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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 35  |  Issue : 1  |  Page : 20-25

Plasma highly upregulated in liver cancer as a biomarker for the detection of hepatocellular carcinoma


1 Clinical Pathology Department, National Liver Institute, University of Menoufia, Shebin Elkom, Egypt
2 Hepatology Department, National Liver Institute, University of Menoufia, Shebin Elkom, Egypt

Date of Submission03-Dec-2020
Date of Decision14-Jan-2021
Date of Acceptance18-Jan-2021
Date of Web Publication18-Apr-2022

Correspondence Address:
Ghada A Mosaed
Talat Harb street- shebein Elkoom
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_438_20

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  Abstract 


Background
Hepatocellular carcinoma (HCC) is a leading cause of cancer death. Because alpha-fetoprotein (AFP) has low diagnostic accuracy, we studied a novel diagnostic biomarker for HCC diagnosis.
Objectives
To evaluate plasma level of highly upregulated in liver cancer (HULC) long noncoding RNA as a biomarker for HCC diagnosis.
Patients and methods
This case–control study included 75 participants in three groups: patients with HCC on top of hepatitis C virus (group I), hepatitis C virus cirrhotic patients (group II), and healthy controls (group III). Clinical examination, radiological and laboratory investigations were done for all cases. HULC was measured by real-time quantitative PCR. AFP and viral markers were determined by enzyme immunoassay based on electro-chemiluminescence. Liver function tests were done on Cobas 6000 analyzer.
Results
The study included 75 participants in three matched groups of age and sex (no of each group = 25 participants). The parameters that showed significant difference (P ≤ 0.05) by univariate analysis were model for end-stage liver disease, Child–Pugh, lower limb edema, ascites, international normalized ratio, alanine aminotransferase, aspartate aminotransferase, total bilirubin, creatinine, AFP, HULC level, albumin, and platelet count. However, multivariate analysis demonstrated that high AFP and HULC levels were the parameters that could independently diagnose HCC. The mean ± SD of HULC of the three groups was 2.1 ± 1.6, 0.5 ± 0.4, and 0.1 ± 0.05, respectively. HULC level at cutoff point of 1.08 ng/ml had sensitivity of 72% and specificity of 92% to diagnose HCC. AFP at cutoff point of 25.55 ng/ml had sensitivity of 68% and specificity of 88%. HULC level was significantly correlated with HCC tumor size, tumor number, and presence of portal vein thrombosis.
Conclusion
HULC level could act as a novel biomarker for HCC diagnosis.

Keywords: alpha-fetoprotein, hepatitis C virus, hepatocellular carcinoma, highly upregulated in liver cancer, liver cirrhosis


How to cite this article:
Mosaed GA, Fouad TR, Naguib MA, Raia GY, Gaweesh EA. Plasma highly upregulated in liver cancer as a biomarker for the detection of hepatocellular carcinoma. Menoufia Med J 2022;35:20-5

How to cite this URL:
Mosaed GA, Fouad TR, Naguib MA, Raia GY, Gaweesh EA. Plasma highly upregulated in liver cancer as a biomarker for the detection of hepatocellular carcinoma. Menoufia Med J [serial online] 2022 [cited 2024 Mar 29];35:20-5. Available from: http://www.mmj.eg.net/text.asp?2022/35/1/20/343123




  Introduction Top


Hepatocellular carcinoma (HCC), a major type of primary liver cancer, is a leading cause of cancer death worldwide [1]. Alpha-fetoprotein (AFP), the widely used tumor marker for HCC diagnosis, has low sensitivity and specificity. Thus, identification of reliable biomarker for HCC diagnosis with high sensitivity and specificity is needed to improve prognosis of these patients [2].

Noncoding RNAs are classified according to their length into small noncoding RNAs and long noncoding RNAs (lncRNAs), ranging from 200 to over 10 000 nucleotides [3]. LncRNAs play a very important role in complex cellular processes, such as cell death, growth, differentiation, epigenetic regulation, genomic imprinting, and chromatin modification [4],[5],[6].

Several lncRNAs have been identified as reliable biomarkers for early detection and accurate prognosis for various carcinomas [7]. Highly upregulated in liver cancer (HULC) is a lncRNA located on chromosome 6p24.3 with ~500 nucleotides in length, contains two exons, does not have open reading frame and does not give rise to any protein [8].

HULC is upregulated in a wide spectrum of human cancers, including HCC, osteosarcoma, pancreatic cancer, colorectal cancer, gastric cancer, and large B-cell lymphoma [9],[10]. The aim of the work was to evaluate plasma level of HULC lncRNA as a biomarker for HCC diagnosis.


