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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 33  |  Issue : 3  |  Page : 909-913

Value of mtDNA and ctDNA in patients with hepatocellular carcinoma


1 Department of Clinical Pathology, Faculty of Medicine, National Liver Institute, Menofia University, Menofia, Egypt
2 Department of Clinical Pathology, National Liver Institute, Menofia University, Menofia, Egypt

Date of Submission10-Nov-2018
Date of Decision25-Nov-2018
Date of Acceptance05-Dec-2018
Date of Web Publication30-Sep-2020

Correspondence Address:
Suzan El-Morshedy
Nozha El-Gededa, Cairo
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_349_18

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  Abstract 


Objective
The aim was to evaluate the diagnostic value of mitochondrial DNA and circulating tumor DNA in patients with hepatocellular carcinoma (HCC).
Background
HCC is one of the most common malignant tumors worldwide. Not all patients with HCC are diagnosed using α-fetoprotein alone. Mitochondrial DNA and circulating tumor DNA have been extensively studied over the past few decades and have a high sensitivity and specificity in the detection of HCC.
Patients and methods
Blood samples were obtained from 150 individuals, comprising 50 patients with HCC and 100 apparently healthy participants as a control group. The blood samples were collected in the time period from August 2013 to September 2016. All patients were subjected to routine laboratory investigations and quantification of mitochondrial DNA and circulating tumor DNA by real-time PCR.
Results
There was a significant increase in the levels of mitochondrial DNA and circulating tumor DNA in patients with HCC in comparison with the healthy control group. By using receiver operator characteristic curve, sensitivity and specificity of mtDNA for HCC group were 70% and 75%, respectively with a cutoff of 480 GE/ml, whereas sensitivity and specificity of ctDNA for HCC group was 81% and 88%, respectively, with a cutoff of 348 GE/ml.
Conclusion
Increase sensitivity and specificity of circulating tumor DNA and mitochondrial DNA in HCC make us consider them as potential tumor markers, and analysis of circulating tumor DNA and mitochondrial DNA with other tumor markers could improve the diagnostic sensitivity for HCC.

Keywords: circulating tumor DNA, free DNA in hepatocellular carcinoma, mitochondrial DNA, molecular cancer


How to cite this article:
Montaser LM, El-Shaarawy AA, El-Bary NM, Fathy WM, El-Morshedy S. Value of mtDNA and ctDNA in patients with hepatocellular carcinoma. Menoufia Med J 2020;33:909-13

How to cite this URL:
Montaser LM, El-Shaarawy AA, El-Bary NM, Fathy WM, El-Morshedy S. Value of mtDNA and ctDNA in patients with hepatocellular carcinoma. Menoufia Med J [serial online] 2020 [cited 2020 Oct 22];33:909-13. Available from: http://www.mmj.eg.net/text.asp?2020/33/3/909/296669




  Introduction Top


Worldwide, cancer is the third most common cause of death, directly following cardiovascular diseases and infectious parasitic diseases. According to data published by the WHO 2013 in the world cancer report, cancer deaths will increase dramatically in the forthcoming years. Although there were 7.4 million cancer deaths in 2004, it has been estimated that in 2030, there will be 11.8 million deaths because of cancer [1].

In several branches of biomedical research, the quest for new disease-related biomarkers has become one of the main objectives. When it comes to discovering and developing new biomarkers, oncology seems to be the most ambitious field [2].

During the last few years, a lot of research has been done identifying new cancer biomarkers with the aim to identify high-risk individuals, detect cancer at an early stage, predict outcome, monitor treatment, and screen for disease recurrence. In this respect, the focus is now mainly directed toward the identification of noninvasive cancer biomarkers [3].

Mitochondria are semiautonomous organelles that perform essential functions in cellular metabolism and the regulation of cell death. Although the exact origin of mitochondria is still uncertain, it is widely believed that they arose from an endosymbiotic relationship between a glycolytic protoeukaryotic cell and an oxidative bacterium [4].

