Germline mutations in homologous recombination repair genes among Chinese pancreatic ductal adenocarcinoma patients detected using next‐generation sequencing

Abstract Background Genetic testing for pancreatic ductal adenocarcinoma (PDAC) patients is in constant development. However, the status of homologous recombination repair (HRR) genes in unselected Chinese PDAC has not been fully explored. This study aims to characterize the profile of germline mutations in HRR genes in Chinese PDAC patients. Methods A cohort of 256 PDAC patients were enrolled at Zhongshan Hospital Fudan University between 2019 and 2021. Germline DNA was analyzed by next‐generation sequencing using a multigene panel of the 21 HRR genes. Results The germline pathogenic (P)/likely pathogenic (LP) variant rates were 7.0% (18/256) in unselected patients with pancreatic cancer. Among them, 1.6% (4/256) were identified as harboring BRCA2 variants, and 5.5% (14/256) patients carried non‐BRCA variants. Variants were detected in eight non‐BRCA genes, including ATM (4, 1.6%), PALB2 (4, 1.6%), ATR (1, 0.4%), BRIP1 (1, 0.4%), CHEK2 (1, 0.4%), MRE11 (1, 0.4%), PTEN (1, 0.4%), and STK11 (1, 0.4%). ATM, BRCA2, and PALB2 were the most prevalent variant genes. If only BRCA1/2 was tested, 5.5% of P/LP variants would have been lost. Further, we found that the landscape of P/LP HRR variants in various population cohorts was quite different. However, no significant difference was found in clinical characteristics between germline HRR P/LP carriers and non‐carriers. In our study, one case carrying a germline PALB2 variant showed a long‐time response to platinum‐based chemotherapy and PARP inhibitor. Conclusion This study comprehensively depicts the prevalence and characteristics of germline HRR mutations in unselected Chinese PDAC patients. Our findings show the clinical utility of a multigene panel may increase the detection of P/LP HRR carriers.


| INTRODUCTION
Pancreatic ductal adenocarcinoma (PDAC) represents the most common form of pancreatic cancer. It is the seventh leading cause of global cancer death, with an estimated 9% 5-year survival rate (Grossberg et al., 2020). The limited therapeutic option is one of the key factors that contribute to poor outcomes for PDAC patients. Recent studies are focused on identifying new therapeutic targets and investigating the genomic landscape of PDAC (Lai et al., 2021).
Genetic testing for PDAC patients is in constant development, and specific genetic alterations indicate personalized managing strategies. Germline pathogenic (P)/likely pathogenic (LP) BRCA1/2 variants are associated with significantly increased risk of PDAC, which are found in approximately 5%-10% of cases of familial PDAC and approximately 3% of cases of apparently sporadic PDAC (Blair et al., 2018). Poly(ADP-ribose) Polymerase (PARP) inhibitors have shown remarkable efficacy in the clinical management of several BRCA-mutated tumors. The phase III trial showed that maintenance treatment with olaparib after chemotherapy lead to a significantly longer progression-free survival compared with placebo in PDAC with germline BRCA1 and BRCA2 (gBRCA1/2) variants (7.4 months vs. 3.8 months, p = 0.004, HR = 0.53) (Golan et al., 2019). According to this study, olaparib was approved by FDA as a maintenance treatment for patients with P/LP variants in gBRCA1/2, and platinum-sensitive, metastatic pancreatic adenocarcinoma. However, gBRCA1/2 variants are found in approximately 5%-10% of cases of familial PDAC and approximately 3% of cases of apparently sporadic PDAC (Blair et al., 2018).
Emerging evidence suggests that patients with P/LP variants in several additional homologous recombination repair (HRR) related genes (e.g., ATM, PALB2, ATR, RAD 51, and CHEK2) might benefit from PARP inhibitors (Reiss et al., 2021;Urbina-Jara et al., 2019). Recently, a Phase II Study (NCT03140670) investigating rucaparib, another PARP inhibitor, showed promising efficacy in patients with BRCA1/2 as well as PALB2 variants (Reiss et al., 2021). Hence, panel genetic testing of germline P/ LP HRR variants is meaningful to identify PDAC patients who will derive clinical benefits from PARP inhibition. On the other hand, germline P/LP variants of DNA repair genes have been associated with an increased risk for PDAC and other tumors (Hu et al., 2018;Yang et al., 2020). Therefore, the National Comprehensive Cancer Network (NCCN) currently recommends genetic counseling and genetic testing to all patients with PDAC as well as to firstdegree relatives of patients with PDAC.
However, the prevalence of germline HRR variants in the Chinese population is rarely reported. Herein, we applied next-generation sequencing (NGS) technologies to test DNA from 256 whole blood samples. Germline deleterious variants, covering 22 core HRR genes, were identified in Chinese patients with PDAC, and the relationship between genetic alterations and clinical parameters was analyzed.

