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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 34  |  Issue : 4  |  Page : 1244-1248

Plasma circulating cell-free DNA integrity as a noninvasive diagnostic tool in hepatocellular carcinoma


Clinical Pathology Department, Faculty of medicine, Menoufia University, Egypt

Date of Submission07-Aug-2021
Date of Decision18-Sep-2021
Date of Acceptance21-Sep-2021
Date of Web Publication24-Dec-2021

Correspondence Address:
Sara M Eldeeb
MD, 32511, Clinical and Chemical Pathology Department, Faculty of Medicine, Menoufia University
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_140_21

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  Abstract 


Background
Hepatocellular carcinoma (HCC) is the world's sixth and Egypt's fourth most common cancer, representing the third leading cause of cancer-related deaths worldwide. Circulating cell-free DNA is a type of cell-free nucleic acid derived from apoptotic, necrotic, and living eukaryotic cells. It carries genetic information consistent with tumor cells and has potential for molecular diagnosis of tumors.
Objectives
To evaluate the clinical significance of plasma circulating cell-free DNA integrity (cfDNAi) as a diagnostic biomarker in HCC compared with alpha-fetoprotein (AFP).
Patients and methods
This case–control study was carried out on 90 participants who were equally classified into three groups: benign liver diseases, HCC groups, and a control group of apparently healthy adults with matched age and sex. Serum AFP and plasma cfDNAi assays were performed for all participants.
Results
Patients with HCC had lower plasma cfDNAi than those with benign liver diseases (P = 0.002) and apparently healthy individuals (P = 0.001). The combination of cfDNAi and AFP in differentiation between the HCC group and healthy individuals revealed a sensitivity, specificity, accuracy, positive predictive value, and negative predictive value of 97, 100, 98, 100, and 97%, respectively.
Conclusion
Plasma cfDNAi can be used as a promising biomarker in the diagnosis of HCC where the combination of cfDNAi and AFP improves the diagnostic performance for HCC diagnosis.

Keywords: alpha-fetoprotein, cell-free DNA, hepatocellular carcinoma, integrity, liquid biopsy


How to cite this article:
Eldeeb SM, Afandy AI, Ahmed O, Khodeer S, Younes F, Hosny D. Plasma circulating cell-free DNA integrity as a noninvasive diagnostic tool in hepatocellular carcinoma. Menoufia Med J 2021;34:1244-8

How to cite this URL:
Eldeeb SM, Afandy AI, Ahmed O, Khodeer S, Younes F, Hosny D. Plasma circulating cell-free DNA integrity as a noninvasive diagnostic tool in hepatocellular carcinoma. Menoufia Med J [serial online] 2021 [cited 2024 Mar 29];34:1244-8. Available from: http://www.mmj.eg.net/text.asp?2021/34/4/1244/333226




  Introduction Top


Hepatocellular carcinoma (HCC) is considered the sixth common cancer all over the world, and it accounts for 75–85% of primary liver cancers [1]. It is considered one of the most aggressive types of human cancer worldwide [2]. An early detection of HCC, followed by effective treatment, is critical for improving the prognosis and reducing the associated economic burden [3].

One of the most commonly used tumor markers for HCC is alpha-fetoprotein (AFP). With a low sensitivity of 62.4% and a cut-off value of 20 ng/ml, AFP is not sensitive and accurate enough for early detection and may reveal false-negative results [4].

Liquid biopsy has rapidly evolved as a diagnostic and monitoring tool in cancer research. The term 'liquid biopsy' encompasses circulating tumor DNA/plasma cell-free DNA (cfDNA), circulating tumor cells, circulating miRNAs, and exosomes [5].

cfDNA are degraded DNA fragments released into the bloodstream. It has been demonstrated to be a promising biomarker, and its concentration was reported to be higher in patients with cancer than in healthy individuals [6].

Cell-free DNA integrity (cfDNAi) was measured as the ratio of longer to shorter DNA fragments [6]. It could be used as a potential prognostic biomarker for solid tumors [7].

The aim of the study was to evaluate the clinical significance of plasma-circulating cfDNAi as a diagnostic biomarker in HCC compared with AFP.


  Patients and methods Top


This study was conducted on 90 patients. The patients were selected from the internal medicine outpatient clinics and inpatient departments of Menoufia University Hospitals.

Informed written consent was attained from all participants, and the study was approved by the Research Ethics Committee of Medical Research, Faculty of Medicine, Menoufia University.

