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Insulin-like growth factor-II is a useful marker to detect hepatocellular carcinoma?
European Journal of Internal Medicine 23 (2012) e157–e161
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Original article
Insulin-like growth factor-II is a useful marker to detect hepatocellular carcinoma? C. Morace a,⁎, M. Cucunato a, R. Bellerone a, G. De Caro b, S. Crinò a, A. Fortiguerra a, F. Spadaro c, A. Zirilli d, A. Alibrandi d, P. Consolo a, C. Luigiano e, M.L. Resta a, O. Ferraù a, A. Spadaro a a
Dipartimento di Medicina Interna, UOC di Medicina interna ad Indirizzo Gastroenterologico, Pad. C, 2° piano, AOU Policlinico Universitario, Via Consolare Valeria, 1‐98125 Messina, Italy Dipartimento Area Medica, Servizio di Endoscopia Digestiva, Treviglio, Italy Dipartimento Farmaco-Biologico, Facoltà di Farmacia, Università di Messina, Vill. SS Annunziata, 98168 Messina, Italy d Dipartimento di Scienze Economiche, Finanziarie, Sociali, Ambientali, Statistiche e del Territorio, Università di Messina, Via dei Verdi, 75‐98100 Messina, Italy e Unità di Gastroenterologia ed Endoscopia Digestiva, Ospedale Bellaria-Maggiore—Bologna, Italy b c
a r t i c l e
i n f o
Article history: Received 12 March 2012 Received in revised form 19 April 2012 Accepted 24 April 2012 Available online 24 May 2012 Keywords: Alpha-fetoprotein Diagnosis Hepatocellular carcinoma Insulin-like growth factor-II Liver cirrhosis
a b s t r a c t Background: Hepatocellular carcinoma (HCC) is a typical hypervascular tumor. The utility of serum alphafetoprotein (α-FP) in its detection is questionable. Over-expression and high circulating levels of insulinlike growth factor-II (IGF-II) were reported in tissue and in serum of patients with HCC. We investigated the diagnostic application of IGF-II in the diagnosis of HCC. Methods: Serum IGF-II and α-FP levels were measured in 178 patients (82 with HCC and 96 with liver cirrhosis) and in 30 healthy controls. Spearman test, non parametric combination test and confidence interval analysis were used for statistical evaluation of data. Results: The best cut-off values selected by ROC curves were 796 ng/ml for IGF-II and 132 ng/ml for α-FP. IGF-II mean values were higher in patients with HCC than in those with liver cirrhosis (LC) (p = 0.0001) but lower in LC than in controls (p = 0.0001). Serum IGF-II levels above cut-off were found in 22% of patients with HCC, in 9.3% of those with cirrhosis and in 20% of controls. α-FP serum levels >132 ng/ml were observed in 48% of HCC, in 3.1% of LC and in none of control group. By correlation study, serum IGF-II levels were significantly correlated with serum α-FP levels (r = 0.427, p = 0.0001) and with nodules' diameter (r = 0.252, p = 0.0130) but not with nodules' number (p > 0.050). Finally, IGF-II showed lower sensitivity, specificity and predictive values than α-FP. Conclusion: Circulating IGF-II is not a useful marker for HCC. Further researches are however needed to evaluate its diagnostic accuracy before and after nutritional adjustment. © 2012 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC) is the most frequent of liver cancer with increasing worldwide incidence [1,2]. HCC usually occurs in cirrhotic livers but may also arise in absence of cirrhosis [3]. It originates from hepatocyte cells whose growth is regulated by various factors, such as insulin, insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), hepatocyte growth factor (HGF) [4,5]. The hepatocarcinogenesis is a complex and multistage process inducing several changes in the cellular genoma [6,7] through steps of induction, promotion and progression [8–10]. In the induction stage genetic changes, caused by chemical (aflatoxin B, drugs etc.) or viral agents, lead to malignant transformation of the hepatocytes and induce activation and impaired expression of oncogenes of “ras,” ⁎ Corresponding author at: UOC di Medicina interna ad indirizzo gastroenterologico, Pad. C, 2° piano, AOU Policlinico Universitario, Dipartimento di Medicina Interna—Via Consolare Valeria, 1‐98125, Messina, Italy. Tel.: +39 0902212333, +39 3280449314 (cell); fax: + 39 090693917. E-mail address: [email protected] (C. Morace).
