Germline and tumor BRCA1/2 pathogenic variants in Chinese triple-negative breast carcinomas

BRCA1/2 screening for all triple-negative breast cancer (TNBC) patients younger than 60 years may still be an economic burden in China. Further evidences that include incidence and outcome of BRCA1/2 pathogenic variants (PV) screened based on younger age or family history (FH) are worth discussing for improving the cost-effectiveness of BRCA1/2 testing in Chinese TNBC. We aimed to investigate the prevalence of germline and tumor BRCA1/2 PV based on age screening in Chinese TNBC patients. Paired blood and tumor DNA from 124 unselected Chinese TNBC patients with less than or equal to 55 years were collected and analyzed for BRCA1/2 PV. Clinicopathological characteristics including age at diagnosis, FH and follow-up data were collected for further analysis. The entire frequency of germline and tumor BRCA1/2 PV was 21.0 and 25%, respectively. Among them, 20 (16.1%) germline and 5 (4.0%) somatic BRCA1/2 single-nucleotide variant/insertion/deletions were found by NGS testing, 6 (4.8%) BRCA1 large genomic rearrangements were detected in blood DNA by MPLA. There was significant correlation between FH of HBOC and germline BRCA1/2 PVs among these patients. Patients with tumor BRCA1/2 PVs had significant improvements than non-carriers in PFS (p = 0.047). No significant impacts were found between various mutation status in OS outcomes. No significant differences were found between BRCA1 or BRCA2 and non-carriers in PFS or OS. There is a high incidence of germline and tumor BRCA1/2 PVs in Chinese TNBC patients with less than or equal to 55 years old. Tumor BRCA1/2 PV carriers showed an improved survival outcome. Our results suggest that BRCA1/2 PVs testing addressed within each specific clinical scenario could be more cost-effective for patients.


Introduction
Triple-negative breast cancer (TNBC) is a subtype of breast cancer lacking the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2), accounting for about 15% of all breast cancer (Denkert et al. 2017). It is characterized as aggressive, heterogeneous, and less differentiated, thereby making it difficult to intervene with a targeted therapy and often leads to poor prognosis (Bianchini et al. 2016).
The breast cancer susceptibility genes 1 and 2 (BRCA1 and BRCA2) are tumor suppressor genes involved in homologous recombination repair (HRR). Inactivation of BRCA1/2 may lead to early onset and increase the cumulative risk of breast cancer (Miki, 1994;Wooster, 1994). A recent study estimated the average risk of developing breast cancer by age 80 years to be 72% for BRCA1 and 69% for BRCA2 carriers (Kuchenbaecker, 2017). Cells with either germline or somatic BRCA1/2 pathogenic variant (PV) in allele can survive despite the defect of HRR, but it comes at the basis of alternative repair pathway that relies heavily on poly ADP-ribose polymerase (PARP) function (Yates and Campbell 2012). Synthetic lethality effect would occur when HR-deficient cells treated with PARP inhibitors (PARPi). Because of this, BRCA1/2 PV detection provides an important basis for clinical selection of PARPi therapy.
On the other hand, BRCA1/2 screening was recommended for TNBC patients diagnosed ≤ 60 years according to NCCN guideline (Daly, 2020). Currently, in China, the expert consensus on BRCA1/2 screening also complies with this standard. Nonetheless, the high costs associated with genetic analyses still impose a great economic burden, especially when BRCA1/2 testing is offered by the National Health Service (Toss, 2020). Finding new strategies, such as using family history (FH) or younger than the age (60 years) recommended by current guideline as screening criteria to further improve the cost-effectiveness of BRCA1/2 testing in TNBC, is still a valuable attempt. Previous studies have found that onset age was one of the clinicopathological factors associated with BRCA1/2 status. Patients with BRCA1/2 PVs were significantly younger than non-carriers (Couch, 2015;Lang, 2017;Shimelis 2018). According to a survey from Chinese National Office for Cancer Prevention and Control, the peak onset age for breast cancer in Chinese women was about 50-55 years, which was 5-10 years younger than that in women from Western countries (Zuo et al. 2017). These data suggest the probability to find a high frequency of BRCA1/2 PV in Chinese breast cancer patients less than 55 years. It may also be a worthwhile discussion to adjust the existing criteria according to the national conditions in China.
To our knowledge, previous studies on the prevalence of BRCA1/2 PV in TNBC mainly evaluated germline single-nucleotide variant (SNV)/insertion/deletion (indel), but usually lacked large genomic rearrangement (LGR) detection, also none specifically for patients less than 55 years (Couch et al. 2015;Lang et al. 2017;Shimelis et al. 2018). Besides that, studies on tumor BRCA1/2 PV are still limited (Gonzalez-Angulo, 2011;Li, 2017;Winter, 2016;Zhong, 2016). In this study, we took advantage of next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) technologies to simultaneously investigate germline and tumor BRCA1/2 PVs in Chinese TNBC patients with less than or equal to 55 years and analyze their association with clinicopathological factors and survival outcomes.

