Background Genetic testing for BRCA1 and BRCA2 is offered typically to selected women based on age of onset and family history of cancer. However, current internationally accepted genetic testing referral guidelines are built mostly on data from cancer genetics clinics in women of European descent. To evaluate the appropriateness of such guidelines in Asians, we have determined the prevalence of germ line variants in an unselected cohort of Asian patients with breast cancer and healthy controls.
Methods Germ line DNA from a hospital-based study of 2575 unselected patients with breast cancer and 2809 healthy controls were subjected to amplicon-based targeted sequencing of exonic and proximal splice site junction regions of BRCA1 and BRCA2 using the Fluidigm Access Array system, with sequencing conducted on a Illumina HiSeq2500 platform. Variant calling was performed with GATK UnifiedGenotyper and were validated by Sanger sequencing.
Results Fifty-five (2.1%) BRCA1 and 66 (2.6%) BRCA2 deleterious mutations were identified among patients with breast cancer and five (0.18%) BRCA1 and six (0.21%) BRCA2 mutations among controls. One thousand one hundred and eighty-six (46%) patients and 97 (80%) carriers fulfilled the National Comprehensive Cancer Network guidelines for genetic testing.
Conclusion Five per cent of unselected Asian patients with breast cancer carry deleterious variants in BRCA1 or BRCA2. While current referral guidelines identified the majority of carriers, one in two patients would be referred for genetic services. Given that such services are largely unavailable in majority of low-resource settings in Asia, our study highlights the need for more efficient guidelines to identify at-risk individuals in Asia.
- breast cancer
- national comprehensive cancer network
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Genetic testing for mutations in BRCA1 and BRCA2 has led to the identification of individuals at higher risk of breast cancer, enabled risk-stratified approaches for management of risk in relatives and enabled the selection of individuals who may benefit from therapies targeting the DNA damage response.1 The majority of studies have hitherto screened high-risk patients with breast cancer selected on the basis of age, family history and, some studies, tumour subtype, such as oestrogen receptor negative or triple negative breast cancer (TNBC).2 These studies have reported the prevalence of deleterious germ line variants in BRCA1 and BRCA2 among Asian high-risk patients with breast cancer is similar to that in other populations, ranging between 10% and 20%.2–6 However, it is estimated that less than 1% of the 560 000 patients with breast cancer diagnosed in 14 Asian countries each year benefit from genetic testing services, because of high cost and limited accessibility.7 In such resource-limited settings, it is critical to have appropriate guidelines for referral for genetic testing. While internationally accepted clinical criteria for referral can be obtained from the National Comprehensive Cancer Network (NCCN) guidelines on genetic/familial high-risk assessment,8 such guidelines has been developed primarily from data from population of European ancestry. There are established differences in breast cancer epidemiology between Asian and Caucasian individuals,9 but the appropriateness of such guidelines in identifying mutation carriers have hitherto not been assessed in Asian populations.
To evaluate current genetic testing referral guidelines, we have conducted an analysis of BRCA1 and BRCA2 in a multiethnic cohort of unselected patients with breast cancer of Chinese, Malay and Indian ethnicity from Malaysia. Our study provides data on the appropriateness of current guidelines for identifying individuals at higher risk of carrying germ line variants in BRCA1 and BRCA2 and lays the foundation for developing risk assessment tools for Asian populations.
We included patients with breast cancer and control subjects who participated in the Malaysian Breast Cancer Genetic Study between October 2002 and March 2015. Incident and prevalent cases and controls were recruited from two hospitals: University Malaya Medical Centre and Sime Darby Medical Centre.10 11 Of the 2870 patients with breast cancer and 2999 control subjects recruited, 2575 and 2809 cases and controls, respectively, were included in this study (see tables 1 and 2 in the online supplementary file 1 for exclusion criteria). Of these 2575 cases, 887 (34%) women were considered to be a priori high or moderate risk and had been previously tested for germ line alterations in BRCA1 and BRCA2 by Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) analysis as described.12–15 All study participants provided written informed consent. The study was approved by the Medical Ethics Committee of University Malaya Medical Centre (application number: 842.9) and the Independent Ethics Committee of Sime Darby Medical Centre (application numbers: 201109.4 and 201208.1).
