Detection and quantification of mutations in the plasma of patients with colorectal tumors

Abstract

The early detection of cancers through analysis of circulating DNA could have a substantial impact on morbidity and mortality. To achieve this goal, it is essential to determine the number of mutant molecules present in the circulation of cancer patients and to develop methods that are sufficiently sensitive to detect these mutations. Using a modified version of a recently developed assay for this purpose, we found that patients with advanced colorectal cancers consistently contained mutant adenomatous polyposis coli (APC) DNA molecules in their plasma. The median number of APC DNA fragments in such patients was 47,800 per ml of plasma, of which 8% were mutant. Mutant APC molecules were also detected in >60% of patients with early, presumably curable colorectal cancers, at levels ranging from 0.01% to 1.7% of the total APC molecules. These results have implications for the mechanisms through which tumor DNA is released into the circulation and for diagnostic tests based on this phenomenon.

Fig. 1.

Effect of the PCR amplicon size on plasma DNA concentration and mutation frequency. (A) The concentration of total APC fragments (WT plus mutant) of various sizes was determined by using digital PCR of plasma DNA from three different patients (patients 29, 30, and 32). (B) The fraction of mutant APC fragments was determined by digital sequencing of PCR products.

Fig. 2.

Schematic of the BEAMing-based assay. (A) Extended beads were prepared by modifications of the BEAMing procedure described by Dressman et al. (16). (B) Single base extensions were performed on the extended beads. Normal DNA sequences contained a G at the queried position; mutant sequences contained an A.

Fig. 3.

Processing of flow cytometry data obtained by BEAMing. (A) Dot plot of forward-scatter (FSC) and side-scatter (SSC) signals of beads. (B) Histogram of single beads with regard to PE signal. (C) Dot plot showing the Cy5 and FITC fluorescence intensity profiles of PE-positive beads. The beads clustered in three distinct populations colored red, green, and blue. Sequencing of individual beads sorted from each population showed that the red and green beads contained homogeneous WT and mutant sequences, respectively; the blue beads contained a mixture of WT and mutant sequences.

Fig. 4.

Examples of flow cytometric profiles of beads generated from plasma DNA (patient 16). Cy5 and FITC fluorescence intensity profiles of PE-positive beads from four patients are shown. The patients, mutations, and fraction of mutant APC fragments are indicated.

Fig. 5.

Fraction of mutant APC gene fragments in the plasma of patients with various colorectal tumors [adenomas (Ad) and Dukes' stage A, B, and D carcinomas]. In each mutation analyzed, DNA from normal lymphoid cells or plasma DNA from healthy donors was used as a control (Normal). The “mutants” observed in assays with normal cellular DNA represent errors generated during the PCR process rather than mutations present in the template DNA (see text). The red lines represent the mean, minimum, and maximum values of the normal controls.