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Cell-Free DNA in Hepatocellular Carcinoma: Comparison
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Please note this is a comparison between Version 2 by Dean Liu and Version 1 by Pattapon Kundirek.

Cell-free DNA (cfDNA) has been used as a non-invasive biomarker for detecting cancer-specific mutations. 

  • hepatocellular carcinoma
  • cell-free DNA
  • whole-exome sequencing
  • biomarker
  • Thailand
  • Oxford Nanopore Technologies
  • copy-number variants

1. Introduction

Hepatocellular carcinoma (HCC) is the most common type of liver cancer (80% of liver cancer). Liver cancer is considered the sixth most common cancer and the second leading cause of death worldwide. A high incidence of HCC has been observed in Eastern Africa and Southeast Asia, including Thailand [1]. HCC frequently develops in the context of underlying chronic liver diseases such as chronic hepatitis or cirrhosis. Most HCC patients are diagnosed at an advanced stage of HCC and experience a short survival time after diagnosis. However, early HCC diagnosis can improve survival due to the efficacy of therapeutic approaches—resection and transplantation are effective therapeutics, but only for early-stage HCC [2]. Therefore, early diagnosis is the key to a good prognosis. Currently, serum alpha-fetoprotein (AFP) is the most widely used biomarker for HCC screening. However, serum AFP assay has a low sensitivity (62.4%) with a high false-negative rate for early HCC diagnosis [3].
Cell-free DNA (cfDNA) consists of short fragments of double-stranded DNA, with lengths ranging from 160 to 200 base pairs. It can be released into plasma from apoptotic or necrotic tumor cells as circulating tumor DNA [4]. Therefore, cfDNA has been used for the non-invasive diagnosis of cancers to provide comprehensive information regarding cancer-associated genetic profiles such as single nucleotide variants (SNVs), copy number variants (CNVs), and epigenetic patterns [4]. In previous studies, the potential utility of cfDNA levels and mutations as a potential clinical biomarker for HCC were investigated, as reviewed by Howell et al. [5]. Based on the eight genes associated with HCC identified from the COSMIC database [6], Howell et al. [7] reported mutations in ARID1A (11.7%), CTNNB1 (7.8%), and TP53 (7.8%) to occur frequently in HCC cfDNA in a European population. The somatic mutations in cfDNA, including SNVs and CNVs, were used to monitor for HCC recurrence in a long-term followup study of a Chinese population [8]. cfDNA was considered a secondary alternative to tumor biopsy to observe genomic alterations of intratumoral heterogeneity (ITH) in HCC that may preclude the need for repeated biopsies [9,10][9][10]. Recently, it was shown that the mutated genes of cfDNA represented cancer-associated genes in 63% (19/30) of patients, and cfDNA could be used for tumor genetic profiling when a biopsy is unavailable [11]. These studies indicated that cfDNA could be a tumor marker for diagnostic and real-time malignancy monitoring to help adjust or guide treatment plans. However, the utility of cfDNA quantification and somatic mutation detection for HCC in a Thai population has not been investigated at the genome-wide level.
HCC is highly heterogeneous in terms of genome composition and genes mutated [12]. This malignancy commonly presents with the molecular anomalies of mutations in the TERT promoter (60% of the patients in the study), TP53 (35–50%), CTNNB1 (20–40%), AXIN1 (9–13%), LAMA2 (5–12%), ARID1A (12%), WWP1 (9%), and RPS6KA3 (8%) genes [13]. However, ethnicity could contribute to global differences in the molecular profile of HCC due to the presence of various risk factors such as hepatitis B virus, hepatitis C virus, alcohol, and metabolic syndrome [14]. Some somatic mutations of HCC in Thailand were consistent with those identified in COSMIC HCC data, but many mutated genes in Thai HCC patients were not found in the COSMIC data [15]. Thus, it is important to characterize the mutational profiles of cfDNA from HCC patients in Thailand even though previous studies of HCC cfDNA have been performed [7,8,9,11,16,17][7][8][9][11][16][17]. Further, most studies performed targeted sequencing (~140 genes), thus missing many potential mutations that might be important in HCC. Therefore, we reasoned that whole-exome sequencing (WES) could provide more comprehensive data to investigate the mutational landscape of cfDNA.

