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ORIGINAL ARTICLE
Year : 2018  |  Volume : 31  |  Issue : 3  |  Page : 1094-1098

Evaluation of serum Midkine as a novel marker in hepatocellular carcinomas


1 Department of Clinical Pathology, Faculty of Medicine, National Liver Institute, Menoufia University, Shebin El-Kom, Menoufia, Egypt
2 Department of Clinical Pathology, National Liver Institute, Menoufia University, Shebin El-Kom, Menoufia, Egypt

Date of Submission22-Aug-2015
Date of Acceptance13-Sep-2015
Date of Web Publication31-Dec-2018

Correspondence Address:
Doaa A Eisaa
Al-Amir Street, Berkit Elsabaa, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_335_15

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  Abstract 


Objectives
To evaluate the clinical significance of serum Midkine (MDK) levels in the diagnosis of hepatocellular carcinoma (HCC) compared with alpha-fetoprotein (AFP).
Background
It was demonstrated that serum MDK levels are increased in cancer patients. So, it was suggested that MDK would likely be a novel marker for diagnosing tumors. In this study, we evaluated the significance of serum MDK levels in the diagnosis of HCC and early-stage HCC compared with AFP.
Patients and methods
The study was conducted on 90 patients who were divided into three groups: group I is the HCC group (n = 50), group II the liver cirrhosis group (n = 20), and group III is the control group (n = 20). Serum samples were obtained from all patients and were used to measure AFP and MDK levels. Receiver operating characteristic curves were drawn for both AFP and MDK in the diagnosis of HCC and early-stage HCC. Area under the curves (AUC) were calculated and were used to measure the sensitivity, specificity, and accuracy of both markers in the diagnosis of HCC and early HCC.
Results
AFP and MDK levels were increased in HCC over cirrhosis groups; and in HCC over control groups with a highly significant difference. MDK had the largest AUC in the diagnosis of both HCC (AUC = 1.00) and early-stage HCC (AUC = 0.829). In the diagnosis of HCC, serum MDK at a cutoff point of 0.62 ng/ml was 98.9% accurate compared with an accuracy of 73.3% for AFP at a cutoff point of 20.2 ng/ml. At a cutoff point of 0.92 ng/ml, serum MDK was 73.3% accurate in the diagnosis of early HCC compared with an accuracy of 65.6% for AFP at a cutoff point of 19.5 ng/ml.
Conclusion
Serum MDK could be a novel diagnostic tumor marker for the detection of HCC, particularly in patients at an early stage.

Keywords: diagnosis, hepatocellular carcinoma, Midkine, tumor markers


How to cite this article:
EI-Edel RH, Tawfik GA, Noreldin RI, El-Jaky MA, Eisaa DA. Evaluation of serum Midkine as a novel marker in hepatocellular carcinomas. Menoufia Med J 2018;31:1094-8

How to cite this URL:
EI-Edel RH, Tawfik GA, Noreldin RI, El-Jaky MA, Eisaa DA. Evaluation of serum Midkine as a novel marker in hepatocellular carcinomas. Menoufia Med J [serial online] 2018 [cited 2019 Mar 20];31:1094-8. Available from: http://www.mmj.eg.net/text.asp?2018/31/3/1094/248740




  Introduction Top


Hepatocellular carcinoma (HCC) is the most common form of liver cancer and is the third leading cause of cancer-related deaths worldwide. The treatment options for HCC are very limited, as it is often diagnosed at a late stage[1]. The global distribution varies by region due to factors at the origin of the disease. HCC is an end result of some chronic infections with hepatitis B virus or hepatitis C virus[2]. Since HCC is among cancers with the worst prognosis, early diagnosis and treatment are the keys for effective treatment of patients with HCC. The use of serological markers in patients at the highest risk for developing HCC may thus decrease HCC mortality and reduce medical costs[3]. The progression of liver disease into liver cancer is primarily monitored by serum levels of the oncofetal glycoprotein, alpha-fetoprotein (AFP), or the core fucosylated glycoform of AFP (AFP-L3). However, AFP can be produced under many circumstances, including other liver diseases and is not present in all those with HCC. Therefore, the use of AFP as a primary screening test for HCC has been questioned, so more sensitive and specific serum biomarkers for HCC are desired[4]. Studies have identified Midkine (MDK) as an HCC serum marker. MDK also known as neurite growth-promoting factor 2 is a protein that in humans is encoded by the MDK gene[5]. MDK, a 13-kDa small heparin-binding growth factor, was originally discovered in embryonal carcinoma cells and is involved in the early stage of retinoic acid-induced differentiation. MDK was identified as one of the five important potential novel biomarkers for early detection of HCCs. In addition, mounting evidence has indicated that MDK plays a significant role in carcinogenesis-related activities, such as proliferation, migration, anti-apoptosis, mitogenesis, transformation, and angiogenesis, in many types of solid tumors, including HCCs[6].

The aim of the study was to evaluate the clinical significance of serum MDK levels in the diagnosis of HCC and compare it with AFP.


