- Email: [email protected]
Role of Osteopontin and CD44s Expression for Patients With Hepatocellular Carcinoma Undergoing Liver Transplantation or Resection
OUTCOMES
Role of Osteopontin and CD44s Expression for Patients With Hepatocellular Carcinoma Undergoing Liver Transplantation or Resection S. Beckebaum, X. Chen, G.C. Sotiropoulos, A. Radtke, M. Daoudaki, H.A. Baba, J. Wohlschlaeger, C.E. Broelsch, G. Gerken, and V.R. Cicinnati ABSTRACT Background. Detection of new biomarkers for hepatocellular carcinoma (HCC) is needed to estimate prognosis after liver transplantation (OLT) or hepatic resection. Osteopontin (OPN) is a secreted, calcium-binding, phosphorylated, acidic glycoprotein that is overexpressed in various cancers. Cluster differentiation 44 standard isoform (CD44s) is one of the primary receptors of OPN; it may contribute to metastatic tumor spread. Materials and Methods. Tumor tissue and surrounding hepatic parenchyma were obtained from 53 HCC patients who underwent liver resection. Their RNA was extracted from nitrogen-frozen tissues, and OPN mRNA levels were estimated by quantitative reverse transcription–polymerase chain reactions. Formalin-fixed, paraffin-embedded tissues were obtained from the same patients, and additionally from 60 OLT HCC patients to perform expression analysis for OPN and CD44s by standard avidin-biotin immunostaining methods. Results. Expression of OPN and CD44s was significantly higher among HCC compared with adjacent nontumor tissue. The OPN mRNA expression and protein abundance correlated positively; OPN overexpression was associated with high tumor grade. A positive correlation existed between OPN and CD44s expression; both proteins were significantly overexpressed in HCC lesions with positive lymph nodes. No significant correlation existed between patient survival and OPN and CD44s expression. Conclusion. Expression of both OPN and CD44s in HCC is associated with advanced tumor stage, thus possibly contributing prognostic information when evaluated together with classical clinicopathological parameters.
From the Department of Gastroenterology and Hepatology (S.B., G.G., V.R.C.), Department of General, Visceral, and Transplantation Surgery (S.B., X.C., G.C.S., A.R., M.D., C.E.B., V.R.C.), and Institute of Pathology and Neuropathology (H.A.B., J.W.), University Hospital Essen, University of Duisburg, Essen, Germany.
This work was supported in part by grant program 107-05710/ IFORES (V.R.C.) and grant program 107-05470/IFORES (S.B.). Address reprint requests to Susanne Beckebaum, MD, Interdisciplinary Liver Transplant Unit, University Hospital Essen, OPZ 2, Ebene A1, Hufelandstr. 55, 45122 Essen, Germany. E-mail: [email protected]
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.08.034
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3182
Transplantation Proceedings, 40, 3182–3184 (2008)
OSTEOPONTIN AND CD44S EXPRESSION
H
EPATOCELLULAR carcinoma (HCC) represents the third most common cause of cancer death in the world; its incidence is continuously increasing.1 Recurrence of HCC after surgical therapy, either liver resection or transplantation, is a major factor limiting patient survival.2 Therefore, the identification of new biomarkers to predict tumor spread and survival remains a subject of fundamental importance. Osteopontin (OPN), a secreted, calcium-binding, phosphorylated, acidic glycoprotein, has been implicated in tumorigenesis; it is recognized as a potential biomarker in a variety of human cancers.3 In a subgroup of patients with HCC and underlying hepatits B virus (HBV) infections, higher OPN levels correlated significantly with tumor stage.4,5 However, the role of OPN as a biomarker for HCC warrants further investigation to confirm its utility in all patients with HCC. OPN can engage a number of receptors, including cluster differentiation 44 (CD44). Moreover, OPN binding to CD44 has been shown to promote tumor cell spread and migration.6 CD44, a glycoprotein widely expressed on the cell surface, is involved in cell– cell and cell–matrix interactions. The standard isoform (CD44s), the smallest form of CD44, has been implicated in lymphocyte homing, tissue regeneration, and tumor cell dissemination.7 The aim of this study was to investigate the role of OPN and CD44s expression among HCC patients undergoing liver transplantation or resection. MATERIALS AND METHODS Patients and Specimens Formalin-fixed, paraffin-embedded tissues were obtained to evaluate the expression of OPN and CD44s among 60 HCC patients who were 16 to 67 years old (median age, 55 years) and who had undergone liver transplantation between 1989 and 2004. Tumor tissue and surrounding hepatic parenchyma were obtained from 53 HCC patients who underwent liver resection (range, 30 –79 years; median, 64 years). The male-to-female ratio was 3:1 in both study groups. The follow-up period of 78 months included 106 of 113 patients (93.8%). The study was approved by the local ethics committee; all patients provided written informed consent.
