Next-Generation Sequencing (NGS) allows high throughput mutation analysis of oncogenes and tumor suppressor genes by targeted resequencing. However, increased sensitivity for mutation detection due to the spatial heterogeneity of the solid tumor or temporal heterogeneity found within the circulating free DNA (cfDNA) may be needed to identify the key biomarkers that may guide the personalized treatment for patients in the era of precision medicine.

ICE COLD-PCR (ICP) technology is capable of simultaneous enrichment of mutations targeted by a ∼50 base reference oligonucleotide in a modified PCR reaction followed by standard Sanger sequencing. In this study, we sought to establish a high sensitivity and high throughput mutation detection platform using ICP combined with NGS. ICP products from low frequency mutation mixtures were enzymatically treated, ligated to the Ion Torrent adapters and analyzed by the Ion Torrent Personal Genome Machine (PGM) and the MiSeq NGS platforms. The ICP-NGS analysis of low-level mutations provided a virtual increase in depth of coverage ahead of NGS analysis that increased the sensitivity of NGS detection.

High sensitivity detection of cancer genetic biomarkers from cfDNA has the potential to facilitate cancer diagnosis and provide guidance for personalized cancer treatment by serving as a liquid biopsy when there is no available tumor tissue and as a process for monitoring treatment effectiveness and emergence of drug resistance. To explore the clinical utility of the ICP-NGS platform in detecting cancer mutations in cfDNA, 26 plasma DNA samples harvested from patients with carcinomas were analyzed simultaneously with NGS for PIK3CA exons 9 and 20, KRAS exon 2, and EGFR exons 18 and 19 with or without prior ICP enrichment for mutants. For the 15 variants (P<0.01 and freq>5%) detected by ICP-NGS, only 3 variants were observed in NGS without ICP; even so, the mutant frequency for the 3 variants was low (∼5-10%) without the prior ICP enrichment but was enriched to ∼80-90% after the ICP. More importantly, 12 of the 15 variants detected by ICP-NGS were only detectable in NGS analysis because of the prior ICP enrichment. These results support the application of ICP in front of NGS in high-sensitivity and high-throughput detection of mutants in cfDNA.

In conclusion, we have established a platform of mutant enrichment by ICE COLD-PCR followed by NGS analysis that is capable of high sensitivity and high throughput detection of cancer genetic biomarkers. ICP-NGS detected the 80% of mutations in cfDNA that were undetectable without ICP enrichment, suggesting that ICP-NGS has the potential to detect and monitor patients' genetic biomarker in real-time and provide guidance for the personalized cancer treatment.

Citation Format: Rui Lin, Sarah Cherubin, Courtney Cubrich, Grant Wu, Ben Legendre, Katherine Richardson. Mutant enrichment by ICE COLD-PCR prior to the next-generation sequencing enables high sensitivity and high throughput detection of cancer biomarkers in patient samples. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1506. doi:10.1158/1538-7445.AM2014-1506