Pasquale Raffaella, Forgione Laura, Roma Cristin, Fenizia Francesca, Bergantino Francesca, Rachiglio Anna M, De Luca Antonella, Gallo Marianna, Maiello Monica R, Palumbo Giuliano, Morabito Alessandro, Azzaro Rosa, Normanno Nicola
Cell Biology and Biotherapy, Istituto Nazionale Tumori "Fondazione G. Pascale" - IRCCS, Via M. Semmola, Naples, Italy.
Medical Oncology, Ospedale Santa Maria della Pietà, Casoria, Italy.
Transl Lung Cancer Res. 2020 Feb;9(1):61-70. doi: 10.21037/tlcr.2020.01.01.
Sequencing artifacts, clonal hematopoietic mutations of indeterminate potential (CHIP) and tumor heterogeneity have been hypothesized to contribute to the low concordance between tissue and cell-free DNA (cfDNA) molecular profiling with targeted sequencing.
We analyzed by targeted sequencing cfDNA from 30 healthy individuals, and cfDNA and matched tumor samples from 30 EGFR-mutant and 77 EGFR wild-type metastatic non-small-cell lung cancer (mNSCLC) patients. Discordant cases were solved by droplet digital PCR (ddPCR).
By testing cfDNA from healthy donors, we developed an algorithm to recognize sequencing artifacts. Applying this method to cfDNA from mNSCLC patients, EGFR mutations were detected with a good sensitivity (76.7%) and specificity (97.4%). In contrast, sensitivity and specificity for KRAS variants were 61.5% and 93.8%, respectively. All EGFR and KRAS variants detected in plasma but not in tissue were confirmed by ddPCR, thus excluding sequencing artifacts. In a fraction of cases, KRAS mutations found in plasma samples were confirmed in tumor tissue suggesting tumor heterogeneity. KRAS variants were found to be more likely sub-clonal as compared with EGFR mutations, and a correlation between clonal origin and frequency of detection in plasma was found. In a case with both EGFR and KRAS variants in cfDNA, we could demonstrate the presence of the KRAS variant in tumor tissue associated with lack of response to tyrosine kinase inhibitors (TKIs).
Although sequencing artifacts can be identified in targeted sequencing of cfDNA, tumor heterogeneity and CHIP are likely to influence the concordance between plasma and tissue testing.
测序假象、潜在不确定性的克隆性造血突变（CHIP）和肿瘤异质性被认为是导致组织与游离DNA（cfDNA）分子谱靶向测序之间一致性较低的原因。
我们对30名健康个体的cfDNA以及30例表皮生长因子受体（EGFR）突变和77例EGFR野生型转移性非小细胞肺癌（mNSCLC）患者的cfDNA及匹配的肿瘤样本进行了靶向测序分析。通过液滴数字PCR（ddPCR）解决不一致的情况。
通过检测健康供体的cfDNA，我们开发了一种识别测序假象的算法。将该方法应用于mNSCLC患者的cfDNA，EGFR突变检测具有良好的敏感性（76.7%）和特异性（97.4%）。相比之下，KRAS变异的敏感性和特异性分别为61.5%和93.8%。血浆中检测到但组织中未检测到的所有EGFR和KRAS变异均通过ddPCR得到证实，从而排除了测序假象。在部分病例中，血浆样本中发现的KRAS突变在肿瘤组织中得到证实，提示存在肿瘤异质性。发现KRAS变异比EGFR突变更可能是亚克隆性的，并且发现克隆起源与血浆中检测频率之间存在相关性。在一例cfDNA中同时存在EGFR和KRAS变异的病例中，我们证实肿瘤组织中存在KRAS变异与对酪氨酸激酶抑制剂（TKIs）无反应相关。
虽然在cfDNA靶向测序中可以识别测序假象，但肿瘤异质性和CHIP可能会影响血浆和组织检测之间的一致性。
