Advantage of Next-Generation Sequencing in Dynamic Monitoring of Circulating Tumor DNA over Droplet Digital PCR in Cetuximab Treated Colorectal Cancer Patients

Abstract

Epidermal growth factor receptor (EGFR) blockade resistance is common in the treatment of RAS wide type colorectal cancer (CRC). During the treatment of cetuximab, acquired resistant genomic alterations always occurs earlier than disease progression observed by medical images. Identification of genomic alterations dynamically might have certain clinical significance. Because of the limitation of repeated tissue biopsy, liquid biopsy is increasingly recognized. Droplet digital polymerase chain reaction (ddPCR) is the main detection methods for circulating tumor DNA (ctDNA), however, the application of next-generation sequencing (NGS) for ctDNA detection becomes more and more popular. Here we develop a NGS-based ctDNA assay and evaluated its sensitivity and specificity while using ddPCR as control. These two technologies were both used for genomic alteration detection for the peripheral blood samples from cetuximab-treated colorectal cancer patients dynamically. Fifteen patients were enrolled in this study, including eight males and seven females. The sensitivity and specificity of our NGS assay were 87.5% and 100% respectively, and liner regression analysis comparing variant allele frequency (VAF) revealed high concordance between NGS and ddPCR (R² = 0.98). NGS actually found more mutation information than ddPCR such as the additional dynamic changes of TP53 which were observed in the disease progression patients. Moreover, the variant allele fraction of TP53 was also found by NGS to be changed along with the clinical efficacy evaluation dynamically during the whole treatment process. In conclusion, our newly developed NGS-based ctDNA assay shows similar performance with ddPCR but have more advantages of its high throughput of multigenetic detection for the dynamic monitoring during the treatment of cetuximab in metastasis CRC patients.

Figure 1

Comparison of the NGS and ddPCR ctDNA analysis. (A) Linear regression from the comparison of NGS and ddPCR VAFs (R² = 0.9867). (B) Bland–Altman plot of the differences (NGS (VAF) - ddPCR (VAF)).

Figure 2

Comparison of mutation profiles between baseline and progression. Columns with red (RAS), orange (ERBB2), golden (EGFR), green (PI3K pathway), blue (ALK, ROS1 and MET), pink (CDKN2A and RB1), purple (TP53) and gray (any genes) indicate the presence of gene alterations. Columns with white indicate wild type. PFS, progression-free survival (months). The number in each box indicates the number of mutations of each gene or pathways.

Figure 3

Dynamic changes of mutations and mutation abundance tested by NGS during treatment in four patients. During the treatment of cetuximab, the abundance of mutations detected in the ctDNA at the baseline showed a downward trend with the treatment process. The corresponding imaging to the blood collection time showed that new drug-resistant mutations were detected in blood in advance of imaging development.