We thank Dr. Hyuk Soo Eun for his interest in our recent publication “Aberrant fragmentomic features of circulating cell-free mitochondrial DNA enable early detection and prognosis prediction of hepatocellular carcinoma” [1] and their thoughtful editorial [2]. Previous studies have demonstrated the potential of circulating cell-free nuclear DNA (ccf-nDNA) fragmentomics in detecting cancers [3-6]. In our previous study, we comprehensively and systematically described the characteristics of ccf-mtDNA fragmentomics for the first time, and innovatively proposed the theory that tumors have unique characteristics of ccf-mtDNA fragmentomics [7]. In the Editorial, the shortcomings pointed out by Hyuk Soo Eun are invaluable feedback. In the following, we would like to respond to and discuss several key issues.

As Professor Hyuk Soo Eun mentioned, we also acknowledge the importance of the study on how ccf-mtDNA changes quantitatively and qualitatively before and after surgical management. Therefore, we analyzed ccf-mtDNA multiple features (including copy number, number of mutations, and fragmentomics) in 30 patients with hepatocellular carcinoma (HCC) before and after the first-time transarterial chemoembolization (TACE) treatment using capture-based ccf-mtDNA sequencing data. We found that the ccf-mtDNA features can be used for prognosis prediction and efficacy evaluation in HCC patients undergoing TACE treatment (unpublished data). Additionally, we are conducting another study to evaluate the clinical value of ccf-mtDNA fragmentomics for detecting minimal residual disease in patients with HCC by collecting plasma samples before surgery and on days 1, 3, and 7 post-surgery.

In the Editorial, Professor Hyuk Soo Eun raised the point that most of the HCC patients enrolled in this study were hepatitis B virus (HBV) origin, and the proportion of non-HBV-related HCC was relatively low. In China, 85% of HCC cases are attributed to HBV infection [8,9]. And the disease progression typically follows a path from chronic hepatitis B (CHB) to liver cirrhosis (LC) and ultimately to HCC [10,11]. To focus on detection of HBV-related HCC, our study only included patients with HBV-related cirrhosis as control. In the future, we aim to broaden our research scope to encompass non-HBV-related HCCs, including those arising from chronic hepatitis C, alcohol-related liver disease, and metabolic dysfunction-associated steatotic liver disease.

We acknowledge that the correlation between ccf-mtDNA fragmentomics and both the histologic types and degrees of tumor differentiation in HCC remains incompletely determined, representing a crucial area of ongoing research. As emphasized by Professor Hyuk Soo Eun, the histologic features of a tumor, being intrinsic properties, exert a significant influence on the prognosis of patients. Therefore, further studies are necessary to explore the association between cf-mtDNA fragmentomics and the histologic type and tumor differentiation of HCC.

We appreciate Professor Hyuk Soo Eun for insightful comments. Both distinguishing whether lesions that appear as degenerative or regenerative nodules on imaging have histologically progressed to early HCC and monitoring cirrhotic nodules that have not yet developed into HCC hold significant clinical importance for early detection of malignancy, assessing disease progression, and guiding treatment decisions. However, in clinical practice, patients with low or high grade degenerative nodule (DN) need to be diagnosed by liver biopsy and pathological analysis, which is an invasive examination and not clinically acceptable to most patients. Therefore, in the present study, patients with CHB/LC were only diagnosed by patient’s history, clinical manifestations and signs, laboratory test results and imaging findings, without diagnosis information of low or high grade DN available. We fully recognize that the ultimate goal of serological diagnosis for HCC is to replace radiological diagnosis. It is warranted for further studies on differential diagnosis between low or high grade DN and HCC, and monitoring cirrhotic nodules that have not yet developed into HCC.

In conclusion, we once again express our gratitude to Professor Hyuk Soo Eun for the insightful comments. We appreciate for the opportunity to discuss the clinical application value of ccf-mtDNA fragmentomics in HCC. We anticipate significant advances to improve the prognosis of the patients with HCC.