Sequencing library preparation and sequencing
Fluidigm D3 design software (Fluidigm, San Francisco, California, USA) was used to design a targeted sequencing panel that included the coding sequences and intron/exon boundaries of coding exons from 31 known or suspected breast cancer susceptibility genes, including BRCA1 and BRCA2. Target sequence enrichment was performed using 48.48 Fluidigm Access Arrays (Fluidigm, San Francisco, California, USA) then sequenced on Illumina Hi-Seq2500 instrument (Illumina, San Diego, California, USA) according to the manufacturer’s protocol as previously described.16 The median read depth across the 261 amplicons covering the BRCA1 and BRCA2 coding sequence was 673 (IQR 534–909).
Sequenced reads were demultiplexed and converted from the Illumina binary format into FASTQ format. Next, adaptor sequences were trimmed using Cutadapt (https://pypi.python.org/pypi/cutadapt). Sequenced reads were then aligned against the human genome reference sequence (hg19) with Burrows-Wheeler Aligner.17 Subsequent local insertion/deletion (indel) realignment and base quality score recalibration were performed using the Genome Analysis Toolkit (GATK; https://www.broadinstitute.org/gatk). Genetic variants were called with GATK Unified Genotyper using the default parameters except –minIndelFrac (set to 0.05).18 Variants were annotated using ANNOVAR (http://www.openbioinformatics.org/annovar)19 and missense variants were further annotated using Align-GVGD (http://agvgd.iarc.fr).20 Nonsense, frameshift, canonical splice site variants (positions −2 and −1 upstream of an exon start and +1 and+2 downstream of an exon end) and single nucleotide variants classified as Class 4 or Class 5 according to BRCA Mutation Database (http://arup.utah.edu/database/BRCA/) or Leiden Open Variation Database were considered deleterious, except for variants located at the C-terminal of BRCA1 and BRCA2 (amino acid position 1856–1863 and 3326–3385, respectively). All deleterious and non-C0 variants as per Align-GVGD were validated by Sanger sequencing.
Analyses were based on the variants identified through the analysis of the Next generation sequencing data only. Carriers of large genomic rearrangement (LGR), non-LGR deleterious variants and variants of unknown significance (VUS) previously identified but not detected in this sequencing study were considered as non-carriers. Categorical and continuous variables were compared using χ2 test and t-test, respectively. Statistical tests were considered significant based on two-sided hypothesis tests with p<0.05.
NCCN guidelines and MyCPG for BRCA1 and BRCA2 testing
The NCCN guidelines V.1.2017 and Malaysian Clinical Practice Guidelines (MyCPG)9 for genetic testing of BRCA1 and BRCA2 for BRCA-related breast and ovarian cancer syndrome were used to identify patients with breast cancer and BRCA1 and BRCA2 carriers whom met testing criteria for BRCA1 and BRCA2 screening. The NCCN guidelines are a statement of evidence and consensus of the authors regarding their views of currently accepted approaches to treatment. The MyCPG are meant to be guides for clinical practice in Malaysia based on the best available evidence at the time of development. BRCA1 and BRCA2 testing criteria for both guidelines used in this study are described in table 1.
Comparisons of the characteristics of breast cancer cases and the healthy women attending opportunistic screening mammography are shown in table 2 and table 3 in the online supplementary file 1. Approximately two-thirds of cases and controls were of Chinese ancestry. Patients with breast cancer were, on average, younger than the controls and enriched for family history of breast cancers up to second degree.