“myc,” “fos” families with consequent cell cycle dysregulation [11–14]. In this first stage hepatocarcinogenesis is down-regulated by increased rate of hepatocyte apoptosis [15]. The successive promotion stage is characterized by a selective increase of cell proliferation, in presence of a constant or repeated liver injury producing inflammation, cell death, regeneration and cirrhosis [16]. Last stage, progression, may derive either from clonal malignant cell proliferation (increased by growth factors as IGF-II, TGF-α, TGF-β) or from mutations of the p53 tumor suppressor gene, with consequent imbalance of the proliferation-apoptosis process [17–21]. Liver cirrhosis, characterized by increased cell proliferation, is a premalignant condition which needs accurate surveillance [22,23]. Serum alpha-fetoprotein (α-FP) level is a useful marker for the detection and monitoring of HCC [24–27] but its diagnostic value is poor because of a low sensitivity and a low positive predictive value [28–30]. The diagnostic utility of other serum markers of HCC, such as glypican-3, des-γ-carboxy prothrombin (DCP), insulin-like growth factor-1 (IGF-I), human hepatocyte growth factor (HGF), cromogranin A (CgA), nociceptin/orphanin (N/OFQ) has been investigated with unsatisfactory results [31–38].
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IGF-II is a 67 amino-acid polypeptide closely related to insulin, produced by liver cells, predominantly active in physiological fetal liver growth [39,40]. Pronounced alterations in the expression of components of IGF axis in hepatocarcinogenesis and abnormal IGF-II, free IGF-II mRNA and IGF-II nRNA in serum and in neoplastic tissue of patients with HCC strongly suggest its role in neoplastic hepatocyte proliferation [41–45]. In the present study we investigated the utility of circulating IGF-II levels, alone and in combination with serum α-FP, in the diagnosis of HCC and their possible correlation with number and/or size of the neoplastic nodules.
the examined groups we used the non parametric combination test, NPC [47]. p b 0.05 was considered statistically significant. Sensitivity, specificity, diagnostic accuracy test, positive (PPV) and negative (NPV) predictive values, with relative confidence interval at 95%, were evaluated [48]. Cut-off values for α-FP and IGF-II were defined using ROC curves [49]. The statistical packages used were the following: Methodologica S.R.L. (2001) for non parametric analysis NPC test; Confidence Interval Analysis (CIA) Windows version 2.0 (2000) for sensitivity and specificity evaluation and CI; SPPS, Windows 11.0 (2001) for Spearman correlation test.
2. Materials and methods 3. Results 2.1. Patients and controls Serum IGF-II values in a population of 178 patients, 82 with HCC and 96 with liver cirrhosis (LC), consecutively referring to the Unit of Clinica Medica, Messina University during a 3 year period (October 2006–October 2009), and in 30 healthy subjects (control group) were evaluated. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the local ethical committee. Informed consent was obtained from each participant. The diagnosis of HCC was based on imaging findings and/or histological confirmation and none of patients had yet undergone any locoregional treatment. The diagnosis of LC was clinical and/or histological. Severity of liver disease was classified by Child–Pugh score. The demographic and clinical characteristics of the population studied are shown in Table 1. 2.2. Assays Circulating IGF-II levels were detected in stored frozen sera (−20 °C), using a commercial enzyme-linked immunosorbent assay (IGF-II direct ELISA, DRG Diagnostics, Germany). Samples were incubated in microtitration wells coated with antiIGF-II antibody. After incubation and washing, the wells were treated with another anti-IGF-II detection antibody labeled with the enzyme horseradish peroxidase (HRP). After a second incubation and washing acidic stopping solution was then added and the degree of enzymatic turnover of the substrate was determined by dual wavelength absorbance measurement of 450 and 620 nm. The absorbance measured was directly proportional to the concentration of IGF-II present. AFP serum values were determined using a routine radioimmunoassay.
No significant differences in age, gender and, in liver patients, Child–Pugh score were found among the three examined groups (Table 1). As shown in Figs. 1 and 2 both α-FP and IGF-II mean values were higher in patients with HCC than in those with LC (p = 0.0001). Mean levels of α-FP in patients with liver cirrhosis were also significantly higher than in control group (p = 0.0002). Conversely, IGF-II mean values were lower in cirrhotic patients than in healthy controls (p = 0.0001). In patients with HCC we carried out a correlation study with regard to variables α-FP, IGF-II, nodules' number and diameter. Serum IGF-II levels were significantly correlated with serum α-FP levels (r = 0.427, p = 0.0001) and with nodules' diameter (r = 0.252, p = 0.0130) but not with nodules' number (p > 0.050). On the other hand, α-FP serum levels were correlated both with nodules' number (r = 0.358, p = 0.0001) and with nodules' diameter (r = 0.209, p = 0.0410) (Table 2). In order to estimate the optimal cut-off values for IGF-II and α-FP, we used a ROC curve. The best cut-off values selected by the ROC curves were 796 ng/ml for IGF-II and 132 ng/ml for α-FP (Fig. 3). The calculated area under the ROC curve was 0.547 for IGF-II and 0.687 for α-FP. For both markers the sensitivity was calculated at several specificity levels.
2.3. Statistical analysis Correlations among the studied variables were calculated using non-parametric Spearman test [46]. In order to evaluate statistically significant differences in the IGF-II and α-FP distribution between
Table 1 Clinical-demographic characteristics of the populations studied.
