Study Populations

Table 1. Table 1. Demographic Characteristics of the Patients with Prostate Cancer.

Seven case series of men with metastatic prostate cancer across multiple institutions in the United States and United Kingdom, including a total of 692 patients, were analyzed. All the patients had a diagnosis of metastatic prostate cancer and were not selected on the basis of family history, age, or any knowledge of genetic background. The demographic characteristics of the men in each series are summarized in Table 1. Detailed information on the specific germline mutations and on clinical features of mutation carriers in each series is provided in Tables S1, S2, and S3 in the Supplementary Appendix, available with the full text of this article at NEJM.org.

Case Series 1, the Stand Up to Cancer–Prostate Cancer Foundation (SU2C-PCF) International Prostate Cancer Dream Team discovery series, was made up of 150 patients for whom data were previously reported in the SU2C-PCF study of molecular stratification of metastatic prostate cancer.18 Case Series 2, the SU2C-PCF validation series, was made up of 84 patients who were newly enrolled in the SU2C-PCF study and for whom data had not been reported previously. Case Series 3, Royal Marsden Prostate Cancer Genomics series, included 131 patients who were considered for enrollment in clinical trials at the Royal Marsden Hospital from January 2013 through July 2015. Case Series 4 consisted of 91 consecutive patients included in the University of Washington rapid autopsy program from 1997 through 2013. Case Series 5 included 69 consecutive patients who were enrolled in the Weill Cornell Medical College precision medicine program. Case Series 6 was made up of 43 consecutive patients from the University of Michigan rapid autopsy program. Case Series 7, from the Memorial Sloan Kettering Cancer Center, included 124 consecutive patients who were enrolled through the Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) study.

The protocols for these case series were approved by the local institutional review boards, and written informed consent was obtained from all patients at the local sites before enrollment. Correlative clinical data were collected at each site with the use of electronic patient records and were entered into deidentified databases. The study was designed by the Stand Up To Cancer–Prostate Cancer Foundation International Prostate Cancer Dream Team investigators. The study sponsors had no role in the design of the study, the collection or analysis of the data, or the preparation of the manuscript. The manuscript was written by four of the authors. All authors reviewed the manuscript, agreed to submit the manuscript for publication, and vouch for the accuracy and completeness of the data and for the fidelity of the study to the protocol.

Sequencing and Bioinformatics Analysis

For the analysis involving Case Series 1, 2, and 6, whole-exome sequencing of germline and tumor DNA was performed as described previously.18 Germline DNA from buccal swabs, buffy coats, or whole blood was isolated with the use of the QIAGEN DNeasy Blood and Tissue Kit. Whole-exome sequencing was performed on the Illumina HiSeq 2500 in paired-end mode.

For the analysis of Case Series 3, germline DNA was extracted from saliva or buccal swab samples with the use of the Oragene kit (DNA Genotek). Libraries for targeted sequencing were constructed with a customized GeneRead DNaseq Panel (Qiagen) covering 53 genes and run on the Illumina MiSeq sequencer, as described previously.16

For the analyses of Case Series 4 and 5, germline DNA was extracted from peripheral blood or nontumor tissue and from matched tumor DNA, as described previously.19 Targeted deep sequencing was performed with the BROCA panel of 53 DNA-repair pathway genes. The bioinformatics pipeline has been described previously.20,21 For tumors from Case Series 5, analyses were performed by means of exome sequencing, as described previously.22 For Case Series 7, tumor and germline genomic sequencing was performed as described previously, with the use of the MSK-IMPACT hybrid capture-based next-generation sequencing assay.23,24

The mean sequencing depth of coverage was more than 100× for all case series, with the exception of sequencing of BAP1, BARD1, BRIP1, and FAM175A, which were not included on the Royal Marsden Hospital panel, and GEN1, which was not included on the Royal Marsden Hospital or Memorial Sloan Kettering panel. Data from the Royal Marsden Hospital and Memorial Sloan Kettering cases were censored for analyses of these genes. In addition, data were censored for CHEK2 in 158 cases for which exon sequencing coverage was incomplete. The depth of coverage for each gene according to site is provided in Table S4 in the Supplementary Appendix.

To compare our results with data from a large series of patients with localized prostate cancer, we analyzed public data from the Cancer Genome Atlas prostate cancer study.25 Paired-end reads (100 bp) were aligned to the hg19 reference human genome with the use of the Burrows–Wheeler Aligner. Annotations were defined with ANNOVAR (http://annovar.openbioinformatics.org/en/latest). Population allele frequencies were extracted from the Exome Aggregation Consortium ExAC Browser (http://exac.broadinstitute.org/), 1000 Genomes (www.1000genomes.org), and the single-nucleotide polymorphism database of the National Center for Biotechnology Information (dbSNP), version 138 (www.ncbi.nlm.nih.gov/projects/SNP).

Interpretation of Variants

Table 2. Table 2. Germline Mutations in Metastatic Cases as Compared with the General Population and Primary Cases.

Our analysis focused on variants identified among 20 genes associated with autosomal dominant cancer-predisposition syndromes that involve maintenance of DNA integrity (Table 2). The pathogenicity of germline variants was determined according to established American College of Medical Genetics and Genomics and Association for Molecular Pathology consensus criteria and International Agency for Research on Cancer guidelines.24,26 At least two independent expert reviewers evaluated all variants against published literature and public databases, including ClinVar and variant-specific databases, in addition to population frequency databases, including 1000 Genomes and the Exome Aggregation Consortium. Expected high-penetrance or moderate-penetrance variants classified as mutations that are pathogenic or likely to be pathogenic are reported here. Low-penetrance variants, such as CHEK2 p.I157T, were excluded.

Statistical Analysis

Associations between DNA-repair gene mutation status and age, race, or Gleason score strata were evaluated with the use of two-sided Fisher’s exact tests. The frequencies of DNA-repair gene mutations among the 692 patients with metastatic prostate cancer were evaluated relative to the expected frequencies from the Exome Aggregation Consortium (53,105 persons) or the Cancer Genome Atlas cohort (499 persons) with the use of two-sided exact binomial tests. We also performed analyses in which the 150 men from the previously reported Case Series 1 were excluded18 (Table S5 in the Supplementary Appendix). No adjustments were made for multiple comparisons; P values of less than 0.05 were considered to indicate statistical significance.