Supplementary file 1
BRCA1 and BRCA2 mutations and VUS
Of the 2575 patients with breast cancer, 55 (2.1%) carried deleterious variants in BRCA1 and 66 (2.6%) had deleterious variants in BRCA2 (table 3). The frequency of deleterious variants was similar in Indian (7.5%) and Malay patients (6.7%), but lower in Chinese patients (3.5%, p<0.01). BRCA2 deleterious variants were more common than BRCA1 deleterious variants among Chinese patients (2.3% vs 1.2%) but less common in Indian patients (2.8% vs 5.0%), while the frequencies were similar in Malay patients (3.3% vs 3.5%; p<0.01 for difference in BRCA1:BRCA2 ratio).
Of 2809 control subjects, five (0.18%) had deleterious variants in BRCA1 and six (0.21%) had deleterious variants in BRCA2 (table 3). The deleterious variant frequencies of BRCA1 and BRCA2 in the controls were similar to those in the Exome Aggregation Consortium East Asian population with reported deleterious variant frequencies of 0.16% and 0.21% for BRCA1 and BRCA2, respectively.
Deleterious variants in BRCA1 and BRCA2 were significantly more common in breast cancer cases compared with control subjects, with estimated ORs for breast cancer of 12.6 (95% CI 5.0 to 31.4) and 12.6 (95% CI 5.4 to 29.0) for BRCA1 and BRCA2, respectively.
VUS in BRCA1 were reported in 12 cases (0.47%) versus four controls (0.14%) (p=0.03). In contrast, there was no difference in the frequency of VUS in BRCA2 in cases versus controls (30 (1.2%) cases versus 39 (1.4%) controls (p=0.70); tables 4 and 5 in the online supplementary file 1).
One hundred and twenty-five of 887 a priori moderate-risk to high-risk patients previously screened had BRCA1 germ line variants (nine LGR, 54 non-LGR deleterious variants and 63 missense, intronic, synonymous and inframe variants) and 242 had BRCA2 germ line variants (four LGR, 49 non-LGR variants and 191 missense, intronic, synonymous and inframe variants). Of these, 98 BRCA1 and 221 BRCA2 variants were detected using this amplicon-based method, giving a sensitivity of 89% (95% CI 86% to 92%, not inclusive of LGR). When examined, the variants missed by the amplicon sequencing method all showed preferential amplification of the wild-type allele (and hence were excluded due to high allelic imbalance) or had low amplicon coverage. Sensitivity for non-LGR deleterious variants was similar (90%; 95% CI 85% to 96%) with 49 of 54 BRCA1 and 44 of 49 BRCA2 deleterious variants detected.
Types and spectrum of deleterious variants
Ninety-seven distinct deleterious variants (41 BRCA1 and 56 BRCA2) and 11 distinct deleterious variants (five BRCA1 and 6 BRCA2) were identified in breast cancer cases and control subjects, respectively (online supplementary file 1). Notable recurrent variants were BRCA1 c.68_69delAG, BRCA1 c.2635G>T and BRCA2 c.262_263CT. BRCA1 c.68_69delAG was observed exclusively in the Indians and constituted 4 of 17 (24%) of BRCA1 deleterious mutations reported in Indian breast cancer cases. BRCA1 c.2635G>T, a reported mutation among Southern Chinese,21 was identified in two Chinese and one Malay breast cancer cases. Interestingly, principal component analysis derived from previous genome-wide genotyping data suggested that this Malay individual is of mixed Chinese and Malay descent (data not shown).22 BRCA2 c.262_263CT contributed 7 of 16 (44%) of BRCA2 variants found in the Malay patients with breast cancer and one in two (50%) BRCA2 variants in Malay control subjects.
