Multiplex KRASG12/G13 mutation testing of unamplified cell-free DNA from the plasma of patients with advanced cancers using droplet digital polymerase chain reaction

Abstract

Background: Cell-free DNA (cfDNA) from plasma offers easily obtainable material for KRAS mutation analysis. Novel, multiplex, and accurate diagnostic systems using small amounts of DNA are needed to further the use of plasma cfDNA testing in personalized therapy.

Patients and methods: Samples of 16 ng of unamplified plasma cfDNA from 121 patients with diverse progressing advanced cancers were tested with a KRASG12/G13 multiplex assay to detect the seven most common mutations in the hotspot of exon 2 using droplet digital polymerase chain reaction (ddPCR). The results were retrospectively compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care.

Results: Eighty-eight patients (73%) had KRASG12/G13 mutations in archival tumor specimens collected on average 18.5 months before plasma analysis, and 78 patients (64%) had KRASG12/G13 mutations in plasma cfDNA samples. The two methods had initial overall agreement in 103 (85%) patients (kappa, 0.66; ddPCR sensitivity, 84%; ddPCR specificity, 88%). Of the 18 discordant cases, 12 (67%) were resolved by increasing the amount of cfDNA, using mutation-specific probes, or re-testing the tumor tissue, yielding overall agreement in 115 patients (95%; kappa 0.87; ddPCR sensitivity, 96%; ddPCR specificity, 94%). The presence of ≥ 6.2% of KRASG12/G13 cfDNA in the wild-type background was associated with shorter survival (P = 0.001).

Conclusion(s): Multiplex detection of KRASG12/G13 mutations in a small amount of unamplified plasma cfDNA using ddPCR has good sensitivity and specificity and good concordance with conventional clinical mutation testing of archival specimens. A higher percentage of mutant KRASG12/G13 in cfDNA corresponded with shorter survival.

Keywords: cell-free DNA; KRAS; droplet digital PCR; multiplex.

Figure 1.

(A) Among the 121 patients whose cfDNA samples were tested for KRAS^G12/G13 mutations, the median overall survival (OS) duration of the 82 patients with a KRAS^G12/G13-mutant cfDNA percentage of <6.2% (7.5 months; 95% confidence interval [CI], 5.6–9.4 months; blue) was significantly longer than that of the 39 patients with a KRAS^G12/G13-mutant cfDNA percentage of ≥6.2% (5.4 months; 95% CI, 3.7–7.1; red; P = 0.001). (B) In a separate analysis that included only the 88 patients with KRAS^G12/G13 mutations in formalin-fixed, paraffin-embedded tumor samples, the median OS duration of the 44 patients with a KRAS^G12/G13-mutant cfDNA percentage of <4.0% (7.3 months; 95% CI, 3.6–11.0; blue) was significantly longer than that of the 44 patients with a KRAS^G12/G13-mutant cfDNA percentage of ≥4.0% (5.5 months; 95% CI, 4.3–6.7; red; P = 0.017).

Figure 2.

Dynamic changes in KRAS^G12/G13-mutated cfDNA in various patients with heavily pretreated KRAS^G12/G13-mutated advanced cancers. In each panel, the top graph depicts the KRAS^G12/G13 mutation allelic frequency (MAF) in the wild-type background, and the bottom graph depicts the number of KRAS^G12/G13 copies per 1 ml of plasma. Red arrows indicate the onset of progressive disease. Shown are sequential measurements of KRAS^G12/G13 (blue); cancer antigen 125 (CA125) levels (panels A and C), cancer antigen 15-3 (CA15-3) levels (panel B), carcinoembryonic antigen (CEA) levels (panels D–F; orange); and the sum (cm) of target lesions on imaging (grey bars) in (A) a patient with metastatic ovarian carcinoma who received targeted therapy with an ERK inhibitor; (B) a patient with metastatic breast carcinoma who received therapy with an mTOR inhibitor, HDAC inhibitor, and anti-VEGF antibody; (C) a patient with metastatic ovarian carcinoma who received targeted therapy with PI3K and MEK inhibitors; (D) a patient with metastatic ampullary carcinoma who received targeted therapy with a MEK inhibitor.

Figure 2.

(E) a patient with colorectal cancer who received an intratumoral autologous dendritic cell vaccine; and (F) a patient with non–small cell lung cancer who received targeted therapy with a FGFR inhibitor.

Figure 3.

(A) KRAS^G12/G13 mutant allele frequencies (MAFs) at baseline (median, 0.50%), during therapy (median, 0.22%), and at disease progression (median, 1.13%) differed significantly (P = 0.04). (B) KRAS^G12/G13 copy numbers at baseline (median, 14), during therapy (median, 17), and at disease progression (median, 273) differed significantly (P = 0.03).