Abstract
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.
Keywords: asian, breast cancer, brca1, brca2, national comprehensive cancer network
Introduction
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.
Methods
Study 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).
Bioinformatics analysis
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.
Statistical analysis
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.
Table 1.
Comparison of screening criteria between NCCN and MyCPG
| Category | NCCN and MyCPG | |
| Personal history of cancer | Ovarian cancer | |
| Bilateral breast cancer ≤50 years old | ||
| Family history of cancer | Male breast cancer | |
| Ovarian cancer | ||
| Proband ≤50 years old +≥1 close blood relative with breast cancer | ||
| Category | NCCN | MyCPG |
| Personal history of cancer | Primary breast cancer ≤45 years old | Primary breast cancer ≤35 years old |
| Family history of cancer | Proband any age +≥1 close blood relative with breast cancer ≤50 years old | Proband any age +≥2 close blood relative with breast cancer ≤50 years old |
| Proband any age +≥2 close blood relative with breast cancer | Proband any age +≥3 close blood relative with breast cancer | |
| Proband ≤50 years old +≥1 close blood relative with pancreatic cancer | ||
| Proband any age +≥2 close blood relative with pancreatic cancer | ||
| Pathology | TNBC≤60 years old | TNBC≤50 years old |
MyCPG, Malaysian Clinical Practice Guidelines; NCCN, National Comprehensive Cancer Network; TNBC, triple negative breast cancer.
Results
Study population
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.
Table 2.
Demographic characteristics and known breast cancer risk factors of study participants*
| Category | Cases (n=2575) | Controls (n=2809) | p Value |
| Demographic factors | |||
| Age (year±SD) | 50.0±10.8 | 52.6±8.2 | <0.001 |
| Age distribution | |||
| <30 | 67 (2.6) | 0 | <0.001 |
| 30–39 | 351 (13.9) | 10 (0.4) | |
| 40–49 | 821 (32.4) | 1101 (39.3) | |
| 50–59 | 804 (31.7) | 1087 (38.8) | |
| ≥60 | 490 (19.3) | 607 (21.6) | |
| Ethnicity | |||
| Chinese | 1726 (67.0) | 1686 (60.0) | <0.001 |
| Malay | 490 (19.0) | 547 (19.5) | |
| Indian | 359 (13.9) | 576 (20.5) | |
| Family history | |||
| Number of first-degree relatives with breast cancer | |||
| 0 | 2224 (86.4) | 2454 (87.5) | 0.061 |
| 1 | 309 (12.0) | 304 (10.8) | |
| 2 | 35 (1.4) | 45 (1.6) | |
| 3 | 7 (0.3) | 1 (0.04) | |
| Number of second-degree relatives with breast cancer | |||
| 0 | 2322 (90.2) | 2640 (94.2) | <0.001 |
| 1 | 219 (8.5) | 148 (5.3) | |
| 2 | 30 (1.2) | 14 (0.5) | |
| 3 | 3 (0.1) | 2 (0.1) | |
| 4 | 1 (0.04) | 0 | |
*Unless otherwise specified, data are presented in no. (%). For each data type, the total number of subjects may differ because of missing or incomplete data.
jmedgenet-2017-104947supp001.pdf (297.2KB, pdf)
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).
Table 3.
Mutation frequencies of BRCA1 and BRCA2 in breast cancer cases compared with population*
| Class | Cases (n=2575) | Controls (n=2809) | ExAC EA (n=4327) | OR (95% CI)† | OR (95% CI)‡ |
| Non-carriers | 2412 (93.7%) | 2755 (98.1%) | 4259 (98.4%) | 1.00 (reference) | 1.00 (reference) |
| BRCA1 | |||||
| Deleterious | 55 (2.1) | 5 (0.2) | 7 (0.2) | 12.6 (5.0 to 31.4) | 13.9 (6.3 to 30.5) |
| VUS | 12 (0.5) | 4 (0.1) | 6 (0.1) | 3.4 (1.1 to 10.6) | 3.5 (1.3 to 9.4) |
| BRCA2 | |||||
| Deleterious | 66 (2.6) | 6 (0.2) | 9 (0.2) | 12.6 (5.4 to 29.0) | 12.9 (6.4 to 26.0) |
| VUS | 30 (1.2) | 39 (1.4) | 46 (1.1) | 0.9 (0.5 to 1.4) | 1.2 (0.7 to 1.9) |
*Unless otherwise specified, data are presented in no. (%)
†Cases versus controls.