They were divided into three age-matched and sex-matched groups: group I (control group) included 30 apparently healthy participants, group II (benign liver disease group) included 30 patients diagnosed with benign liver diseases, and group III (HCC group) included 30 newly diagnosed patients with HCC confirmed by triphasic computed tomography with elevated AFP levels [8].

All patients and control groups were subjected to detailed history taking, a full clinical examination, and laboratory investigations in the form of complete liver function tests [serum total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase, serum albumin, and prothrombin concentration] and complete blood count with platelet count. Complete blood count was performed using an XT-1800i hematology analyzer (SYSMEX, Kobe, Japan). Serum total bilirubin, ALT, aspartate aminotransferase, and serum albumin were determined using a Beckman AU480 Chemistry analyzer (Beckman Instruments Inc., Carlsbad, California, USA). Prothrombin concentration was done by a STAGO analyzer (Stago Canada Ltd, Mississauga, Canada). Abdominal ultrasonography for tumor size, triphasic computed tomography abdomen, and Child–Pugh classification were done for the HCC group. Measurement of AFP was done by a Cobas e 601 analyzer (Roche Diagnostics, Rotkreuz, Switzerland).

cfDNA was extracted from the plasma using the QIAamp Circulating Nucleic Acid kit (Qiagen, Hilden, Germany.) according to the manufacturer's instructions, and then DNA concentration was measured. Amplification and quantification of 115 and 247-bp ALU amplicons was performed according to the published protocol [6]. cfDNAi was calculated as the ratio of ALU 247 to ALU 115 [9].

Statistical analysis

Data were collected and submitted for statistical analysis using the Statistical Program for Social Sciences, version 19 (SPSS Inc., Chicago, Illinois, USA). The Kruskal–Wallis test is used for comparison between three or more groups not normally distributed having quantitative variables. The analysis of variance test is used for comparison between three or more groups having quantitative variables. Spearman's correlation is used to measure the association between two quantitative variables not normally distributed. Odds ratio describes the probability that people who are exposed to a certain factor will have a disease compared with people who are not exposed to the factor. The receiver operating characteristic curves are used to evaluate the diagnostic utility of cfDNAi. The cutoff value with the highest accuracy was selected as the diagnostic cutoff point. A P value 0.05 was considered significant.


  Results Top


The HCC group showed a significant reduction in cfDNAi in comparison with the benign liver disease and control groups (P = 0.002 and 0.001, respectively) [Table 1].
Table 1 The demographic, clinical, and biochemical characteristics of studied groups

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There was a significant positive correlation between cfDNAi and prothrombin time concentration (P = 0.049, r = 0.363). However, there was no significant correlation between cfDNAi and other parameters [Table 2].
Table 2 Correlation between cell-free DNA integrity and different measured parameters of the hepatocellular carcinoma group

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Age (P = 0.018), ALT (0.002), AFP (0.006), and cfDNAi (P = 0.001) were independent predictors of HCC [Table 3].
Table 3 Association of cell-free DNA integrity and other measured parameters with prediction of hepatocellular carcinoma

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The evaluation of the diagnostic value of cfDNAi in combination with AFP level for discrimination between HCC group and control healthy group revealed that sensitivity, specificity, accuracy, positive predictive value (PPV), and negative predictive value were 97, 100, 98, 100, and 97%, respectively [Table 4].
Table 4 Sensitivity and specificity of cell-free DNA integrity and alpha-fetoprotein in detection of hepatocellular carcinoma among the studied groups

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The evaluation of the diagnostic value of cfDNAi in combination with AFP level for discrimination between HCC group and benign liver disease group revealed that sensitivity, specificity, accuracy, PPV, and negative predictive value were 100, 80, 90, 83, and 100%, respectively [Table 4] and [Figure 1].
Figure 1: (a) ROC curve for sensitivity and specificity of cell-free DNA (cfDNA) integrity in detection of hepatocellular carcinoma (HCC) among benign and HCC groups. (b) ROC curve for sensitivity and specificity of AFP in detection of HCC among benign and HCC groups. (c) ROC curve for sensitivity and specificity of cfDNA integrity in detection of HCC among benign and control groups. (d) ROC curve for sensitivity and specificity of AFP in detection of HCC among control and HCC groups. AFP, alpha-fetoprotein; ROC, receiver operating characteristic.

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  Discussion Top


HCC is considered as one of the most common malignant tumors worldwide with a poor prognosis as it is diagnosed only in advanced stages. The molecular profiling for patients with HCC is obtained from resected tumor materials or biopsies which is limited, so dynamic monitoring of patients cannot be performed. Compared with invasive procedures, circulating tumor DNA has been proposed as an alternative source to perform molecular profiling of tumor DNA in patients with cancer [5].

