Mutation profiling of tumor DNA from plasma and tumor tissue of colorectal cancer patients with a novel, high-sensitivity multiplexed mutation detection platform

Abstract

Background: Circulating tumor DNA (ctDNA) holds promise as a non-invasive means for tumor monitoring in solid malignancies. Assays with high sensitivity and multiplexed analysis of mutations are needed to enable broad application.

Methods: We developed a new assay based on sequence-specific synchronous coefficient of drag alteration (SCODA) technology, which enriches for mutant DNA to achieve high sensitivity and specificity. This assay was applied to plasma and tumor tissue from non-metastatic and metastatic colorectal cancer (CRC) patients, including patients undergoing surgical resection for CRC liver metastases.

Results: Across multiple characterization experiments, the assay demonstrated a limit of detection of 0.001% (1 molecule in 100,000) for the majority of the 46 mutations in the panel. In CRC patient samples (n=38), detected mutations were concordant in tissue and plasma for 93% of metastatic patients versus 54% of non-metastatic patients. For three patients, ctDNA identified additional mutations not detected in tumor tissue. In patients undergoing liver metastatectomy, ctDNA anticipated tumor recurrence earlier than carcinoembryonic antigen (CEA) value or imaging.

Conclusions: The multiplexed SCODA mutation enrichment and detection method can be applied to mutation profiling and quantitation of ctDNA, and is likely to have particular utility in the metastatic setting, including patients undergoing metastatectomy.

Figures

Figure 1. Multiplexed SCODA mutation enrichment and detection assay

A) Method. Following plasma preparation (1), plasma DNA is extracted (2a) and spiked with randomly barcoded internal controls (2b) representing every mutation in the panel, and amplified with a limited number of cycles in a multiplex PCR reaction (3). PCR primers are designed to amplify regions of the genome containing the 46 mutations of interest, and carry universal linker sequences and sample barcodes to enable subsequent sample processing and multiplexing. Amplified DNA and internal controls are enriched for the 46 mutations of interest by SCODA cyclic electrophoresis (4) in a gel containing hybridization probes against all mutations of interest. DNA enriched for mutant sequences (5a) is amplified through additional PCR as per conventional Illumina library construction, pooled with additional samples, and sequenced (6). B) Assay characterization. Each graph denotes results from samples created by titrating synthetic DNA carrying the specified mutation into reference wild-type (WT) DNA. Horizontal axes denote estimated input copies per sample, while the vertical axes denote the number of copies detected by the assay. Limit of Detection (LOD) is calculated as the greater of a single copy of mutant DNA or three standard deviations above the average background detected for each mutation in wild-type DNA samples. Input copies are limited to 10 and greater to limit the effect of sampling fluctuation in the titration. Each sample is 300 ng of DNA, such that 10 copies are equivalent to an allele fraction of ~ 0.01%.

Figure 1. Multiplexed SCODA mutation enrichment and detection assay

A) Method. Following plasma preparation (1), plasma DNA is extracted (2a) and spiked with randomly barcoded internal controls (2b) representing every mutation in the panel, and amplified with a limited number of cycles in a multiplex PCR reaction (3). PCR primers are designed to amplify regions of the genome containing the 46 mutations of interest, and carry universal linker sequences and sample barcodes to enable subsequent sample processing and multiplexing. Amplified DNA and internal controls are enriched for the 46 mutations of interest by SCODA cyclic electrophoresis (4) in a gel containing hybridization probes against all mutations of interest. DNA enriched for mutant sequences (5a) is amplified through additional PCR as per conventional Illumina library construction, pooled with additional samples, and sequenced (6). B) Assay characterization. Each graph denotes results from samples created by titrating synthetic DNA carrying the specified mutation into reference wild-type (WT) DNA. Horizontal axes denote estimated input copies per sample, while the vertical axes denote the number of copies detected by the assay. Limit of Detection (LOD) is calculated as the greater of a single copy of mutant DNA or three standard deviations above the average background detected for each mutation in wild-type DNA samples. Input copies are limited to 10 and greater to limit the effect of sampling fluctuation in the titration. Each sample is 300 ng of DNA, such that 10 copies are equivalent to an allele fraction of ~ 0.01%.

Figure 2. Mutations detected in tissue and plasma with the multiplexed SCODA mutation enrichment and detection assay

In highlighted cells, the detected mutation allele (or alleles) is specified, and for plasma, the number of detected copies (cp) of mutant DNA is also provided, normalized for a 5 mL input volume of plasma. ND, not detected.

Figure 3. Perioperative dynamics of plasma mutation levels in patients with stage IV colorectal cancer

Panels A-D show plasma levels of detected mutations in the circulating tumor DNA (ctDNA, left y-axis) as well as CEA values (right y-axis) in patients that underwent surgery with the intent of complete metastasis resection. The value at time point 0 represents the preoperative ctDNA level. ctDNA levels (cp, copies) are normalized for 5 mL input volume of plasma. After blood draw, surgery was performed at day 0. Arrows indicate imaging assessments by computed tomography (CT). In panels B, C and D, colored shading indicates the administration of chemotherapy. PD, progressive disease; NED, no evidence of disease. CEA normal range is 0-5 ng/mL.