  Patients and methods Top


This is a case–control study that included patients (>18 years old) with HCC on top of hepatitis C virus (HCV), HCV cirrhotic patients, and apparently healthy participants (control). This study was done at National Liver Institute Hospital, Menoufia University, from March 2018 till December 2019. We excluded patients with hepatitis B virus infection, other etiologies of HCC, and cancer of any organ other than the liver. Our study was approved by the ethical committee of the National Liver Institute, Menoufia University.

Diagnosis of HCC was based on positive triphasic computed tomography scan. Diagnosis of liver cirrhosis was based on clinical examination, ultrasonography, and laboratory parameters.

CBC was done using Sysmex XT-1800i (Sysmex Corporation, Kobe, Japan) automated hematology analyzer. Liver function tests (aspartate transaminase, alanine transaminase, serum albumin, total and direct bilirubin) were done on the Cobas 6000analyser (c501 module; Roche Diagnostics, GmbH, Mannheim, Germany). AFP and viral markers (HCV antibodies, hepatitis B surface antigen and hepatitis B core antibodies) were determined by electrochemiluminescence-based enzyme immunoassay 'ECLIA' using Cobas 6000 immunoassay analyzer (e 601 model).

Plasma level of HULC lncRNA analysis was assessed by real-time quantitative (RT-qPCR).

Whole blood samples collected in EDTA tubes were centrifuged at 1000 rpm for 3 min to spin down the blood cells. Total RNA was extracted from plasma using MiRNeasy Mini Kit (Cat. no. 217004; Qiagen, Hilden, Germany) followed by reverse transcription using Thermo-Fisher Scientific High Capacity cDNA. The RT-qPCR was performed using Qiagen Rotor GENE Q RT-PCR system according to the manufacturer's instructions. The reaction mixture included 12.5 μl of thermo scientific Maxima SYBR Green qPCR Master Mix Kit (Cat. K0251), 1.25 μl of forward primer, 1.25 μl of reverse primer, 8 μl of nuclease free water, and 2 μl of template DNA (cDNA). The HULC primers were F: 5′ATC TGC AAG CCA GGA AGA GTC 3′, R: 5′- CTT GCT TGA TGC TTT GGT GTGT 3′and GADPH primers were; F: 5′ CAT CAC CAT CTT CCA GGA GCG 3′, R: 5′ TGA CCT TGC CCA CAG CCTT 3′. GAPDH was used as a housekeeping gene. The cycling conditions of the reactions were as follows: 95°C for 15 min, followed by 40 cycles at 94°C (15 s), then 52 and 55°C were annealing temperatures of HULC and GAPDH, respectively, and finally 70°C for 30 s. Expression level of HULC in each sample was normalized to that of the internal control, GAPDH. The fold change of HULC expression in tumor samples versus healthy controls was calculated by delta-Ct method algorithms.

Statistical analysis

All data were statistically analyzed using Statistical Package of Social Science (SPSS version 22.0; IBM/SPSS Inc., Chicago, Illinois, USA).

Qualitative variables were compared using a χ2 test, whereas quantitative continuous data were compared using analysis of variance test. P value less than 0.05 was considered statistically significant. Univariate analysis with linear Pearson correlation was done. Multivariate logistic regression analysis was done to variables that were significantly related to HCC. The area under the receiver operating characteristic (ROC) curve was done for variables that were significant by multivariate analysis.


  Results Top


Our study was carried on three matched groups in age and sex, with each group including 25 participants.

There was a statistically significant difference (P ≤ 0.05) among the three studied groups regarding model for end-stage liver disease (MELD), Child–Pugh grading, lower limb edema, and ascites [Table 1].
Table 1: Demographic and clinical parameters of studied groups

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There was no significant statistical difference (P > 0.05) among the three studied groups regarding age and sex [Table 1].

There was a statistically significant difference (P ≤ 0.05) among the three studied groups regarding international normalized ratio (INR), alanine aminotransferase, aspartate aminotransferase, total bilirubin, creatinine, albumin, platelet count, AFP, and HULC level [Table 2].
Table 2: Laboratory parameters of studied groups

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There was no statistically significant difference (P > 0.05) among the three studied as regarding hemoglobin and direct bilirubin [Table 2].

These results are measured by ETI correlation test.

There was a significant correlation (P ≤ 0.05) between HULC level and each of the following: tumor size, tumor number, and portal vein thrombosis in patients with HCC [Table 3].
Table 3: Correlation between highly upregulated in liver cancer level and different tumor characters among hepatocellular carcinoma group patients

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There was no significant correlation (P > 0.05) between HULC level and tumor site in patients with HCC [Table 3].