2. Cell-Free DNA in Hepatocellular Carcinoma

cfDNA is released from apoptotic and necrotic cells into the blood circulation [25][18]. In normal conditions, the clearance of cfDNA is conducted by immune cells. However, in tumor conditions, the clearance of cfDNA is not efficient, leading to an accumulation of cellular debris such as DNA [26][19]. cfDNA can be detected in both cancer patients and healthy patients, but the levels of cfDNA differ between the two [27][20]. An increase in cfDNA in the blood circulation was observed primarily in patients with tumoral mass compared with non-tumor patients [27][20]. In concordance with previous studies in HCC [28[21][22],29], the levels of cfDNA were significantly higher in patients with HCC than in patients with CH and were associated with worse clinical parameters, including tumor size and BCLC stage. Specifically, the high levels of cfDNA were found in patients with advanced-stage HCC (BCLC stage C) compared with early-stage (stage A) and intermediate stage (stage B). These results suggest that the level of cfDNA reflects tumor progression to a certain extent. Currently, the conventional biomarker for detecting HCC and its recurrence (serum AFP) has limited sensitivity to detect early HCC and can also be elevated in other disease states. A previous study demonstrated that cfDNA could improve the diagnosis of HCC when combined with serum AFP [30][23]. In agreement with this report, the combination of plasma cfDNA and serum AFP assays increased the performance of HCC screening over either marker alone. These results imply that cfDNA could increase the efficiency of discriminating HCC patients from non-cancer patients. In addition to analyzing the cfDNA concentration, we performed WES on cfDNA from patients with HCC and analyzed for genetic alterations that could reflect the genetic profile of the tumor mass [11]. To the best of our knowledge, this is the first study that demonstrated genetic alterations in cfDNA from patients with HCC in Southeast Asia (Thailand), where there is a high incidence of HCC [1]. There have been a few studies that reported genetic alterations in cfDNA from a small number of HCC patients using WES [9,22][9][24]. In agreement with these reports, we found the same genes to be frequently mutated, including TP53 (detected in most of the cancers), FLG, TTN, and ADAMTS12, and WES analysis of cfDNA could be used to detect mutated genes in all HCC patients. When comparing with targeted sequencing of cfDNA, WES analysis provides more comprehensive data on the entire set of mutated genes in samples and does not require previous knowledge of the mutational profile [31][25]. Importantly, the sensitivity of low variant detection is inverse to the proportion of the size of gene panel to sequencing cost [32][26]. Although we performed WES to analyze cfDNA, the lowest mutation allele frequency was around 0.6–1% in this study. However, the gene alterations identified in cfDNA from patients with HCC were partially concordant with those identified in another WES study on cfDNA and tumoral tissues from Chinese HCC patients [9]. Interestingly, a previous study of cfDNA without prior knowledge of the mutation profile in biopsy tissues demonstrated that 27% of mutations in cfDNA were present in the biopsy [33][27]. This was similar to our study in that we found 31% concordance between mutated genes in cfDNA and HCC tissue in Thai patients [15]. Furthermore, although our cohort consisted of cfDNA and germline DNA from patients who underwent nonoperative treatment (meaning we were unable to access tumor tissue), we still found the mutations in cfDNA in concordance with other studies [9,15,22][9][15][24] of HCC tissues. These data indicate that cfDNA data could be used to reflect the tumor genome when tumor DNA is not available [33][27]. In this study, we found that the eight most frequently mutated genes in cfDNA from HCC were also frequently mutated in HCC tissue from TCGA data, including TP53 (33%), TTN (30%), FLG (17%), OBSCN (16%), HRNR (13%), and ADAMTS12 (4%). Indeed, previous studies demonstrated that TP53, TTN, FLG, and OBSCN were frequently mutated genes in HCC patients [34,35][28][29]. Regarding FLG and OBSCN, these mutations were found in Asian patients with HCC, and FLG was altered in Asian patients more than in any other ethnicity [35][29]. Interestingly, in our study, ZNF814 and ZNF492 