High Prevalence of Germline Mutations in Cancer Susceptibility Genes in Thai Patients with Clinical Spectrum of Hereditary Breast-Ovarian Cancer Syndrome

Background: Germline genetic mutation plays a signicant role in breast cancer susceptibility. The strength of such predisposition varies among ethnic groups across the globe, and clinical data from Asian population to develop a strategic approach to who should undergo a genetic test are lacking. Methods: We performed a multigene test with next generation sequencing in our 5-year hereditary breast-ovarian cancer spectrum cohort consists of 306 breast cancer patients, 62 ovarian cancer patients, 14 pancreatic cancer patients and 7 prostatic cancer patients. Results: There were 83 pathogenic/likely pathogenic (P/LP) variants identied in 104 patients, 44 of these P/LP variants were novel. We reported a high rate of germline P/LP variants in breast cancer (24%), ovarian cancer (37%), pancreatic cancer (14%), and prostatic cancer (29%). Germline P/LP variants in BRCA1 and BRCA2 accounted for 80% of P/LP variants found in breast cancer and 57% of P/LP variants found in ovarian cancer. The detection rate of NCCN 2019 guideline for genetic/familial high-risk assessment of breast and ovarian cancers was 22-40%. Conclusion: Overall, the data from this study strongly support the consideration of multigene panel test as a diagnostic tool for patients with hereditary breast-ovarian cancer spectrum in Thailand.


Introduction
Breast cancer is the second most common cancer and the second leading cause of cancer-related death in the US [1]. Genetic predisposition accounts for 10-30% of breast cancer cases, and its rate of nding germline pathogenic variants in BRCA1 or BRCA2 (gBRCA) was 3-5% [2,3]. In recent review, prevalence of BRCA1/2 status in breast cancer varied across the globe. Mutations in gBRCA were found in 3% of unselected breast cancer, while the prevalence could be above 20% in selected group [4]. Following the discovery of BRCA1 and BRCA2, several breast cancer genes with various degree of penetrance were identi ed [2]. BRCA1, BRCA2, CDH1, STK11 and TP53 are generally considered high-penetrance genes for breast cancer and the moderate-penetrance genes included ATM, BRIP1, CHEK2, and PALB2, though the gene lists can be dynamic [5,6]. It is comprehensible that testing more genes could identify more patients with heritable form of breast cancer and provide bene t on cancer screening or prevention for at-risk individuals. With higher throughput and cheaper cost of next generation sequencing, multigene panel testing has been widely adopted for patients with breast cancer [7].
Though, speci c guidelines for each causative gene are increasingly available, consensus on breast cancer germline testing strategy among medical community is lacking. Various approaches on test eligibility are ranging from a population-based screening campaign to an individual-based program [8]. Multiple models to estimate the likelihood of having gBRCA mutations and different testing criteria for patients with breast cancer have been used based on population data and national healthcare policies. Successful clinical implementation of germline testing also requires data from ethnically diverse population. Unfortunately, existing models and test criteria are mostly suitable for Western population, while data on other ancestries are very limited.
This study aims to investigate prevalence and diversity of mutations from multigene panel testing of Thai patients with breast cancer and other cancer in the hereditary breast-ovarian cancer spectrum and compare the clinical phenotype of patients with detectable mutations to a widely accepted clinical guideline.

