A novel KRAS exon 2 drop-off digital PCR assay for mutation detection in cell-free DNA of cancer patients

Abstract

Background: KRAS exon 2 mutations are highly prevalent in human malignancies, making them attractive targets for detection and monitoring in cell-free DNA (cfDNA) of cancer patients. Drop-off assays designed for digital polymerase chain reaction (ddPCR drop-off) span entire mutational hotspots and detect any mutated allele within the covered region, overcoming a major limitation of mutation-specific ddPCR assays. We therefore set out to develop a novel KRAS codon 12/13 ddPCR drop-off assay for the robust, highly sensitive and specific detection of KRAS exon 2 hotspot mutations in cfDNA.

Methods: We designed, optimized and extensively validated a KRAS codon 12/13 ddPCR drop-off assay. We compared assay performance to a commercially available KRAS multiplex assay. For clinical validation, we analyzed plasma samples collected from patients with KRAS-mutated gastrointestinal malignancies.

Results: Limit of detection of the newly established ddPCR drop-off assay was 0.57 copies/µL, limit of blank was 0.13 copies/µ. The inter-assay precision (r²) was 0.9096. Our newly developed KRAS ddPCR drop-off assay accurately identified single nucleotide variants in 35/36 (97.2%) of circulating tumor DNA-positive samples from the patient validation cohort. Assay cross-validation showed that the newly established KRAS codon 12/13 ddPCR drop-off assay outperformed a commercially available KRAS multiplex ddPCR assay in terms of specificity. Moreover, the newly developed assay proved to be suitable for multiplexing with mutation-specific probes.

Conclusion: We developed and clinically validated a highly accurate ddPCR drop-off assay for KRAS exon 2 hot-spot detection in cfDNA with broad applicability for clinic and research.

Keywords: KRAS; Cell-free DNA (cfDNA); Circulating-tumor DNA (ctDNA); Drop-off assay; Droplet digital polymerase chain reaction (ddPCR); Liquid biopsy; Precision medicine.

Fig. 1

Technical validation - In-house designed KRAS ddPCR drop-off assay. Primers and locked nucleic acid (LNA)-based probes were designed, and the assay optimized individually regarding cycling conditions. drop-off assay ddPCR approach with only wild-type (WT) DNA (A), only mutant (MUT) DNA (B), both WT and MUT DNA (C) and a no template control (NTC) (D). Green: droplets positive for KRAS WT, Blue: MUT KRAS droplets, Orange: double positive droplets (WT + MUT), Gray: empty droplets. Green circle: In the case of a wild-type sequence, both probes bind, resulting in a double-positive signal. Blue circle: In the case of a mutation, only the reference probe binds to the DNA, resulting in a single FAM-positive signal. Figure 1B depicts a mutation with 100% variant allele frequency, while Fig. 1C shows a mixture of mutant and WT DNA. Based on the four scenarios (WT only, MUT only, WT + MUT, NTC), individual clusters were identified using Crystal Miner software. In our KRAS drop-off assay, an orange cluster was included to represent double-positive droplets, indicating the co-encapsulation of both double-positive and FAM-only positive droplets within a single droplet. This co-encapsulation results in the formation of a new cluster. DNA, deoxyribonucleic acid; FAM, 6-carboxyfluorescein; HEX, hexachlorofluorescein

Fig. 2

Technical validation - Screening for KRAS mutations with generic drop-off assay. Detection of KRAS G12 V/D/R/A/C/S and G13D with the KRAS drop-off assay is shown. Green: droplets positive for KRAS WT, Blue: MUT KRAS droplets, Orange: double positive droplets (WT + MUT), Gray: empty droplets. Based on the controls performed for the ddPCR experiment (WT only, MUT only, WT + MUT, NTC), the individual clusters were formed using the Crystal Miner software. In our KRAS drop-off assay, an orange cluster was included to represent the double-positive droplets, indicating co-encapsulation of both double-positive and FAM-only positive droplets within a single droplet. This co-encapsulation results in the formation of a new cluster. FAM, 6-carboxyfluorescein; HEX, hexachlorofluorescein; MUT, mutant; NTC, no template control; WT, wild-type

