Abstract A29: Mutation profiling of colorectal cancer ctDNA using AmpliSeq CHP2 cancer panel

Introduction: Analysis of ctDNA has potential applications in targeted therapy selection and disease monitoring in the clinical management of colorectal cancer. Here we use an amplicon based target enrichment method, AmpliSeq v2 (CHP2), to study a set of tumor/plasma pairs (n = 13) collected from stage IV colon cancer patients to determine whether amplicon based NGS can be used to profile mutations in ctDNA from CRC patients. Methods: The CHP2 is designed to survey 2800 mutations in 50 cancer-related genes. Matched tumor and plasma samples were purchased from Indivumed. DNA was extracted from 3 or 5 ml plasma using QIAamp DSP circulating NA kit and eluted in 10 μl buffer per ml plasma. The sample input used in the CHP2 reactions was between 3.8 - 55 ng ctDNA. The background noise level of hotspot nt positions was calculated from 10 healthy donor plasma samples. A custom data analysis program was developed to detect variants clearly different from background noise. ddPCR mutation assays were purchased from BioRad and performed using the QX200. ddPCR data was analyzed using QuantaSoft software. Results: The ctDNA samples can be divided into two groups based on the DNA concentration measured by Qubit (range of 0.16-13 ng/ul): 6 with >1 ng/ul and 7 with KRAS, NRAS, PIK3CA, TP53, BRAF, APC, SMAD4 and FBXW7 . Eleven out of 13 ctDNA samples showed expected somatic mutations found in the matched tumors. Twenty eight out of 35 somatic mutations including 8 truncating mutations, mostly in APC and a 3bp insertion in SMAD4 , were detected in 13 tumor/plasma pairs. ctDNA mutations can be grouped into 3 levels: allele frequencies >10% (N = 7), NRAS Q60L and PIK3CA E545G) were detected in two ctDNA samples, while mutations in other genes TP53, APC and GNAQ were found only in the matched tumors, probably due to differential roles of these genes in cancer progression and metastasis. The sequencing read depth of these mutations ranged from 1528-17364. Four mutations with low allele frequency between 0.06-0.2% were reproducibly detected in an independent run. The background error rate at these nt positions was zero. The somatic mutations with >10% allele frequency were detected in 3 ctDNA samples (ctDNA concentration > 1ng/ul) that have co-existing mutations in KRAS and PIK3CA and/or APC gene, suggesting polyclonal evolution in multiple oncogenic pathways. Several Germline SNPs were detected in multiple genes and excluded from concordance calculation between tumor and plasma. A small subset of ctDNA samples, including 1 negative and 2 positive samples ( KRAS G12D 1.4% and PIK3CA E545G 0.9%) was also analyzed by ddPCR and results were consistent with those obtained using CHP2. Conclusions: AmpliSeq CHP2 cancer panel achieved 0.06-0.2% allele frequency sensitivity and 85% (11/13) tumor/plasma concordance, which are comparable with previous reports using alternative panels. We also demonstrate the efficiency of target enrichment by AmpliSeq based multiplex PCR, which can detect as low as two mutant copies from wild type background. This high sensitivity method is ideal for clinical sample testing where the amount of plasma-derived DNA is limited. Citation Format: WeiHua Liu, Zhenyu Yan, Paul Labrousse, Peng Fang, Jennifer Biroschak, Cindy Spittle, Chad Galderisi, Jin Li. Mutation profiling of colorectal cancer ctDNA using AmpliSeq CHP2 cancer panel. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A29.