Study population
The study protocols were approved by the Siriraj Hospital Institutional Review Board Protocol No.474/2562(EC1) and 418/2562(EC2). The study was conducted according to the Good Clinical Practice and the Declaration of Helsinki. All Thai patients who were diagnosed with primary breast, ovarian, pancreas, or prostate cancers and treated at Siriraj Hospital, whose blood were sent for germline cancer susceptibility gene testing between 2016 to 2020 were included. We also included patients who had a report of pathogenic variants or likely pathogenic variants (P/LP variants) in genes for breast cancer listed in Table 1 as a secondary nding. We excluded patients with known clinical or molecular diagnosis of genetic diseases (e.g. neuro bromatosis type 1), patients referred for testing of only speci c mutations, or asymptomatic individuals with known affected family members. We recruited a total of 377 unrelated patients. Three hundred and six patients had breast cancer, of which 19 of them also had primary ovarian cancer. Forty-three patients had primary ovarian cancer without breast cancer. There were 14 patients with pancreatic cancer and 7 patients with prostate cancer. Their tumor histological statuses, age of onset, and family history were comprehensively reviewed with the 2019 National Comprehensive Cancer Network (NCCN) guideline for genetic/familial high-risk assessment of breast and ovarian cancers. Descriptive statistics was used to calculate the rate of P/LP variants or variants of undetermined signi cance (VUS) across different indications. There were an additional of 7 patients who were recruited because they harboured P/LP variants in genes for breast cancer (either ATM, BRCA1 or BRCA2) as a secondary nding. Multigene panel targeted sequencing did expand spectrum of germline mutations in our database.
From 219 VUS identi ed in this study, only 27 VUS (12.3%) were found in BRCA1 and BRCA2 while 192 VUS belonged to other genes. ATM was the most commonly identi ed gene with VUS, followed by APC and MSH6. There were 7 putative loss-of-function (pLOF) variants (frameshift, stop gain, start loss, and splice site variants) in 6 genes (APC, BRCA2, MSH2, RECQL, RAD51C, and XRCC2) with insu cient data to be designated as P/LP.
The details of identi ed P/LP variants, patient's phenotype and familial history were shown in Table 2.
We also included 7 patients who had a report of P/LP variants in genes for breast cancer as a secondary nding in Table 2. Details of VUS with putative loss-of-function prediction and its patient's phenotype were listed in Table 3. All P/LP variants in BRCA1/2 were illustrated in a lollipop plot in Fig. 1.  Prostate cancer patients that warranted further genetic test by NCCN 2019 guideline were described as metastatic prostate cancer or having high-grade prostate cancer (Gleason score ≥ 7) with family history of certain cancer. In our 7 patients with prostate cancer, there were 3 patients who had evidence of metastasis, and 4 patients who had Gleason score ≥ 7). Patient with BRCA1 P/LP variants had Gleason score of 6 with familial history of gastric, thyroid, prostate and pancreas. The other patient with BRCA2 P/LP variants had prostate cancer Gleason score of 9, with another primary cancer included male breast cancer and squamous cell carcinoma of esophagus. He had no familial history of cancer.