Number of patients Male/female n Age (range) Child–Pugh score n (%) A B C HCV n (%) HBV n (%) Alcool n (%)
HCC
Cirrhosis
Controls
p Value
82 65/17 62 (38–81)
96 75/21 63 (34–78)
30 24/6 62 (37–80)
>0.05 >0.05 >0.05 >0.05
48 (58) 26 (32) 8 (10) 48 (58) 18 (22) 16 (20)
55 28 13 60 18 18
(57) (29) (14) (62) (19) (19)
>0.05 >0.05 >0.05
Fig. 1. Distribution of α-FP values in groups studied. Serum levels of ln(α-FP) in 82 patients with HCC, in 96 patients with liver cirrhosis and in 30 healthy controls. The logarithmic transformation was applied to better view the graphical representation of α-FP distribution. Box- and whiskers plots express medians, and interquartile and overall ranges. HCC and CIRRHOTIC patients have significantly higher serum α-FP mean levels than controls (p = 0.0001 and p = 0.0002 respectively, NPC test). HCC has significantly higher mean α-FP levels than CIRRHOTIC patients (p = 0.0001, NPC test).
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ROC Curve 1.00 0.80 0.60 0.40 0.20 0.00 0.00
0.20
0.40
0.60
IGF-II
Fig. 2. Distribution of IGF-II values in groups studied. Serum levels of IGF-II in 82 patients with HCC, in 96 patients with liver cirrhosis and in 30 healthy controls. Boxand whiskers plots express medians, and interquartile and overall ranges. HCC and CIRRHOTIC patients have significantly lower serum IGF-II mean levels than controls (p = 0.0001 for both comparisons, NPC test) but HCC has significantly higher mean IGF-II levels than CIRRHOTIC patients (p = 0.0001, NPC test).
Serum IGF-II levels above the best cut-off were found in 18 patients with HCC (22%), in 9 patients with liver cirrhosis (9.3%) and in 6 healthy subjects (20%); on the other hand, serum α-FP levels >132 ng/ml were observed in 39 patients with hepatocellular carcinoma (48%), in 3 patients with LC (3.1%) and in none of control group. Sensitivity, specificity and predictive values for IGF-II and α-FP in the diagnosis of HCC are showed in Table 3. In particular, using the optimal serum cut-off for α-FP (132 ng/ml), we obtained a specificity of 96.9% and a sensitivity of 40.6%, with a diagnostic accuracy of 68.7%. When the best cut-off for IGF-II was used (796 ng/ml), the resulting specificity was 90.6% and sensitivity was 18.9%, with a diagnostic accuracy of 54.7%. Finally when both markers were determined in parallel the sensitivity was 46.9%, the specificity was 90.6% and the diagnostic accuracy was 68.8%.
4. Discussion Hepatocellular carcinoma (HCC) is the leading cause of death in patients with liver cirrhosis [50] and early detection is of utmost importance for the treatment outcomes of this cancer. Periodic surveillance for diagnosis of HCC in patients with cirrhosis is recommended but serum α-FP level, although is a useful marker for monitoring of HCC, showed high false-negative rate for small size HCC [51]. The diagnostic accuracy of other serologic markers such as glypican-3, des-γ-carboxy prothrombin (DCP), insulin-like growth factor-1 (IGF-I), human hepatocyte growth factor (HGF), cromogranin A (CgA), nociceptin/orphanin (N/OFQ) is not yet clarified [31–38]. Recent researches have demonstrated changes in the IGF axis that affect the pathogenesis of HCC [2]. Table 2 p Value relative to correlation among: α-FP, IGF-II, nodules' number and diameter. α-FP α-FP IGF-II Nodules Diameter
Rs p-Value Rs p-Value Rs p-Value Rs p-Value
1.0000 0.4270 0.0001 0.3580 0.0001 0.2090(*) 0.0410
IGF-II 0.4270 0.0001 1.0000 − 0.1680 0.1020 0.2520(*) 0.0130
Nodules
Diameter
0.3580 0.0001 − 0.1680 0.1020 1.0000
0.2090 0.0410 0.2520 0.0130 − 0.0370 0.7240 1.0000
− 0.0370 0.7240
0.80
1.00
α-FP
Fig. 3. ROC curves for best cut-off. Receiver operating characteristic curve to identify the best cut-off for both α-FP and IGF-II serum levels for the diagnosis of HCC. The X-axis shows the different values of sensitivity, the Y-axis shows the corresponding values of 1-specificity. In the ROC curve the true positive rate (sensitivity) is plotted in function of the false positive rate (1-specificity) for different cut-off points, both for α-FP (in blue) and IGF-II (in red). Each point on the ROC curve represents a sensitivity/ specificity pair corresponding to a particular decision threshold. The area under the ROC curve is a measure of how well each parameter (α-FP and IGF-II) can distinguish between the two diagnostic groups (HCC/cirrhosis).
