Clinical Significance of Germline Pathogenic Variants among 51 Cancer Predisposition Genes in an Unselected Cohort of Italian Pancreatic Cancer Patients

Simple Summary Multigene germline panel testing data, extended beyond BRCA, in unselected pancreatic cancer patients, are missing in the Italian population. We aimed here to determine the prevalence and impact of pathogenic variants in 51 pancreatic cancer candidate susceptibility genes in an unselected cohort of Italian pancreatic cancer patients. We found that 17% were carriers. CDKN2A was the most frequently mutated gene, followed by BRCA2 and ATM. Carriers showed better overall survival. A total of 41% of them had no family history. All CDKN2A carriers were older than 50 years, and BRCA1/2 carriers were younger than 70 years. CDKN2A and ATM should be added to current BRCA1/2 testing independently of family history in our population. Abstract Multigene germline panel testing is recommended for Pancreatic Cancer (PC) patients; however, for non-BRCA1/2 genes, the clinical utility is unclear. A comprehensive multi-gene assessment in unselected Italian PC patients is missing. We evaluated the prevalence and impact of Pathogenic Variants (PV) in 51 PC susceptibility genes in a real-world series of 422 Italian PC patients unselected for Family History (FH), compared the clinical characteristics and conducted survival analyses. 17% of patients had PVs (70/422), mainly in BRCA1/2 (4.5%, all <70 y), CDKN2A (4.5%, all >50 y), ATM (2.1%). PV carriers were younger (64 vs. 67; p = 0.02) and had more frequent personal/FH of PC, melanoma and breast/ovarian cancer (all p < 0.05). The Overall Survival (OS) was longer in patients carrying PVs (HR 0.78; p = 0.090), comprising ATM carriers (HR 0.33; p = 0.054). In the oxaliplatin-treated subset, PV carriers showed better control of the disease, although this was not statistically significant (67% vs. 56%). CDKN2A, BRCA2 and ATM were the most frequently altered genes. ATM PVs were positively associated with OS in 41% of PV carriers, 60% of whom carried CDKN2A, BRCA2 or ATM PVs, had negative FH and would have been missed by traditional referral. Thus, CDKN2A and ATM should be added to BRCA1/2 testing regardless of FH.


Introduction
Exocrine Pancreatic Cancer (PC) is one of the deadliest solid tumors, with an average 5-year survival rate of 5-10% [1]. In 2020, 495,773 new cases and 466,003 deaths were Italian Medicines Agency (AIFA) recently withdrew Olaparib from the approved treatments for PCs.
The POLO study also confirmed the well-known platinum sensitivity of patients carrying germline PVs causing DNA Damage Repair-Homologous Recombination Deficiency (DDR-HRD), such as BRCA1/2 [27]. However, a comprehensive analysis by MGP in unselected Italian PC patients is lacking. Preliminary data from different cohorts of unselected Italian PC patients focusing on CDKN2A and BRCA1/2 showed a high prevalence of CDKN2A PVs, regardless of familial status [28] and of BRCA1/2 PVs-these latter being observed only in patients < 74 y [29].
This study aimed to evaluate the prevalence and impact on outcomes of PVs in 51 PC susceptibility genes in a retrospective/prospective series of 422 Italian PC patients recruited at our Institution and unselected for FH.

Patients Selection and Characteristic
From 2020 to 2022, we recruited a consecutive series of 111 PC patients referred to our center for molecular analysis. Moreover, we retrospectively included an additional series of 311 non-consecutive PC patients who underwent molecular analysis at our center. Patients with neuroendocrine pancreatic tumors were excluded. All patients (N = 422) were unselected for age, FH or personal history, and there were no differences in terms of patients' characteristics (age, family, personal history and prevalence of PVs) between retrospective and prospective groups, except for the stage and Overall Survival (OS). This may be explained by stage migration and better management of patients in recent years. All patients signed an informed consent according to local ethics committee protocols and answered a standardized family history questionnaire on the presence and type of cancers in the family. One hundred twenty-two patients have been described in previous publications; however, the analysis was limited to CDKN2A or BRCA1-2 [28,30], as well as Mismatch Repair (MMR) genes for a subset of high-risk patients [30,31]. In addition, 31 patients, either positive for CDKN2A PVs or negative but considered high-risk because of PC family history, had been previously tested by Whole-Exome Sequencing (WES) [32] and were re-tested here by MGP.
The NGS libraries were constructed using the Ion AmpliSeq Kit for Chef DL8 (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's protocol starting from 20 ng of genomic DNA. According to the manufacturer, the libraries were sequenced with an Ion Torrent S5 GeneStudio Plus system using 530 Chip (Thermo Fisher Scientific). The S5 sequencing data were analyzed by the Ion Torrent Software Suite (Thermo Fisher Scientific) using the plugin Variant Caller and Ion Reporter (Thermo Fisher Scientific). The filtered variants were analyzed and classified according to the American College of Medical Genetics and Genomics (ACMG) classification [35]. Only ACMG class 4 (Likely Pathogenic) and 5 (Pathogenic) variants were considered as PVs in this study.

