Introduction
Colorectal cancer (CRC) is the third most diagnosed cancer worldwide1, with a similar prevalence observed in Denmark, where it ranks as the third most frequent malignancy2. In recent decades, the incidence of early-onset colorectal cancer—defined as CRC diagnosed in individuals under 50 years of age—has been rising3,4. Since CRC is often asymptomatic in the early stages, late-stage diagnosis is associated with high mortality rates5. Therefore, it is crucial to develop new strategies to identify risk factors and early tumour formation, enabling timely prevention, treatment, and improved patient outcomes6. While colonoscopy remains the gold standard, it is invasive and often unnecessary; data from Region Zealand show that nearly 60% of individuals undergoing colonoscopy after a positive faecal immunochemical test (FIT) do not have lesions requiring treatment7. The development of molecular diagnostics, such as faecal DNA-based screening, offers a promising alternative that could significantly improve early detection while reducing the number of unnecessary colonoscopies, particularly in high-risk populations such as those with inflammatory bowel disease (IBD), previous CRC history, or genetic predispositions.
Aims & Methods
We evaluated two alternative approaches for detecting colorectal cancer-related mutations from faecal-derived human DNA: short-read next-generation sequencing (NGS) and digital PCR (dPCR). The aim was to assess whether these methods could support a non-invasive screening strategy that does not rely on the detection of occult blood. Faecal samples were collected from patients in Region Zealand who tested positive in the Danish national colorectal cancer (CRC) screening programme between January 2022 and January 2023 (FIT ≥100 ng/mL).
Faecal DNA was extracted using the QIAamp Fast DNA Stool Mini Kit (Qiagen) with RNase treatment. Formalin-fixed paraffin-embedded (FFPE) tumour samples were processed with the GeneRead DNA FFPE Kit (Qiagen). For next-generation sequencing (NGS), libraries were prepared with the QIAseq Targeted DNA Pro kit (Qiagen), targeting regions commonly mutated in CRC. Digital PCR (dPCR) was performed using the QIAcuity Probe PCR Kit and a panel targeting six validated single-nucleotide variants (SNVs) in CRC driver genes.
Results
Of the 71 sequencing DNA libraries generated from faecal samples, only three yielded more than 100 high-quality human reads, highlighting the challenge of recovering host DNA from stool material due to high gut microbiome content and inhibitory substances. Across the 33 patients successfully analysed, a total of 163 faecal DNA variants were detected by either NGS or dPCR. Of these, 21 were classified as oncogenic or likely oncogenic in colorectal cancer, and five were pathogenic variants not directly linked to CRC. These combined results demonstrate the feasibility of faecal DNA-based mutation detection as a non-invasive screening tool and underscore the importance of adequate DNA recovery for reliable variant identification.
Conclusion
Our study provides compelling evidence that mutation-based faecal DNA screening is feasible and may augment current CRC detection strategies. While current sensitivity remains suboptimal, the high PPV of dPCR under stringent quality control is encouraging. Our findings contribute to a growing body of literature supporting the transition toward precision diagnostics in cancer screening. When implemented within a personalised framework, faecal DNA mutation analysis may reduce unnecessary colonoscopies and enable early detection of high-risk lesions.
References
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Disclosure
The authors declare no competing interests.