Insulin-like growth factor-II (IGF-II) is an autocrine and paracrine mitogen for liver cancer cell proliferation and IGF-II mRNA is distributed in the cytoplasm of hepatocytes and overexpressed in HCC tissues [52,53]. In the last years many researches have shown that IGF-II and IGF-II mRNA expressions were higher in HCC cells than in adjacent non-cancerous hepatic tissues [54–57]. Mitogenic activity of IGF-II is reduced by IGF binding protein 3 (IGFBP-3), and is reported undetectable or low in human HCC samples [53,58,59]. Furthermore reduced expression of IGFBP-3 was also significantly correlated with clinicopathological characteristics of HCC (tumor size, histological differentiation, capsular and portal venous invasion) [60]. HCC is a hypervascular tumor and IGF-II seems to play an important pathogenetic role by increasing vascular endothelial growth factor (VEGF)-mRNA and protein levels and, consequently, the hypervascularization, in a timedependent manner in human hepatoma cells [61]. Recently Dong et al. reported circulating free IGF-II levels significantly higher in patients with HCC than those with chronic hepatitis or liver cirrhosis; in addition circulating IGF-II mRNA was detected only in HCC patients and it was correlated with clinical stage and with presence of distant metastases of HCC [62]. In order to investigate if circulating IGF-II levels represent a valid indicator of HCC, we evaluated serum IGF-II and α-FP concentrations in patients with liver cirrhosis, with and without HCC, comparing the diagnostic value of the two markers. In our study circulating free IGF-II levels above normal range were found in 22% of patients with HCC and in 9% of patients with liver cirrhosis, showing low sensibility and high specificity (90.6%) of this marker. Furthermore determination in parallel of IGF-II and α-FP did not significantly increase the diagnostic accuracy in the detection of HCC.
Table 3 Diagnostic accuracy tests.
α-FP IGF-II α-FP + IGF-II
Sensitivity %
Specificity %
Accuracy%
PPV%
NPV%
LR+
LR−
40.6 18.9 46.9
96.9 90.6 90.6
68.7 54.7 68.8
92.9 66.7 83.3
62 52.7 63
13 2 5
0.61 0.90 0.59
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Also Tsai et al. investigated the diagnostic utility of serum IGF-II and α-FP levels in patients with HCC and with liver cirrhosis alone and reported IGF-II and α-FP levels higher in the former in comparison with the latter; moreover, in accordance with our results, they reported IGF-II mean values lower in cirrhotic patients than in healthy controls but, contrary to data obtained in our study, determination in parallel of both markers significantly increased the diagnostic accuracy and sensitivity (96.5% and 97.9%, respectively), with a high specificity (95.1%) [63]. More recently same authors demonstrated that serum IGF-II can be used as an independent serologic marker or a complementary marker to α-FP for the diagnosis of small HCC [64]. The discrepancy of our data with those of Tsai et al. may be related to the different methods of detecting of serum IGF-II levels; in both studies, in fact, Tsai et al. assessed IGF-II levels together with prealbumin values, after adequate nutritional adjustment. Thus, IGF-II values were expressed in mg/g prealbumin. On the contrary, in our study we used a method of detection (expressed in ng/ml) lacking in nutritional adjustment and, therefore, with better performance in order to time and costs. As we know HCC is a hypervascular tumor and angiogenesis is a critical finding for its growth and progression. Kim et al. suggested that hypoxia represents a very important stimulus for the new vessel formation and is associated to an overexpression of IGF-II in HCC tissue; secreted IGF-II, in addiction, may act as an angiogenic factor directly as well as indirectly by stimulating expression of VEGF gene [65]. In our study, when we applied NPC test, we observed a statistically significative correlation between circulating IGF-II levels and tumor's size (p = 0.013), probably in relation to a greater vascularization of cancer. In conclusion, among the many growth factors closely associated with development and growth of HCC, insulin-like growth factor-II (IGF-II) seems to have an important role, both directly and indirectly. However in our experience, circulating IGF-II cannot be recommended as a marker for detection of HCC in cirrhotic patients. Anyway the high specificity of IGF-II (90%) may suggest its use as confirmatory test in diagnosis of HCC. Learning points • IGF-II is a 67 amino-acid polypeptide closely related to insulin, produced by liver cells, predominantly active in physiological fetal liver growth. • Over-expression and high circulating levels of insulin-like growth factor-II (IGF-II) were reported in tissue and in serum of patients with HCC. • In this study we investigated the utility of circulating IGF-II levels, alone and in combination with serum α-FP, in the diagnosis of HCC and their possible correlation with number and/or size of the neoplastic nodules. • Circulating IGF-II is not a useful marker for HCC. Conflict of interest statement All authors have no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within 3 years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work. References [1] Colombo M. Malignant neoplasm of the liver. In: Schiff ER, Sorrel MF, Maddrey WC, editors. Schiff's Disease of the Liver. 9th ed. Philadelphia: Lippincott William & Wilkins; 2003. p. 1377–404. [2] Scharf J-G, Dombrowski F, Ramadori G. The IGF axis and hepatocarcinogenesis. J Clin Pathol Mol Pathol 2001;54:138–44.
