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Background <p><i>KRAS</i> 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 <i>KRAS</i> codon 12/13 ddPCR drop-off assay for the robust, highly sensitive and specific detection of <i>KRAS</i> exon 2 hotspot mutations in cfDNA.</p> Methods <p>We designed, optimized and extensively validated a <i>KRAS</i> codon 12/13 ddPCR drop-off assay. We compared assay performance to a commercially available <i>KRAS</i> multiplex assay. For clinical validation, we analyzed plasma samples collected from patients with <i>KRAS-</i>mutated gastrointestinal malignancies.</p> Results <p>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 (r<sup>2</sup>) was 0.9096. Our newly developed <i>KRAS</i> 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 <i>KRAS</i> codon 12/13 ddPCR drop-off assay outperformed a commercially available <i>KRAS</i> multiplex ddPCR assay in terms of specificity. Moreover, the newly developed assay proved to be suitable for multiplexing with mutation-specific probes.</p> Conclusion <p>We developed and clinically validated a highly accurate ddPCR drop-off assay for <i>KRAS</i> exon 2 hot-spot detection in cfDNA with broad applicability for clinic and research.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年5月24日

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  • Bianca Addamo-De Nard,
  • Meret Geissmann,
  • Dilara Akhoundova,
  • Clelia Pistoni,
  • Tomas Brezina,
  • Martin Zoche,
  • Achim Weber,
  • Saskia Hussung,
  • Ralph Fritsch

摘要

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.