‡Cases versus ExAC EA.
EA: East Asian; ExAC: Exome Aggregation Consortium; VUS, variants of unknown significance.
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).
Table 4.
Association between BRCA1 and BRCA2 mutation status and clinicopathological characteristics a
| Clinical variables | BRCA1 carriers (n=55) | BRCA2 carriers (n=66) | Non-carriers (n=2454) | p Value† | p Value‡ |
| Age (year±SD) | 40.8±10.6 | 45.7±10.8 | 50.3±10.7 | <0.001 | 0.001 |
| Age distribution | |||||
| <30 | 9 (16.4) | 2 (3.1) | 56 (2.3) | <0.001 | 0.001 |
| 30–39 | 19 (34.5) | 20 (30.8) | 312 (12.9) | ||
| 40–49 | 15 (27.3) | 21 (32.3) | 785 (32.5) | ||
| 50–59 | 10 (18.2) | 12 (18.5) | 782 (32.4) | ||
| ≥60 | 2 (3.6) | 10 (15.4) | 478 (19.8) | ||
| Family history of breast cancer up to first degree | |||||
| No | 37 (67.3) | 47 (71.2) | 2143 (87.3) | <0.001 | <0.001 |
| Yes | 18 (32.7) | 19 (28.8) | 311 (12.7) | ||
| Family history of breast cancer up to second degree | |||||
| No | 32 (58.2) | 41 (62.1) | 1952 (79.5) | <0.001 | 0.001 |
| Yes | 23 (41.8) | 25 (37.9) | 502 (20.5) | ||
| Family history of ovarian cancer up to first degree | |||||
| No | 50 (90.9) | 63 (95.5) | 2429 (99.0) | <0.001 | 0.007 |
| Yes | 5 (9.1) | 3 (4.5) | 25 (1.0) | ||
| Family history of ovarian cancer up to second degree | |||||
| No | 49 (89.1) | 63 (95.5) | 2413 (98.3) | <0.001 | 0.078 |
| Yes | 6 (10.9) | 3 (4.5) | 41 (1.7) | ||
| Bilateral breast cancer | |||||
| No | 45 (81.8) | 60 (90.9) | 2321 (94.6) | <0.001 | 0.197 |
| Yes | 10 (18.2) | 6 (9.1) | 133 (5.4) | ||
| Ovarian cancer | |||||
| No | 53 (96.4) | 65 (98.5) | 2439 (99.4) | 0.007 | 0.362 |
| Yes | 2 (3.6) | 1 (1.5) | 15 (0.6) | ||
| Grade (%) | |||||
| I | 1 (2.8) | 0 | 236 (12.2) | <0.001 | 0.030 |
| II | 8 (22.2) | 25 (52.1) | 957 (49.4) | ||
| III | 27 (75.0) | 23 (47.9) | 746 (38.5) | ||
| Lymph node | |||||
| Negative | 30 (63.8) | 24 (44.4) | 1241 (56.6) | 0.320 | 0.076 |
| Positive | 17 (36.2) | 30 (55.6) | 953 (43.4) | ||
| Stage | |||||
| I | 10 (23.8) | 12 (23.5) | 614 (30.1) | 0.311 | <0.001 |
| II | 21 (50.0) | 18 (35.3) | 1042 (51.1) | ||
| III | 10 (23.8) | 13 (25.5) | 291 (14.3) | ||
| IV | 1 (2.4) | 8 (15.7) | 93 (4.6) | ||
| Oestrogen receptor | |||||
| Negative | 38 (80.9) | 11 (19.6) | 759 (33.6) | <0.001 | 0.028 |
| Positive | 9 (19.1) | 45 (80.4) | 1497 (66.4) | ||
| Progesterone receptor | |||||
| Negative | 36 (83.7) | 23 (44.2) | 875 (43.0) | <0.001 | 0.857 |
| Positive | 7 (16.3) | 29 (55.8) | 1161 (57.0) | ||
| Human epidermal growth factor 2 | |||||
| Negative | 43 (93.5) | 44 (84.6) | 1512 (70.5) | 0.001 | 0.027 |
| Positive | 3 (6.5) | 8 (15.4) | 632 (29.5) | ||
| Triple negative breast cancer | |||||
| No | 9 (22.0) | 43 (87.8) | 1626 (82.9) | <0.001 | 0.369 |
| Yes | 32 (78.0) | 6 (12.2) | 336 (17.1) | ||
| Ki-67 | |||||
| Low | 2 (33.3) | 4 (57.1) | 277 (64.1) | 0.119 | 0.703 |
| High | 4 (66.7) | 3 (42.9) | 155 (35.9) | ||
*Unless otherwise specified, data are presented in no. (%). For each data type, the total number of subjects may differ because of missing or incomplete data.
