Background and Objective

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the occurrence of pathogenic variants in BRCA1/2 in 5–6% of patients. Biallelic loss of BRCA1/2 enriches for response to platinum agents and poly (ADP-ribose) polymerase 1 inhibitors. There is a dearth of evidence on the mechanism of inactivation of the wild-type BRCA1 allele in PDAC tumors with a germline BRCA1 (gBRCA1) pathogenic or likely pathogenic variant (P/LPV). Herein, we examine promotor hypermethylation as a “second hit” mechanism in patients with gBRCA1-PDAC.

Methods

We evaluated patients with PDAC who underwent Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) somatic and germline testing from an institutional database. DNA isolated from tumor tissue and matched normal peripheral blood were sequenced by MSK-IMPACT. In patients with gBRCA1-PDAC, we examined the somatic BRCA1 mutation status and promotor methylation status of the tumor BRCA1 allele via a methylation array analysis. In patients with sufficient remaining DNA, a second methylation analysis by pyrosequencing was performed.

Results

Of 1012 patients with PDAC, 19 (1.9%) were identified to harbor a gBRCA1 P/LPV. Fifteen patients underwent a methylation array and the mean percentage of BRCA1 promotor methylation was 3.62%. In seven patients in whom sufficient DNA was available, subsequent pyrosequencing confirmed an unmethylated BRCA1 promotor. Loss of heterozygosity was detected in 12 of 19 (63%, 95% confidence interval 38–84) patients, demonstrating loss of heterozygosity is the major molecular mechanism of BRCA1 inactivation in PDAC. Two (10.5%) cases had a somatic BRCA1 mutation.

Conclusions

In patients with gBRCA1-P/LPV-PDAC, loss of heterozygosity is the main inactivating mechanism of the wild-type BRCA1 allele in the tumor, and methylation of the BRCA1 promoter is a distinctly uncommon occurrence.

中文翻译：

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甲基化分析揭示启动子高甲基化是胚系 BRCA1 相关胰腺导管腺癌“二次打击”的罕见原因

背景和目标

胰腺导管腺癌 (PDAC) 的特征是5-6% 的患者出现BRCA1/2致病性变异。BRCA1/2的双等位基因缺失增强了对铂类药物和聚（ADP-核糖）聚合酶 1 抑制剂的反应。在具有种系BRCA1 ( gBRCA1 ) 致病性或可能致病性变异 (P/LPV) 的PDAC 肿瘤中，野生型 BRCA1 等位基因失活的机制缺乏证据。在此，我们检查启动子高甲基化作为gBRCA1 -PDAC患者的“二次打击”机制。

方法

我们评估了患有 PDAC 的患者，这些患者接受了来自机构数据库的纪念斯隆凯特琳癌症靶点综合突变分析 (MSK-IMPACT) 体细胞和种系检测。通过 MSK-IMPACT 对从肿瘤组织和匹配的正常外周血中分离的 DNA 进行测序。在患有gBRCA1 -PDAC 的患者中，我们通过甲基化阵列分析检查了肿瘤BRCA1等位基因的体细胞BRCA1突变状态和启动子甲基化状态。在具有足够剩余 DNA 的患者中，通过焦磷酸测序进行第二次甲基化分析。

结果

在 1012 名 PDAC 患者中，有 19 名 (1.9%) 被发现携带gBRCA1 P/LPV。15 名患者接受了甲基化阵列， BRCA1启动子甲基化的平均百分比为 3.62%。在有足够 DNA 的 7 名患者中，随后的焦磷酸测序证实了未甲基化的BRCA1启动子。在 19 名患者中的 12 名（63%，95% 置信区间 38-84）中检测到杂合性缺失，表明杂合性缺失是PDAC 中BRCA1失活的主要分子机制。两个 (10.5%) 病例有体细胞BRCA1突变。

结论

在gBRCA1- P/LPV-PDAC患者中，杂合性缺失是肿瘤中野生型BRCA1等位基因的主要失活机制，而BRCA1启动子的甲基化非常罕见。