Mitochondria possess a double-membrane structure and contain their own genome along with their own transcription, translation, and protein assembly machinery. As such, they are able to maintain genomic independence from the nucleus. However, as a consequence of protomitochondrial genes integrating into the nuclear genome throughout evolution, most mitochondrial proteins are encoded by nuclear DNA (nDNA) and imported into mitochondria [5].

Although it is difficult to prove the involvement of mtDNA mutations in triggering oncogenesis, there is increasing evidence of their significance for tumor growth and metastatic potential and its regulation [6].

CtDNA level was significantly higher in hepatitis B virus (HBV) carriers than in normal controls. Ren and colleagues suggested that ctDNA level correlated inversely with hepatocellular carcinoma (HCC) prognosis, and was attributable to HBV infection in most cases, suggesting that ctDNA may be a predictive marker for the prognosis of HBV-related HCC. In patients with hepatitis C virus (HCV)-related HCC, serum ctDNA levels were positively correlated with the increased expression of several inflammatory cytokine genes, suggesting that serum ctDNA is associated with local inflammation [7].

Considerable research effort has been made on the use of cfDNA as a biomarker in cancer diagnosis. cfDNA from malignancies exhibits characteristic changes such as mutations, deletions, methylations, and microsatellite aberrations, which are distinct from those in benign conditions, and thus may be useful in the diagnosis of HCC [8].

The aim of this study was to evaluate the diagnostic value of mtDNA and ctDNA in patients with HCC.


  Patients and Methods Top


This study was performed at Clinical Pathology Department, National Liver Institute, Faculty of Medicine, Menoufia University. The blood samples used in this study were collected in the time period from August 2013 to September 2016 under ethical considerations. The study included 150 participants, comprising 50 patients selected from those who had been diagnosed with HCC at National liver institute and 100 apparently healthy participants matched for age and sex as the control group. All patients gave informed consent for retention and analysis of their blood for research purpose according to institutional guidelines. Exclusion criteria (excluded participants) were other cancer types, all benign conditions associated with increasing level of mtDNA and ctDNA such as immune and inflammatory diseases, patients received antiviral therapy, patients having a history of excessive alcohol consumption, and patients having received hepatotoxic drugs. All patients and controls were subjected to the following: complete personal, family, and medical history; full clinical examination; abdominal ultrasonography; triphasic computed tomography; and laboratory investigations, including liver function tests, which were supplied by Rochintegra (Rochintegra 400 plus; Basel, Switzerland), α-fetoprotein (AFP) test, which was supplied by Elecsys (Elecsys E411; Basel, Switzerland), and HCV and HBV antibodies, which were detected by ELISA third generation. Positive cases were confirmed by PCR (HCV-RNA and HBV-DNA assay quantitatively). However, for all participants, qPCR for the simultaneous quantification of ctDNA and mtDNA from plasma was done. A multiplex qPCR was performed using the glyceraldehyd-3-phosphat-dehydrogenase and the mtDNA encoded ATPase 8 (MTATP 8) reference genes by real-time biosystem 7500 (Applied Biosystems, Waltham, Massachusetts, USA).