Statistical Analysis
Descriptive characteristics were reported as mean (standard deviation) or absolute frequency (relative frequency). Patients carrying PVs were compared to the non-carriers using T-test for continuous variables and Chi-squared or Fisher's exact test for categorical variables. Univariable Cox proportional hazards models were used to identify characteristics associated with prognosis in terms of overall survival. Subsequently, we presented multivariable Cox proportional hazards models adjusting for confounders. Additionally, as a sensitivity analysis, age-adjusted Cox proportional hazards models were performed to specifically assess the impact of each type of mutation.
Within the subsample of patients who received oxaliplatin-based treatment, the association between PVs and disease progression was evaluated by a Chi-squared test. Finally, an age-adjusted Cox proportional hazards model was performed to compare responding and not-responding patients. All statistical analyses were conducted using Stata version 16.0 (Stata Corporation, College Station, TX, USA), and p-values < 0.05 were considered statistically significant.

Results
We recruited 422 patients belonging to a real-world series of patients diagnosed with Pancreatic cancer, unselected for family history and analyzed them using a multigene panel containing 51 genes. Table 1 shows the patients' characteristics according to the germline status. Overall, 52% were females, and the mean age was 67 years (range 30-92). As expected, germline PV carriers were significantly younger than non-carriers (p-value = 0.0254). Overall, 13% of patients had a first or second-degree relative with PC. A family history of PC was more frequent in carriers than in non-carriers (26% vs. 11%; p = 0.001); the same pattern was confirmed in cases with a personal and/or family history of either breast and ovarian cancer (36% vs. 14%, p < 0.001), melanoma (19% vs. 5%; p < 0.01) and personal history of other tumors (32% vs. 13%, p < 0.001).

Prevalence of Pathogenic Variants in Pancreatic Cancer Susceptibility Genes: Correlation with Family History and Age at Diagnosis
Seventy out of 422 enrolled patients (17%) carried one or more pathogenic variant (PV) in at least one of the 51 genes included in the panel (Table 1). CDKN2A was the most frequently mutated gene (19/422; 4.5%), confirming the founder p.Gly101Trp as the most frequent PV in our series (11/19 = 57.9%) [36]. For this reason, CDKN2A in our cohort shows a higher frequency compared to the literature data [37] (Figure 1).

Prevalence of Pathogenic Variants in Pancreatic Cancer Susceptibility Genes: Correlation with Family History and Age at Diagnosis
Seventy out of 422 enrolled patients (17%) carried one or more pathogenic variant (PV) in at least one of the 51 genes included in the panel (Table 1). CDKN2A was the most frequently mutated gene (19/422; 4.5%), confirming the founder p.Gly101Trp as the most frequent PV in our series (11/19 = 57.9%) [36]. For this reason, CDKN2A in our cohort shows a higher frequency compared to the literature data [37] (Figure 1).