Clinicopathological characteristics of deleterious variant carriers
BRCA1 and BRCA2 carriers were more likely to be diagnosed at a younger age compared with non-carriers (table 4; mean ages at diagnosis 41, 46, and 50 years old, respectively). While 49% of patients with breast cancer were diagnosed before the age of 50, 72% of BRCA1 and BRCA2 carriers were diagnosed before the age of 50 (78% and 66% for BRCA1 and BRCA2, respectively). BRCA1 and BRCA2 carriers were also significantly more likely to have family history of breast or ovarian cancer and high grade tumours (grade III). In addition, BRCA1 carriers were more likely to have bilateral breast cancer, personal history of ovarian cancer and TNBC, whereas BRCA2 carriers were more likely to have oestrogen receptors positive breast cancers, human epidermal growth factor receptor negative breast cancer and later stage of breast cancer presentation (stage IV). Further comparison of the clinical and pathological characteristics of BRCA1 and BRCA2 carriers showed no differences in these variables among the different ethnic groups (see table 10 in the online supplementary file 1).
Predictive value of testing guidelines
In order to determine the appropriateness of using age of onset, family history and pathological features of breast cancer to identify women who may benefit most from genetic testing, we determined the proportion of women and carriers who fulfilled the criteria for the NCCN Genetic/Familial High-Risk Assessment: Breast and Ovarian (V.2.2017) and compared it with those who fulfilled the criteria for MyCPG for BRCA1 and BRCA2 testing. Both criteria included women with breast and ovarian cancer, bilateral breast cancer under the age of 50, male breast cancer and strong first-degree relative with breast cancer. However, the criteria differ in age of primary breast cancer (≤45 vs ≤35), age of onset of TNBC (≤60 vs ≤50) and the significance of family history of breast and other cancers. In the present study, 46% of patients with breast cancer, 91% of BRCA1 carriers and 71% of BRCA2 carriers fulfilled the NCCN criteria, whereas 24% of patients with breast cancer, 73% of BRCA1 carriers and 50% of BRCA2 carriers fulfilled the MyCPG criteria.
The prevalence of BRCA1 and BRCA2 deleterious variant carriers among Asian breast cancer cases has hitherto been largely investigated in a priori high-risk cohorts selected on the basis of age of diagnosis, family history of breast and ovarian cancer and to a limited extent, pathological features of the cancers.2 To the best of our knowledge, this is the largest study involving full exon screening of BRCA1 and BRCA2 in an unselected series of Asian patients with breast cancer. We found BRCA1 and BRCA2 deleterious variants in 4.7% (95% CI 3.9% to 5.5%) of patients with breast cancer in this unselected hospital-based series, with the frequencies of BRCA1 and BRCA2 deleterious variants being similar. Comparison with previous clinical testing, including analysis of LGR, indicates a sensitivity of 90%, suggesting that the true prevalence would be approximately 5%–6%.
The population frequencies of BRCA1 and BRCA2 deleterious variant carriers in the controls were similar to that observed in the Exome Aggregation Consortium East Asians, at approximately 0.2% for each gene. Our results were also consistent with previous estimates of 0.4% BRCA1 and BRCA2 mutation carrier frequency in Caucasian population.23 24 The estimated breast cancer ORs associated with BRCA1 and BRCA2 deleterious variants (12.6 for both genes) were similar to those estimated in European populations.25 These results suggest that BRCA1 and BRCA2 mutations are associated with similar relative risks in Asian and European populations, which would imply that the absolute risk of breast cancer in carriers would be lower in Asian women. However, the OR estimates have wide confidence limits, and larger studies will be needed to provide more precise estimates.
Consistent with previous studies,5 6 26 27 we show that carriers of both BRCA1 and BRCA2 deleterious variants were more likely than non-carriers to be diagnosed at a younger age, have family history of breast or ovarian cancer and high tumour grade. In addition, bilateral breast cancer, personal history of ovarian cancer and TNBC pathology were significantly associated with BRCA1 deleterious variant carriers.
Full exon sequencing on an unselected series of patients with breast cancer allowed us to evaluate how often BRCA1 and BRCA2 deleterious variant carriers might be missed in clinical practice in a typical resource-constrained Asian country such as Malaysia. Using the more stringent MyCPG genetic testing criteria, only 24% of patients with breast cancer would be offered genetic counselling, but 40% of deleterious variant carriers would be missed. On the other hand, using the NCCN genetic testing criteria, 80% of deleterious variant carriers fulfilled the criteria and would therefore be offered genetic counselling, but nearly half (46%) of all patients with breast cancer would also need genetic counselling, making this a costly and potentially unaffordable risk-stratified management approach.
