In the present study, we investigated the serum levels of IGF-1, IGF-2 and IGFBP-3 in early, intermediate and late stages of cirrhosis, assessed by the CP score, as well as in HCC patients. We also studied their correlation with the HCC stage, and with AFP serum levels. In this study, we focused on HCV-induced cirrhosis since the majority of HCC cases in Egypt develop from chronic hepatitis C infection. We found that serum levels of IGF-1, IGF-2, IGFBP-3 were reduced significantly in cirrhosis and in HCC patients in comparison to the controls. Moreover, the reduction in IGF-1, IGFBP-3, but not IGF-2 levels was significant in HCC in comparison to patients with cirrhosis. The reduction in IGF-1, IGF-2 and IGFBP-3 levels negatively correlated with the CP scores (A, B and C) and only IGFBP-3 levels showed a significant negative correlation with AFP levels and a negative correlation with the HCC stage, which was not statistically significant.
In agreement with these results, Aishima et al
) showed an insignificant negative correlation between HCC staging and serum IGFBP-3 and IGF-1 levels. Moreover, in the present study, IGFBP-3 levels significantly discriminated between cirrhosis and HCC at a sensitivity of 87%, specificity of 80% and a cut-off value of <682.6 ng/ml. The PPV and NPV at the optimal IGFBP-3 cut-off value were 78 and 88%, respectively. Experts in risk prediction encourage the use of predictive values to assess the clinical relevance of biomarkers (21
). IGF-1 and IGF-2 had an AUC of 0.78 and 0.74, respectively, and their combined detection did not increase their AUC, sensitivity or specificity. By contrast, when IGFBP-3 values were combined with those of either IGF-1 or IGF-2, their AUCs were increased to 0.93 and 0.9, respectively, with a concomitant increase in both the sensitivity and specificity. Therefore, concomitant to AFP, IGFBP-3 is a promising marker for the prediction of HCC developing from HCV-cirrhosis in Egyptian patients.
Our results are consistent with several published reports on the serum levels of IGFBP-3 in cirrhosis and in HCC (15
) and in chronic hepatitis prior to developing cirrhosis (25
). Furthermore, in HCC patients with or without cirrhosis, IGFBP-3 mRNA levels (13
) and protein levels (28
) were lower than those in non-tumor tissues. The decrease in IGFBP-3 mRNA correlated with a tumor-specific IGFBP-3 promoter hypermethylation (26
). Findings of other studies have shown that IGFBP-3 protein and IGF-1R were lost or underexpressed in poorly differentiated HCC, but overexpressed in well-differentiated HCC in comparison to normal liver tissue and that IGFBP-3 levels significantly correlated with tumor size, histological differentiation, capsular and portal invasion (20
), establishing a role for IGFBP-3 in negatively regulated cell proliferation. The same authors reported that the addition of exogenous IGFBP-3 markedly blocked IGF-1- and IGF-2-stimulated proliferation of KYN-2 and HepG2 cells, and also suppressed IGF-1-induced invasion in KYN-2 cells. The results of Aishima et al
have shown that the serum levels of IGFBP-3 correlate with the tissue levels of IGFBP-3 protein (20
). Thus, in the present study, IGFBP-3 serum levels serve not only as a biomarker for the prediction of hepatic dysfunction and progression towards malignancy, but may have a pathophysiological significance. The molecular mechanism underlying the reduction in serum IGFBP-3 levels in HCV-induced HCC remains to be elucidated. Previous reports demonstrated the association between the hepatitis B virus oncogenic protein (HBx) modulation of DNA methylation and the downregulation of IGFBP-3 expression in cell lines, animal models and human tumor samples (29
). Furthermore, it has been shown that HBx recruits histone deacetylase 1 to repress IGFBP-3 transcription (30
). Moreover, IGFBP-3 has been shown to trigger intracellular signalling: Stimulation of phosphotyrosine phosphatase and phosphatidylinositol-3 kinase activities and increase in intracellular calcium (31
) potentially through its own ‘putative’ receptor. However, no underlying genetic polymorphisms have been detected in IGFBP-3 to contribute to its downregulation in HCC (34
In agreement with our results, Wu et al
) showed a significant reduction of serum IGF-1, IGF-2 and IGFBP-3 levels in patients with cirrhosis compared to controls. Progressive reduction in IGF-2 levels observed in the present study in cirrhosis and in HCC patients are consistent with El-Houseini et al
). In partial disagreement with our results, a recent study investigated the same parameters in similar patient groups and reported that IGF-2, despite being lower in HCC cases than in healthy controls, was significantly higher compared to patients with liver cirrhosis and, therefore, high serum IGF-2 levels can be used as a tumor marker to discriminate HCC from cirrhosis (36
). We did not find a significant difference in the serum levels of IGF-2 in patients with cirrhosis in comparison to those with HCC, and both exhibited lower serum levels than in healthy controls. The differences between those findings and ours may be due to the sample size, as well as the methods used to measure IGF-2. Rehem and El-Shikh (36
) conducted their studies on a smaller sample size (60 patients with liver cirrhosis, 20 HCC patients and 20 controls) compared to our sample size (72 patients with liver cirrhosis, 62 HCC patients and 100 healthy controls). It is an established fact that a larger sample size results in better assessment with lower bias. Furthermore, we have used ELISA to measure serum IGF-2 levels, while in the other study radioimmunoassay (RIA) was used (36
). Moreover, Rehem and El-Shikh (36
) did not report any data concerning the diagnostic accuracy of IGF-1 and IGFBP-3, nor did they demonstrate any significant correlation between IGF-2 and AFP in these cases. However, the authors concluded that IGF-2 and AFP may be used as complementary tumor markers to discriminate HCC from cirrhosis. In contrast, our analysis of the sensitivity, specificity and diagnostic accuracy of the three IGF members studied revealed that IGFBP-3 was a more effective predictor for the development of HCC than IGF-1 and IGF-2.