  Patients and Methods Top


The study was conducted on 90 patients who were divided into three groups; 70 patients were selected from the inpatient wards and the outpatient clinic at National Liver Institute, Menoufia University and 20 patients were selected as controls. Informed written consent was obtained from all patients. The study has been approval by the ethics committee of the National Liver Institute.

Patients included in this study were classified into three groups:

  1. Group I HCC (n = 50)


  2. This group included 50 patients with HCC: 44 men and six women. Their ages ranged from 40 to 70 years with a mean ± SD of 55.9 ± 6.9

  3. Group II liver cirrhosis (n = 20)


  4. This group included 20 patients with liver cirrhosis; 15 males and five females. Their ages ranged from 40 to 60 years with a mean ± SD of 52.6 ± 6.3

  5. Group III control group (n = 20)


  6. This group included 20 apparently healthy patients: 15 men and five women served as controls, their ages ranged from 30 to 60 years with a mean ± SD of 53.1 ± 5.3.


All patients and controls were subjected to the following:

  1. Full history taking and thorough clinical examination
  2. Abdominal ultrasonography and ultrasound-guided liver biopsy were performed by true-cut needle or liver biopsy gun for the cirrhotic patients when possible
  3. Triphasic computed tomographic in patients with focal lesions
  4. The following laboratory investigations were done:


    1. Liver function tests including alanine transaminase, aspartate transaminase, serum albumin, total, and direct bilirubin
    2. Prothrombin concentration and international normalized ratio
    3. Serum AFP was measured using Quantikine sandwich enzyme-linked immunosorbent assays (ELISAs) (R&D Systems, Abingdon, UK)[7]
    4. Hepatitis markers were done by ELISA and confirmed with quantitative PCR for positive cases
    5. Determination of MDK: it was determined by Human Midkine ELISA Kit provided by EIAab (Wuhan EIAab Science Co. Ltd, Wuhan, China)[8].


Test principle of serum Midkine measurement using human Midkine enzyme-linked immunosorbent assay kit

The microtiter plate provided in this kit has been precoated with an antibody specific to MDK. Standards or samples were then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for MDK. Avidin conjugated to horseradish peroxidase was then added to each microplate well and incubated. Then a TMB substrate solution was added to each well. Only those wells that contained MDK, biotin-conjugated antibody, and enzyme-conjugated avidin had exhibited a change in color. The enzyme-substrate reaction was terminated by the addition of sulfuric acid solution and the color change was measured spectrophotometrically at a wavelength of 450 ± 2 nm. The concentration of MDK in the samples was then measured[8].

Barcelona Clinic Liver Cancer staging of hepatocellular carcinoma patients

The HCC patients were classified into different Barcelona Clinic Liver Cancer (BCLC) stages (0–D) for further statistical subgroup analysis[9].

Statistical analysis

The data were collected, tabulated, and analyzed by the statistical package for the social sciences, version 22.0 (SPSS Inc., Chicago, Illinois, USA), on an IBM compatible computer.

Two types of statistics were done:

Descriptive statistics: for example, percentage (%), mean, and SD.

Analytic statistics:

  1. χ2: was used to study the association between two qualitative variables
  2. Student's t test: was used for comparison between two groups having quantitative variables
  3. Mann–Whitney test: was used for comparison between two groups with nonparametric distribution having quantitative variables
  4. Analysis of variance (f) test: was used for comparison between three or more groups having quantitative variables
  5. Kruskal–Wallis test (nonparametric test): was used for comparison between three or more groups not normally distributed having quantitative variables
  6. The receiver operating characteristic curves: These are graphic representations of the relationship between sensitivity and specificity at different cutoff points for a diagnostic test[10]. They were used in this study to measure the diagnostic accuracy of AFP and MDK in the diagnosis of HCC and early-stage HCC.



  Results Top


AFP and MDK levels were increased in HCC over liver cirrhosis groups; and in HCC over control groups with a highly significant difference. However, there was no significant difference between liver cirrhosis and control groups regarding AFP and MDK levels [Table 1].
Table 1: Comparison between the studied groups regarding alpha-fetoprotein and Midkine

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There was no significant difference between different stages of BCLC within the HCC group regarding AFP and MDK levels [Table 2].
Table 2: Comparison between stages of Barcelona Clinic Liver Cancer regarding alpha-fetoprotein and Midkine

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AFP had an area under the receiver operating characteristic curve (AUC) of 0.826 in the diagnosis of HCC with a P value of less than 0.001. At a cutoff point of 20.2 ng/ml, AFP was 64% sensitive, 85% specific, and had a positive predictive value (PPV) of 84.2%, a negative predictive value (NPV) of 65.4% and an overall accuracy of 73.3% in the diagnosis of HCC, while MDK had an AUC of 1.00 in the diagnosis of HCC with a P value of less than 0.001. At a cutoff point of 0.60 ng/ml, MDK was 100% sensitive, 97.5% specific, and had a PPV of 98.04%, an NPV of 100% and an overall accuracy of 98.9% in HCC diagnosis [Table 3].
Table 3: Evaluation of alpha-fetoprotein and Midkine in the diagnosis of hepatocellular carcinoma