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Original article
Insulin-like growth factor-II is a useful marker to detect hepatocellular carcinoma? C. Morace a,⁎, M. Cucunato a, R. Bellerone a, G. De Caro b, S. Crinò a, A. Fortiguerra a, F. Spadaro c, A. Zirilli d, A. Alibrandi d, P. Consolo a, C. Luigiano e, M.L. Resta a, O. Ferraù a, A. Spadaro a a
Dipartimento di Medicina Interna, UOC di Medicina interna ad Indirizzo Gastroenterologico, Pad. C, 2° piano, AOU Policlinico Universitario, Via Consolare Valeria, 1‐98125 Messina, Italy Dipartimento Area Medica, Servizio di Endoscopia Digestiva, Treviglio, Italy Dipartimento Farmaco-Biologico, Facoltà di Farmacia, Università di Messina, Vill. SS Annunziata, 98168 Messina, Italy d Dipartimento di Scienze Economiche, Finanziarie, Sociali, Ambientali, Statistiche e del Territorio, Università di Messina, Via dei Verdi, 75‐98100 Messina, Italy e Unità di Gastroenterologia ed Endoscopia Digestiva, Ospedale Bellaria-Maggiore—Bologna, Italy b c
a r t i c l e
i n f o
Article history: Received 12 March 2012 Received in revised form 19 April 2012 Accepted 24 April 2012 Available online 24 May 2012 Keywords: Alpha-fetoprotein Diagnosis Hepatocellular carcinoma Insulin-like growth factor-II Liver cirrhosis
a b s t r a c t Background: Hepatocellular carcinoma (HCC) is a typical hypervascular tumor. The utility of serum alphafetoprotein (α-FP) in its detection is questionable. Over-expression and high circulating levels of insulinlike growth factor-II (IGF-II) were reported in tissue and in serum of patients with HCC. We investigated the diagnostic application of IGF-II in the diagnosis of HCC. Methods: Serum IGF-II and α-FP levels were measured in 178 patients (82 with HCC and 96 with liver cirrhosis) and in 30 healthy controls. Spearman test, non parametric combination test and confidence interval analysis were used for statistical evaluation of data. Results: The best cut-off values selected by ROC curves were 796 ng/ml for IGF-II and 132 ng/ml for α-FP. IGF-II mean values were higher in patients with HCC than in those with liver cirrhosis (LC) (p = 0.0001) but lower in LC than in controls (p = 0.0001). Serum IGF-II levels above cut-off were found in 22% of patients with HCC, in 9.3% of those with cirrhosis and in 20% of controls. α-FP serum levels >132 ng/ml were observed in 48% of HCC, in 3.1% of LC and in none of control group. By correlation study, serum IGF-II levels were significantly correlated with serum α-FP levels (r = 0.427, p = 0.0001) and with nodules' diameter (r = 0.252, p = 0.0130) but not with nodules' number (p > 0.050). Finally, IGF-II showed lower sensitivity, specificity and predictive values than α-FP. Conclusion: Circulating IGF-II is not a useful marker for HCC. Further researches are however needed to evaluate its diagnostic accuracy before and after nutritional adjustment. © 2012 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved.
1. Introduction Hepatocellular carcinoma (HCC) is the most frequent of liver cancer with increasing worldwide incidence [1,2]. HCC usually occurs in cirrhotic livers but may also arise in absence of cirrhosis [3]. It originates from hepatocyte cells whose growth is regulated by various factors, such as insulin, insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), hepatocyte growth factor (HGF) [4,5]. The hepatocarcinogenesis is a complex and multistage process inducing several changes in the cellular genoma [6,7] through steps of induction, promotion and progression [8–10]. In the induction stage genetic changes, caused by chemical (aflatoxin B, drugs etc.) or viral agents, lead to malignant transformation of the hepatocytes and induce activation and impaired expression of oncogenes of “ras,” ⁎ Corresponding author at: UOC di Medicina interna ad indirizzo gastroenterologico, Pad. C, 2° piano, AOU Policlinico Universitario, Dipartimento di Medicina Interna—Via Consolare Valeria, 1‐98125, Messina, Italy. Tel.: +39 0902212333, +39 3280449314 (cell); fax: + 39 090693917. E-mail address: [email protected] (C. Morace).
“myc,” “fos” families with consequent cell cycle dysregulation [11–14]. In this first stage hepatocarcinogenesis is down-regulated by increased rate of hepatocyte apoptosis [15]. The successive promotion stage is characterized by a selective increase of cell proliferation, in presence of a constant or repeated liver injury producing inflammation, cell death, regeneration and cirrhosis [16]. Last stage, progression, may derive either from clonal malignant cell proliferation (increased by growth factors as IGF-II, TGF-α, TGF-β) or from mutations of the p53 tumor suppressor gene, with consequent imbalance of the proliferation-apoptosis process [17–21]. Liver cirrhosis, characterized by increased cell proliferation, is a premalignant condition which needs accurate surveillance [22,23]. Serum alpha-fetoprotein (α-FP) level is a useful marker for the detection and monitoring of HCC [24–27] but its diagnostic value is poor because of a low sensitivity and a low positive predictive value [28–30]. The diagnostic utility of other serum markers of HCC, such as glypican-3, des-γ-carboxy prothrombin (DCP), insulin-like growth factor-1 (IGF-I), human hepatocyte growth factor (HGF), cromogranin A (CgA), nociceptin/orphanin (N/OFQ) has been investigated with unsatisfactory results [31–38].