Other studies have demonstrated a significant increase in IGF-2 mRNA expression in human cirrhotic liver, in liver cancers and in the peripheral blood of HCC, in human hepatoma cell lines, in comparison to that of normal adult liver (37
). This discrepancy may be attributed to the fact that in the present study we measured only the free IGF-2 levels in serum but not mRNA. IGF-2 levels may be lower than the mRNA since the bulk of the former is bound to IGFBPs. We believe that serum IGF-2 levels may not be used as a marker for HCC progression, as in our setting IGF-2 had an AUC of 0.74, whereas it has been previously reported that ROC curves with an AUC≤0.75 are not clinically useful (41
). A comprehensive study by Tovar et al
) demonstrated overexpression of IGF-2 mRNA resulting from reactivation of the fetal promoters P3 and P4, downregulation of IGFBP-3, allelic loss of IGF-2R and activation of IGF-1R in a specific subclass of human HCC; the ‘proliferation’ subclass. It is expected that a reduction in the serum levels of IGF-1 and to a lesser extent IGF-2 would occur in cirrhotic liver, since the main pool of circulating IGF-1 is synthesized in liver parenchyma (42
), whose mass is greatly reduced in cirrhotic liver, whereas IGFBP-3 is produced by Küpffer, endothelial and hepatic stellate cells, which also synthesize IGF-2 (43
). Liver damage is apparently not the only cause for reduced IGF-1 levels in HCC, as reduced IGF-1 levels were reported in males with HCC without a history of liver cirrhosis and in virus-free metastatic liver cancer (47
), and did not correlate with the CP score in patients with liver cirrhosis followed up until the development of HCC (11
). In the present study, the gradual decrease in circulating IGF-1 levels from HCC precursors to malignancy is consistent with our previous observation, which indicated the initial overexpression of IGF-1R in preneoplastic stages is followed by a gradual decrease and complete loss in HCC in rats (7
). Moreover, our findings are in concordance with reports by Huynh et al
) and Aishima et al
), who showed lower levels of IGF-1R expression in a significant number of human HCC by immunohistochemistry. Low serum levels of IGF-1 do not prove a specific function of IGF-1 in hepatocarcinogenesis per se
, but from this and previous reports from human and experimental hepatocarcinogenesis the following may be speculated: i) the low levels of IGF-1 in cirrhosis and in HCC may be caused by separate events since chronic liver disease is known to be an IGF-1 deficiency state, as reviewed by Bonefeld and Moller (49
); however, the cause of low IGF-1 serum levels in HCC patients with no history of cirrhosis remains to be elucidated. ii) IGF-1 may only be required in initiating events of hepatocarcinogenesis; iii) the autocrine and paracrine effects of IGF-1 may be more relevant to cell transformation than its endocrine effect, and iv) IGFBP-3 may have other roles in modulating IGF-1 effects than its binding capacity.
In conclusion, although our results have showm that serum IGF-1, IGF-2 and IGFBP-3 are reduced with the progression of hepatic dysfunction, only IGFBP-3 may be considered as the most promising serological marker for the prediction of the development of HCC in Egyptian chronic HCV patients with liver cirrhosis. Future studies should be directed towards understanding the association between serum and tissue growth factor levels.