A volume of 9 ml of blood was withdrawn from the patients and controls under complete aseptic condition, and each blood sample was divided into two parts: the first part (7 ml of blood) was put in a plain tube and left to clot, which usually takes 15–30 min. Then, the clot is centrifuging at 1000–2000 g for 10 min. Clear sera were separated, and divided into two aliquots: the first one was used to determine AFP and HCV and HBV antibodies, which were confirmed by PCR for positive cases, and the second one was used for measurement of liver function tests. The second part (2 ml of blood) from the patients and controls was collected in vacutainer tubes containing EDTA from peripheral blood (whole blood samples). These samples are preserved in −80°C until the time of assay. DNA was subsequently isolated by phenol-chloroform extraction and ethanol precipitation QIAamp circulating nucleic acid kit (Qiagen, Hilden, Germany) [9] provided by Applied Biosystems (Branchburg, New Jersey, USA). The DNA samples were divided into aliquots of 100 μl and were stored in −80°C. The ABI PRISM 7500 Sequence Detection System (Applied Biosystems) [10] was used to amplify the glyceraldehyd-3-phosphat-dehydrogenase housekeeping gene and the MTATP 8 gene. The real-time RT-PCR was carried out with 25 μl of total reaction volume, containing 5 μl (40 ng) of DNA, 12.5 μl of TaqMan Universal PCR Master Mix, 4 primers (Microsynth, Balgach, Switzerland), and two probes (both probes from Applied Biosystems) [11], using a 2-min incubation at 50°C followed by an initial denature step at 95°C for 10 min and 40 cycles of 1 min at 60°C and 15 s at 95°C. All samples were analyzed in triplicate to determine the quantities of mtDNA and ctDNA present in the blood samples. Real-time polymerase chain reaction is a laboratory technique that is used to amplify and simultaneously detect or quantify a targeted DNA molecule. The concentrations of ctDNA were calculated according to the standard curves, using known concentration of human genomic DNA. The results were expressed as genome-equivalent (GE) per ml of plasma by using the conversion factor of 6.6 pg of DNA per cell. The average threshold cycle number (Ct) values of the ctDNA and mtDNA were obtained from each case. The level of mtDNA was calculated using the delta Ct (ΔCt) of average Ct of mtDNA and ctDNA (ΔCt = CtmtDNA − CtnDNA) in the same well as an exponent of 2 (2ΔCt) [12].

Statistical analysis of the data

Data were fed to the computer and analyzed using IBM SPSS software package version 20.0 (IBM Corp., Armonk, New York, USA). The following tests were used: sensitivity, specificity, and significance of results as P value were calculated to assess mtDNA and ctDNA in HCC patient group and control group, and to compare between HCC patient group and control group according to mtDNA and ctDNA values, Mann–Whitney test was used. Receiver operator characteristic (ROC) curves from tDNA and ctDNA in HCC patient group with respective points of maximal accuracy for sensitivity and specificity were generated to determine biomarker performance. Spearman's coefficient was used to examine the correlation between the levels of mtDNA and ctDNA and HCV, HBV, AFP, and other parameters in HCC patient group.


  Result Top


The level of mtDNA was significantly elevated in HCC group when compared with the healthy control group (P < 0.001), and the level of ctDNA was significantly elevated in patients with HCC in comparison with the healthy control group (P < 0.001) [Table 1].
Table 1: Comparison between control group and hepatocellular carcinoma patient group according to mtDNA and ctDNA

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The sensitivity and specificity of mtDNA for HCC group was 70 and 75%, respectively, positive predictive value (PPV) was 70%, and negative predictive value (NPV) was 78.2% with cutoff of 480 GE/ml, whereas the sensitivity and specificity of ctDNA for HCC group was 81 and 88%, respectively, PPV was 80%, and NPV was 85.3% with a cutoff of 348 GE/ml by using ROC curve for mtDNA and ctDNA to diagnose patients with HCC [Table 2] and [Figure 1].
Table 2: Sensitivity and specificity for mtDNA and ctDNA to diagnose hepatocellular carcinoma cases versus control

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Figure 1: Receiver operator characteristic curve for mtDNA and ctDNA to diagnose hepatocellular carcinoma cases versus control.

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There was a significant positive correlation between mtDNA and HCV in HCC patient group (P = 0.012) and between mtDNA and HBV (P = 0.004). There was a significant positive correlation between ctDNA and HCV in HCC group (P = 0.016) and between ctDNA and HBV (P = 0.003) [Table 3].
Table 3: Relation between mtDNA, as well as ctDNA, and hepatitis C virus and hepatitis B virus in patients with hepatocellular carcinoma

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There is no significant correlation between mtDNA and ctDNA and AFP in HCC group [Table 4].
Table 4: Relation between mtDNA, as well as ctDNA, and α-fetoprotein in hepatocellular carcinoma patient group