†BRCA1 carriers versus non-BRCA1/2 carriers.
‡BRCA2 carriers versus non-BRCA1/2 carriers.
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.
Discussion
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.
Conclusion
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.
Acknowledgments
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.
Footnotes
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.
References
- 1. Easton DF, Pharoah PD, Antoniou AC, Tischkowitz M, Tavtigian SV, Nathanson KL, Devilee P, Meindl A, Couch FJ, Southey M, Goldgar DE, Evans DG, Chenevix-Trench G, Rahman N, Robson M, Domchek SM, Foulkes WD. Gene-panel sequencing and the prediction of breast-cancer risk. N Engl J Med 2015;372:2243–57. 10.1056/NEJMsr1501341 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Kwong A, Shin VY, Ho JC, Kang E, Nakamura S, Teo SH, Lee AS, Sng JH, Ginsburg OM, Kurian AW, Weitzel JN, Siu MT, Law FB, Chan TL, Narod SA, Ford JM, Ma ES, Kim SW. Comprehensive spectrum of BRCA1 and BRCA2 deleterious mutations in breast cancer in Asian countries. J Med Genet 2016;53:15–23. 10.1136/jmedgenet-2015-103132 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Buys SS, Sandbach JF, Gammon A, Patel G, Kidd J, Brown KL, Sharma L, Saam J, Lancaster J, Daly MB. A study of over 35,000 women with breast cancer tested with a 25-gene panel of hereditary cancer genes. Cancer 2017;123:1721–30. 10.1002/cncr.30498 [DOI] [PubMed] [Google Scholar]
- 4. Couch FJ, Hart SN, Sharma P, Toland AE, Wang X, Miron P, Olson JE, Godwin AK, Pankratz VS, Olswold C, Slettedahl S, Hallberg E, Guidugli L, Davila JI, Beckmann MW, Janni W, Rack B, Ekici AB, Slamon DJ, Konstantopoulou I, Fostira F, Vratimos A, Fountzilas G, Pelttari LM, Tapper WJ, Durcan L, Cross SS, Pilarski R, Shapiro CL, Klemp J, Yao S, Garber J, Cox A, Brauch H, Ambrosone C, Nevanlinna H, Yannoukakos D, Slager SL, Vachon CM, Eccles DM, Fasching PA. Inherited mutations in 17 breast cancer susceptibility genes among a large triple-negative breast cancer cohort unselected for family history of breast cancer. J Clin Oncol 2015;33:304–11. 10.1200/JCO.2014.57.1414 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Zhang J, Sun J, Chen J, Yao L, Ouyang T, Li J, Wang T, Fan Z, Fan T, Lin B, Xie Y. Comprehensive analysis of BRCA1 and BRCA2 germline mutations in a large cohort of 5931 Chinese women with breast cancer. Breast Cancer Res Treat 2016;158:455–62. 10.1007/s10549-016-3902-0 [DOI] [PubMed] [Google Scholar]
- 6. Lang GT, Shi JX, Hu X, Zhang CH, Shan L, Song CG, Zhuang ZG, Cao AY, Ling H, Yu KD, Li S, Sun MH, Zhou XY, Huang W, Shao ZM. The spectrum of BRCA mutations and characteristics of BRCA-associated breast cancers in China: screening of 2,991 patients and 1,043 controls by next-generation sequencing. Int J Cancer 2017;141:129–42. 10.1002/ijc.30692 [DOI] [PubMed] [Google Scholar]
- 7. Nakamura S, Kwong A, Kim SW, Iau P, Patmasiriwat P, Dofitas R, Aryandono T, Hu Z, Huang CS, Ginsburg O, Rashid MU, Sarin R, Teo SH. Current status of the management of hereditary breast and ovarian cancer in Asia: first report by the Asian BRCA consortium. Public Health Genomics 2016;19:53–60. 10.1159/000441714 [DOI] [PubMed] [Google Scholar]
