Circulating Cell-Free DNA from Colorectal Cancer Patients May Reveal High KRAS or BRAF Mutation Load

Abstract

We used a novel method based on allele-specific quantitative polymerase chain reaction (Intplex) for the analysis of circulating cell.free DNA (ccfDNA) to compare total ccfDNA and KRAS- or BRAF-mutated ccfDNA concentrations in blood samples from mice xenografted with the human SW620 colorectal cancer (CRC) cell line and from patients with CRC. Intplex enables single-copy detection of variant alleles down to a sensitivity of ≥0.005 mutant to wild-type ratio. The proportion of mutant allele corresponding to the percentage of tumor-derived ccfDNA was elevated in xenografted mice with KRAS homozygous mutation and varied highly from 0.13% to 68.7% in samples from mutation-positive CRC patients (n = 38). Mutant ccfDNA alleles were quantified in the plasma of every patient at stages II/III and IV with a mean of 8.4% (median, 8.4%) and 21.8% (median, 12.4%), respectively. Twelve of 38 (31.6%) and 5 of 38 (13.2%) samples showed a mutation load higher than 25%and 50%, respectively. This suggests that an important part of ccfDNA may originate from tumor cells. In addition, we observed that tumor-derived (mutant) ccfDNA was more fragmented than ccfDNA from normal tissues. This observation suggests that the form of tumor-derived and normal ccfDNA could differ. Our approach revealed that allelic dilution is much less pronounced than previously stated, considerably facilitating the noninvasive molecular analysis of tumors.

Figures

Figure 1

Primer designs used in the study: primers targeting a KRAS region (A) or a BRAF region (B) for quantifying ccfDNA WT and mutated concentrations.

Figure 2

Quantification by Q-PCR of ccfDNA derived from malignant and nonmalignant cells in the mouse model. Tumor weight is represented by the red curve (right axis). (A) Concentration of ccfDNA derived from mouse (normal) cells (mWT ccfDNA) in control (not grafted) mice (Mouse Nos. 1–3) and in athymic nude mice (Mouse Nos. 4–11) xenografted with the SW620 colorectal human cells, determined using a primer set targeting a mouse KRAS second intron WT sequence (Table W2). (B) Concentration of ccfDNA derived from human cells (hWT ccfDNA) using a primer set targeting a human KRAS second intron WT sequence. (C) Concentration of ccfDNA derived from human cells (hBRAFm ccfDNA) using a primer set targeting a human BRAF 15th exon sequence containing the V600E point mutation (Table W2). (D) Concentration of ccfDNA derived from human cells (hKRASm ccfDNA) using a primer set targeting a human KRAS second exon sequence that contains the G12V point mutation. (E) Concentration of total ccfDNA (mWT ccfDNA + hWT ccfDNA). (F) Proportion of hKRASm ccfDNA in total ccfDNA. (G) Proportion of tumor-derived ccfDNA and KRAS-mutated tumor-derived ccfDNA versus nontumor-derived ccfDNA expressed as the percentage of the total ccfDNA (derived from both mouse and human cells as quantified here). Concentration values are described in Table W3.

Figure 3

Histogram of the proportion of mutant allele (mA%) in plasma samples from CRC patients with KRAS- or BRAF-mutated tumor.

Figure 4

Comparison of the ccfDNA fragment size distribution of mutant (black bars) and nonmutant (gray bars) ccfDNA in plasma samples from patients with CRC bearing a point mutation in the KRAS exon 2. Data are expressed as ng of ccfDNA/ml plasma. ccfDNA from the plasma of patients CRC6 (KRAS G12D), CRC3 (KRAS G12D), and CRC1 (KRAS G13D) was quantified by amplifying KRAS exon 2 sequences of increasing size (82, 138, 200, 300, 355, and 390 bp; Figure W1 and Table W2). Detection of the resulting amplicons of various sizes enables quantification of the size fractions of ccfDNA contained in a mononucleosome (180–200 bp) and a dinucleosome (360–380 bp) [27,28]. Plasma of CRC6, CRC3, and CRC1 were chosen because of their high total ccfDNA content and high mutant ccfDNA proportion (Table 1).

Figure 5

Histogram representation of the various parameters indicative of ccfDNA fragment size pattern. Size fraction is expressed as a percentage of the highest value obtained in each size set. KRAS second exon primer systems enable quantification of mutant ccfDNA and ccfDNA harboring the WT sequence at the mutation location, respectively. Numbers under the histograms: 6, 3, 1, and 2, plasma from CRC patients with a positive mutational status for KRAS (CRC1–3 and CRC6).