| Patients and samples
A cohort of 256 PDAC patients who were admitted to the Zhongshan Hospital Fudan University between June 2019 and June 2021 was included in the study. Clinical information was retrieved from medical records. The study complied with the ethical standards of the Declaration of Helsinki and was reviewed and approved by the institutional ethics committee (Zhongshan Hospital Fudan University; B2021-056). Written informed consent was obtained from all participants.

| Clinical characteristics collection
Demographic and clinical information, including age, gender, tumor location, tumor stage, and KRAS status of tumor tissue were retrieved from the hospital information system. The definition of family history is these firstdegree relatives with a history of any solid malignancy.

| NGS sequencing
A 10-mL blood sample was collected from each patient in an EDTA blood collection tube. Germline DNA (gDNA) was extracted using Blood DNA kit (Amoy Diagnostics) and quantified using Qubit (Life Technologies). Germline DNA was analyzed using a handle HRR NGS panel (Amoy Diagnostics) according to manufacturer's instructions, which covered single-nucleotide variants (SNV) and insertions/deletions (Indel) variants in protein-coding K E Y W O R D S germline mutations, homologous recombination, next-generation sequencing, pancreatic ductal adenocarcinoma regions and intron/exon boundaries of 21 genes in- (NM_001142548.1), STK11 (NM_000455.4), and TP53 (NM_000546.5). This panel allowed the detection of copy number variations (CNV) of BRCA1/2. Briefly, DNA libraries were constructed by molecular inversion probebased capture, and then sequenced to a depth of ×100 on the MiSeq platform (Illumina, San Diego, USA).

| Variant calling and bioinformatics analysis
Sequence data were aligned to the UCSC hg19 human reference genome sequence. The interpretation of all identified variants was based on current evidence from the scientific literature and public databases, including ClinVar, Ensembl, Varsome, LOVD, dbSNP, Genome Aggregation Database (gnomAD), and Exome Aggregation Consortium (ExAC). The clinical classification of the variants was carried out according to the American College of Medical Genetics and Genomics (ACMG) recommendations with the 5-tier system: benign (B), likely benign (LB), variant of uncertain significance (VUS), LP, and P (Richards et al., 2015).

| Statistical analysis
The distribution of each categorical variable was summarized in terms of its frequencies and percentages. The comparisons of categorical data were done using Fisher's exact test. A p-value <0.05 (2-sided) was considered statistically significant. Statistical analysis was performed using Statistical Product and Service Solutions (SPSS) Statistics 20.0 (IBM Corporation).

| Patient characteristics
From July 2019 to July 2021, a total of 256 Chinese PADC patients were included in this study. The median age at diagnosis was 64 years old (range 32-82), and 161 (62.9%) patients were male. Ninety-one patients (35.5%) had signs of metastasis at the time of diagnosis, and 20 patients (7.8%) had a history of multiple cancer. Family history of cancer within third-degree relatives was confirmed in nine patients (3.5%). Patient detailed clinicopathological characteristics are summarized in Table 1.

| Landscape of germline HRR gene alterations
The overall landscape of unique alterations in each patient is summarized in Figure 1. HRR P/LP variants were identified in 7.0% (18/256) of patients in our study, whereas 53 patients were carriers of VUS (22.3%). The majority of P/LP variants occurred in ATM, BRCA2, and PALB2 ( Figure 2). Of the 18 P/LP variants, nine were frameshifts, six were nonsense SNVs, one were splice sites, and two were missense SNVs (Table 2, Figure 3). Most of these variants were absent or rare in East Asian from gnomAD database.