- 8. Daly MB, Pilarski R, Axilbund JE, Buys SS, Crawford B, Friedman S, Garber JE, Horton C, Kaklamani V, Klein C, Kohlmann W, Kurian A, Litton J, Madlensky L, Marcom PK, Merajver SD, Offit K, Pal T, Pasche B, Reiser G, Shannon KM, Swisher E, Voian NC, Weitzel JN, Whelan A, Wiesner GL, Dwyer MA, Kumar R. National Comprehensive Cancer Network. Genetic/familial high-risk assessment: breast and ovarian, version 1.2014. J Natl Compr Canc Netw 2014;12:1326–38. [DOI] [PubMed] [Google Scholar]
- 9. Gomez SL, Quach T, Horn-Ross PL, Pham JT, Cockburn M, Chang ET, Keegan TH, Glaser SL, Clarke CA. Hidden breast cancer disparities in Asian women: disaggregating incidence rates by ethnicity and migrant status. Am J Public Health 2010;100(Suppl 1):S125–31. 10.2105/AJPH.2009.163931 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Wen WX, Soo JS, Kwan PY, Hong E, Khang TF, Mariapun S, Lee CS, Hasan SN, Rajadurai P, Yip CH, Mohd Taib NA, Teo SH. Germline APOBEC3B deletion is associated with breast cancer risk in an Asian multi-ethnic cohort and with immune cell presentation. Breast Cancer Res 2016;18:56 10.1186/s13058-016-0717-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Mariapun S, Li J, Yip CH, Taib NA, Teo SH. Ethnic differences in mammographic densities: an Asian cross-sectional study. PLoS One 2015;10:e0117568 10.1371/journal.pone.0117568 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Phuah SY, Looi LM, Hassan N, Rhodes A, Dean S, Taib NA, Yip CH, Teo SH. Triple-negative breast cancer and PTEN (phosphatase and tensin homologue) loss are predictors of BRCA1 germline mutations in women with early-onset and familial breast cancer, but not in women with isolated late-onset breast cancer. Breast Cancer Res 2012;14:R142 10.1186/bcr3347 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Ng PS, Wen WX, Fadlullah MZ, Yoon SY, Lee SY, Thong MK, Yip CH, Mohd Taib NA, Teo SH. Identification of germline alterations in breast cancer predisposition genes among Malaysian breast cancer patients using panel testing. Clin Genet 2016;90:315–23. 10.1111/cge.12735 [DOI] [PubMed] [Google Scholar]
- 14. Thirthagiri E, Lee SY, Kang P, Lee DS, Toh GT, Selamat S, Yoon SY, Taib NA, Thong MK, Yip CH, Teo SH. Evaluation of BRCA1 and BRCA2 mutations and risk-prediction models in a typical Asian country (Malaysia) with a relatively low incidence of breast cancer. Breast Cancer Res 2008;10:R59 10.1186/bcr2118 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Kang P, Mariapun S, Phuah SY, Lim LS, Liu J, Yoon SY, Thong MK, Mohd Taib NA, Yip CH, Teo SH. Large BRCA1 and BRCA2 genomic rearrangements in Malaysian high risk breast-ovarian cancer families. Breast Cancer Res Treat 2010;124:579–84. 10.1007/s10549-010-1018-5 [DOI] [PubMed] [Google Scholar]