Figure 1.
Comparison between the relative frequency of candidate susceptibility genes PVs in our cohort and literature data according to the systematic review by Astiazaran-Symonds E and Goldstein AM [37].
Three patients showed multiple PVs: two sporadic PC patients had two (in BRCA2 and CHEK2) and three (in ATM, CDKN2A and NBN) pathogenic variants, respectively. The third, who developed both melanoma and PC, showed 2 PVs (in CDKN2A and NBN). Interestingly, two out of three patients carrying NBN PVs had a concomitant PV in a high penetrance predisposition gene (CDKN2A or ATM).
Seven PVs were found in CHEK2 (1.7%), four of whom had no FH. Five PVs (1.2%) were found in the COL7A1 gene; among these patients, only two showed a family history of PC and breast cancer. Finally, we detected two PVs in MSH2, in 2 patients with suspected Lynch Syndrome. No variants were identified in the PALB2 gene. The complete list of LPs and PVs is described in Supplementary Table S1.
As expected, PVs in BRCA1/2 were more frequent in subjects with a personal and/or family history for breast/ovarian cancer (12/76, 15.8%). However, a 1.8% frequency was observed even in sporadic subjects (5/283). CDKN2A PVs were frequent in patients with a family history of PC (8/55, 14.5%) and with a personal or family history of melanoma (6/30, 20%); however, the rate remains high (8/283, 2.8%) in sporadic subjects. Notably, ATM PVs frequency was 5.4% in patients with a family history of PC (3/55) and 3.3% in patients with personal or family history of melanoma (1/30). ATM PVs mutation frequency was as high as that of BRCA-2 (1.8%) in sporadic patients.
Overall, 11.3% of PV were found in sporadic patients (32/283) and would have been missed if we tested patients based only on family history (Table 2). When we analyzed age at diagnosis comparing patients according to specific PVs, we found that the overall frequency of PVs was 22.2% among patients ≤50 y, 24.4% among patients 51-60 y, 17.5% among patients 61-70 y and 11.6% for those who were >70 y. The highest frequency of BRCA1/2 PV was found among the youngest patients (≤50, 13.9%), while no PVs were found in patients older than 70.
Conversely, the highest CDKN2A mutation rate was found in patients aged 61-70 y (6.3%) and was 3.8% in patients > 70 y. No PVs in CDKN2A were found in patients younger than 50 y (Supplementary Table S2). Overall, patients with BRCA1/2 genes were younger (mean age = 58) than CDKN2A patients (mean age = 67) (Supplementary Table S2). Patients carrying any PVs showed better OS compared to non-carriers in the univariable analysis (HR 0.78; 95% CI 0.59-1.04; p = 0.090) and was confirmed in the multivariable analysis (HR 0.81; 95% CI 0.61-1.09; p = 0.160). In the sensitivity analysis we also evaluated the impact of PVs in single gene and we found that all nine patients carrying ATM PVs had a better prognosis compared to non-carriers in terms of median OS (15 vs. 11 months; age-adjusted HR 0.33; 95% CI 0.10-1.02; p = 0.054). No significant differences were observed between patients with PVs in other genes and non-carriers. setting, 24% (N = 6) had a Progression of Disease (PD) as the Best Response (BR) to oxaliplatin, 28% (N = 7) had a partial response and 32% (N = 8) had stable disease. In the metastatic setting, 42% (N = 34) had a PD as the best response to oxaliplatin, 21% (N = 17) had a partial response, and 30% (N = 24) had a stable disease. Patients carrying a PV more frequently showed a good response to treatment (67%) compared to non-carriers (56%) but differences were not statistically significant (p = 0.381). When we focused on patients with BRCA or ATM PVs, all nine patients showed control of disease as BR to oxaliplatin.

