用于区分各种肝腺癌的 DNA 甲基化生物标志物面板，包括肝细胞癌、胆管癌、结直肠癌肝转移和胰腺腺癌肝转移。
DNA methylation biomarker panels for differentiating various liver adenocarcinomas, including hepatocellular carcinoma, cholangiocarcinoma, colorectal liver metastases and pancreatic adenocarcinoma liver metastases.
机构信息
Faculty of Medicine, Institute of Pathology, University of Ljubljana, Ljubljana, Slovenia.
出版信息
Clin Epigenetics. 2024 Nov 4;16(1):153. doi: 10.1186/s13148-024-01766-z.
BACKGROUND
DNA methylation biomarkers are one of the most promising tools for the diagnosis and differentiation of adenocarcinomas of the liver, which are among the most common malignancies worldwide. Their differentiation is important because of the different prognoses and treatment options. This study aimed to validate previously identified DNA methylation biomarkers that successfully differentiate between liver adenocarcinomas, including the two most common primary liver cancers, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), as well as two common metastatic liver cancers, colorectal liver metastases (CRLM) and pancreatic ductal adenocarcinoma liver metastases (PCLM), and translate them to the methylation-sensitive high-resolution melting (MS-HRM) and digital PCR (dPCR) platforms.
METHODS
Our study included a cohort of 149 formalin-fixed, paraffin-embedded tissue samples, including 19 CRLMs, 10 PCLMs, 15 HCCs, 15 CCAs, 15 colorectal adenocarcinomas (CRCs), 15 pancreatic ductal adenocarcinomas (PDACs) and their paired normal tissue samples. The methylation status of the samples was experimentally determined by MS-HRM and methylation-specific dPCR. Previously determined methylation threshold were adjusted according to dPCR data and applied to the same DNA methylation array datasets (provided by The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO)) used to originally identify the biomarkers for the included cancer types and additional CRLM projects. The sensitivities, specificities and diagnostic accuracies of the panels for individual cancer types were calculated.
RESULTS
In the dPCR experiment, the DNA methylation panels identified HCC, CCA, CRC, PDAC, CRLM and PCLM with sensitivities of 100%, 66.7%, 100%, 86.7%, 94.7% and 80%, respectively. The panels differentiate between HCC, CCA, CRLM, PCLM and healthy liver tissue with specificities of 100%, 100%, 97.1% and 94.9% and with diagnostic accuracies of 100%, 94%, 97% and 93%, respectively. Reevaluation of the same bioinformatic data with new additional CRLM projects demonstrated that the lower dPCR methylation threshold still effectively differentiates between the included cancer types. The bioinformatic data achieved sensitivities for HCC, CCA, CRC, PDAC, CRLM and PCLM of 88%, 64%, 97.4%, 75.5%, 80% and 84.6%, respectively. Specificities between HCC, CCA, CRLM, PCLM and healthy liver tissue were 98%, 93%, 86.6% and 98.2% and the diagnostic accuracies were 94%, 91%, 86% and 98%, respectively. Moreover, we confirmed that the methylation of the investigated promoters is preserved from primary CRC and PDAC to their liver metastases.
CONCLUSIONS
The cancer-specific methylation biomarker panels exhibit high sensitivities, specificities and diagnostic accuracies and enable differentiation between primary and metastatic adenocarcinomas of the liver using methylation-specific dPCR. High concordance was achieved between MS-HRM, dPCR and bioinformatic data, demonstrating the successful translation of bioinformatically identified methylation biomarkers from the Illumina Infinium HumanMethylation450 BeadChip (HM450) and lllumina MethylationEPIC BeadChip (EPIC) platforms to the simpler MS-HRM and dPCR platforms.
背景
DNA 甲基化生物标志物是诊断和区分肝腺癌的最有前途的工具之一，肝腺癌是全球最常见的恶性肿瘤之一。它们的区分很重要，因为它们的预后和治疗选择不同。本研究旨在验证先前确定的 DNA 甲基化生物标志物，这些生物标志物可成功区分肝腺癌，包括两种最常见的原发性肝癌，肝细胞癌（HCC）和胆管癌（CCA），以及两种常见的转移性肝癌，结直肠癌肝转移（CRLM）和胰腺导管腺癌肝转移（PCLM），并将其转化为甲基化敏感的高分辨率熔解（MS-HRM）和数字 PCR（dPCR）平台。
方法
我们的研究包括 149 例福尔马林固定、石蜡包埋的组织样本，包括 19 例 CRLM、10 例 PCLM、15 例 HCC、15 例 CCA、15 例结直肠腺癌（CRC）、15 例胰腺导管腺癌（PDAC）及其配对的正常组织样本。通过 MS-HRM 和甲基化特异性 dPCR 实验确定样本的甲基化状态。根据 dPCR 数据调整先前确定的甲基化阈值，并应用于最初用于确定包括癌症类型和其他 CRLM 项目的生物标志物的相同 DNA 甲基化阵列数据集（由癌症基因组图谱（TCGA）和基因表达综合数据库（GEO）提供）。计算了各癌症类型的面板的灵敏度、特异性和诊断准确性。
结果
在 dPCR 实验中，DNA 甲基化面板分别以 100%、66.7%、100%、86.7%、94.7%和 80%的灵敏度识别 HCC、CCA、CRC、PDAC、CRLM 和 PCLM。该面板以 100%、100%、97.1%和 94.9%的特异性区分 HCC、CCA、CRLM、PCLM 和健康肝组织，以 100%、94%、97%和 93%的诊断准确性区分。用新的附加 CRLM 项目重新评估相同的生物信息学数据表明，较低的 dPCR 甲基化阈值仍然可以有效地区分所包括的癌症类型。生物信息学数据对 HCC、CCA、CRC、PDAC、CRLM 和 PCLM 的灵敏度分别为 88%、64%、97.4%、75.5%、80%和 84.6%。HCC、CCA、CRLM、PCLM 和健康肝组织之间的特异性分别为 98%、93%、86.6%和 98.2%，诊断准确率分别为 94%、91%、86%和 98%。此外，我们证实从原发性 CRC 和 PDAC 到其肝转移，所研究的启动子的甲基化是被保留的。
结论
癌症特异性甲基化生物标志物面板具有较高的灵敏度、特异性和诊断准确性，可使用甲基化特异性 dPCR 区分原发性和转移性肝腺癌。MS-HRM、dPCR 和生物信息学数据之间具有高度一致性，证明了从 Illumina Infinium HumanMethylation450 BeadChip（HM450）和 Illumina MethylationEPIC BeadChip（EPIC）平台生物信息学确定的甲基化生物标志物成功转化为更简单的 MS-HRM 和 dPCR 平台。
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