Study patients and samples
A total of 124 female Chinese patients who got surgery in the year of 2015 in Shanghai Cancer Center were sequentially selected for our study population. 21.5% (124/577) of all TNBC patients were enrolled because they met all of the following inclusion criteria: (1) pathologically confirmed TNBC by two separate clinical pathologists (Xiaoyan Zhou, Qianming Bai). TNBC was defined as ER (-), PR (-) and HER2 (-). ER and PR statuses were determined by standard immunohistochemistry (IHC) method. HER2 status was determined by IHC and/or by fluorescence in situ hybridization (FISH). Results of IHC and FISH were interpreted according to NCCN guideline (Wolff, 2018); (2) age with 18-55 years; (3) regional tumor cells accounted for more than 20% and sufficient formalin-fixed paraffin-embedded (FFPE) sections for somatic testing; (4) blood sample for germline testing; (5) quality control was qualified in the process of testing and bioinformatics analysis; and (6) willingness to provide signed consent in advance of the trial. Patients not meeting all these inclusion criteria were excluded. Blood and paired FFPE tissue samples were collected and retrieved from our biobank and the Department of Pathology for BRCA1/2 testing. Genomic DNAs extracted from tumor tissue sections and peripheral blood were performed using QIAamp DNA MiniKit and QIAamp DNA MidiKit (QIAgen, Valencia, CA), respectively. Clinicopathological parameters, treatment strategies and patient's disease status were electronically retrieved from the Hospital Information System (HIS) of Fudan University Shanghai Cancer Center. Disease relapse or progression was determined by medical imaging, serology, or histology. Progression-free survival (PFS) was measured from diagnosis to local or systemic recurrence or the last follow-up, while OS was measured from diagnosis to death or the last follow-up. Patients have one or more family members within two generations with breast or ovarian cancer were considered having FH of HBOC. No kinship was found among them according to information of genetic counseling. The study was approved by Medical Ethics Committee of Fudan University Shanghai Cancer Center (Shanghai, China) and informed consent was obtained from all individual participants included in the study.

BRCA1/2 SNV/indel detection by NGS
For targeted NGS analysis, total 265 primer pairs in two pools (133 pairs in pool 1, and 132 pairs in pool 2) were used in Oncomine ™ BRCA Research Assay (Thermo Fisher Scientific, Schaumburg, IL, USA), which can amplify the entire coding regions and 20 bp upstream or downstream of exon-intron boundaries of BRCA1/2 genes. Multiplex PCR was performed using 20 ng genomic DNA with the following cycling conditions: 99 °C × 2 min, 20 cycles of 99 °C × 15 s, and 60 °C × 4 min. The amplicons were treated with 2 µL FuPa reagent to partially digest primers and phosphorylate the amplicons with the following conditions: 50 °C × 10 min, 55 °C × 10 min, and 60 °C × 20 min. The diluted barcodes (Thermo Fisher Scientific) were ligated with the following conditions: 22 °C × 30 min, and 68 °C × 5 min, and 72 °C × 10 min. Libraries were purified using Agencourt AMPure XP reagents (Beckman Coulter, Brea, CA, USA). Concentration was measured using an Ion Library Quantitation Kit (Thermo Fisher Scientific), then the same amount of 100 pmol/L libraries was pooled in one sequencing reaction. Emulsion PCR was implemented with the Ion OneTouch ™ 2.0 System and Hi-Q ™ View OT2 reagents (Thermo Fisher Scientific) following the manufacturer's instructions. The template-positive particles were purified using Ion OneTouch ™ ES system and MyOne ™ Streptavidin C1 Beads (Thermo Fisher Scientific). Parallel sequencing was performed on a Personal Genome Machine (PGM) sequencer using the Ion PGM™ Hi-Q ™ Sequencing Kit according to the manufacturer's instructions. Sequencing was performed using 500 flow runs that generated ~ 200 bp reads.
LGRs detection by MLPA Genomic DNAs extracted from all of the enrolled patient's peripheral blood patients were detected with BRCA1/2 LGRs following the MLPA instructions (MRC-Holland, Amsterdam, The Netherlands). Probe mix P002 and P087 were used for the detection or confirmation of BRCA1 LGRs. Similarly, probe mix P045 and P090 were used for BRCA2 LGRs. Briefly, 100 ng genomic DNA dissolved in 5 µl TE buffer (Thermo Fisher Scientific) was used for denaturation with 98 °C × 5 min, then MLPA probe mix was added for hybridization with the following conditions: 95 °C × 1 min, and 6 0 °C × 16-20 h. After hybridization, a Ligase-65 master mix was added for ligation with the following conditions: 54 °C pause, 54 °C × 15 min, and 98 °C × 5 min. Multiplex PCR was performed with the following cycling conditions: 35 cycles of 95 °C × 30 s, 60 °C × 30 s, 72 °C × 60 s and an additional 72 °C × 20 min. Finally, electrophoresis and data analysis were performed on ABI 3500 machine (Thermo Fisher Scientific) and Coffalyser.NET software v140721.1958 (MRC-Holland).