| Correlations between clinical characteristics and P/LP HRR variant
HRR variants were observed more frequently in patients with a family history of cancer (including esophageal cancer, breast cancer, colorectal cancer, gastric cancer, lung cancer, pancreatic cancer, and liver cancer), whereas there was no significant difference (22.2% vs. 6.5%, p = 0.250). Of the 18 patients harboring P/LP HRR variants, only two had a family history of cancer. We found no statistically significant association between HRR mutation status and age, gender, KRAS status, tumor location, tumor status, or multiple cancer history (Table 1).

| Germline PALB2 variant is a promising biomarker for platinum-based chemotherapy and PARP inhibitor
In this study, we observed that one patient with a germline PALB2 variant exhibited a long-time response to platinum-based chemotherapy and PARP inhibitor (Figure 4). The patient underwent pancreatiplenectomy in November 2017. Five months postoperatively, multiple metastases appeared involving the lung and peritoneum. He received a standard gemcitabine/nabpaclitaxel regimen as first-line chemotherapy. After 10-month treatment, PET-CT revealed progression disease (PD). Since February 2019, he received 2 cycles of FOLFIRI. However, the patients underwent rapid PD. Then, he received FOLFIRINOX, which included oxaliplatin (a platinum-based drug), as third-line therapy, and follow-up images revealed partial response (PR). Mutational profiling analysis using targeted NGS revealed that the patient carried a germline PALB2 variant. Olaparib (a PARP inhibitor) was thereby administered as maintenance therapy. Surprisingly, he showed a significant response to olaparib, and a PFS of 23 months was observed. This case indicated that PDAC patients with germline PALB2 variants may derive benefits from both platinum-based chemotherapy and PARP inhibitor.

| DISCUSSION
Alterations in HRR genes are emerging as novel targets for treatment in different cancers and, particularly, for personalized therapies. In the present study, we performed germline testing by next-generation sequencing using a multigene panel of the 21 HRR genes in 256 Chinese PDAC patients. Using this expanded gene panel, we found that homologous recombination deficiency in PDAC may be underreported. In our study, when restricting our analysis to BRCA1/2 variants, we found that only 1.6% of patients are represented. When including non-BRCA HRR variants, this number increased to 7.0%. Multigene panel testing could identify HR defects in an additional 5.5% of patients, expanding the population who may benefit from DNA damage response agents. The overall incidence of germline variants in our cohort was similar to what has been reported in previous studies. Yurgelun et al. found that 7.3% of patients carried the germline variants in 24 detected DNA damage response and repair (DDR) genes in an American PDAC cohort (Yurgelun et al., 2019).
Other studies have demonstrated that 3.9%-19.8% of patients with PDAC carry germline P/LPVs in cancerpredisposing genes (Brand et al., 2018;Cremin et al., 2020;Hu et al., 2018;Shindo et al., 2017;Yurgelun et al., 2019). However, we found the differences in variant rates among studies mainly caused by gene panel involvement and ethnic specificity. A systematic review included 60 studies with 21,842 participants who reported that the main contribution to homologous recombination deficiency (HRD) was through BRCA2, BRCA1, and ATM, followed by FANC genes, CHEK2, and PALB2 in PDAC patients (Casolino et al., 2021). Based on the Chinese population, we found that the most frequently deleterious mutated genes were ATM, BRCA2, and PALB2. However, differences were found in the mutation spectrum among different population cohort. BRCA1 deleterious variant was more prevalent in non-hispanic white and Greek PDAC population (0.65% and 0.4%, respectively), but it was not found in our cohort study.
Currently, little is known about the molecular differences that might exist between PDAC patients with mutated HRR genes and those with wild-type HRR genes. In our study, we analyzed the association between germline HRR variants and tumor KRAS status. However, no significant correlation was observed. Seeber A et al. presented a large study investigating the molecular landscape of patients with BRCA-mutated and PALB2-mutated pancreatic cancer. No differences were found in the prevalence of KRAS alterations. However, patients with BRCA/PALB2 variants were associated with the expression of biomarkers T A B L E 2 Mutation details of P/LP gene variants.  that are potentially associated with response to immunotherapy such as tumor mutation burden (TMB), PD-L1 expression, and microsatellite instability (MSI) status (Seeber et al., 2020). Previous research showed that PDAC patients belonging to families with known BRCA1/2 variants are a decade younger (Golan et al., 2014). There is no unified definition regarding the age of early-onset pancreatic cancer. In our study, a cut-off of 50 years old was used according to several previous research (Okur & Chung, 2017;Brune et al., 2010). We found that the HRR gene variant rate was similar between early-onset and non-early-onset patients. Although there was a slightly higher variant rate in PDAC patients with a family history of cancer or multiple cancer history, no significant difference was observed (22.2% vs. 6.9%, P = 0.137; 15.0% vs. 6.8%, P = 0.176). A previous study also reported that the rate of germline variants in HRD genes is similar in sporadic and familial PDAC patients (Casolino et al., 2021). These findings highlighted that onset of age, family history, and history of multiple cancer were not predictors of an underlying genetic factor in PDAC patients. Therefore, current recommendations are that all newly diagnosed patients should undergo germline testing, particularly as a subset of these variants are therapeutically actionable (Lowery et al., 2018).

