Circulating Methylated SEPT9 as a Novel Biomarker for Early Detection of Hepatocellular Carcinoma in Cirrhotic Patients

Highlights

• Circulating methylated SEPT9 demonstrates superior diagnostic accuracy compared to AFP alone for hepatocellular carcinoma (HCC) detection in patients with cirrhosis.
• Combining methylated SEPT9 with AFP markedly increases sensitivity for early-stage (BCLC 0-A) HCC detection, achieving a 3.2-fold sensitivity improvement over AFP alone.
• Use of a tiered biomarker strategy (disjunction for sensitivity, conjunction for specificity) enables tailored diagnostic approaches maximizing either early detection or confirmatory specificity.
• This biomarker combination holds promise to address current surveillance gaps, potentially facilitating earlier curative interventions and improved patient outcomes.

Background

Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide, predominantly arising on a background of cirrhosis. Surveillance programs deploying ultrasonography plus alpha-fetoprotein (AFP) testing are standard but hampered by suboptimal sensitivity, especially in early-stage HCC (Barcelona Clinic Liver Cancer [BCLC] stage 0-A). Given that early-stage detection critically impacts prognosis by expanding curative treatment options, improving surveillance modalities remains an unmet clinical need.

AFP, although widely used, suffers from limited sensitivity and false positives that compromise its standalone utility. Recent advances in liquid biopsy approaches have identified circulating DNA methylation markers with potential for cancer detection. Among these, methylated septin 9 (SEPT9), a gene involved in cytoskeletal dynamics and oncogenic pathways, has emerged as a promising plasma biomarker for various cancers including HCC. Prior small-scale and retrospective studies have suggested diagnostic utility of methylated SEPT9, but rigorously designed prospective investigations were lacking.

Key Content

Study Design and Patient Population

The referenced prospective, cross-sectional diagnostic accuracy study enrolled 574 patients with cirrhosis undergoing routine HCC surveillance across two French academic centers from February 2018 to October 2024. The cohort was predominantly male (72.1%) with a median age of 63 years. HCC diagnosis was established per international consensus via centralized radiologic review blinded to methylated SEPT9 results, ensuring methodologic robustness.

Methodology of Biomarker Assessment

Plasma methylated SEPT9 was assessed from three independent aliquots per patient, with positivity classified ordinally as single-, double-, or triple-positive based on replicate detection, allowing graded confidence in methylation presence. Serum AFP was measured with a clinically relevant cutoff of 20 ng/mL. Composite biomarker analysis used two logical frameworks: tier 1 (disjunction; either positive for SEPT9 or AFP) to maximize sensitivity and tier 2 (conjunction; both positive) to maximize specificity.

Diagnostic Performance of Methylated SEPT9 and AFP

Methylated SEPT9 alone outperformed AFP, with an area under the receiver operating characteristic curve (AUROC) of 0.79 (95% CI, 0.74–0.84) versus 0.71 (95% CI, 0.66–0.76) for AFP (p=0.002, posterior probability of superiority >99.8%). This signifies a meaningful improvement in diagnostic discrimination.

Tier 1a analysis incorporating single-positive SEPT9 or AFP >20 ng/mL achieved 87.8% sensitivity (95% CI, 81.6–93.5) and a robust negative likelihood ratio of 0.2, successfully recovering 78% of HCC cases missed by AFP alone. Importantly, sensitivity for early-stage BCLC 0-A tumors was substantially higher with tier 1a (74.5%) compared to AFP alone (23.5%), representing over threefold improvement.

Conversely, tier 2 (requiring triple-positive SEPT9 and elevated AFP) conferred exceptional specificity (99.6%), a positive likelihood ratio of 76.0, and a very high diagnostic odds ratio (112.1), ideal for confirmatory diagnostic settings minimizing false positives.

Subgroup and Stage-Stratified Findings

The enhanced performance of methylated SEPT9 and combination tiers was particularly pronounced in early-stage HCC (BCLC 0-A), underscoring its potential to improve detection when curative treatments are most effective. The study’s stratified analysis confirms that methylated SEPT9 maintains discriminative value regardless of liver disease severity or cirrhosis etiology.

Expert Commentary

The data underpin the clinical promise of methylated SEPT9 as a robust liquid biopsy biomarker that complements AFP and ultrasound in cirrhotic patients. Mechanistically, SEPT9 methylation likely reflects tumorigenic epigenetic dysregulation, offering biological plausibility for its diagnostic value.

Current surveillance guidelines (EASL, AASLD) emphasize ultrasound and AFP but acknowledge these have imperfect sensitivity, often failing early tumor detection. This study’s prospective design and rigorous assessment of SEPT9 across a large, representative cirrhosis population provide compelling evidence to reconsider biomarker panels.

By combining SEPT9 and AFP in a tiered strategy, clinicians could triage patients according to diagnostic priorities—employing sensitive detection to trigger further imaging or using high specificity criteria to confirm malignancy before invasive procedures. Nonetheless, limitations include the need for validation in diverse populations, cost-effectiveness analyses, and assessment of real-world integration with ultrasound performance.

Further translational research is needed to clarify SEPT9 methylation dynamics in hepatocarcinogenesis and explore its utility in monitoring treatment response or recurrence. Implementation studies are also crucial to test whether improved detection yields survival benefit through earlier interventions.

Conclusion

The study by Oussalah et al. represents a major advance in HCC surveillance, demonstrating that circulating methylated SEPT9 significantly improves diagnostic performance alone and in combination with AFP in cirrhotic patients. Particularly for early-stage disease, this approach enhances sensitivity beyond conventional markers, potentially enabling earlier curative therapies.

Future prospective trials should evaluate the impact of incorporating methylated SEPT9 into established surveillance algorithms on patient outcomes and healthcare utilization. This biomarker-based refinement of HCC detection sets the stage for precision oncology in chronic liver disease surveillance, with important implications for reducing HCC mortality worldwide.

References

• Oussalah A, Silva Rodriguez M, Conroy G, et al. Circulating Methylated SEPT9 for Detection of Hepatocellular Carcinoma in Cirrhosis. JAMA Oncol. 2026 Jul 2. PMID: 42390849. https://pubmed.ncbi.nlm.nih.gov/42390849/
• European Association for the Study of the Liver. EASL Clinical Practice Guidelines: Management of hepatocellular carcinoma. J Hepatol. 2018 Jul;69(1):182-236. PMID: 29628281.
• American Association for the Study of Liver Diseases. AASLD Practice Guidance on Hepatocellular Carcinoma. Hepatology. 2018 Jan;67(1):358-380. PMID: 28799425.
• Sun Z, Yu X, Wang X, et al. Methylated SEPT9 Gene as a Noninvasive Biomarker for Hepatocellular Carcinoma Detection: A Meta-Analysis. Front Oncol. 2022;12:882826. PMID: 35918673.