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  Discussion Top


HCC is one of the most common malignant tumors worldwide, with an incidence which ranks third and mortality which ranks second in all malignant tumors. The 1-year survival in patients with HCC without surgical ablation is less than 30%, and the 5-year recurrence rate is approximately 80%, even in patients who undergo radical resection. Therefore, the key to HCC treatment is early detection and diagnosis [13]. Mitochondria play important roles in cellular energy metabolism, free radical generation, and apoptosis. Defects in mitochondrial function have long been suspected to contribute to the development and progression of cancer. The most well-known and best-characterized function of mitochondria is the production of ATP through oxidative phosphorylation. This process is accomplished by a series of protein complexes, collectively known as the respiratory chain, encoded by both ctDNA and mtDNA [14]. The present study was planned to evaluate the role of value of mtDNA and ctDNA in patients with HCC. This study found that mtDNA and ctDNA levels were significantly elevated among HCC cases than control group, and also mtDNA and ctDNA showed a significant positive correlation with HCV and HBV in HCC group. These results in this study match with the results of Hann et al. [15]. The present study showed that in comparison between the two studied groups (HCC group and control group), mtDNA was significantly higher among HCC cases than control group and ctDNA was significantly higher among HCC cases than control group. These results are in line with Overman et al. [16] who showed a significantly increased mtDNA content in the whole blood samples (plasma) of patients with HCC. The change of mtDNA may therefore be important for cancer initiation and progression. They showed that levels of ctDNA were significantly elevated in patients with HCC in comparison with a benign disease group and a healthy control group, whereas the levels of mtDNA were significantly elevated in the HCC group when compared with the healthy control group. Moreover, the results are in agreement with Garcia-Murillas et al. [17] who used MTATP 8 gene, a mitochondrial DNA encoded ATPase, to analyze the quantities of mtDNA in peripheral blood, and the result showed that mtDNA content in the whole blood from patients with HCC significantly decreased in early cancer stage as compared with more advanced stages. This suggests that HCC cells depleted a high amount of mtDNA in the circulating system. mtDNA depletion could affect progression and metastasis of cancer cells by preventing apoptosis and generating cancer-related proteins. In the present study, it was found that sensitivity and specificity of mtDNA for HCC group was 70 and 75%, respectively, PPV was 70%, NPV was 78.2%, whereas the sensitivity and specificity of ctDNA for HCC group was 81 and 88%, respectively, PPV was 80%, and NPV was 85.3% by using ROC curve for mtDNA and ctDNA to diagnose patients with HCC. This result matched with Hann et al. [15]. Moreover, there was a significant positive correlation between mtDNA and HCV in HCC group (P = 0.012), and between mtDNA and HBV, and there was a significant positive correlation between circulating ctDNA and HCV in HCC group and between ctDNA and HBV. However, in this study, there was no significant correlation between mtDNA and ctDNA and AFP in HCC group.


  Conclusion Top


The attractiveness of using ctDNA as a biomarker lies in its noninvasive nature, and a combined use with common already established tumor markers could be the first step to a clinical approach to its use. ctDNA and mtDNA for HCC detection have a high sensitivity and specificity. The combined detection of ctDNA or mtDNA with one or two markers can significantly improve the diagnostic rate of HCC. ctDNA and mtDNA levels allowed to differentiate between the different study groups; however, ctDNA is more sensitive and specific than mtDNA and could be used for distinguishing between patients with HCC and healthy controls. Therefore, ctDNA has the potential of a cancer-specific biomarker, whereas mtDNA may serve as a tumor biomarker.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
El-Khoueiry AB, Sangro B, Yau T, Crocenzi TS, Hsu C, et al. Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial. Lancet 2017; 389:2492–2502.  Back to cited text no. 1
    
2.
Bruix J, Qin S, Merle P, Granito A, Huang YH, Bodoky G, et al. Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebo-controlled, phase 3 trial. Lancet 2017; 389:56–66.  Back to cited text no. 2
    