0953-6205/$ – see front matter © 2012 European Federation of Internal Medicine. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.ejim.2012.04.014
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IGF-II is a 67 amino-acid polypeptide closely related to insulin, produced by liver cells, predominantly active in physiological fetal liver growth [39,40]. Pronounced alterations in the expression of components of IGF axis in hepatocarcinogenesis and abnormal IGF-II, free IGF-II mRNA and IGF-II nRNA in serum and in neoplastic tissue of patients with HCC strongly suggest its role in neoplastic hepatocyte proliferation [41–45]. In the present study we investigated the utility of circulating IGF-II levels, alone and in combination with serum α-FP, in the diagnosis of HCC and their possible correlation with number and/or size of the neoplastic nodules.
the examined groups we used the non parametric combination test, NPC [47]. p b 0.05 was considered statistically significant. Sensitivity, specificity, diagnostic accuracy test, positive (PPV) and negative (NPV) predictive values, with relative confidence interval at 95%, were evaluated [48]. Cut-off values for α-FP and IGF-II were defined using ROC curves [49]. The statistical packages used were the following: Methodologica S.R.L. (2001) for non parametric analysis NPC test; Confidence Interval Analysis (CIA) Windows version 2.0 (2000) for sensitivity and specificity evaluation and CI; SPPS, Windows 11.0 (2001) for Spearman correlation test.
2. Materials and methods 3. Results 2.1. Patients and controls Serum IGF-II values in a population of 178 patients, 82 with HCC and 96 with liver cirrhosis (LC), consecutively referring to the Unit of Clinica Medica, Messina University during a 3 year period (October 2006–October 2009), and in 30 healthy subjects (control group) were evaluated. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the local ethical committee. Informed consent was obtained from each participant. The diagnosis of HCC was based on imaging findings and/or histological confirmation and none of patients had yet undergone any locoregional treatment. The diagnosis of LC was clinical and/or histological. Severity of liver disease was classified by Child–Pugh score. The demographic and clinical characteristics of the population studied are shown in Table 1. 2.2. Assays Circulating IGF-II levels were detected in stored frozen sera (−20 °C), using a commercial enzyme-linked immunosorbent assay (IGF-II direct ELISA, DRG Diagnostics, Germany). Samples were incubated in microtitration wells coated with antiIGF-II antibody. After incubation and washing, the wells were treated with another anti-IGF-II detection antibody labeled with the enzyme horseradish peroxidase (HRP). After a second incubation and washing acidic stopping solution was then added and the degree of enzymatic turnover of the substrate was determined by dual wavelength absorbance measurement of 450 and 620 nm. The absorbance measured was directly proportional to the concentration of IGF-II present. AFP serum values were determined using a routine radioimmunoassay.
No significant differences in age, gender and, in liver patients, Child–Pugh score were found among the three examined groups (Table 1). As shown in Figs. 1 and 2 both α-FP and IGF-II mean values were higher in patients with HCC than in those with LC (p = 0.0001). Mean levels of α-FP in patients with liver cirrhosis were also significantly higher than in control group (p = 0.0002). Conversely, IGF-II mean values were lower in cirrhotic patients than in healthy controls (p = 0.0001). In patients with HCC we carried out a correlation study with regard to variables α-FP, IGF-II, nodules' number and diameter. Serum IGF-II levels were significantly correlated with serum α-FP levels (r = 0.427, p = 0.0001) and with nodules' diameter (r = 0.252, p = 0.0130) but not with nodules' number (p > 0.050). On the other hand, α-FP serum levels were correlated both with nodules' number (r = 0.358, p = 0.0001) and with nodules' diameter (r = 0.209, p = 0.0410) (Table 2). In order to estimate the optimal cut-off values for IGF-II and α-FP, we used a ROC curve. The best cut-off values selected by the ROC curves were 796 ng/ml for IGF-II and 132 ng/ml for α-FP (Fig. 3). The calculated area under the ROC curve was 0.547 for IGF-II and 0.687 for α-FP. For both markers the sensitivity was calculated at several specificity levels.
2.3. Statistical analysis Correlations among the studied variables were calculated using non-parametric Spearman test [46]. In order to evaluate statistically significant differences in the IGF-II and α-FP distribution between
Table 1 Clinical-demographic characteristics of the populations studied.
