Recently, the paper titled ‘Aberrant fragmentomic features of circulating cell-free mitochondrial DNA enable early detection and prognosis prediction of hepatocellular carcinoma’ was featured in Clinical and Molecular Hepatology, and it provided a very interesting discussion on the usefulness of circulating cell free-mitochondrial DNA (ccf-mtDNA) in the diagnosis and prognosis assessment of hepatocellular carcinoma (HCC) [1]. In response, I wrote an editorial titled ‘Circulating cell-free mitochondrial DNA for diagnosing hepatocellular carcinoma and assessing prognosis,’ and the authors provided detailed replies to it [2]. Therefore, I was pleased to receive the authors’ correspondence and am also very happy to engage in an active discussion with them about this study again. In particular, I would like to express my sincere gratitude to the authors for their detailed discussion of my editorial and for their thorough responses.

First, the authors mentioned the qualitative or quantitative changes of ccf-mtDNA before and after treatment for HCC [3]. A previous study has examined quantitative evaluation based on circulating tumor DNA (ctDNA) in tumors and qualitative evaluation based on serial blood collection [4]. Specifically, when single quantification of ctDNA in tumors was performed, tumor staging, prognostication, genomic analysis, and the selection of targeted therapies were evaluated [4]. Additionally, longitudinal analysis was conducted by quantitatively tracking ctDNA for tumor burden monitoring in response to treatment or qualitatively tracing ctDNA to compare changes in genomic profiles and clonal evolution over time [4]. The study introduced earlier focuses on ctDNA, but it is believed that a similar approach can be applied to ccf-mtDNA by analyzing it before and after treatment, thereby allowing for its potential clinical application. As the authors mentioned, it is believed that the fragmentomics pattern of ccf-mtDNA may change before and after manipulation of HCC tumors, such as transarterial chemoembolization or radiofrequency ablation. The authors are already conducting follow-up studies on this, suggesting that it could be used for prognostic evaluation, and the upcoming results are eagerly anticipated.

Furthermore, the authors also mentioned minimal residual disease (MRD) in HCC, which is crucial as it leads to recurrence during follow-up after surgical resection, ultimately impacting recurrence-free survival in HCC patients [3]. According to a previous study, to detect ctDNA for MRD, 27 genes with a mutation frequency greater than 5% were evaluated through ctDNA profiling in a Chinese HCC cohort [5]. Among them, 14 mutated genes associated with relapse after two years were excluded, while 13 genes associated with early relapse were identified as MRD monitoring genes [5]. These genes were developed into a plasma-only MRD panel [5]. By applying this method, if patients with HCC are categorized into two groups—those who relapse after two years and those who relapse within two years—and the fragmentomic pattern of ccf-mtDNA is analyzed, it is expected that meaningful results will emerge.

Secondly, I agree that the authors mentioned HCC patients with hepatitis B were predominantly included in this study, as it is the most common cause of HCC in Southeast Asia, including Korea and China [3]. While chronic viral hepatitis remains the leading cause of HCC in Asia and Africa, non-viral causes, such as metabolic dysfunction-associated steatotic liver disease and alcoholic liver disease, are more prevalent in North America, Europe, and Australia [6]. Therefore, validating this study’s findings for HCC patients with non-viral chronic liver disease is crucial for the global application of ccf-mtDNA in early HCC diagnosis and prognosis.

Thirdly, as noted by the authors in their correspondence, the changes in ccf-mtDNA fragmentomics according to histological type are still under investigation, with promising results anticipated [3]. In HCC, recurrence is more frequent in the clear cell subtype, and a poor prognosis is associated with the macrotrabecular massive subtype [7]. Therefore, further research linking the clinicopathological features of HCC with ccf-mtDNA is warranted.

Fourth, in cases of high-grade degenerative nodule (DN), which has not yet progressed to HCC but may be considered a precursor stage, instances have been reported where a high-grade DN is identified on radiology but later diagnosed as HCC after surgery, or where HCC is diagnosed on radiology but is found to be high-grade DN on pathology. By leveraging the finding that ccf-mtDNA fragmentation variability is significantly higher, and its correlation with the ccf-mtDNA fragmentation profile of median HC is significantly lower in HCC patients compared to those with chronic viral hepatitis B or liver cirrhosis, as demonstrated in this study, it is anticipated that medical costs associated with the differential diagnosis and treatment of high-grade DN could be reduced. Therefore, ccf-mtDNA could be a valuable tool in preventing delays in surgery for HCC due to misdiagnosis as high-grade DN, as well as preventing unnecessary surgery when high-grade DN is misdiagnosed as HCC.

The authors’ current study extends beyond utilizing the fragmentomic pattern of ccf-mtDNA for the early diagnosis and prognosis of HCC, proposing its potential as a valuable tool for predicting the clinicopathological characteristics of HCC. Therefore, the scope of its application is expected to broaden further through the authors’ future follow-up studies.