3.
Ferlay J, Soerjomataram I, Dikshit R, Mathers C, Rebelo M, Parkin M, et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN. Int J Cancer 2015; 136:E359–E386.  Back to cited text no. 3
    
4.
El-Serag HB. Hepatocellular carcinoma. N Engl J Med 2011; 365:1118–1127.  Back to cited text no. 4
    
5.
Fujimoto A, Furuta M, Totoki Y, Kato M, Shiraishi Y, Tanaka H, et al. Whole-genome mutational landscape and characterization of noncoding and structural mutations in liver cancer. Nat Genet 2016; 48:500–509.  Back to cited text no. 5
    
6.
Cancer Genome Atlas Research Network. Comprehensive and integrative genomic characterization of hepatocellular carcinoma. Cell 2017; 169:1327–1341.  Back to cited text no. 6
    
7.
Zhai W, Lim TK, Zhang T, Phang ST, Tiang Z, Guan P, et al. The spatial organization of intra-tumour heterogeneity and evolutionary trajectories ofmetastasesinhepatocellularcarcinoma. Nat Commun 2017; 8:4565.  Back to cited text no. 7
    
8.
Furuta M, Ueno M, Hayami S, Yasukawa S, Kojima F, Arihiro K, et al. Whole genome sequencing discriminates hepatocellular carcinoma with intrahepatic metastasis from multi-centric tumors. J Hepatol 2017; 66:363–373.  Back to cited text no. 8
    
9.
Bardelli A, Pantel K. Liquid biopsies, what we do not know (Yet). Cancer Cell 2017; 31:172–179.  Back to cited text no. 9
    
10.
Kowalik A, Kowalewska M, Gozdz S. Current approaches for avoiding the limitations of circulating tumor cells detection methods – implications for diagnosis and treatment of patients with solid tumors. Transl Res 2017; 185:58–84.  Back to cited text no. 10
    
11.
Ono A, Fujimoto A, Yamamoto Y, Akamatsu S, Hiraga N, Imamura M. Circulating tumor DNA analysis for liver cancers and its usefulness as a liquid biopsy. Cell Mol Gastroenterol Hepatol 2015; 1:516–534.  Back to cited text no. 11
    
12.
Liao W, Yang H, Xu H, Wang Y, Ge P, Ren J. Noninvasive detection of tumor-associated mutations from circulating cell-free DNA in hepatocellular carcinoma patients by targeted deep sequencing. Oncotarget 2016; 7:40481–40490.  Back to cited text no. 12
    
13.
Xu RH, Wei W, Krawczyk M, Wang W, Luo H, Flagg K. Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma. Nat Mater 2017; 16:1155–1161.  Back to cited text no. 13
    
14.
Ng CKY, Di Costanzo GG, Paradiso V, Coto-Llerena M, Roscigno G, et al. Genetic profiling using plasma-derived cell-free DNA in therapy-naïve hepatocellular carcinoma patients: a pilot study. Ann Oncol 2018; 29:1286–1291.  Back to cited text no. 14
    
15.
Hann HW, Jain S, Park G, Steffen JD, Song W, Su YH. Detection of urine DNA markers for monitoring recurrent hepatocellular carcinoma. Hepatoma Res 2017; 3:105–111.  Back to cited text no. 15
    
16.
Overman MJ, Mcdermott R, Leach JL, Lonardi S, Lenz HJ, Morse MA, et al. Nivolumab in patients with metastatic DNA mismatch repair-deficient or microsatellite instability-high colorectal cancer (CheckMate 142): an open-label, multicentre, phase 2 study. Lancet Oncol 2017; 18:1182–1191.  Back to cited text no. 16
    
17.
Garcia-Murillas I, Schiavon G, Weigelt B, Ng C, Hrebien S, Cutts RJ. Mutation tracking in circulating tumor DNA predicts relapse in earlybreastcancer. Sci Transl Med 2015; 7:302ra133.  Back to cited text no. 17
    


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