215Trp)
BOB21578WB	NBN	c.643C>T (p.Arg215Trp)
BOB17928	P16	c.9_32del (p.Ala4_Pro11del)
BOB21134WB	P16	c.430C>T (p.Arg144Cys)
BOB20888WB	PALB2	c.400G>A (p.Asp134Asn)
BOB20592WB	PALB2	c.656A>G (p.Asp219Gly)
BOB20988WB	PALB2	c.656A>G (p.Asp219Gly)
BOB18125	PALB2	c.3037A>G (p.Ile1013Val)
BOB20870WB	PALB2	c.3350+4A>G
BOB21672WB	PALB2	c.298C>T (p.Leu100Phe)
BOB21660WB	PALB2	c.1564C>T (p.Pro522Ser)
BOB20037	PALB2	c.22C>A (p.Pro8Thr)
BOB21587WB	PALB2	c.109C>T (p.Arg37Cys)
BOB17440	PALB2	c.3296C>G (p.Thr1099Arg)
BOB18667	PALB2	c.950C>T (p.Thr317Ile)
BOB18594	PALB2	c.1430C>T (p.Thr477Ile)
BOB20237	PMS2	c.1092T>A (p.Asp364Glu)
BOB17523	PMS2	c.1417G>A (p.Glu473Lys)
BOB17824	PMS2	c.86G>C (p.Gly29Ala)
BOB20760WB	PMS2	c.86G>C (p.Gly29Ala)
BOB24634	PMS2	c.53T>C (p.Ile18Thr)
BOB20970WB	PMS2	c.935T>C (p.Met312Thr)
BOB21149WB	PMS2	c.1723A>G (p.Asn575Asp)
BOB17523	PMS2	c.58C>G (p.Arg20Gly)
BOB20041	PMS2	c.1567T>A (p.Ser523Thr)
BOB17196	PMS2	c.2149G>A (p.Val717Met)
BOB20894WB	PMS2	c.2149G>A (p.Val717Met)
BOB21149WB	PMS2	c.2386G>A (p.Val796Ile)
BOB17992	PTEN	c.210-7_210-3del
BOB20892WB	RAD51C	c.-13A>C
BOB20047	RAD51C	c.428A>G (p.Gln143Arg)
BOB20233	RAD51C	c.601C>G (p.Leu201Val)
BOB18416	RAD51C	c.605A>G (p.Asp202Gly)
BOB18729	RAD51C	c.752A>G (p.Asp251Gly)
BOB21394WB	RAD51C	c.790G>A (p.Gly264Ser)
BOB18729	RAD51C	c.7G>A (p.Gly3Arg)
BOB20042	RAD51D	c.491T>C (p.Leu164Pro)
BOB21149WB	RAD51D	c.620C>T (p.Ser207Leu)
BOB21391WB	RAD51D	c.972G>T (p.Gln324His)
BOB21979WB	SMAD4	c.1448-18C>A
BOB18288	SMAD4	c.667+3G>A
BOB20958WB	STK11	c.1040C>G (p.Ala347Gly)
BOB17218	STK11	c.1211C>T (p.Ser404Phe)
BOB18813	TP53	c.139C>T (p.Pro47Ser)
BOB22178WB	TP53	c.139C>T (p.Pro47Ser)
BOB17207	TP53	c.329G>A (p.Arg110His)
BOB23508WB	TP53	c.75-18T>G
BOB20974WB	TP53	c.845G>A (p.Arg282Gln)
BOB20610WB	TP53	c.877G>T (p.Gly293Trp)

APC NM_000038.5 ATM NM_000051.3 BARD1 NM_000465.3 BMPR1A NM_004329.2 BRCA1 NM_007294.3 BRCA2 NM_000059.3 BRIP1 NM_032043.2 CDH1 NM_004360.3 CDK4 NM_000075.3 CHEK2 NM_007194.3

EPCAM NM_002354.2 MLH1 NM_000249.3 MSH2 NM_000251.2 MSH6 NM_000179.2 MUTYH NM_001128425.1 NBN NM_002485.4 P14ARF NM_058195.3

P16 NM_000077.4 PALB2 NM_024675.3 PMS2 NM_000535.5 PTEN NM_000314.4 RAD51C NM_058216.2 RAD51D NM_002878.3 SMAD4 NM_005359.5 STK11 NM_000455.4

TP53 NM_000546.5

AUTHOR CONTRIBUTIONS

Conception and design: Nadine Tung, Nancy U. Lin, Brian A. Allen, Anne-Renee Hartman, Eric P. Winer, Judy E. Garber

Financial support: Richard J. Wenstrup

Provision of study materials or patients: Nancy U. Lin, Eric P. Winer, Judy E. Garber

Collection and assembly of data: Nancy U. Lin, Eric P. Winer, Judy E. Garber

Data analysis and interpretation: Nadine Tung, Nancy U. Lin, John Kidd, Brian A. Allen, Nanda Singh, Richard J. Wenstrup, Anne-Renee Hartman, Judy E. Garber

Manuscript writing: All authors

Final approval of manuscript: All authors

AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

Frequency of Germline Mutations in 25 Cancer Susceptibility Genes in a Sequential Series of Patients With Breast Cancer

The following represents disclosure information provided by authors of this manuscript. All relationships are considered compensated. Relationships are self-held unless noted. I = Immediate Family Member, Inst = My Institution. Relationships may not relate to the subject matter of this manuscript. For more information about ASCO's conflict of interest policy, please refer to www.asco.org/rwc or jco.ascopubs.org/site/ifc.

Nadine Tung

Research Funding: Myriad Genetics

Nancy U. Lin

No relationship to disclose

John Kidd

Employment: Myriad Genetics

Stock or Other Ownership: Myriad Genetics

Brian A. Allen

Employment: Myriad Genetics

Stock or Other Ownership: Myriad Genetics

Nanda Singh

Employment: Myriad Genetics

Stock or Other Ownership: Myriad Genetics

Richard J. Wenstrup

Employment: Myriad Genetics

Leadership: Myriad Genetics

Stock or Other Ownership: Myriad Genetics

Anne-Renee Hartman

Employment: Myriad Genetics

Stock or Other Ownership: Myriad Genetics

Eric P. Winer

No relationship to disclose

Judy E. Garber

Consulting or Advisory Role: Novartis (I), Pfizer (I), Pfizer, Sequenom, SV Life Sciences (I)

Research Funding: Myriad Genetics, Novartis (I), Pfizer (I), Ambry Laboratories