The advantages of application of circulating molecular markers in peripheral blood (liquid biopsies) are valuable because they are easily accessible, reliable, reproducible, and early detectable in cancer. Blood-based biomarkers such as circulating DNA, circulating tumor cells, and miRNAs have been applied in the diagnosis of many cancers [9].

DNA integrity, the ratio of longer to shorter fragments [10], has been identified as an effective diagnostic biomarker with high pooled sensitivity and specificity in different types of cancer, most notably in colorectal and breast carcinomas, suggesting the importance of liquid biopsy [10],[11].

cfDNA is released during apoptosis in healthy individuals and is uniformly truncated into fragments of around 200 bp. However, in patients with cancer, cfDNA originates from both apoptotic and necrotic sources and is shorter and might thus increase the proportion of shorter DNA copies and lead to decreased cfDNAi in malignancies [6].

In the current study, the clinical significance of plasma cfDNAi as a diagnostic biomarker for HCC in comparison with AFP was evaluated.

It was realized that the cfDNAi of the HCC group was significantly lower than that of the benign disease and healthy control groups, with a significant decrease in the benign group than the control one. Huang et al. [6] reported that cfDNAi significantly decreased in patients with liver malignancies, whereas patients with benign liver diseases and healthy individuals had comparable size distribution.

Such a decrease in cfDNAi can be explained by increased necrosis within tumors resulting in increased fragmented DNA copies released into circulation [12]. The clearance of apoptotic and necrotic debris by infiltrating phagocytes is hampered during tumor growth, thus resulting in its accumulation and secretion into the circulation [13]. Furthermore, Suzuki et al. [14] found that cfDNAi sharply increased after resection of liver cancers.

There was no significant correlation between cfDNAi and age, tumor number, and other laboratory parameters except prothrombin concentration. According to Wang et al. [15], there is a positive linear correlation between total plasma cfDNA and prothrombin time. However, the current study showed no significant correlation between cfDNAi and serum albumin.

In the current study, age, ALT, AFP, and cfDNAi were independent predictors of HCC.

Yan et al. [16] revealed that age and cfDNA, rather than AFP and ALT, were independent predictors of HCC. They also constructed an HCC index (combination biomarker model including age, cfDNA and AFP), which presented the optimal performance for HCC screening.

Elevated serum AFP levels (>20 ng/ml) correlate with an increased risk for HCC development. Although the sensitivity of AFP is excellent, its specificity is low [17]. Moreover, AFP may be elevated in cirrhosis or hepatitis cases and not all HCCs secrete AFP. So, the combination of multiple biomarkers can be used to effectively improve diagnostic efficacy [3].

Regarding the ability of cfDNAi and AFP level in differentiating patients with HCC from healthy individuals and the benign liver disease group, it was found that the area under the curve value of cfDNAi is lower than that of AFP.

However, the combination of cfDNAi and AFP yielded better diagnostic performance in differentiation between the HCC group and healthy individuals. This is in contrast to Yan et al. [16], who reported that the area under the curve value of cfDNAi was better than that of AFP. However, comparison between the two markers in benign liver disease and HCC groups revealed that cfDNAi showed better sensitivity and PPV than AFP. Yan et al. [16] reported that the diagnostic performance can be improved when cfDNA is used in combination with age and AFP.

Huang et al. [6] reported that the application of cfDNAi in early detection of cancer is impractical. The application of novel biomarkers for early cancer detection should undergo five phases, and AFP is the only biomarker that has finished all five phases and been approved by the Food and Drug Administration [18]. Limitation to our study was that cfDNA is not specific and is elevated in other malignancies and pathologies, such as in acute bacterial or viral infections and severe chronic disease. Another limitation was the relatively small size of samples. So, we recommend further studies including a large sample size to determine whether cfDNAi assay is a useful tool in early detection of patients with HCC hoping to prevent delayed diagnosis of such disease. Moreover, we recommend evaluation of cfDNAi in combination with other biomarkers like lens culinaris agglutinin and des-gamma-carboxy prothrombin for the diagnosis of HCC.


  Conclusion Top


cfDNAi is significantly decreased in patients with HCC. It is better to use cfDNAi with AFP to improve the diagnostic performance for HCC diagnosis. However, its application as a diagnostic biomarker needs more high-quality studies to further explore its diagnostic performance in HCC diagnosis.

Conflicts of interest

None declared.



