Department of Gastroenterology and Human Nutrition Unit, All India Institute of Medical Sciences, New Delhi, 110029, India.
Department of Gastrointestinal Surgery, All India Institute of Medical Sciences, New Delhi, India.
J Cancer Res Clin Oncol. 2020 Apr;146(4):897-907. doi: 10.1007/s00432-020-03169-y. Epub 2020 Mar 7.
Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive cancer. There are various sub-cellular events (both genetic and epigenetic) that get dysregulated leading to tumorigenesis. Methylation in promoters of tumor suppressor genes is one of these epigenetic phenomena contributing to the pathogenesis of cancer. Genes analyzed for promoter methylation status in this study namely SPARC (Secreted Protein Acidic and Rich in Cysteine, UCHL1 (ubiquitin carboxy-terminal hydrolase L1), NPTX2 (neuronal pentraxin 2), PENK (proenkephalin) had been studied in pancreatic cancer, but there is a need to check methylation in these genes as circulatory non-invasive markers. This study analyzed the absolute quantification of methylation levels of SPARC, UCHL1, PENK, and NPTX2 genes promoters in PDAC patients as well as in chronic pancreatitis (CP) patients and healthy subjects (HC) and evaluated its clinical significance in PDAC.
The study included 65 PDAC patients, 25 CP patients, and 25 healthy controls. DNA was extracted from their plasma samples and subsequently given bisulfite treatment. Absolute quantization of methylated and unmethylated copies of gene promoters of all the four genes was performed using real-time PCR (SYBR green) by the standard curve method. Methylation levels were expressed as methylation index (MI) for each gene in each patient. MI was calculated from absolute copy numbers as follows: MI-methylated copy number/methylated copy number + unmethylated copy number). These indices were used to compare gene methylation levels within different groups and to correlate with clinicopathological features and survival of pancreatic cancer patients. An appropriate statistical analysis was applied.
Methylation indices for all the four genes in PDAC cases were found to be significantly higher as compared to that in healthy individuals. SPARC MI values were found to differentiate early-stage PDAC patients from CP patients. PDAC patients with the metastasized disease and stage IV disease were found to have high MI for the SPARC gene as well as for the NPTX2 gene, while a higher UCHL1 methylation index was found to correlate with an advanced stage of the disease. Higher MI values for SPARC and NPTX2 genes were found to associate with poor survival in patients with PDAC.
Methylation load in the form of MI for each of the four genes assessed in plasma may emerge as a non-invasive biomarker to differentiate pancreatic cancer from healthy individuals. But only SPARC and NPTX2 hypermethylation were able to distinguish pancreatic cancer from chronic pancreatitis. Association of aberrant methylation in SPARC and NPTX2 gene with metastasis and poor survival of patients suggest the role of methylation in these genes as prognostic markers.
胰腺导管腺癌（PDAC）是一种非常侵袭性的癌症。有各种亚细胞事件（遗传和表观遗传）失调导致肿瘤发生。肿瘤抑制基因启动子的甲基化是导致癌症发病机制的表观遗传现象之一。本研究分析了 SPARC（富含半胱氨酸的酸性分泌蛋白，UCHL1（泛素羧基末端水解酶 L1）、NPTX2（神经元五肽 2）、PENK（前啡肽）启动子甲基化状态的绝对定量在 PDAC 患者以及慢性胰腺炎（CP）患者和健康受试者（HC）中，并评估了其在 PDAC 中的临床意义。
该研究纳入了 65 名 PDAC 患者、25 名 CP 患者和 25 名健康对照者。从他们的血浆样本中提取 DNA，然后进行亚硫酸氢盐处理。使用实时 PCR（SYBR 绿色）通过标准曲线法对所有四个基因的启动子的甲基化和未甲基化拷贝的绝对定量进行了分析。在每个患者中，甲基化水平表示为每个基因的甲基化指数（MI）。MI 是根据绝对拷贝数计算的，公式为：MI-甲基化拷贝数/甲基化拷贝数+未甲基化拷贝数）。这些指标用于比较不同组之间的基因甲基化水平，并与胰腺癌细胞的临床病理特征和生存相关。应用了适当的统计分析。
与健康个体相比，PDAC 病例中所有四个基因的 MI 值均明显升高。SPARC MI 值可区分早期 PDAC 患者与 CP 患者。转移性疾病和 IV 期疾病的 PDAC 患者发现 SPARC 基因和 NPTX2 基因的 MI 值较高，而 UCHL1 甲基化指数升高与疾病的晚期相关。SPARC 和 NPTX2 基因的较高 MI 值与 PDAC 患者的不良生存相关。
评估血浆中四种基因的 MI 值的甲基化负荷可能成为一种非侵入性生物标志物，用于将胰腺癌与健康个体区分开来。但只有 SPARC 和 NPTX2 的过度甲基化能够将胰腺癌与慢性胰腺炎区分开来。SPARC 和 NPTX2 基因的异常甲基化与转移和患者不良生存相关，表明这些基因的甲基化作为预后标志物的作用。
