Abstract
Background/Objectives:
Pancreatic cancer is a lethal disease with a poor 5-year survival rate. Pathogenic germline variants in the coding regions of ATM, BRCA1, and BRCA2 are found in up to 4.8% of pancreatic cancer patients. Germline promoter methylation and gene silencing arising from a germline variant or through other mechanisms have been described as a cause of tumor suppressor gene inactivation.
Methods:
We measured the level of promoter methylation of the ATM, BRCA1, and BRCA2 genes in peripheral blood lymphocytes from 655 patients with pancreatic cancer using real-time PCR.
Results:
No evidence of germline promoter methylation of any of these genes was found. Promoter methylation levels were minimal with no patient having promoter methylation greater than 3.4%, 3.3%, and 7.6% for ATM, BRCA1 and BRCA2, respectively, well below levels found in patients who have inherited promoter methylation (~50%).
Conclusions:
We found no evidence of germline promoter methylation of the pancreatic susceptibility genes ATM, BRCA1 and BRCA2 in patients with pancreatic cancer. This study reveals that constitutive germline methylation of promoter CpG islands is rare in pancreatic cancer.
Keywords: Inherited, Pancreas, Cancer, Methylation, CpG Island
Introduction
Approximately 1.5% of people will development pancreatic cancer by age 851. Pathogenic germline variants in pancreatic cancer susceptibility genes including ATM, BRCA1, BRCA2, CDKN2A, MLH1, MSH2, MSH6, PALB2, PMS2, PRSS1, STK11, and others, are associated with an increased risk of pancreatic cancer2, 3. Sadly, the prognosis for patients with pancreatic cancer is dismal, with a reported 5-year survival rate only around 9%4. However, understanding the genetic basis of inherited risk of pancreatic cancer can impact patient care. Specifically, individuals with a pathogenic germline variant in a pancreatic cancer susceptibility gene and a family history of pancreatic cancer are eligible for clinical surveillance and cancers may be detected early when outcomes improved5. Furthermore, patients with pancreatic cancer and a pathogenic germline variant in a pancreatic cancer susceptibility gene may have targeted therapies available to them. For example, patients with a pathogenic germline variant in BRCA1, BRCA2, and PALB2 may have a tumor susceptible to treatment with Poly(ADP-ribose) Polymerase (PARP) inhibitors6. Similarly, patients with pancreatic cancer and a pathogenic germline variant in a mismatch repair gene may have a tumor responsive to immune checkpoint inhibitors7.
To date, pathogenic germline variants in pancreatic cancer susceptibility genes have been identified in 5–10% of all patients with pancreatic cancer8, 9 and this number rises to 15–20% of patients with a first-degree relative with pancreatic cancer10. Heritability estimates for pancreatic cancer range from 21–36%11, 12. While common genetic variants also explain a portion of the heritability of pancreatic cancer (4.1%), rare variation not accounted for by the established susceptibility genes listed before may also play a role.
Hypermethylation of promoter regions in peripheral blood has previously been associated with an increased risk of breast cancer13. In particular, hypermethylation of ATM and BRCA1 promoters in peripheral blood is associated with a 1.9–3.2- and 1.7–3.5-fold increased risk of breast cancer respectively14–18. Recently, a germline variant in the 5’ untranslated region (UTR) of BRCA1 (NC_000017.11:g.43125358A>T; NM_007294.3:c.−107A>T) was reported to result in constitutive hypermethylation of the BRCA1 promoter CpG island in two families with breast and ovarian cancer. Specifically, the c.−107A>T germline variant in the BRCA1 5’ UTR resulted in 50% methylation of 10 CPGs in the BRCA1 core promoter, suggesting methylation of one allele. RNA sequencing confirmed loss of expression of the methylated allele. Importantly, the BRCA1 5’ UTR variant (c.−107A>T) segregated with hypermethylation and allele-specific expression19.
These studies highlight the intriguing possibility that a germline variant in a pancreatic cancer susceptibility gene could be associated with an increased risk of cancer as a result of constitutive methylation and silencing of one allele. However, the contribution of ATM and BRCA1 promoter CpG island methylation to inherited risk of pancreatic cancer is yet-to-be established. Similarly, the germline promoter methylation status of BRCA2, an integral component of double strand break repair, similar to BRCA1, with pathogenic germline variants frequently identified in patients with pancreatic cancer, is unknown.