Discussion
We analyzed a panel of 51 genes in a retrospective/prospective cohort of 422 Italian PC patients unselected for family history, stage or age of onset. To the best of our knowledge, this is the largest cohort of unselected PC Italian patients analyzed with a MGP comprehensive of all PC susceptibility genes described to date. Overall, 70 PVs (17%) were found, a relatively high frequency compared to similar studies. However, due to the heterogeneity in cohort selection and dissimilar sequencing approaches across studies, the prevalence of PVs in PC susceptibility genes cannot be easily quantified, ranging from 5% to nearly 20% in different studies and reviews [18,34,[37][38][39][40][41][42].
The latest frequency confirms recent findings from an extended melanoma family study in the Italian population showing a 12.4% of PVs in melanoma families with PC cases [47]. However, the mutation rate remains high (2.8%) even in sporadic subjects. This may reflect the presence of founder CDKN2A PVs in our population [36].
As expected, BRCA1 and BRCA2 PV rate nearly doubled in cases with a FH of PC nearly compared to sporadic subjects (3.6% vs. 1.7%), while it ranged from 1.7% to 15.8% in subjects with a personal or family history of breast/ovarian cancer (Table 2). Again, these data confirm previously observed BRCA1/2 mutation rates in Italian families with history of both breast/ovarian and PC [40,41].
Carriers of BRCA1/2 PVs showed a lower age of onset compared to CDKN2A carriers. While CDKN2A PVs are distributed only in the age categories above 50 years, BRCA1/2 are the most common PVs in the age category below 50 years (13.9%). In our cohort, the oldest patient carrier of a germline BRCA1/2 PVs was 70 years old. This supports the results obtained in a different unselected cohort of Italian PC patients tested for BRCA1/2 only, showing that BRCA1/2 mutations were found in patients < 74 y [29].
ATM was the first most frequently mutated gene in the age category between 51 and 60 years and the third mutated gene overall (2.13%). Interestingly, ATM PV/LPVs frequency was second to CDKN2A only in patients with a family history of PC (5.4%) and was found at a frequency of 3.3% in patients with familiarity for melanoma. These data also confirm recent figures observed for ATM germline PVs in melanoma cases [47][48][49].
Overall, CHEK2 and COL7A1 PVs were found with a frequency above 1%. Further investigation will be needed to elucidate their role in pancreatic cancer predisposition. However, our findings are in line with those from the PAN Cancer [34]. A recent paper found that the combined frequency of heterozygous carriers of CHEK2 in the general population was 1.2% [50]. Interestingly, PVs in TP53 and MMR genes were below 1% and were found in patients with a personal and/or FH of breast/ovarian and colon cancer, respectively.
In our study, we observed that patients carrying any PVs had a better OS compared to non-carriers (HR = 0.66; 95% CI 0.47-0.93; p = 0.017). However, when we focused on PVs in specific genes, only patients carrying ATM PVs (N = 9) showed better OS compared to WT patients (N = 345) (HR = 0.33). Our results confirm previous reports that suggested ATM alterations as prognostic for improved outcomes in patients with PC [51].
Despite efforts in extensive molecular profiling in PC for actionable genetic alterations or pathways, targeted therapy showed to provide limited clinical benefit for patients with this disease to date [52]. One exception is alterations in DNA damage repair (DDR) genes, especially BRCA1/2, since BRCA-associated tumors, including breast, prostate and ovarian tumors, respond to poly-(adenosine diphosphate-ribose) polymerase (PARP) inhibitors and platinum-based therapy.
However, the efficacy of PARP inhibitors in DDR-related genes other than BRCA is less clear. Recent two phase II nonrandomized clinical trials showed that Olaparib was well tolerated. However, it showed limited antitumor activity in patients with advanced, platinum-sensitive PC with DDR-genetic alterations, most of them carried ATM alterations [53]. Unfortunately, no patients with ATM alterations were treated with PARP inhibitors in our cohort. However, we observed a trend in better disease control in PV carriers treated with oxaliplatin, although not statistically significant.
More data in larger cohorts are needed to confirm these results. Although the prevalence of DNA MMR is <1%, these patients may benefit from immunotherapy [24], thus we believe that evaluation of MMR system (somatic and/or germline) is crucial in PC patients as well as in all other solid tumor types [54]. BRCA1/2, CDKN2A and ATM, account for an overall PV/LPV prevalence of 11.1% in our cohort.
Had we selected PC patients for genetic testing according to their personal and family history suggestive for familial PC or hereditary cancer syndromes, we would have missed 41% of patient carriers of PVs in candidate gens. Sixty% of them were carriers of PVs in CDKN2A, BRCA2 and ATM. At least CDKN2A and ATM, should be added to BRCA1/2 testing by MGP independently of FH.
These PVs are clinically actionable since they result in hereditary cancer syndromes for which international guidelines support surveillance aimed to reduce mortality among relatives. Although such guidelines are not of proven efficacy for PC mortality reduction, identification of probands may still reduce mortality for other cancers through 'ad hoc' prevention protocols. However, several studies and meta-analyses have shown that the diagnostic yield of pre-malignant or malignant lesions (including earlier stage) in high risk individuals undergoing screening/surveillance is much higher than the lifetime risk of PC in the general population and this might be considered a surrogate for mortality reduction [55][56][57][58][59].
In Italy, the AISP (Italian Association for the Study of the Pancreas), developed a registry of families at high risk of PC to be included in surveillance protocols [59,60]. As previously mentioned, an extended MGP testing-beyond BRCA1 and 2-in all PC patients in not standard of care in Italy and an estimate of a real world MGP frequency in unselected PC patients was missing. The registry is currently recruiting families at high-risk because of family history or positive mutation testing. Our data support an extended MGP testing (at least to CDKN2A and ATM) to identify positive high-risk carriers to be included in registry surveillance protocols, also independently of family history.
We acknowledge that our study presents some limitations, mainly because of a nonconsecutive and retrospective enrollment of part of the cohort. In addition, few data are available on treatments (oxaliplatin-based, anti-PARP and/or immunotherapy) in PV carriers. No correlation with somatic tumor samples was conducted. Moreover, stage migration and changes in patient management were observed during the long patient recruitment period.

Conclusions
In conclusion, our study showed that the universal use of MGP testing in PC patients allowed us to identify a high PV prevalence (17%). These data might address unmet clinical need to identify actionable mutations and provide the opportunity to identify well-known hereditary cancer syndromes. Finally, 40% of patients with PVs did not have suspected criteria within our cohort and would have been missed with standard selection criteria.
Thus, we propose extending the offer of genetic testing beyond BRCA1 and 2 at least to CDKN2A and ATM, independently of family history, with no age restrictions to identify mutations and actionable not only for target therapy in affected members but also for surveillance research protocols in carriers, including unaffected relatives.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cancers14184447/s1, Table S1: List of Likely Pathogenic and Pathogenic Variants; Table S2: Overall mutation rate and related to a single gene. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.