Fig. 3

Technical validation - drop-off ddPCR setting with specific detection of KRAS G12C. Simultaneous screening for genomic KRAS alterations at exon 2 codon 12/13 of the KRAS gene and specific detection of KRAS G12C. Green: droplets positive for KRAS WT, Blue: MUT KRAS G12/G13 droplets, Gray: empty droplets, Red: MUT KRAS G12C positive droplets, Yellow: double positive droplets (WT and KRAS G12C mutant). Based on the controls performed for the ddPCR experiment (WT only, MUT only, WT + MUT, NTC), the individual thresholds for the fluorescence channels were determined using the Crystal Miner software. Green lines: Threshold between HEX negative and positive droplets, blue lines: Threshold between FAM negative and positive droplets, red lines: Threshold between Cy5 negative and positive droplets. Cy5, cyanin-5; FAM, 6-carboxyfluorescein; HEX, hexachlorofluorescein; MUT, mutant; NTC, no template control; WT, wild-type

Fig. 4

Technical validation - Inter-assay precision of the KRAS ddPCR drop-off assay.cfDNA samples from pancreatic cancer patients were analyzed with different assays as indicated. (A) drop-off assay (left column) vs. corresponding single-target assays (right column) vs. the commercial KRAS kit from ID Solutions^® (middle column). The commercial kit shows a distinct subclone population in the HEX channel, visible as a second population within the double-positive (WT + MUT) droplet cloud region (green), likely caused by off-target amplification from a homologous region on chromosome 6. Analysis of sample 283 shows a more accurate estimation of VAF and KRAS wild type allele frequency by the drop-off assay. Green: droplets positive for KRAS WT, Blue: MUT KRAS droplets, Orange: double positive droplets (WT + MUT), Gray: empty droplets. Green lines: Threshold between HEX negative and positive droplets, blue lines: Threshold between FAM negative and positive droplets. (B) Variant allele frequencies (VAFs) were plotted against each other. cfDNA, cell-free DNA; FAM, 6-carboxyfluorescein; HEX, hexachlorofluorescein; MUT, mutant; WT, wild-type

Fig. 5

Clinical validation - Clinical response prediction by mutant KRAS cfDNA kinetics. cfDNA samples from three different pancreatic cancer patients during palliative systemic treatment were analyzed with the KRAS ddPCR drop-off assay. Sample 1 is the baseline sample taken before starting systemic treatment. (A) Tumor progression during palliative first-line treatment. (B) Treatment response indicated by decreasing mutant copies/µl. (C) Example of specific KRAS G12C detection in cfDNA. Green: droplets positive for KRAS WT, Blue: MUT KRAS droplets, Orange: double positive droplets (WT + MUT), Gray: empty droplets. Green lines: Threshold between HEX negative and positive droplets, red lines: Threshold between Cy5 negative and positive droplets. cfDNA, cell-free DNA; Cy5, cyanin-5; FAM, 6-carboxyfluorescein; HEX, hexachlorofluorescein; MUT, mutant; WT, wild-type

Fig. 6

Clinical validation - Overall survival (OS) analyses for gastrointestinal cancer patients undergoing systemic treatment. Top left: Kaplan-Meier estimate of OS for gastrointestinal cancer patients stratified by KRAS mutant cfDNA positivity (ctDNA positive) during systemic treatment. Top right: Kaplan-Meier estimate of OS for gastrointestinal cancer patients stratified by KRAS mutant cfDNA changes during systemic treatment: ctDNA decrease/stable versus increase. Bottom left: Kaplan-Meier estimate of OS for gastrointestinal cancer patients stratified by CA 19 − 9 positivity during systemic treatment. Bottom right: Kaplan-Meier estimate of OS for gastrointestinal cancer patients stratified by CA 19 − 9 changes during systemic treatment: CA 19 − 9 decrease/stable versus increase