Pancreatic ductal adenocarcinoma (PDAC) is expected to become the second leading cause of cancer-related deaths in the US before 2030 [1
]. In the NCCN (National Comprehensive Cancer Network) guidelines, germline testing is recommended for patients with PDAC, using comprehensive gene panels for hereditary cancer syndromes [2
]. The genes commonly associated with pathogenic germline alterations are BRCA 1
, and TP53
]. Among them, the frequency of detected BRCA 1
(breast cancer susceptibility gene-1 and -2) is 4% to 7% [4
]. The risk for pancreatic cancer is elevated two- to six-fold in these patients [6
Recently, the POLO trial showed the benefit of poly ADP ribose polymerase (PARP) inhibitors in BRCA
genes encode proteins involved in homologous recombination repair, and cells with mutations are sensitive to PARP inhibitors. However, there was no difference in overall survival between the PARP inhibitor and placebo groups (p
= 0.68) [8
]. Furthermore, in the real world, it is difficult for clinicians to change regimens in patients who are tolerant to FOLFIRINOX (oxaliplatin, irinotecan, folinic acid, and fluorouracil) chemotherapy.
In PDAC, FOLFIRINOX and nab-paclitaxel are recommended as first-line chemotherapy regimens. The guidelines recommend FOLFIRINOX in patients who are young and with better performance status (ECOG 0–1) [2
]. However, there are limited data to predict the efficacy of the FOLFIRINOX regimen in patient outcomes [9
]. Platinum-based chemotherapy is tolerable and responsible in patients with DNA damage repair gene mutations [10
]. However, data are still limited, and no Asian data are available yet [12
Here, we sought to investigate the proportion of germline BRCA 1/2 mutations in patients with germline blood tests. Finally, we investigated the treatment response of FOLFIRINOX in patients with a BRCA 1/2 mutation.
2. Materials and Methods
2.1. Study Population
This dual institutional retrospective analysis was performed on all patients diagnosed with PDAC who underwent a germline blood test between January 2012 and February 2020. We identified 66 patients who underwent a germline blood test. Of these, two patients were excluded from the study on account of insufficient clinical data (n = 2). One patient was diagnosed and treated at another hospital, and one patient died shortly after diagnosis due to deterioration of the condition. The remaining 64 patients were included in the analysis. This study was performed in accordance with the Declaration of Helsinki, as reflected by the institutional review board of Severance Hospital (approval number 4-2021-1151).
We evaluated patient characteristics, laboratory variables, tumor characteristics, progression-free survival (PFS), overall survival (OS), and overall response rate (ORR). Patient demographics and clinical characteristics, including age, sex, personal and family history of cancer, hypertension, diabetes mellitus, smoking history, body mass index (BMI), systemic chemotherapy, and response to treatment, were obtained from medical records and imaging studies. BMI, defined as body weight divided by the square of the height, was categorized following the guidelines of the World Health Organization (WHO 2000) (BMI < 18.5, underweight; 18.5–24.9, normal range; ≥25.0, overweight; and ≥30.0, obese). Tumor characteristics (location, extent, and number of metastatic organs) and laboratory characteristics (carbohydrate antigen [CA] 19-9) were also investigated.
The date of death and the date of the last follow-up were reviewed to estimate the OS and PFS. We observed both survival and follow-up data until 5 March 2021. OS was defined as the interval from the start of FOLFIRINOX until death. PFS was defined as the interval from the start of FOLFIRINOX to progressive disease (PD) or death. Patients who remained without death or PD were censored at the time of the last follow-up. Responses were determined using RECIST (response evaluation criteria in solid tumors) v1.1. ORR was defined as the percentage of patients who had a best response rating of complete response (CR) or partial response (PR) at any time point during treatment with chemotherapy. Patients without measurable disease at baseline were excluded from the ORR analysis.
2.3. DNA Extraction and Sequencing
Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini Kit (Qiagen, Venlo, The Netherlands). The amount of input DNA was approximately 500 ng. DNA was fragmented into segments between 150 and 250 bp using the Bioruptor®
Pico sonication system (Diagenode, Liege, Belgium), end-repaired, and ligated to Illumina adapters (Illumina, San Diego, CA, USA) and indices. Sequencing libraries were hybridized with capture probes (Celemic, Seoul, Korea). The enriched DNA was then amplified, and clusters were generated and sequenced on a NextSeq 550 instrument (Illumina) with 2 × 151 bp reads [17
]. Pathogenicity interpretations of the variants were performed according to the 2015 American College of Medical Genetics and Genomics guidelines by professional medical geneticists, using evidence from variant type assessments, population allele frequency, prediction algorithm results, and searches within databases such as ClinVar.
2.4. Statistical Analysis
The baseline demographics and characteristics of the patients were analyzed using descriptive statistics. The differences in baseline characteristics and ORR between BRCA-positive and BRCA-negative groups were analyzed using the chi-square test for categorical variables and the Student’s t-test for continuous variables. We estimated the median OS and PFS according to BRCA mutations using Kaplan–Meier curves and compared them using the log-rank test. A time-dependent Cox regression analysis was applied to estimate hazard ratios (HRs) with 95% confidence intervals (CIs) of pancreatic cancer mortality associated with BRCA mutations. Statistical significance was set at p < 0.05. All analyses were conducted using SPSS version 26.0 (SPSS, Chicago, IL, USA).
