Abstract 985: An effective cfDNA methylation-based assay in discriminating primary liver cancer from cirrhosis or chronic hepatitis virus infection: marker discovery, phase I pilot, and phase II clinical validation

Abstract Background: Early detection of primary liver cancer (PLC) in individuals with liver cirrhosis (LC) or chronic hepatitis virus infection (CHVI) improves survival. Timely, effective, and affordable tools with high sensitivity are urgently needed in clinical practice. Methods: Tissue and/or plasma samples from 159 healthy individuals and 89 PLC, LC, or CHVI patients were sequenced by a targeted methylation panel (~70, 000 CpGs) to screen candidate methylated DNA markers (MDMs). In phase I, the performance of each selected MDM was validated in 175 plasma samples (PLC, n=101; LC/CHVI, n=74) by a CO-methylation aMplification rEal-Time PCR (COMET) assay. Logistic models were then trained and validated in phase II with 310 plasma samples (hepatocellular carcinoma HCC, n=212; combined hepatocellular-cholangiocarcinoma cHCC-CC, n=12; LC/CHVI, n=106; training vs. validation, 2: 1). Results: The methylation levels of eleven selected MDMs with top performance showed a significant increase in PLC compared with LC/CHVI in both tissue and plasma samples (P0. 05). In phase I, eight of the above eleven MDMs with an area under the curve (AUC) over 0. 80 to differentiate PLC and LC/CHVI were chosen for further investigation. In phase II, the MDM-based logistic model achieved sensitivity of 87. 2% (95% confidence interval CI, 80. 8%-92. 4%) and 88. 0% (78. 4%-94. 4%), at respective specificity of 97. 1% (90. 1%-99. 7%) and 100% (90. 3%-100%) in the training and validation sets. In the validation set, sensitivity in patients with BCLC stage 0, diameter3 cm, AFP-negative, and PIVKA-II-negative was 90. 0% (55. 5%-99. 7%), 88. 9% (65. 3%-98. 6%), 80. 6% (64. 0%-91. 8%), and 81. 3% (54. 4%-96. 0%), respectively. The classifier achieved similar sensitivity in patients with or without HBV/HCV infection (88. 2% vs 90. 0%). Additionally, our model detected 19 of 24 (79. 3%, 57. 8%-92. 9%) intrahepatic cholangiocarcinoma. Combining AFP and PIVKA-II, the model achieved higher sensitivity of 93. 3% (85. 1%-97. 8%) and specificity of 100. 0% (90. 3%-100%). Conclusion: In conclusion, the present study demonstrated the feasibility of using an easy-to-implement cfDNA methylation assay (COMET) to discriminate early-stage liver cancer from other liver diseases, with superior accuracy over AFP and PIVKA-II. Notably, the combination of the COMET, AFP, and PIVKA-II increased the overall sensitivity, indicating the potential benefit of integrating these approaches in clinical practices. Moving forward, further investigation is needed to validate these findings in larger prospective clinical studies. Keywords: primary liver cancer, early detection, liver cirrhosis, chronic hepatitis virus infection, methylated plasma DNA marker. Citation Format: Yang Tian, Yanan Wang, Mingxin Pan, Lei Zhang, Qiancheng You, Bingsi Li, Shangli Cai, Feng Shen, Guoyue Lv. An effective cfDNA methylation-based assay in discriminating primary liver cancer from cirrhosis or chronic hepatitis virus infection: marker discovery, phase I pilot, and phase II clinical validation abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 985.