Biological heterogeneity is a key feature of malignancies that significantly contributes to disease progression and therapy resistance. Residual/relapsed tumor foci may represent genetically divergent subclones, which remain uncovered as repeated and multiple tumor sampling is usually limited. The analysis of circulating free DNA (cfDNA) from the peripheral blood plasma (also called a liquid biopsy, LB) is a new achievement that provides an effective tool for follow-up monitoring of cancer-related genetic status. The present study highlights the phenomenon of mutational variability observed in patients with metastatic KRAS mutant colorectal cancer (mCRC) during treatment with bevacizumab in combination in a longitudinal fashion.

The prospective study included 490 mCRC patients evaluated between 2020 and 2022 in our institution. Out of the 211 KRAS mutant cases (43.06%) 12 tumors were identified with multiple KRAS gene variants (5.68%). Detailed follow-up investigations were possible in 3 of these patients including the genotyping of the primary and available metastatic tumors, and the peripheral blood cfDNA. cfDNA was collected from three different time points before and between cycles of combined treatment with bevacizumab chemotherapy. KRAS gene variants were identified using reverse-hybridization strips, and next-generation sequencing (NGS), and confirmed by conventional Sanger sequencing.

Interestingly, surgery and multiple treatment cycles reorganized the mutational profiles in the selected cases. The effect of the treatments resulted either in the overrepresentation of one of the pre-existing gene variants or in the appearance of new KRAS variants absent in the primary sample, according to the plasma cfDNA findings. Besides the KRAS variants demonstrated by targeted analysis, NGS mutational profiling identified some additional pathogenic variants from the cfDNA samples (including NRAS and MET alterations).

In conclusion, plasma cfDNA sampling enables the monitoring of mutational heterogeneity and subclonal dynamics of the actual metastatic tumor mass in mCRC. The pattern of molecular profile potentially reflects a differential drug response determining further progression.

生物异质性是恶性肿瘤的一个关键特征，它会显着促进疾病进展和治疗耐药性。残留/复发的肿瘤病灶可能代表遗传上不同的亚克隆，由于重复和多次肿瘤取样通常是有限的，因此仍未被发现。外周血浆中循环游离 DNA (cfDNA) 的分析（也称为液体活检，LB）是一项新成果，为癌症相关遗传状态的后续监测提供了有效工具。本研究突出了在转移性KRAS突变结直肠癌 (mCRC) 患者中观察到的突变变异现象，该患者在纵向联合使用贝伐珠单抗治疗期间。

前瞻性研究包括 490 名 mCRC 患者，这些患者在 2020 年至 2022 年间在我们机构接受了评估。在 211 个KRAS突变病例 (43.06%) 中，12 个肿瘤被鉴定为具有多个KRAS基因变异 (5.68%)。对其中 3 名患者进行了详细的随访调查，包括原发性和可用转移性肿瘤的基因分型，以及外周血 cfDNA。在贝伐单抗化疗联合治疗周期之前和之间的三个不同时间点收集 cfDNA。KRAS基因变异使用反向杂交条带和下一代测序 (NGS) 进行鉴定，并通过传统的 Sanger 测序进行确认。

有趣的是，手术和多个治疗周期重组了所选病例的突变谱。根据血浆 cfDNA 的发现，这些处理的效果要么导致一种预先存在的基因变异的过度表达，要么导致原始样本中不存在的新KRAS变异的出现。除了通过靶向分析证明的KRAS变异外，NGS 突变分析还从 cfDNA 样本中识别出一些额外的致病变异（包括NRAS和MET变异）。

总之，血浆 cfDNA 采样能够监测 mCRC 中实际转移性肿瘤块的突变异质性和亚克隆动力学。分子谱的模式可能反映了决定进一步进展的不同药物反应。