- 16. Decker B, Allen J, Luccarini C, et al. . Rare, protein-truncating variants in ATM, CHEK2 and PALB2, but not XRCC2, are associated with increased breast cancer risks. J Med Genet 2017;54:732–41. 10.1136/jmedgenet-2017-104588 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009;25:1754–60. 10.1093/bioinformatics/btp324 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo MA. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 2010;20:1297–303. 10.1101/gr.107524.110 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19. Wang K, Li M, Hakonarson H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res 2010;38:e164 10.1093/nar/gkq603 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20. Tavtigian SV, Deffenbaugh AM, Yin L, Judkins T, Scholl T, Samollow PB, de Silva D, Zharkikh A, Thomas A. Comprehensive statistical study of 452 BRCA1 missense substitutions with classification of eight recurrent substitutions as neutral. J Med Genet 2006;43:295–305. 10.1136/jmg.2005.033878 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Kwong A, Ng EK, Wong CL, Law FB, Au T, Wong HN, Kurian AW, West DW, Ford JM, Ma ES, Ek N, Es M. Identification of BRCA1/2 founder mutations in Southern Chinese breast cancer patients using gene sequencing and high resolution DNA melting analysis. PLoS One 2012;7:e43994 10.1371/journal.pone.0043994 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Michailidou K, Hall P, Gonzalez-Neira A, Ghoussaini M, Dennis J, Milne RL, Schmidt MK, Chang-Claude J, Bojesen SE, Bolla MK, Wang Q, Dicks E, Lee A, Turnbull C, Rahman N, Fletcher O, Peto J, Gibson L, Dos Santos Silva I, Nevanlinna H, Muranen TA, Aittomäki K, Blomqvist C, Czene K, Irwanto A, Liu J, Waisfisz Q, Meijers-Heijboer H, Adank M, van der Luijt RB, Hein R, Dahmen N, Beckman L, Meindl A, Schmutzler RK, Müller-Myhsok B, Lichtner P, Hopper JL, Southey MC, Makalic E, Schmidt DF, Uitterlinden AG, Hofman A, Hunter DJ, Chanock SJ, Vincent D, Bacot F, Tessier DC, Canisius S, Wessels LF, Haiman CA, Shah M, Luben R, Brown J, Luccarini C, Schoof N, Humphreys K, Li J, Nordestgaard BG, Nielsen SF, Flyger H, Couch FJ, Wang X, Vachon C, Stevens KN, Lambrechts D, Moisse M, Paridaens R, Christiaens MR, Rudolph A, Nickels S, Flesch-Janys D, Johnson N, Aitken Z, Aaltonen K, Heikkinen T, Broeks A, Veer LJ, van der Schoot CE, Guénel P, Truong T, Laurent-Puig P, Menegaux F, Marme F, Schneeweiss A, Sohn C, Burwinkel B, Zamora MP, Perez JI, Pita G, Alonso MR, Cox A, Brock IW, Cross SS, Reed MW, Sawyer EJ, Tomlinson I, Kerin MJ, Miller N, Henderson BE, Schumacher F, Le Marchand L, Andrulis IL, Knight JA, Glendon G, Mulligan AM, Lindblom A, Margolin S, Hooning MJ, Hollestelle A, van den Ouweland AM, Jager A, Bui QM, Stone J, Dite GS, Apicella C, Tsimiklis H, Giles GG, Severi G, Baglietto L, Fasching PA, Haeberle L, Ekici AB, Beckmann MW, Brenner H, Müller H, Arndt V, Stegmaier C, Swerdlow A, Ashworth A, Orr N, Jones M, Figueroa J, Lissowska J, Brinton L, Goldberg MS, Labrèche F, Dumont M, Winqvist R, Pylkäs K, Jukkola-Vuorinen A, Grip M, Brauch H, Hamann U, Brüning T, Radice P, Peterlongo P, Manoukian S, Bonanni B, Devilee P, Tollenaar RA, Seynaeve C, van Asperen CJ, Jakubowska A, Lubinski J, Jaworska K, Durda