Development and Validation of a Targeted ‘Liquid’ NGS Panel for Treatment Customization in Patients with Metastatic Colorectal Cancer

Abstract

The detection of actionable mutations in tumor tissue is a prerequisite for treatment customization in patients with metastatic colorectal cancer (mCRC). Analysis of circulating tumor DNA (ctDNA) for the identification of such mutations in patients’ plasma is an attractive alternative to invasive tissue biopsies. Despite having the high analytical sensitivity required for ctDNA analysis, digital polymerase chain reaction (dPCR) technologies can only detect a very limited number of hotspot mutations, whilst a broader mutation panel is currently needed for clinical decision making. Recent advances in next-generation sequencing (NGS) have led to high-sensitivity platforms that allow screening of multiple genes at a single assay. Our goal was to develop a small, cost- and time-effective NGS gene panel that could be easily integrated in the day-to-day clinical routine in the management of patients with mCRC. We designed a targeted panel comprising hotspots in six clinically relevant genes (KRAS, NRAS, MET, BRAF, ERBB2 and EGFR) and validated it in a total of 68 samples from 30 patients at diagnosis, first and second disease progression. Results from our NGS panel were compared against plasma testing with BEAMing dPCR regarding the RAS gene status. The overall percent of agreement was 83.6%, with a positive and negative percent agreement of 74.3% and 96.2%, respectively. Further comparison of plasma NGS with standard tissue testing used in the clinic showed an overall percent agreement of 86.7% for RAS status, with a positive and negative percent agreement of 81.2% and 92.8%, respectively. Thus, our study strongly supports the validity and efficiency of an affordable targeted NGS panel for the detection of clinically relevant mutations in patients with mCRC.

Circulating Tumor DNA in the Management of Early-Stage Breast Cancer

“…It should be noted that the BEAMing assay has been commercialized and it can only be performed by trained personnel in accredited centers. Based on our experience [ 38 ], it is a rather costly technique that involves a complicated and quite laborious protocol, which is probably hard to implement in everyday clinical practice.…”

“…It should be noted that the BEAMing assay has been commercial-ized and it can only be performed by trained personnel in accredited centers. Based on our experience [38], it is a rather costly technique that involves a complicated and quite laborious protocol, which is probably hard to implement in everyday clinical practice. Digital PCR (dPCR) is an exceptionally powerful technique that allows for accurate quantification of rare mutations as it was first demonstrated by Kinzler and Vogelstein who coined the term [32].…”

Genomic and Transcriptomic Research in the Discovery and Application of Colorectal Cancer Circulating Markers

“…The rate of concordance of these two platforms amounted to more than 98% in the ctDNA from mCRC patients [31]. Kastrisiou et al [35] developed an original cost-effective NGS gene panel based on the hotspots in six genes: KRAS, NRAS, MET, BRAF, ERBB2, and EGFR. Validation of the panel was made using 68 blood plasma samples from 30 mCRC patients with a diagnosis of the first and second disease progression stages.…”

Special Issue: Next-Generation Sequencing in Tumor Diagnosis and Treatment II

“…Liquid biopsy is a useful tool not only for the management of NSCLC. Kastrisiou and colleagues had developed a targeted, cost-effective NGS gene panel that could be easily integrated in the day-to-day clinical routine starting from the plasma of patients with metastatic colorectal carcinomas (mCRC) [ 15 ]. The panel allows for the analysis of the hotspots in six clinically mCRC-relevant genes ( KRAS , NRAS , MET , BRAF , ERBB2 , and EGFR ) with a negative and positive agreement of RAS Testing in Tissue and Plasma specimens of 92.8% and 81.2%, respectively [ 15 ].…”

“…As reported above, NGS multi-gene panel may be useful also to better characterize clinically interesting cases. De Falco and colleagues described two patients with CRC at different stages (pT2N0M0 and pT4cN1cM1), but both of them harbored double concurrent KRAS pathogenic mutations (p.G12D and p.G13D) that are normally mutually exclusive [ 15 ]. Moreover, using an NGS panel, the authors observed that the two tumors also harbored other mutations in PIK3CA , SMAD4 , NOTCH1 , ERBB2 , and EGFR genes, but all of them were different in the two tumors [ 15 ].…”