There was a significant difference (P < 0.05) among the three studied groups regarding AFP and HULC [Table 4].
Table 4: Logistic regression analysis of variables with significant difference in univariate analysis (reference category is control group)

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There was no significant difference (P > 0.05) among the three studied groups regarding MELD score, Child–Pugh, lower limb edema, ascites, INR, alanine aminotransferase, aspartate aminotransferase, total bilirubin, creatinine, albumin, and platelet count [Table 4].

HULC level has sensitivity of 72% and specificity of 92% at cutoff point of 1.08 ng/ml to diagnose HCC. AFP has sensitivity 68% and specificity 88% at cutoff point of 25.55 ng/ml to diagnose HCC. AUC for HULC and AFP was 0.86 and 0.80, respectively. Accuracy for HULC and AFP was 82 and 78%, respectively [Table 5].
Table 5: Area under curve for highly upregulated in liver cancer and alpha-fetoprotein

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Areas under the ROC curves of HULC and AFP were 0.86 and 0.80, respectively. The greatest area under curve (0.94) is for combination of HULC and AFP [Figure 1].
Figure 1: ROC curves of HULC, AFP, and their combined measurements for discrimination between HCC and cirrhotic groups. AFP, alpha-fetoprotein; HCC, hepatocellular carcinoma; HULC, highly upregulated in liver cancer; ROC, receiver operating characteristic.

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  Discussion Top


HCC is the most common cancer with high mortality rate because of the difficulty in early diagnosis and treatment [11]. Although AFP is the most common tumor marker for HCC diagnosis, it has low diagnostic accuracy [12]. Therefore, we studied HULC lncRNA as a promising marker for HCC diagnosis.

LncRNAs are involved in the regulation of various cell processes, including gene expression, alternative splicing, and transcriptional regulation [13]. LncRNAs expression is associated with tumor occurrence, invasion, and metastasis [14]. Many lncRNAs can be used as biomarkers in various human cancers [15],[16],[17].

Many studies have reported that lncRNAs were up-regulated or down-regulated in tumor tissues in contrast to nontumor tissues [18],[19]. Few studies have measured lncRNAs expression in plasma of HCC patients because of their unstable expression and easily degradation in plasma.

Previous researchers studied HULC expression in patients with hepatitis B virus-related HCC, but in our study, we tested HULC expression in plasma of patients with HCV-related HCC. Our results showed that HULC is significantly overexpressed in patients with HCC compared with cirrhotic and control groups. In addition, HULC was significantly correlated with tumor size, tumor number, and the presence of portal vein thrombosis. These results are in agreement with Li et al.[20] who reported that HULC expression in plasma was significantly correlated with tumor size and tumor capsular. Hence, HULC levels might reflect progression of the tumor.

The parameters that showed significant difference between groups of our study by univariate analysis were high MELD score, Child–Pugh grading, lower limb edema, ascites, INR, total bilirubin, creatinine, AFP, HULC level, albumin, and platelet count. On the contrary, multivariate analysis showed that AFP and HULC were the parameters that could independently predict HCC. These results indicate that expression of HULC in plasma could be used as a diagnostic biomarker for HCC.

ROC curve analysis for HULC showed sensitivity, specificity, and accuracy of 72, 92, and 82%, respectively, at a cutoff of 1.08 ng/ml. These results are in agreement with those obtained by Xie et al., 2013 [21]. Li et al.[20] reported slightly different results. We believe that their study was conducted on patients with HCC who underwent hepatic resections and healthy volunteers as controls, whereas our study was conducted on patients with HCC without any surgical management, cirrhotic patients, and healthy volunteers as controls.

ROC curves of HULC and AFP demonstrated that HULC had higher sensitivity and specificity than those of AFP. Areas under the ROC curves of HULC and AFP were 0.86 and 0.80, respectively. Combination of both HULC and AFP got the highest AUC (0.94). Thus, combination of HULC and AFP gives the highest prediction value of HCC diagnosis. These results are in agreement with those obtained by Huang et al. [22].

Based on these results, expression level of HULC in plasma can be used as a promising diagnostic biomarker for HCC. However, our study is limited by the small sample size. Further large-scale studies are needed to confirm these findings.


  Conclusion Top


HULC is highly expressed in plasma of patients with HCC on top of HCV related to liver cirrhosis with significant difference compared with HCV-related cirrhotic patients and healthy controls. These findings indicate that the expression of HULC in plasma can be used as a novel and a rapid diagnostic biomarker for HCC.

Financial support and sponsorship

Nil.

Conflicts of interest

None declared.



 
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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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