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With the evaluation of AFP among early HCC group, AFP had an AUC of 0.707 and a P value of 0.003. At a cutoff point of 19.5 ng/ml, AFP had a sensitivity and specificity of 66.7 and 65.2%, respectively, PPV was 41.03%, NPV was 84.3%, and the overall accuracy was 65.6%. With evaluation of MDK among early HCC group, MDK had an AUC of 0.829 and a P value of less than 0.001. At a cutoff point of 0.92 ng/ml, MDK had a sensitivity and specificity of 91.2 and 66.7%, respectively; PPV was 50%; NPV was 95.7%; and the overall accuracy was 73.3% [Table 4] and [Figure 1], [Figure 2].
Table 4: Evaluation of alpha-fetoprotein and Midkine in the diagnosis of early hepatocellular carcinoma

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Figure 1: ROC curve demonstrates the sensitivity and specificity of AFP and Midkine in the HCC group. AFP, alpha-fetoprotein; HCC, hepatocellular carcinoma; ROC, receiver operating characteristic.

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Figure 2: ROC curve demonstrates the sensitivity and specificity of AFP and Midkine in early HCC group. AFP, alpha-fetoprotein; HCC, hepatocellular carcinoma; ROC, receiver operating characteristic.

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  Discussion Top


HCC is the most common form of liver cancer and is the third leading cause of cancer-related deaths worldwide, preceded only by lung and stomach cancers[11]. Serum AFP is the golden standard of diagnostic markers for HCC. However, its diagnostic value is still debatable[12]. Therefore, identification of new specific and sensitive biomarkers for early diagnosis is urgently needed[13]. Many alternative novel biomarkers, such as AFP-L3, des-γ-carboxy-prothrombin, Golgi protein 73, and Glypican-3, have been investigated; however, their diagnostic values about early HCCs remain controversial. Thus, it is necessary to identify new serologic biomarkers with both sufficient sensitivity and specificity to detect HCCs at an early stage and/or with negative AFP[14]. MDK was identified as one of the five important potential novel biomarkers for early detection of HCCs[15].

In the present study, we compared the levels of MDK and AFP in the serum samples from 90 patients with various benign and malignant liver diseases and healthy people as controls to determine its clinical significance in the detection of HCC in general and early-stage HCC in particular compared with AFP.

In this study, serum levels of AFP were significantly elevated in chronic liver diseases and even more elevated in HCC cases with statistically significant difference (P < 0.001).

The results agreed with those of El Shafie et al.[16] who reported that the serum levels of AFP were significantly elevated in chronic liver diseases and more elevated in HCC cases (P < 0.001).

Ikematsu et al.[4] found that the level of MDK expression was increased in many types of human carcinomas. An enzyme-linked immunoassay, which utilizes a combination of rabbit and chicken antibodies, revealed that serum MDK level is significantly elevated in cancer patients.

Shaheen et al.[17] reported that serum MDK was significantly elevated in the HCC group compared with cirrhotic and healthy control groups.

In our study, the sensitivity of MDK at a cutoff value of 0.62 ng/ml was found to be significantly higher when compared with that of AFP at a cutoff value of 20.2 ng/ml, and the specificity of MDK at the same cutoff value was significantly higher than that of AFP at a cutoff value of 20.2 ng/ml.

This agreed with Zhu et al.[18] who found that the optimal cutoff value of MDK was 0.654 ng/ml, while 20 ng/ml, the currently recommended clinical cutoff value, was used for AFP. At a cutoff value of 0.654 ng/ml, the sensitivity of MDK for HCC diagnosis was 86.9%, which was much higher than that of AFP (51.9%).

In this study, AFP had an AUC of 0.826 while MDK had an AUC of 1.00. This agreed with Shaheen et al.[17] who found that the AUC of MDK (0.941) was much larger than that of serum AFP (0.671).

In the current study, there was no significant difference in serum MDK levels and different BCLC stages within the HCC Group (group I).

Consistent with our results, Shaheen et al.[17] also found that there was no significant association between serum MDK and neither BCLC stage, tumor diameter nor tumor numbers.

Kadomatsu and Muramatsu[19] reported that the blood MDK level is frequently elevated in advanced human carcinomas, decreased after surgical removal of the tumors, and was correlated with prognostic factors. Thus, they stated that it was a good marker for evaluating the progress of carcinomas.

In this study, the evaluation of MDK among the early HCC group had an accuracy of 73.3% while AFP had an accuracy of 65.6%.

Muramatsu et al.[20] found that the MDK levels detected by anti-MDK antibodies in HCC patients were significantly enhanced at the early stage of HCC.


  Conclusion Top


Serum MDK could be a novel diagnostic tumor marker for the detection of HCC, particularly in patients at an early stage. In conclusion, serum MDK can be a novel diagnostic tumor marker for the detection of HCCs, particularly in patients at an early stage.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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    Figures

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