 
  References Top

1.
Rashed WM, Kandeil MAM, Mahmoud MO, Ezzat S. Hepatocellular carcinoma (HCC) in Egypt: a comprehensive overview. J Egypt Natl Canc Inst 2020; 32:5.  Back to cited text no. 1
    
2.
Huang X, Sun L, Wen S, Deng D, Wan F, He X, et al. RNA sequencing of plasma exosomes revealed novel functional long noncoding RNAs in hepatocellular carcinoma. Cancer Sci 2020; 111:3338–3349.  Back to cited text no. 2
    
3.
Wang T, Zhang KH. New blood biomarkers for the diagnosis of AFP-negative hepatocellular carcinoma. Front Oncol 2020; 10:1316.  Back to cited text no. 3
    
4.
Okajima W, Komatsu S, Ichikawa D, Miyamae M, Ohashi T, Imamura T, et al. Liquid biopsy in patients with hepatocellular carcinoma: circulating tumor cells and cell-free nucleic acids. World J Gastroenterol 2017; 23:5650–5668.  Back to cited text no. 4
    
5.
Wu X, Li J, Gassa A, Buchner D, Alakus H, Dong Q, et al. Circulating tumor DNA as an emerging liquid biopsy biomarker for early diagnosis and therapeutic monitoring in hepatocellular carcinoma. Int J Biol Sci 2020; 16:1551–1562.  Back to cited text no. 5
    
6.
Huang A, Zhang X, Zhou SL, Cao Y, Huang XW, Fan J, et al. Plasma circulating cell-free DNA integrity as a promising biomarker for diagnosis and surveillance in patients with hepatocellular carcinoma. J Cancer 2016; 7:1798–1803.  Back to cited text no. 6
    
7.
Ravaioli S. Cell-Free DNA Integrity: Applications. Methods Mol Biol. 2019;1909:77-83. doi: 10.1007/978-1-4939-8973-7_5. PMID: 30580423.  Back to cited text no. 7
    
8.
Murakami T, Kim T, Takahashi S, Nakamura H. Hepatocellular carcinoma: multidetector row helical CT. Abdom Imaging 2002; 27:139–46.  Back to cited text no. 8
    
9.
Cheng J, Tang Q, Cao X, Burwinkel B. Cell-free circulating DNA integrity based on peripheral blood as a biomarker for diagnosis of cancer: a systematic review. Cancer Epidemiol Biomarkers Prev 2017; 26:1595–1602.  Back to cited text no. 9
    
10.
Stötzer OJ, Lehner J, Fersching-Gierlich D, Nagel D, Holdenrieder S. Diagnostic relevance of plasma DNA and DNA integrity for breast cancer. Tumor Biol 2014; 35:1183–91.  Back to cited text no. 10
    
11.
Thakur S, Tobey A, Daley B, Auh S, Walter M, Patel D, et al. Limited utility of circulating cell-free DNA integrity as a diagnostic tool for differentiating between malignant and benign thyroid nodules with indeterminate cytology (Bethesda Category III). Front Oncol 2019; 9:905.  Back to cited text no. 11
    
12.
Wang BG, Huang HY, Chen YC, Bristow RE, Kassauei K, Cheng CC, et al. Increased plasma DNA integrity in cancer patients. Cancer Res 2003; 63:3966–8.  Back to cited text no. 12
    
13.
Diaz LA, Bardelli A. Liquid biopsies: genotyping circulating tumor DNA. J Clin Oncol Off J Am Soc Clin Oncol 2014; 32:579–586.  Back to cited text no. 13
    
14.
Suzuki N, Kamataki A, Yamaki J, Homma Y. Characterization of circulating DNA in healthy human plasma. Clin Chim Acta 2008; 387:55–58.  Back to cited text no. 14
    
15.
Wang D, Hu X, Long G, Xiao L, Wang ZM, Zhou LD. The clinical value of total plasma cell-free DNA in hepatitis B virus-related hepatocellular carcinoma. Ann Transl Med 2019; 7:650.  Back to cited text no. 15
    
16.
Yan L, Chen Y, Zhou J, Zhao H, Zhang H, Wang G. Diagnostic value of circulating cell-free DNA levels for hepatocellular carcinoma. Int J Infect Dis 2018; 67:92–97.  Back to cited text no. 16
    
17.
Zacharakis G, Aleid A, Aldossari KK. New and old biomarkers of hepatocellular carcinoma. Hepatoma Res 2018; 4:65.  Back to cited text no. 17
    
18.
Pepe MS, Etzioni R, Feng Z, Potter JD, Thompson ML, Thornquist M, et al. Phases of biomarker development for early detection of cancer. J Natl Cancer Inst 2001; 93:1054–61.  Back to cited text no. 18
    


    Figures

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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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