K, Mannermaa A, Kataja V, Kosma VM, Hartikainen JM, Bogdanova NV, Antonenkova NN, Dörk T, Kristensen VN, Anton-Culver H, Slager S, Toland AE, Edge S, Fostira F, Kang D, Yoo KY, Noh DY, Matsuo K, Ito H, Iwata H, Sueta A, Wu AH, Tseng CC, Van Den Berg D, Stram DO, Shu XO, Lu W, Gao YT, Cai H, Teo SH, Yip CH, Phuah SY, Cornes BK, Hartman M, Miao H, Lim WY, Sng JH, Muir K, Lophatananon A, Stewart-Brown S, Siriwanarangsan P, Shen CY, Hsiung CN, Wu PE, Ding SL, Sangrajrang S, Gaborieau V, Brennan P, McKay J, Blot WJ, Signorello LB, Cai Q, Zheng W, Deming-Halverson S, Shrubsole M, Long J, Simard J, Garcia-Closas M, Pharoah PD, Chenevix-Trench G, Dunning AM, Benitez J, Easton DF. Breast and Ovarian Cancer Susceptibility Collaboration; Hereditary Breast and Ovarian Cancer Research Group Netherlands (HEBON); kConFab Investigators; Australian Ovarian Cancer Study Group; GENICA (Gene Environment Interaction and Breast Cancer in Germany) Network. Large-scale genotyping identifies 41 new loci associated with breast cancer risk. Nat Genet 2013;45:353–61. 10.1038/ng.2563 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23. Antoniou AC, Cunningham AP, Peto J, Evans DG, Lalloo F, Narod SA, Risch HA, Eyfjord JE, Hopper JL, Southey MC, Olsson H, Johannsson O, Borg A, Pasini B, Passini B, Radice P, Manoukian S, Eccles DM, Tang N, Olah E, Anton-Culver H, Warner E, Lubinski J, Gronwald J, Gorski B, Tryggvadottir L, Syrjakoski K, Kallioniemi OP, Eerola H, Nevanlinna H, Pharoah PD, Easton DF. The BOADICEA model of genetic susceptibility to breast and ovarian cancers: updates and extensions. Br J Cancer 2008;98:1457–66. 10.1038/sj.bjc.6604305 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24. Song H, Cicek MS, Dicks E, Harrington P, Ramus SJ, Cunningham JM, Fridley BL, Tyrer JP, Alsop J, Jimenez-Linan M, Gayther SA, Goode EL, Pharoah PD. The contribution of deleterious germline mutations in BRCA1, BRCA2 and the mismatch repair genes to ovarian cancer in the population. Hum Mol Genet 2014;23:4703–9. 10.1093/hmg/ddu172 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25. Lee AJ, Cunningham AP, Kuchenbaecker KB, Mavaddat N, Easton DF, Antoniou AC. Consortium of Investigators of Modifiers of BRCA1/2; Breast Cancer Association Consortium. BOADICEA breast cancer risk prediction model: updates to cancer incidences, tumour pathology and web interface. Br J Cancer 2014;110:535–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26. Tung N, Lin NU, Kidd J, Allen BA, Singh N, Wenstrup RJ, Hartman AR, Winer EP, Garber JE. Frequency of germline mutations in 25 cancer susceptibility genes in a sequential series of patients with Breast Cancer. J Clin Oncol 2016;34:1460–8. 10.1200/JCO.2015.65.0747 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Atchley DP, Albarracin CT, Lopez A, Valero V, Amos CI, Gonzalez-Angulo AM, Hortobagyi GN, Arun BK. Clinical and pathologic characteristics of patients with BRCA-positive and BRCA-negative breast cancer. J Clin Oncol 2008;26:4282–8. 10.1200/JCO.2008.16.6231 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28. Park B, Dowty JG, Ahn C, Win AK, Kim SW, Lee MH, Lee JW, Kang E, Hopper JL, Park SK. Breast cancer risk for Korean women with germline mutations in BRCA1 and BRCA2. Breast Cancer Res Treat 2015;152:659–65. 10.1007/s10549-015-3495-z [DOI] [PubMed] [Google Scholar]