Discussion
Breast cancer is one of the common cancers associated with heritable mutations. Identifying germline mutation in those patients provides great bene t on treatment selection, prophylactic and screening options for both the patients and their at-risk family members. For the rst time, this study provided prevalence and landscape of germline P/LP variants among Thai patients with breast-ovarian cancer spectrum who were clinically indicated for genetic test. Germline P/LP variants were detected in 24% of breast cancer and 37% of ovarian cancer patients.
The prevalence observed in our breast cancer patients was signi cantly higher than Western patients who underwent genetic testing with similar clinical indication. In 2018, the rate of P/LP variants from multigene-sequencing performed by 4 laboratories across the US in "higher-risk" patients ltered by National Comprehensive Cancer Network (NCCN) guideline was 12.5% [7]. There are some explanations for this double in detection rate in Thai population. First, as both genetic and lifestyle factors are associated with an increased risk of breast cancer, differences in lifestyle could affect the rate. Many lifestyle factors such as hormonal use, obesity, and alcohol consumption among Asian population are less prevalent than Western counterpart. It is therefore possible that genetic factor could play more role on cancer susceptibility in Thai patients whose clinical phenotypes were not totally conformed to common sporadic cancer. Next, it had been noted that approximately half of the patients in our study ful lled more than one NCCN 2019 indication. Our cohort may represent easily recognized patients with higher risk pro le than previous studies. Data of multigene-sequencing in breast cancer in Asian population was limited, and most publication did not recruit patients based on NCCN guideline. Nevertheless, there was one multicentre study in Chinese breast cancer patient that recruited patients using the Breast Cancer Diagnosis and Treatment Guidelines and Standards (Chinese Cancer Society, V2015), of which the criteria were slightly more stringent than the NCCN 2019. The detection rate of germline P/LP observed was comparable to our nding (23.8% vs 23.9% in our study) [10], as well as the proportion of gBRCA (71% vs 79.5% in our study).
The overall rate of P/LP germline mutation in ovarian cancer was 37% (N = 62). gBRCA accounted for 23% in this cancer group (57.5% of overall P/LP variants). The rate was considerably higher than observed ovarian cancer patients tested with multigene panel sequencing in the US laboratory [11] (overall rate of 13.4%, 50.5% of this were gBRCA; N = 663). Data of multigene-sequencing in ovarian cancer in Asian population was also limited. When consider only gBRCA in an Asian population, our rate was comparable to the rate of 14.7% for gBRCA in Japanese ovarian cancer cohort [12] and 22.4% in Chinese cohort [13]. Expansion of genetic test beyond BRCA1/2 nearly double the rate of nding germline P/LP variants, thus multigene panel approach should be fostered in ovarian cancer. The pathological report of high-grade serous cystadenoma contributed to 63.2% of all patients with positive P/LP variants. We support the idea that multigene panel test should be carried out in all patients with ovarian cancer regardless of their pathological nding [12].
An observed rate of 14.3% in pancreatic cancer (N = 14) was also comparable with previous study in pancreatic cancer (10.5%) [11]. We reported gBRCA as a secondary nding in patients in 3 patients with colon cancer, 1 patient with nasopharyngeal cancer and 1 patient with lung cancer in Table 2. Previous report from US laboratory estimated the yield of gBRCA in colon/stomach cancer to be 1.6% and colon/endometrial cancer to be 2.9% [11]. The yield of secondary nding of genes for hereditary breastovarian cancer spectrum in colorectal cancer patients should be reviewed in the future when the number of testing is su cient.
The overall rate of VUS in our breast cancer patients was as high as 40%. This rate fell between observed rates of VUS among different ethnics in the US laboratories [7] (23.7% in white, 44.5% in African-American, and 50.9% in Asian). When looking into speci c genes, the prevalence of VUS in BRCA1/2 in our cohort was lower than previous report [14] (7.2% compare to 15% in European laboratories, and 21% in African-American population). However, there was a report of 0% rate of VUS in BRCA1/2 in Asian population by the US laboratories [7]. The decrease in rate of VUS in our database was likely contributed from the availability of genomic data in Asian population, and the increase of functional studies in recent year [15].
Previous study from South Korea showed that most of VUS in BRCA1/2 (57%) remained unchanged and only 2.7% of the VUS was reclassi ed as likely pathogenic [16]. The reclassi cation of identi ed variants in this study remains to be seen. We have selected variants that almost ful ll the ACMG 2015 guideline for P/LP variant classi cation [9]. There were 8 variants with putative loss of function in Table 3 which included start codon loss in APC and RECQL. Although there were many reports of start codon loss in other diseases [17,18], initiation codon loss in APC, a well-known gene, had never been reported in colorectal cancer cases [19]. Additional genomic data and functional validation might help reclassi cation of VUS in our study.
Absence of copy number variations of BRCA1 and BRCA2 in our cohort may suggest that prevalence of large deletion/duplication in gBRCA among Thai patients is not as common as other population [20].

Utilization Of Nccn Guideline In Thai Population
Declarations -Ethical Approval and Consent to participate: The study protocols were approved by the Siriraj Hospital Institutional Review Board Protocol No.474/2562(EC1) and 418/2562(EC2).
-Consent for publication:

Not Applicable
The prevalence of nding positive germline variants in each speci ed indication from NCCN guideline 2019 ranged between 27%-40% (Fig. 1). Among breast cancer patients, the rate was highest (38%) in breast cancer patient with personal or familial history of primary malignancy in hereditary breast ovarian cancer spectrum. Our breast cancer patients with another primary cancer not in hereditary breast ovarian cancer spectrum also yielded the rate of germline P/LP variants of 22% (N = 18). This scenario could be added as testing indication in Thai population as multigene panel sequencing in patients with pretest probability over 10% was proposed to be cost-effective in US and UK population [21]. The rate of nding P/LP variants did positively correlate with number of indications ful lled (Fig. 2). This warrants a strong recommendation of providing germline genetic test in patient with multiple indication ful lled.

Conclusion
We reported a high diagnostic yield of P/LP variants from multigene panel sequencing in Thai patients with breast cancer (24%), or ovarian cancer (37%) that ful lled NCCN 2019 indication for germline genetic testing. The rate of VUS and the number of identi ed novel variants were high and re ected the need to include more Asian or Thai dataset in genomic database. The results from our study warrant the incorporation of multigene panel sequencing in management of breast cancer and ovarian cancer in Thailand. The data that support the ndings of this study are available from the corresponding author upon reasonable request.

Abbreviations
-Competing interests: The authors declare that they have no competing interests -Acknowledgements: We wish to thank all participants for their cooperation and contribution to our study. We thank all physicians and health professionals for their patient's clinical care.