Changes in Colorectal Carcinoma Genomes under Anti-EGFR Therapy Identified by Whole-Genome Plasma DNA Sequencing

Abstract

Monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR), such as cetuximab and panitumumab, have evolved to important therapeutic options in metastatic colorectal cancer (CRC). However, almost all patients with clinical response to anti-EGFR therapies show disease progression within a few months and little is known about mechanism and timing of resistance evolution. Here we analyzed plasma DNA from ten patients treated with anti-EGFR therapy by whole genome sequencing (plasma-Seq) and ultra-sensitive deep sequencing of genes associated with resistance to anti-EGFR treatment such as KRAS, BRAF, PIK3CA, and EGFR. Surprisingly, we observed that the development of resistance to anti-EGFR therapies was associated with acquired gains of KRAS in four patients (40%), which occurred either as novel focal amplifications ( n = 3) or as high level polysomy of 12p ( n = 1). In addition, we observed focal amplifications of other genes recently shown to be involved in acquired resistance to anti-EGFR therapies, such as MET ( n = 2) and ERBB2 ( n = 1). Overrepresentation of the EGFR gene was associated with a good initial anti-EGFR efficacy. Overall, we identified predictive biomarkers associated with anti-EGFR efficacy in seven patients (70%), which correlated well with treatment response. In contrast, ultra-sensitive deep sequencing of KRAS, BRAF, PIK3CA, and EGFR did not reveal the occurrence of novel, acquired mutations. Thus, plasma-Seq enables the identification of novel mutant clones and may therefore facilitate early adjustments of therapies that may delay or prevent disease progression.

Author Summary

Targeted therapies based on characteristics of the tumor genome are increasingly being offered to patients with cancer. For example, colorectal carcinomas that are wild type for KRAS are frequently treated with monoclonal antibodies targeting the Epidermal Growth Factor Receptor (EGFR). However, almost all patients with clinical response to anti-EGFR therapies develop resistance and underlying mechanisms are poorly understood. Because of the instability of tumor genomes the status of predictive biomarkers, such as the KRAS gene, can change during the course of disease. So-called “liquid biopsies”, e.g. analyses of circulating tumor DNA, provide genetic follow-up data non-invasively from peripheral blood. When using whole genome sequencing of plasma DNA (plasma-Seq) we observed that specific copy number changes of genes, such as KRAS, MET, or ERBB2, can be acquired under therapy and determine responsiveness to therapy. In fact, our data suggest that non-invasive genome profiling is capable of predicting responsiveness or emerging resistance to anti-EGFR therapy in the majority of cases. Hence, non-invasive testing of the current status of the tumor genome can help reduce of harm from erroneous therapeutic decisions and optimize treatment for maximal efficacy and minimal side effects, which is important for decreasing metastasized CRC-related morbidity and mortality.