BMC Cancer · 5 authors, 3 centres
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This study found that total cell-free DNA (cfDNA) levels are significantly higher in colorectal cancer (CRC) patients compared to healthy controls and increase with tumor stage. Mitochondrial cfDNA fragment levels were strongly reduced even in early-stage CRC, suggesting potential as an early diagnostic biomarker. The findings challenge the assumption that increased cfDNA in CRC is driven by necrosis, as long nuclear fragments decreased rather than increased with disease progression.
**Background:** Colorectal cancer (CRC) is among the most common cancers in developed countries. Current screening methods (fecal tests, colonoscopy) have limitations in sensitivity or practicality. Circulating cell-free DNA (cfDNA) in blood plasma ('liquid biopsy') has emerged as a promising diagnostic tool. This study aimed to quantify nuclear and mitochondrial cfDNA fragments in CRC patients versus healthy controls, assess DNA integrity indices, and evaluate diagnostic accuracy using two analytical approaches: equal template concentration (ETC) and normalized to total cfDNA (NTC).
**Methods:** Plasma samples were collected from 80 CRC patients (UICC stage I: n=21, II: n=21, III: n=20, IV: n=18) and 50 healthy individuals. Total cfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit and quantified via Qubit fluorometer. Quantitative real-time PCR (qPCR) was performed targeting short and long fragments of KRAS (67 bp and 305 bp), Alu (115 bp and 247 bp), and MTCO3 (67 bp and 296 bp). Analyses were conducted under ETC (all samples diluted to 0.1 ng/µl template) and NTC (data normalized to total cfDNA concentration). DNA integrity index (DII) was calculated as the ratio of long to short fragments. Diagnostic accuracy was assessed using ROC curves, and predictive models were built using LASSO multinomial logistic regression with tenfold cross-validation.
**Key Results:** Total cfDNA levels were significantly higher in CRC patients (median 10.51 ng/ml, IQR: 6.92–19.83) compared to controls (median 6.10 ng/ml, IQR: 4.50–8.28; P<0.0001) and increased with tumor stage (UICC I: 8.87, II: 9.09, III: 12.46, IV: 20.66 ng/ml; P=0.0084). At ETC, long nuclear fragments (KRAS 305 and Alu 247) were significantly lower in CRC patients (P=0.0021 and P=0.0003, respectively), while short fragments showed no difference. DII for nuclear markers decreased from controls to advanced stages (KRAS 305/67: control median 1.05 vs. CRC 0.63, P=0.0003; Alu 247/115: control 0.22 vs. CRC 0.12, P<0.0001). Mitochondrial cfDNA (MTCO3) fragments were significantly reduced in CRC patients at both ETC and NTC conditions (MTCO3 67: control median 2.61 vs. CRC 0.32 ng/ml, P<0.0001; MTCO3 296: control 1.82 vs. CRC 0.30 ng/ml, P<0.0001). The LASSO model combining ETC and NTC predictors achieved the lowest misclassification error (33.1%) and highest multiclass AUC (0.843), outperforming models using either predictor set alone.
**Clinical Implications:** The study demonstrates that mitochondrial cfDNA markers (MTCO3) have strong diagnostic potential for early-stage CRC detection, with significantly reduced levels even in UICC stage I. The finding that long nuclear cfDNA fragments decrease rather than increase with tumor progression challenges the hypothesis that elevated cfDNA in cancer is primarily due to necrosis. The ETC approach may provide more accurate assessment of fragment-level changes compared to traditional NTC methods. The combined use of nuclear and mitochondrial markers in predictive models shows promise for improving CRC diagnosis, though validation in larger, population-based studies is needed. Limitations include the case-control design and potential confounding by age differences between groups.