Roma Cristin, Sacco Alessandra, Forgione Laura, Esposito Abate Riziero, Lambiase Matilde, Dotolo Serena, Maiello Monica Rosaria, Frezzetti Daniela, Nasti Guglielmo, Morabito Alessandro, De Luca Antonella, Normanno Nicola
Cell Biology and Biotherapy Unit, Istituto Nazionale Tumori-IRCCS-Fondazione G. Pascale, 80131 Naples, Italy.
SSD Innovative Therapies for Abdominal Metastases, Istituto Nazionale Tumori-IRCCS-Fondazione G. Pascale, 80131 Naples, Italy.
Diagnostics (Basel). 2022 Aug 12;12(8):1956. doi: 10.3390/diagnostics12081956.
Targeted sequencing of circulating cell-free DNA (cfDNA) is used in routine clinical diagnostics for the identification of predictive biomarkers in cancer patients in an advanced stage. The presence of KRAS mutations associated with clonal hematopoiesis of indeterminate potential (CHIP) might represent a confounding factor. We used an amplicon-based targeted sequencing panel, covering selected regions of 52 genes, for circulating cell-free total nucleic acid (cfTNA) analysis of 495 plasma samples from cancer patients. The cfDNA test failed in 4 cases, while circulating cell-free RNA (cfRNA) sequencing was invalid in 48 cases. In the 491 samples successfully tested on cfDNA, at least one genomic alteration was found in 222 cases (45.21%). We identified 316 single nucleotide variants (SNVs) in 21 genes. The most frequently mutated gene was TP53 (74 variants), followed by KRAS (71), EGFR (56), PIK3CA (33) and BRAF (19). Copy number variations (CNVs) were detected in 36 cases, while sequencing of cfRNA revealed 6 alterations. Analysis with droplet digital PCR (ddPCR) of peripheral blood leukocyte (PBL)-derived genomic DNA did not identify any KRAS mutations in 39 cases that showed KRAS mutations at cfDNA analysis. These findings suggest that the incidence of CHIP-associated KRAS mutations is relatively rare in routine clinical diagnostics.
循环游离DNA（cfDNA）的靶向测序用于常规临床诊断，以识别晚期癌症患者的预测性生物标志物。与不确定潜能的克隆性造血（CHIP）相关的KRAS突变的存在可能是一个混杂因素。我们使用了一个基于扩增子的靶向测序面板，覆盖52个基因的选定区域，对495例癌症患者的血浆样本进行循环游离总核酸（cfTNA）分析。4例cfDNA检测失败，48例循环游离RNA（cfRNA）测序无效。在491例成功进行cfDNA检测的样本中，222例（45.21%）发现至少一种基因组改变。我们在21个基因中鉴定出316个单核苷酸变异（SNV）。突变最频繁的基因是TP53（74个变异），其次是KRAS（71个）、EGFR（56个）、PIK3CA（33个）和BRAF（19个）。36例检测到拷贝数变异（CNV），而cfRNA测序显示6处改变。对39例在cfDNA分析中显示KRAS突变的外周血白细胞（PBL）来源的基因组DNA进行液滴数字PCR（ddPCR）分析，未发现任何KRAS突变。这些发现表明，在常规临床诊断中，与CHIP相关的KRAS突变的发生率相对较低。