In this study, we determined the prevalence of ATM, BRCA1, and BRCA2 promoter CpG island methylation and therefore, germline variants that result in constitutive methylation of these regions, in 655 patients with pancreatic cancer.
Methods
Study population and ethics statement
This study was approved by the Johns Hopkins University Institutional Review Board and informed consent was obtained from all patients. Patients with pancreatic cancer included in this study were enrolled in the National Familial Pancreatic Tumor Registry at the Johns Hopkins University. 655 patients were included in this study.
DNA extraction
Peripheral lymphocytes were obtained via blood samples collected at the time of clinic visit or registry enrollment. Peripheral blood lymphocytes were used for germline analysis as they were a readily available source of DNA with previous reports of constitutive promoter methylation19. DNA was extracted from peripheral blood lymphocytes using the PAXgene Blood DNA Kit (QIAGEN, catalog no. 761133). DNA concentration was determined using the Qubit dsDNA HS Assay Kit (Invitrogen, catalog no. Q32851) and quality was determined using the NanoDrop One (ThermoFisher Scientific, catalog no. ND-ONE). All patient DNA samples had a concentration >4 μg/mL, a A260/A230 ratio > 1.7, and a A260/A280 ratio > 1.8.
qPCR assay of promoter CpG island methylation
Promoter CpG island methylation was assayed using Qiagen q-PCR MethylScreen technology according to the manufacturer’s protocol. Briefly, DNA samples were digested using the EpiTect II DNA Methylation Enzyme Kit (Qiagen, MD, cat. No. 335452). Digested DNA samples were used as templates for qPCR using EpiTect Methyl II PCR Assay primers targeting promoter CpG islands of ATM (Qiagen, catalog no. EPHS102729–1A), BRCA1 (Qiagen, catalog no. EPHS115453–1A), and BRCA2 (Qiagen, catalog no. EPHS103696–1A). DNA samples were assayed with control primers EP_SEC (Qiagen, catalog no. EPHS115450–1A) and EP_DEC (Qiagen, catalog no. EPHS115451–1A) to ensure efficacy of methylation-specific DNA digestion. qPCR was performed on a StepOnePlus Real-Time PCR System (ThermoFisher Scientific, catalog no. 4376600). Percent methylated DNA for each assayed promoter CpG island was calculated using CT values with the Qiagen data analysis template (Qiagen). A level of methylation below <25% was considered to indicate that neither allele was methylated at levels suggestive of constitutive methylation. A methylated percent of between 25 and 75% was considered to indicate that one allele was uniformly methylated. A methylated percent >75% was considered to indicate that both alleles were methylated.
Statistical analysis
Confidence intervals were calculated with STATA v13 (StataCorp, TX) using a binomial distribution.
Results
655 patients with pancreatic cancer were included in this study (Table 1). In total, 357 patients (54.5%) were male and 298 patients (45.5% female). 113 patients (17.2%) met the criteria for familial pancreatic cancer, that is, patients were part of a family in which at least two affected first-degree relatives had pancreatic cancer.
Table 1.
Demographic information of patients with pancreatic cancer included in study.
| Characteristic | Number of patients | Percent of patients (%) |
|---|---|---|
| Gender | ||
| Male | 357 | 54.5 |
| Female | 298 | 45.5 |
| Age at diagnosis | ||
| ≤40 | 18 | 2.7 |
| 41–50 | 48 | 7.3 |
| 51–60 | 153 | 23.4 |
| 61–70 | 254 | 38.8 |
| 71–80 | 144 | 22.0 |
| ≥81 | 34 | 5.2 |
| Unknown | 4 | 0.6 |
| Type of pancreatic cancer | ||
| Familial | 113 | 17.2 |
| Sporadic | 539 | 82.3 |
| Not reported | 3 | 0.5 |
As variants that result in promoter CpG island methylation may be associated with an increased risk pancreatic cancer, we determined the methylation status of the promoter CpG islands of ATM (chr11:108093211–108093969), BRCA1 (chr17:41277192–41277557), and BRCA2 (chr13:32889533–32889900) in 655 patients to establish the prevalence of germline hypermethylation of these regions in patients with pancreatic cancer. Mean percent methylated DNA of the ATM, BRCA1, and BRCA2 CpG islands was 0.1% (range: 0.0–3.4%), 0.3% (range: 0.0–3.3%), and 0.1% (range: 0.0–7.6%), respectively (Figure 1). No patient had percent methylation of the ATM, BRCA1, and BRCA2 CpG islands greater than 3.4%, 3.3%, and 7.6% respectively, indicating that both alleles of all three genes were unmethylated. The likely prevalence of methylation of these promoter CpG islands, and germline variants that result in constitutive methylation of these promoter CpG islands in patients with pancreatic cancer, is 0 (97.5% one sided confidence interval: 0–0.005).