- 29. Yao L, Sun J, Zhang J, He Y, Ouyang T, Li J, Wang T, Fan Z, Fan T, Lin B, Xie Y. Breast cancer risk in Chinese women with BRCA1 or BRCA2 mutations. Breast Cancer Res Treat 2016;156:441–5. 10.1007/s10549-016-3766-3 [DOI] [PubMed] [Google Scholar]
- 30. Chen S, Parmigiani G. Meta-analysis of BRCA1 and BRCA2 penetrance. J Clin Oncol 2007;25:1329–33. 10.1200/JCO.2006.09.1066 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31. Høberg-Vetti H, Bjorvatn C, Fiane BE, Aas T, Woie K, Espelid H, Rusken T, Eikesdal HP, Listøl W, Haavind MT, Knappskog PM, Haukanes BI, Steen VM, Hoogerbrugge N. BRCA1/2 testing in newly diagnosed breast and ovarian cancer patients without prior genetic counselling: the DNA-BONus study. Eur J Hum Genet 2016;24:881–8. 10.1038/ejhg.2015.196 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32. Grindedal EM, Heramb C, Karsrud I, Ariansen SL, Mæhle L, Undlien DE, Norum J, Schlichting E. Current guidelines for BRCA testing of breast cancer patients are insufficient to detect all mutation carriers. BMC Cancer 2017;17:438 10.1186/s12885-017-3422-2 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33. Zhong X, Dong Z, Dong H, Li J, Peng Z, Deng L, Zhu X, Sun Y, Lu X, Shen F, Su X, Zhang L, Gu Y, Zheng H. Prevalence and prognostic role of BRCA1/2 variants in unselected Chinese breast cancer patients. PLoS One 2016;11:e0156789 10.1371/journal.pone.0156789 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34. Ginsburg OM, Dinh NV, To TV, Quang LH, Linh ND, Duong BT, Royer R, Llacuachaqui M, Tulman A, Vichodez G, Li S, Love RR, Narod SA. Family history, BRCA mutations and breast cancer in Vietnamese women. Clin Genet 2011;80:89–92. 10.1111/j.1399-0004.2010.01545.x [DOI] [PubMed] [Google Scholar]
- 35. DeSantis CE, Bray F, Ferlay J, Lortet-Tieulent J, Anderson BO, Jemal A. International variation in female breast cancer incidence and mortality rates. Cancer Epidemiol Biomarkers Prev 2015;24:1495–506. 10.1158/1055-9965.EPI-15-0535 [DOI] [PubMed] [Google Scholar]
- 36. Marroni F, Aretini P, D’Andrea E, Caligo MA, Cortesi L, Viel A, Ricevuto E, Montagna M, Cipollini G, Federico M, Santarosa M, Marchetti P, Bailey-Wilson JE, Bevilacqua G, Parmigiani G, Presciuttini S. Penetrances of breast and ovarian cancer in a large series of families tested for BRCA1/2 mutations. Eur J Hum Genet 2004;12:899–906. 10.1038/sj.ejhg.5201256 [DOI] [PubMed] [Google Scholar]
- 37. Kurian AW, Gong GD, Chun NM, Mills MA, Staton AD, Kingham KE, Crawford BB, Lee R, Chan S, Donlon SS, Ridge Y, Panabaker K, West DW, Whittemore AS, Ford JM. Performance of BRCA1/2 mutation prediction models in Asian Americans. J Clin Oncol 2008;26:4752–8. 10.1200/JCO.2008.16.8310 [DOI] [PMC free article] [PubMed] [Google Scholar]
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