Data analysis
Pathologic characteristics tabulated by their types or ranges were compared between groups by chi-square test or Fisher's exact test or unpaired two-tailed t test, as appropriate. Survival analysis was performed using log-rank test, Kaplan-Meier analysis. All analyses were performed using SPSS version 19 (SPSS version 19; SPSS, Chicago, IL). p values < 0.05 were considered as statistically significant.
All BRCA1/2 PVs including SNV, indel and LGRs were visualized by IGV software or Coffalyser software. Notablely, no patient in this cohort was found with two or more deleterious PVs showing a mutual exclusion of BRCA1/2 PV. In terms of distribution, all PVs are scattered in various functional domains and protein-binding regions of BRCA1/2 (Fig. 2).

The association between BRCA1/2 PVs and clinicopathological characteristics
Age, FH of HBOC, histology, tumor grade, TNM stage and ki67 expression were included in pathologic characteristics. The median age in this cohort was 46 years (range 24-55 years). A minority of patients (12.9%) had potentially significant FH, and 92.7% of them were invasive ductal cancer (IDC) with tumor grade of 2 or 3. The proportions of both BRCA1/2 germline (46.7%) and tumor PV (46.7%) in  (Fig. 3, p = 0.047). No significant impacts were found between various mutation statuses in OS outcomes. No significant differences were found between BRCA1 or BRCA2 and noncarriers in PFS or OS (Fig. 4). The low number of events precluded Cox regression analysis.