Number of patients Male/female n Age (range) Child–Pugh score n (%) A B C HCV n (%) HBV n (%) Alcool n (%)
HCC
Cirrhosis
Controls
p Value
82 65/17 62 (38–81)
96 75/21 63 (34–78)
30 24/6 62 (37–80)
>0.05 >0.05 >0.05 >0.05
48 (58) 26 (32) 8 (10) 48 (58) 18 (22) 16 (20)
55 28 13 60 18 18
(57) (29) (14) (62) (19) (19)
>0.05 >0.05 >0.05
Fig. 1. Distribution of α-FP values in groups studied. Serum levels of ln(α-FP) in 82 patients with HCC, in 96 patients with liver cirrhosis and in 30 healthy controls. The logarithmic transformation was applied to better view the graphical representation of α-FP distribution. Box- and whiskers plots express medians, and interquartile and overall ranges. HCC and CIRRHOTIC patients have significantly higher serum α-FP mean levels than controls (p = 0.0001 and p = 0.0002 respectively, NPC test). HCC has significantly higher mean α-FP levels than CIRRHOTIC patients (p = 0.0001, NPC test).
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ROC Curve 1.00 0.80 0.60 0.40 0.20 0.00 0.00
0.20
0.40
0.60
IGF-II
Fig. 2. Distribution of IGF-II values in groups studied. Serum levels of IGF-II in 82 patients with HCC, in 96 patients with liver cirrhosis and in 30 healthy controls. Boxand whiskers plots express medians, and interquartile and overall ranges. HCC and CIRRHOTIC patients have significantly lower serum IGF-II mean levels than controls (p = 0.0001 for both comparisons, NPC test) but HCC has significantly higher mean IGF-II levels than CIRRHOTIC patients (p = 0.0001, NPC test).
Serum IGF-II levels above the best cut-off were found in 18 patients with HCC (22%), in 9 patients with liver cirrhosis (9.3%) and in 6 healthy subjects (20%); on the other hand, serum α-FP levels >132 ng/ml were observed in 39 patients with hepatocellular carcinoma (48%), in 3 patients with LC (3.1%) and in none of control group. Sensitivity, specificity and predictive values for IGF-II and α-FP in the diagnosis of HCC are showed in Table 3. In particular, using the optimal serum cut-off for α-FP (132 ng/ml), we obtained a specificity of 96.9% and a sensitivity of 40.6%, with a diagnostic accuracy of 68.7%. When the best cut-off for IGF-II was used (796 ng/ml), the resulting specificity was 90.6% and sensitivity was 18.9%, with a diagnostic accuracy of 54.7%. Finally when both markers were determined in parallel the sensitivity was 46.9%, the specificity was 90.6% and the diagnostic accuracy was 68.8%.
4. Discussion Hepatocellular carcinoma (HCC) is the leading cause of death in patients with liver cirrhosis [50] and early detection is of utmost importance for the treatment outcomes of this cancer. Periodic surveillance for diagnosis of HCC in patients with cirrhosis is recommended but serum α-FP level, although is a useful marker for monitoring of HCC, showed high false-negative rate for small size HCC [51]. The diagnostic accuracy of other serologic markers such as glypican-3, des-γ-carboxy prothrombin (DCP), insulin-like growth factor-1 (IGF-I), human hepatocyte growth factor (HGF), cromogranin A (CgA), nociceptin/orphanin (N/OFQ) is not yet clarified [31–38]. Recent researches have demonstrated changes in the IGF axis that affect the pathogenesis of HCC [2]. Table 2 p Value relative to correlation among: α-FP, IGF-II, nodules' number and diameter. α-FP α-FP IGF-II Nodules Diameter
Rs p-Value Rs p-Value Rs p-Value Rs p-Value
1.0000 0.4270 0.0001 0.3580 0.0001 0.2090(*) 0.0410
IGF-II 0.4270 0.0001 1.0000 − 0.1680 0.1020 0.2520(*) 0.0130
Nodules
Diameter
0.3580 0.0001 − 0.1680 0.1020 1.0000
0.2090 0.0410 0.2520 0.0130 − 0.0370 0.7240 1.0000
− 0.0370 0.7240
0.80
1.00
α-FP
Fig. 3. ROC curves for best cut-off. Receiver operating characteristic curve to identify the best cut-off for both α-FP and IGF-II serum levels for the diagnosis of HCC. The X-axis shows the different values of sensitivity, the Y-axis shows the corresponding values of 1-specificity. In the ROC curve the true positive rate (sensitivity) is plotted in function of the false positive rate (1-specificity) for different cut-off points, both for α-FP (in blue) and IGF-II (in red). Each point on the ROC curve represents a sensitivity/ specificity pair corresponding to a particular decision threshold. The area under the ROC curve is a measure of how well each parameter (α-FP and IGF-II) can distinguish between the two diagnostic groups (HCC/cirrhosis).