Figure 1. Methylation of ATM, BRCA1 and BRCA2 promoter CpG islands in patients with pancreatic cancer.
Percent methylation of ATM, BRCA1 and BRCA2 promoter CpG islands for 655 patients with pancreatic cancer. No patient had promoter methylation greater than 3.4%, 3.3%, and 7.6% respectively, indicating that no patient had inherited variants that promote constitutive methylation of these regions.
Discussion
Understanding the genetic basis of pancreatic cancer risk is important to identify patients that could be treated with targeted therapies such as PARP inhibitors or immune checkpoint inhibitors, as well as, to identify relatives that may benefit from clinical surveillance5–7. Given the limited treatments available for pancreatic cancer, identifying patients who will benefit from these targeted treatments is critical to improving the survival of this deadly cancer. Germline genetic testing focuses on detecting alterations in the coding sequence that impact protein function. However, constitutive methylation of promoter CpG islands can also increase cancer risk through gene silencing. Furthermore, previous studies have demonstrated that somatic methylation of BRCA1 and BRCA2 occurs frequently in breast cancers and ovarian cancers20–22. Germline methylation has also been shown to play a role in breast cancer risk and may be impacted by the underlying DNA sequence as was recently described for a germline variant in the 5’ untranslated region of BRCA1 in families with hereditary breast and ovarian cancer syndrome19. To understand the contribution of such variants to inherited risk of pancreatic cancer, we determined the prevalence of promoter CpG island methylation of ATM, BRCA1, and BRCA2 in 655 patients with pancreatic cancer. We did not identify any patients with methylated promoter CpG islands, indicating that genetic variants that result in constitutive changes in methylation of ATM, BRCA1, or BRCA2 promoter CpG islands are a rare cause of pancreatic cancer.
In our study, we determined methylation of three defined regions of DNA encompassing promoter CpG islands, including the region of the BRCA1 promoter CpG island previously associated with risk of hereditary breast and ovarian cancer19, using a q-PCR method. However, it is possible that additional DNA regions in ATM, BRCA1, and BRCA2, or in genes that were not assayed, are associated with risk of pancreatic cancer. Unbiased approaches such as whole genome bisulfide sequencing, or targeted sequencing of DNA loci hypermutated somatically or in cell free DNA in patients with PDAC, may identify methylated DNA associated with risk of pancreatic cancer. Furthermore, other epigenetic modifications not assessed in our study, for example histone acetylation, may affect expression of pancreatic cancer susceptibility genes and be associated with risk of pancreatic cancer.
In conclusion, we determined methylation of promoter CpG islands of ATM, BRCA1, and BRCA2 in 655 patients with pancreatic cancer and determined that the prevalence of germline methylation of these regions in patients with pancreatic cancer is less than 0.5%.
Acknowledgements
Funding
The Sol Goldman Pancreatic cancer Research Center; Susan Wojcicki and Denis Troper; Rolfe Pancreatic Cancer Foundation; NIH/NCI P50 CA62924; Joseph C Monastra Foundation; The Gerald O Mann Charitable Foundation (Harriet and Allan Wulfstat, Trustees); Art Creates Cures Foundation.
Footnotes
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Conflict of Interest
R.H.H has the right to receive royalty payments from Thrive Earlier Diagnosis for the GNAS in pancreatic cysts invention. The authors declare no other conflicts of interest.
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