Allele frequency in East Asian
In our study, we identified PALB2 as one of the most frequently mutated genes among germline HRR genes in PDAC patients. PALB2 encodes a protein essential for double-strand break repair and homologous recombination by serving as a bridging molecule, which connects the BRCA complex and stimulates the strand invasion of RAD51. Recently, a study investigating rucaparib, a PARP inhibitor, showed promising efficacy in PDAC patients with PALB2 variants. In our study, we observed one patient with PALB2 germline mutation who exhibited a long-time response to platinum-based chemotherapy and PARP inhibitor. The patient remained progression-free on PARP inhibitor therapy for nearly 2 years. This case indicated that metastatic PDAC patients who harbor a germline PALB2 variant may benefit from PARP inhibitor. While we cannot conclusively state that the entirety of patients with HRR variants would derive clinical benefit from PARP inhibition, the predictive value of non-BRCA HRR variants for PARP inhibition is not well established and warrants continued exploration.

F I G U R E 4
A 50-year-old male was diagnosed with pancreatic adenocarcinoma in July 2017. The patient underwent pancreatiplenectomy in November 2017. Five months postoperatively, multiple metastases appeared involving the lung and peritoneum. He received chemotherapy with gemcitabine and nab-paclitaxel as first-line therapy since Apr 2018. PET-CT revealed PD on Feb 2019. Since then, he received 2 cycles of FOLFIRI. However, the patients underwent rapid PD. Then he received FOLFIRINOX as third-line therapy, and follow-up images revealed PR. Mutational profiling analysis using targeted next-generation sequencing revealed that the patient carried a germline PALB2 mutation. Olaparib was thereby administered as maintenance therapy. The patient showed a significant response to Olaparib and a PFS of 23 months was observed. DFS, disease-free survival; PD, progression disease; PFS, progression-free survival; PR, partial response; SD, stable disease.
We acknowledge that there are several limitations in the present study. With a relatively small sample size and retrospective design of this study, any conclusion should be declared with caution. Further, prospective and larger studies are needed to explore the predictive and prognostic role of HRR deleterious variants. In addition, annotations of variants in HRR genes other than BRCA1/2 are seriously inadequate. As a result, more than 20% of patients were identified as VUS carriers. This type of inconclusive result is a hard challenge to face, requiring further functional experiments and pedigree analysis for validation.

| CONCLUSIONS
In the present study, we confirmed the importance of sequencing germline HRR genes in PDAC patients. Prospective studies evaluating the prognostic and predictive role of germline HRR gene variants in PDAC are warranted.

ACKNO WLE DGE MENTS
We sincerely thank all the participants who volunteered to take part in this study.