In this study, BRCA 1
germline mutations predicted the treatment response of FOLFIRINOX in patients with PDAC. The PFS was longer in patients with a BRCA 1
mutation than those with a wild type, even though the difference was not statistically significant. In this study, the rate of BRCA 1
mutations was 13.6%. The data values were slightly higher than previous data (range 4–7%) in the general population [4
]. The higher proportion of BRCA 1
mutations may be due to the change in detection method with the adoption of next-generation sequencing. In addition, considering that a high proportion of patients were previously diagnosed with breast cancer in this study, the results are similar to those of previous studies. The prevalence of BRCA 1
mutations in Asian patients with familial breast cancer and early-onset breast cancer was reported to be 2.8% to 31.8% [18
]. Previous studies showed that BRCA
gene mutations were associated with patients’ survival outcomes [12
]. In this study, patients with BRCA
gene mutations did not show different survival outcomes on account of the small number of patients.
The clinical significance and prognostic value of germline BRCA pathogenic mutations in tumors are well-known, but whether missense variants of uncertain significance (VUS) have clinical impact is not known. Variants in the gene were often classified as VUSs because of an insufficient understanding of the gene’s role. Variants can be reclassified from VUS to likely pathogenic, and further, to pathogenic. Phosphorylation of BRCA 1
mutations plays an important role in their function as regulators of DNA repair, transcription, and cell cycles in response to DNA damage. Tram et al. suggested that VUS have the potential to interfere with the phosphorylation process via abolishing or creating phosphorylation sites on BRCA 1
]. Hu et al. reported that germline VUS variant carriers had superior disease-free survival when compared with wild-type PDAC patients receiving adjuvant chemotherapy (16.5 months vs. 13.1 months, p
= 0.007) [21
]. Previous statistics indicate that between 10–20% of BRCA sequencing results are VUSs, and of these, more than 50% are missense mutations [22
]. In this study, BRCA 1
missense mutations (VUSs) were detected in 15.2% of our cohort (Supplementary Table S1
). The ORR was significantly higher in BRCA-positive patients, including missense mutations of VUS, than in BRCA-negative patients (7/12, 58.3% vs. 3/31, 9.7%, p
= 0.002). With the further accumulation of data in the future, VUS can be reclassified as pathogenic.
Previously, several studies reported on the proportion of BRCA 1
mutations and their impact on patients with PDAC [9
]. Golan et al. showed a difference in survival outcome for stage 3 or 4 PDAC patients with BRCA 1
mutations in platinum-based chemotherapy (22 months vs. 9 months, p
= 0.039) [19
]. Wattenberg et al. reported on the treatment response of platinum-based chemotherapy in PDAC patients with BRCA 1
mutations (58% vs. 21%, p
= 0.002) [12
]. In the present study, patients who received FOLFIRINOX chemotherapy showed a better treatment response in BRCA
-positive patients compared to BRCA
-negative patients. However, patients who received nab-paclitaxel chemotherapy did not show any difference in treatment response, irrespective of BRCA
Recently, several studies attempted to identify patients who benefit from palliative first-line FOLFIRINOX chemotherapy. Transcriptomic analysis showed that the basal type showed a better treatment response to FOLFIRINOX chemotherapy. The immunohistochemistry stained marker KRT81 may be a predictive marker to identify patients in the clinical field [29
]. Circulating blood markers, such as ctDNA and exosomes, were also suggested as predictors for FOLFIRINOX response [30
]. In other studies, protein markers, CES2 expression, and female gender predicted the response to FOLFIRINOX in PDAC [31
]. The ideal predictor is a non-invasive clinically feasible tool during patient treatment. In this study, BRCA 1
was a predictor of the response to FOLFIRINOX. However, the proportion of BRCA
cases was very low in patients with PDAC. Several clinical trials are currently ongoing to identify better blood germline biomarkers (ClinicalTrials.gov NCT04289961; NCT04143152).
Despite the efficacy of BRCA
on treatment response in patients, the present study did not show survival benefits in patients who underwent FOLFIRINOX. Regardless of how good a prognostic or therapeutic predictive marker may be, it cannot outperform clinical parameters, such as cancer stage, age, sex, and metastasis, on their prognosis. Germline mutations can be used to predict FOLFIRINOX treatment response; however, they are still limited in predicting patient prognosis. A previous study by Sehdev et al. and Golan et al. also showed a significant difference in the prognosis of BRCA-positive patients who received platinum-based chemotherapy [16
Our study has strengths. This is the first report of the ORR in numerous patients with BRCA
1/2 mutations following the use of FOLFIRINOX in Asia. In a previous study, less was known about the prevalence and treatment outcomes of FOLFIRINOX involving BRCA
1/2 mutations in Asia [14
]. The high ORR of 71.4% with FOLFIRINOX therapy in BRCA
-positive patients suggests that platinum therapy may be particularly desirable for this subset of patients in clinical scenarios marked by high disease burden and symptomatic disease, and for patients with PDAC. This study may help guide treatment decisions for patients with PDAC.
This study has several limitations. First, this is a retrospective study. Although we adjusted several factors via multivariate analysis, selection and/or information bias could remain. The lack of statistically significant differences in both OS and PFS in this study population may be attributed to the limited number of patients enrolled in the study: only 32 patients in our study were treated with palliative, first-line FOLFIRINOX. Second, although we found no significant difference in the proportion of males between groups, there were fewer males in the BRCA
-positive group (2/9, 22.2% vs. 22/55, 40.0%, p
= 0.464) [32
]. In this study, relatively young patients were enrolled compared to previous studies (median 57.5 years) [12
]. Both findings could plausibly skew bias toward the null hypothesis. Third, we were unable to control for mortality comorbidities that might have affected our results. However, since FOLFIRINOX is indicated for relatively healthier PDAC patients with good performance status, we do not think that the difference in comorbidities is the only explanation for our results.