Discussion
In this study, 124 paired tumor and peripheral blood samples from Chinese TNBC patients with less than or equal to 55 years were analyzed for the entire coding regions and exon-intron boundaries of BRCA1/2. This study provided the first simultaneous overview of the germline and tumor BRCA1/2 PVs including SNV, indel and LGRs in Chinese TNBC patients. Studies on the spectrum and prevalence of BRCA1/2 PVs among TNBC patients have been reported over the years. There have been two large-scale germline BRCA1/2 screening in unselected TNBC cohort in the United States, the prevalence of PVs (SNV and indel) was 8.4% (714/8753) and 10.4% (224/2148), respectively (Couch et al. 2015;Shimelis et al. 2018). In China, there are also two large-scale studies showing that germline BRCA1/2 PV (SNV and indel) prevalence in TNBC ranged from 10.1% (102/1099) to 11.2% (124/1104) (Lang et al. 2017;Sun 2017). Additional study showed that LGRs occurred in 2.4% (13/834) of the familial breast carcinomas and in 1.1% (7/660) of sporadic TNBC patients less than 60 years by MLPA (Su, 2018). For somatic PV, limited studies to date showed that BRCA1/2 PV rate in TNBC ranged from 1.3 to 1.43% (Gonzalez-Angulo et al. 2011;Zhong et al. 2016). In our current work, the incidence of germline and somatic BRCA1/2 PV (SNV and indel) was 16.1 and 4%, respectively, which was a little bit higher than the above studies. One of the reasons for the difference is the deviation that may be caused by our small sample size. Another equally important reason is that the onset age of patients selected in this cohort was less than or equal to 55 years. Since Couch et al. also found that the germline BRCA1/2 PV rates were 16.6% (125/754) and 14.3% (160/1120) in patients under 50 and 60 years, respectively, which were significantly higher than that in patients without regard to age 10.4% (224/2148) (Couch et al. 2015). Our finding suggests a high BRCA1/2 PV frequency in Chinese TNBC patients less than 55 years. Moreover, we also found a significantly higher proportion of both germline and tumor BRCA1/2 PVs in the patients with FH than that in sporadic patients, which provides a basis for the hypothesis that BRCA1/2 PVs testing addressed within each specific clinical scenario could be more cost-effective for patients. In addition to the cost of genetic testing, targeted therapies can in some instances come at a significantly increased cost. Clinical efficacy or clinical utility of BRCA1/2 testing needs to be considered comprehensively. From therapeutic point of view, although the PARPi olaparib is now approved in advanced germline BRCA1/2 breast cancer, this treatment may remain unaffordable to many health care systems and patients for many years (Tutt, 2018). Except for targeted drugs, chemotherapy remains the only available non-investigational systemic treatment option for non-BRCA -mutated advanced TNBC (Cardoso, 2020). There is no specific recommendation on drug types, except for platinum compounds for patients with BRCA1/2 mutations (Cardoso et al. 2020). From the perspective of early diagnosis or prevention of cancer in patients and their families, BRCA1/2 testing will have additional meaning.
Generally, the role of BRCA1/2 gene status on prognosis remains controversial for TNBC patients. In a randomized phase III trial comparing carboplatin with the microtubule-disrupting agent docetaxel in advanced unselected TNBC, they found that patients with germline BRCA1/2 PVs showed a significant advantage in objective response rate and PFS with platinum therapy compared to docetaxel (Tutt et al. 2018). There are also many studies found that there is no significant correlation between BRCA1/2 status and prognosis in breast cancer (Rennert et al. 2007;Yadav, 2018). In a retrospective study, 7915 breast cancer patients were followed up and analyzed for survival, they found that BRCA1 mutation carriers had an unfavorable survival compared with non-carriers (Sun et al. 2017). They drew different conclusions may be due to different subtypes of breast cancer screened and different chemotherapy drugs used. In this study, Cox regression was not available because of the low number of progression and death. We only use univariate analysis to analyze the relationship between BRCA1/2 status and prognosis, and found that BRCA1/2 PV carriers responded better to systemic treatment. Our results suggest that patients with BRCA1/2 PV may be more sensitive to chemotherapy. Further research is needed for specific mechanism.
There are still some deficiencies in this work. First, as we mentioned earlier, the sample size we screened is relatively small, which may lead to partial deviation of PV frequency. However, this deficiency has been verified by comparing with the results of large-scale domestic and international studies, which proves that the BRCA1/2 PV frequencies we obtained by age-based screening are highly reliable. Second, although 4.8% LGRs have been detected in blood samples by MLPA, we did not detect LGRs in tumor samples. Because FFPE or fresh tissue samples are difficult technical challenge for MLPA method, here we make assumptions that all LGRs identified from the blood samples are also present in the tumor tissues based on theory. Finally, HR genes include Fanconi anemia genes (BRIP1, PALB2), the core RAD genes  RAD51D), and genes involved in HR pathways either directly (CHEK2, BARD1, NBN, ATM) or indirectly (CDK12), but is not limited to BRCA1/2 (Boussios, 2020). Multi-gene panel testing may find more potential beneficiaries, it should be attempted in future work.
In summary, there is a high incidence of germline (21.0%) and tumor (25.0%) BRCA1/2 PVs in Chinese TNBC patients with less than or equal to 55 years old.
LGRs and somatic PV detection should be considered in chemotherapy or targeted therapy in TNBC. Tumor BRCA1/2 PV carriers showed an improved survival outcome, which indicates that comprehensive detection of BRCA1/2 PV, including SNV, indel and LGRs, would benefit more patients. Our results suggest that BRCA1/2 PVs testing addressed within each specific clinical scenario could be more cost-effective for patients.