were also frequently mutated at the same sites in cfDNA. Even though these mutations occurred at low frequency in HCC tissue from TCGA data, a recent finding showed that mutations in the ZNF family are associated with human disease, including cancer [36,37][30][31]. Several things could account for the high rates of specific mutations found in the current study. One is that our study was based on an Asian population, but current databases are based primarily on White populations. There are different causes of HCC and different genetic backgrounds, even in Asian countries [12]. On the other hand, the HCC study in Thailand demonstrated that HCC subtypes of different ethnicities were not completely matched between Thai HCC patients and those of other races/ethnicities, and somatic mutations from Thai HCC patients were not entirely in agreement with the COSMIC database [15]. In addition, the co-occurrence of mutations in HRNR and TTN in our study was associated with a worse prognosis in patients with HCC. Thus, the mutations in cfDNA might be prognostic markers for patients with HCC, but this requires further investigation. The detection of mutations in plasma cfDNA in HCC provides exciting possibilities for guiding treatment in patients. We identified patients with an activating hotspot mutation to the CTNNB1 gene in the Wnt/beta-catenin pathway. In previous studies [38[32][33],39], it was demonstrated that the mutation of S33C and S37A in CTNNBB1 might lead to loss of phosphorylation sites in the beta-catenin protein, increasing the expression of CTNNB1 and dysregulating the Wnt/beta-catenin pathway. In this context, a recent study of 31 patients with HCC treated with an immune checkpoint inhibitor demonstrated that the activation of Wnt/beta-catenin signaling was associated with poor response to therapy and shorter survival [40][34]. Moreover, a study of 17 regorafenib-treated HCC patients demonstrated that CTNNB1 mutation was found exclusively in non-responders [41][35]. Sorafenib is used globally as a standard first-line treatment for advanced HCC and targets multiple kinases, including BRAF, a serine/threonine-protein kinase. In a previous study, we identified patients with BRAF mutations, and these correlated with response to the multi-kinase inhibitor sorafenib [42][36]. Thus, cfDNA profiling may allow the use of precision oncology to improve the efficiency of treatments and ultimately the clinical outcome of patients with HCC. In CNV analysis, amplifications in chromosomes 1q, 3q, 7q, 8q, 12p, 15q, and 17q and loss in chromosome 5p-q were observed in 16.67% of cfDNA HCC samples (5/30 samples). Even though the analysis of CNVs in cfDNA was complicated by a high background signal in our study (due to the fragmentation of cfDNA and sequencing bias from WES), the CNVs we identified were similar to those identified in previous studies of HCC tissue and cfDNA, such as gains in chromosomes 1q, 7q, 8q, and 17q [43,44][37][38]. These CNVs of cfDNA were also used for scoring genomic instability, which was associated with tumor progression and overall survival time in patients with HCC [44][38]. These data suggest that CNVs of cfDNA could serve as prognostic markers for HCC. Recently, SMURF-seq was developed to improve the efficiency of CNV analysis by concatenating short fragments into long molecules before sequencing [45][39]. SMURF-seq can be performed with low-coverage reads, shorter time, and at low cost and obtain similar CNV data as short-read sequencing within a day; it also uses a portable device that would be suitable for clinical sites. Therefore, SMURF-seq was used to perform CNV analysis on HCC tissues to compare with CNVs from cfDNA. To our knowledge, this is the first study to perform CNV analysis for HCC using nanopore technology. SMURF-seq clearly identified CNVs in HCC, such as gains in chromosomes 1q and 8q, which are commonly found in HCC cfDNA and tissue [44][38]. These results indicate that gains in chromosomes 1q and 8q were concordant across HCC cfDNA and tissue DNA, as identified with WES and SMURF-seq. Even though SMURF-seq can give a cursory view of CNVs in HCC tissues, greater sequencing depth is still needed to improve the resolution of CNVs to ensure the reliability of CNV detection. （The editor will correct the format and rearrange the reference after the entry is online）

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