A blood-based assay combining cell-free DNA (cfDNA) fragmentomics with droplet digital PCR–based methylation analysis improved detection of early-stage colorectal cancer (CRC) and precancerous lesions, according to findings presented at the 2026 ASCO Breakthrough meeting in Singapore.
In this study, fragmentomics referred to patterns in cfDNA fragments, including fragment length, end motifs, and nucleosome footprinting. Methylation analysis evaluated epigenetic changes that can occur early in colorectal tumorigenesis.
The research, presented by Thi Tuong Vi Van, BSc, of the Medical Genetics Institute in Ho Chi Minh City, Vietnam, evaluated plasma cfDNA samples from 380 patients with nonmetastatic CRC, 121 individuals with precancerous lesions, and 506 healthy controls. The investigators found that cfDNA fragmentomic features could detect CRC, but adding methylation markers improved detection of precancerous lesions, which has remained a key limitation for blood-based CRC screening approaches.
“Cell-free DNA fragmentomic features enable the detection of colorectal cancer, but have limited sensitivity for precancerous lesions,” Ms. Van said during the presentation. She added that multiplex droplet digital PCR methylation analysis of SEPT9 and HS3ST2 may provide complementary information for fragmentomics-based detection.
Rationale for the Study
Colonoscopy remains the standard approach for CRC screening because it can detect cancer and allow removal of precancerous polyps. However, Ms. Van noted that its invasive nature may limit participation in population-level screening. Noninvasive options, including fecal immunochemical testing, are more accessible but may have limited sensitivity for early-stage CRC lesions and precancerous lesions.
Ms. Van said the progression from dysplasia or adenoma to invasive CRC provides “a long window” for early detection. The study was designed around the idea that tumor-derived cfDNA/ctDNA (circulating tumor DNA) may carry distinct fragmentomic and methylation signals that can be detected in blood before or during early cancer development.
“ctDNA carries distinct fragmentomic and methylation patterns from the tumor,” Ms. Van said. “We hypothesized that combining them could improve the detection of both early-stage colorectal cancer and precancerous lesions.”
Study Design
The investigators used shallow whole-genome sequencing to evaluate cfDNA fragment length profiles, nucleosome footprinting patterns, and four-mer fragment end motifs. These features were used to develop a machine-learning fragmentomics model.
For methylation analysis, the researchers selected candidate methylation regions using an in-house cfDNA methylation database and tissue data from The Cancer Genome Atlas. They then developed a multitarget droplet digital polymerase chain reaction panel. SEPT9 and HS3ST2 were selected for their complementary value to the fragmentomics model, and methylation and fragmentomic scores were integrated into a combined model.
“To overcome these challenges, we established an integrated workflow,” Ms. Van said.
In the fragmentomics analysis, CRC samples showed enrichment of short cfDNA fragments. Precancerous lesions had fragment length distributions more similar to those of healthy controls, but showed distinct nucleosome footprinting patterns. CRC and precancerous lesions also showed differences in fragment end motifs.
In the independent test set, the fragmentomics model achieved an area under the curve of 0.89. At 92.4% specificity, sensitivity was 71.1% for CRC and 40.0% for precancerous lesions. In the subsequent integrated analysis fragmentomics alone yielded 77.6% sensitivity for colorectal cancer lesions and 46.7% for precancerous lesions. Adding methylation markers increased sensitivity to 81.6% for colorectal cancer lesions and 73.3% for precancerous lesions, with 91.0% specificity.
Methylation analysis of SEPT9 and HS3ST2 identified additional samples with CRC and precancerous lesions that had been missed by the fragmentomics-based model, Ms. Van said. “It demonstrates their complementary power,” she added.
Discussant Perspective
Discussant Desmond Yip, MBBS, FRACP, of The Canberra Hospital and Australian National University, placed the findings in the context of current CRC screening strategies. He noted that early detection of precursor lesions and early-stage CRC can reduce CRC mortality, but screening participation remains a challenge in many settings.
Blood-based screening may be more acceptable to some patients than stool-based testing or colonoscopy, Dr. Yip said. But improved uptake alone would not solve the screening problem if precursor lesions are missed.
“The major weakness of the available blood-based screening has been the poor advanced precursor lesion detection,” he said.
Current blood-based approaches have generally performed better for CRC detection than for advanced adenomas or other precursor lesions. According to Dr. Yip, newer assays are beginning to address that gap.
“Blood-based cfDNA methylation screening platforms have now progressed beyond the single gene to be multimodal or multidimensional, with improvement in precursor lesion detection,” he said.
Discussing Ms. Van’s study specifically, Dr. Yip highlighted the improvement in precursor-lesion detection after methylation markers were added to the fragmentomics model.
“Their combination fragmentome and methylation improved the detection of precancerous lesions from 47% to 73%,” Dr. Yip said.
He cautioned, however, that the study used enriched cohorts of patients with CRC, individuals with precancerous lesions, and healthy controls, rather than an average-risk screening population. The findings, he suggested, should therefore be viewed as promising but preliminary.
Need for Further Validation
Both Ms. Van and Dr. Yip emphasized that the assay requires additional validation before it can be considered for clinical screening.
Ms. Van said the next steps include prospective validation and assessment of whether the approach can be used for CRC screening in low- and middle-income countries.
“Given the limitations of this retrospective study, we will further validate [the assay] in a prospective cohort and assess its utility for colorectal cancer screening in low- and middle-income countries,” she said.
Dr. Yip said the key next step is testing the approach in real-world screening settings.
“They need prospective validation across different populations and ethnic groups,” he said. “They need validation in [an] average-risk population and compared against colonoscopy, ideally, with demonstration of feasibility and cost-effectiveness.”
DISCLOSURE: Dr. Van reported no disclosures. Dr. Yip reported stock and other ownership interests in Commonwealth Serum Laboratories; honoraria from Servier and Sirtex Limited; and travel, accommodations, and expenses from MSD Oncology and Sirtex Medical.
REFERENCE
1. Van TTV, Tran TT, Tran LS: Integrated fragmentomics and droplet digital PCR–based methylation analysis of cell-free DNA for enhanced detection of precancerous lesions and early colorectal cancer. 2026 ASCO Breakthrough Meeting. Abstract 95. Presented June 26, 2026.