Insulin-like growth factor-II (IGF-II) is an autocrine and paracrine mitogen for liver cancer cell proliferation and IGF-II mRNA is distributed in the cytoplasm of hepatocytes and overexpressed in HCC tissues [52,53]. In the last years many researches have shown that IGF-II and IGF-II mRNA expressions were higher in HCC cells than in adjacent non-cancerous hepatic tissues [54–57]. Mitogenic activity of IGF-II is reduced by IGF binding protein 3 (IGFBP-3), and is reported undetectable or low in human HCC samples [53,58,59]. Furthermore reduced expression of IGFBP-3 was also significantly correlated with clinicopathological characteristics of HCC (tumor size, histological differentiation, capsular and portal venous invasion) [60]. HCC is a hypervascular tumor and IGF-II seems to play an important pathogenetic role by increasing vascular endothelial growth factor (VEGF)-mRNA and protein levels and, consequently, the hypervascularization, in a timedependent manner in human hepatoma cells [61]. Recently Dong et al. reported circulating free IGF-II levels significantly higher in patients with HCC than those with chronic hepatitis or liver cirrhosis; in addition circulating IGF-II mRNA was detected only in HCC patients and it was correlated with clinical stage and with presence of distant metastases of HCC [62]. In order to investigate if circulating IGF-II levels represent a valid indicator of HCC, we evaluated serum IGF-II and α-FP concentrations in patients with liver cirrhosis, with and without HCC, comparing the diagnostic value of the two markers. In our study circulating free IGF-II levels above normal range were found in 22% of patients with HCC and in 9% of patients with liver cirrhosis, showing low sensibility and high specificity (90.6%) of this marker. Furthermore determination in parallel of IGF-II and α-FP did not significantly increase the diagnostic accuracy in the detection of HCC.
Table 3 Diagnostic accuracy tests.
α-FP IGF-II α-FP + IGF-II
Sensitivity %
Specificity %
Accuracy%
PPV%
NPV%
LR+
LR−
40.6 18.9 46.9
96.9 90.6 90.6
68.7 54.7 68.8
92.9 66.7 83.3
62 52.7 63
13 2 5
0.61 0.90 0.59
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Also Tsai et al. investigated the diagnostic utility of serum IGF-II and α-FP levels in patients with HCC and with liver cirrhosis alone and reported IGF-II and α-FP levels higher in the former in comparison with the latter; moreover, in accordance with our results, they reported IGF-II mean values lower in cirrhotic patients than in healthy controls but, contrary to data obtained in our study, determination in parallel of both markers significantly increased the diagnostic accuracy and sensitivity (96.5% and 97.9%, respectively), with a high specificity (95.1%) [63]. More recently same authors demonstrated that serum IGF-II can be used as an independent serologic marker or a complementary marker to α-FP for the diagnosis of small HCC [64]. The discrepancy of our data with those of Tsai et al. may be related to the different methods of detecting of serum IGF-II levels; in both studies, in fact, Tsai et al. assessed IGF-II levels together with prealbumin values, after adequate nutritional adjustment. Thus, IGF-II values were expressed in mg/g prealbumin. On the contrary, in our study we used a method of detection (expressed in ng/ml) lacking in nutritional adjustment and, therefore, with better performance in order to time and costs. As we know HCC is a hypervascular tumor and angiogenesis is a critical finding for its growth and progression. Kim et al. suggested that hypoxia represents a very important stimulus for the new vessel formation and is associated to an overexpression of IGF-II in HCC tissue; secreted IGF-II, in addiction, may act as an angiogenic factor directly as well as indirectly by stimulating expression of VEGF gene [65]. In our study, when we applied NPC test, we observed a statistically significative correlation between circulating IGF-II levels and tumor's size (p = 0.013), probably in relation to a greater vascularization of cancer. In conclusion, among the many growth factors closely associated with development and growth of HCC, insulin-like growth factor-II (IGF-II) seems to have an important role, both directly and indirectly. However in our experience, circulating IGF-II cannot be recommended as a marker for detection of HCC in cirrhotic patients. Anyway the high specificity of IGF-II (90%) may suggest its use as confirmatory test in diagnosis of HCC. Learning points • IGF-II is a 67 amino-acid polypeptide closely related to insulin, produced by liver cells, predominantly active in physiological fetal liver growth. • Over-expression and high circulating levels of insulin-like growth factor-II (IGF-II) were reported in tissue and in serum of patients with HCC. • In this study we investigated the utility of circulating IGF-II levels, alone and in combination with serum α-FP, in the diagnosis of HCC and their possible correlation with number and/or size of the neoplastic nodules. • Circulating IGF-II is not a useful marker for HCC. Conflict of interest statement All authors have no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within 3 years of beginning the submitted work that could inappropriately influence, or be perceived to influence, their work. References [1] Colombo M. Malignant neoplasm of the liver. In: Schiff ER, Sorrel MF, Maddrey WC, editors. Schiff's Disease of the Liver. 9th ed. Philadelphia: Lippincott William & Wilkins; 2003. p. 1377–404. [2] Scharf J-G, Dombrowski F, Ramadori G. The IGF axis and hepatocarcinogenesis. J Clin Pathol Mol Pathol 2001;54:138–44.
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