Notably, both NCCN genetic testing criteria and current risk prediction model underestimated BRCA2 more significantly than BRCA1 carriers. In this study, NCCN referral guidelines underestimated by threefold BRCA2 carriers compared with BRCA1 (29% vs 9%). The underdetection of BRCA1 and BRCA2 carriers by current genetic testing guidelines and risk prediction models may be accounted by the lower absolute risks associated with BRCA1 and BRCA2 mutations in Asians compared with Caucasians,28–30 the higher BRCA2:BRCA1 mutation ratio in Asian patients with breast cancer to that of Caucasian2 5 6 26 31–33 compounded by under-reporting of family history of breast cancer cases in Asian settings,7 34 and lower population incidence rates of breast cancer in Asian compared with Caucasian populations.35 This highlights a need for additional biomarkers or methods to identify Asian women who would benefit from genetic counselling and genetic testing, particularly in families with insignificant family history from resource-constrained settings.
With the availability of Asian-specific estimates of BRCA1 and BRCA2 carrier prevalence in unselected patients with breast cancer and unaffected population, and the risk estimates conferred by these genes, these may guide modifications to existing models and testing guidelines, or development of novel ones, to predict BRCA1 and BRCA2 carriers more accurately in Asian individuals.36 37
One limitation of our study is that LGRs were not included because MLPA was not performed in all patients with breast cancer. Furthermore, some carriers may have been missed due to the sensitivity of our amplicon-based sequencing approach.
Five per cent of unselected Asian patients with breast cancer are carriers of germ line deleterious variants in BRCA1 or BRCA2 and approximately 80% of carriers would have been offered genetic counselling based on current NCCN screening criteria. Our study provides the foundation for developing risk assessment tools for the Asian population, and highlights the need for cost-effective strategies to triage women for genetic counselling and testing in low-resource settings.
The authors would like to thank the participants and their families for taking part in this study. We thank Phuah Sze Yee, Tan Min Min, Norhashimah Hassan, Maheswari Jaganathan, Leelavathy D/O Krishnan, Faridah Binti Bakri and Thong Meow Keong for assistance with recruitment of patients, data cleaning, tissue collections, DNA preparation and helpful discussions.
Handling editor Constantin Polychronakos
Contributors Acquisition of data and analysis and interpretation of data: WXW, JA, KNL, SM, SNH, PSN, DS-CL, SYL, SYY, JL, SYL, BD, KP, LD, CL, CB, DC, PH, JS, CHY, NAMT, WKH, ACA and AMD. Manuscript writing: WXW and SHT. Conception and design: SHT and DFE. All authors equally contributed to the critical review and final approval of manuscript
Funding This study was funded by research grants from the Wellcome Trust (203477/Z/16/Z), Ministry of Higher Education to University Malaya (UM.C/HIR/MOHE/06), Estee Lauder Group of Companies, Cancer Research Malaysia, Cancer Research UK (C1287/A16563 to DFE, C8197/A16565 to AMD and C12292/A20861 to ACA), the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement 634935 (BRIDGES) and the PERSPECTIVE project, funded from the Government of Canada through Genome Canada and the Canadian Institutes of Health Research, the Ministère de l'Économie, de la Science et de l’Innovation du Québec through Genome Québec and the Quebec Breast Cancer Foundation. BD was supported by the Intramural Research Program of the National Human Genome Research Institute.
Competing interests None declared.
Patient consent Obtained.
Ethics approval Medical Ethics Committee of University Malaya Medical Centre (application number: 842.9) and the Independent Ethics Committee of Sime Darby Medical Centre (application numbers: 201109.4 and 201208.1).
Provenance and peer review Not commissioned; externally peer reviewed.
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