Quantitative Reverse Transcription–Polymerase Chain Reaction (qRT–PCR) Tumor and adjacent hepatic tissue were snap-frozen in liquid nitrogen immediately after surgical resection. Total RNA was isolated using the TRIzol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s protocol. The average amount and purity of RNA was determined by spectrophotometry. The qRT–PCR was carried out in ABI 7700 sequence detection system (SDS, Applied Biosystems, Foster City, Calif). Expression of OPN was assessed by relative quantification using the standard curve method.8 Beta-actin mRNA was used as an internal control, and mRNA extracted from HepG2 hepatoma cell line served as the calibrator. Sequence-specific PCR primers (Carl Roth GmbH ⫹ Co. KG, Karlsruhe, Germany) and fluorochrome-labeled internal oligonucleotide TaqMan probes (Qiagen, Hilden, Germany) were: OPN-forward primer, 5=-CGAGGAGTTGAAT GGTGCATAC-3=; OPN-reverse primer, 5=-TTTCAGCACTCTGGTCATCCA-3=; OPN-probe, 5=-ACGAGGACATCACCTCACA-3=; beta-actin-forward primer, 5=-ACCGAGG CCCCCCTG-3=; beta-actin-reverse primer, 5=-GGTCTCAAACATGAT-
3183 CTGGGTCA-3=; beta-actin-probe, 5=-ACCCCAAGGC CA ACCGCGA-3=.
Immunohistochemistry Sections of formalin-fixed, paraffin-embedded tissue samples deparaffinized in xylene were rehydrated in ethanol. Endogenous peroxidase and nonspecific background staining were blocked by incubating slides with 3% aqueous hydrogen peroxide for 30 minutes. The slides were washed with phosphate-buffered saline (PBS; pH 7.4) for 5 minutes. Tissue sections were heated in citrate buffer (10 mmol/L, pH 6.0) in a microwave oven (100°C) for 20 minutes. Slides were then incubated with anti-human OPN mouse monoclonal antibody (MAb, clone 10A16, Immuno-Biological Laboratories, Japan, Cat. No. 10011) and anti-human CD44s mouse MAb (clone DF1485, Novocastra Laboratories, Newcastle-uponTyne, United Kingdom, Cat. No. NCL-CD44-2) overnight at 4°C, followed by incubation with biotinylated secondary antibody for 20 minutes and staining with Cell and Tissue Staining Kit (R&D Systems,) according to the manufacturer’s protocol. The slides were analyzed independently by two observers using light microscopy. Staining results were classified into four groups, according to the percentage of positively stained tumor cells. The staining criteria were negative (⫺); weak (⫹), ⱕ25%; moderate (⫹⫹), 25%–50%; and strong (⫹⫹⫹), ⱖ50%.
Statistical Methods The OPN/beta-actin mRNA expressions in tumor versus adjacent hepatic tissue were compared using the Wilcoxon signed-rank tests. Multivariate correlation analysis was performed to determine the relationship between OPN Virgule/CD44s expression and clinicopathological features. Overall survival curves were calculated using the Kaplan-Meier method. Results are expressed as mean values ⫾ SEM unless otherwise specified. The level of critical significance was assigned at P ⬍ .05. All analyses were performed using SPSS 12.0 (SPSS, Chicago, Ill).
RESULTS
The qRT–PCR analysis revealed that OPN mRNA was overexpressed in 39 of 53 (74%) resected HCC patients. The average OPN/beta-actin ratio in tumor tissue was significantly higher compared with noncancerous tissue from the same patient (4.77 ⫾ 1.74 vs 0.07 ⫾ 0.03, P ⬍ .001). In adjacent nontumor hepatic tissue, the OPN/beta-actin ratio was less than 1.0 in 52 of 53 patients (98%). In contrast, in HCC, OPN/beta-actin ratios were lower than 1.0 in 33 of 53 subjects (62%), between 1.0 and 10.0 in 13 of 53 (25%), and higher than 10.0 in 7 of 53 (13%) patients. OPN was detected in cancer-cell cytoplasm by immunohistochemistry. Bile duct epithelia in adjacent nontumor tissue also stained intensely positive for OPN, whereas it was not detected in normal hepatocytes. We observed a significant correlation between OPN mRNA expression and OPN protein abundance (rho ⫽ 0.3497, P ⬍ .05). The OPN mRNA expression in HCC was significantly higher among patients older than 60 years than those younger than 60 years (P ⬍ .01). The OPN/beta-actin ratio in tumor tissue was less than 1 in 32 of 50 patients (64%) with high tumor differentiation, but greater than 1 in 3 of 3 patients (100%) with low tumor differentiation. The OPN
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protein abundance in tumor tissue of patients with N1 status was significantly greater than that in patients with N0 status (P ⬍ .05). Similarly, OPN/beta-actin ratio in tumor tissue was less than 1 in 32 of 48 patients (67%) with status N0, but more than 1 in 4 of 5 patients (80%) with status N1. No significant correlation was seen between the OPN expression and overall survival or patient characteristics, such as gender, etiology of liver disease, and Child-Turcotte-Pugh classification (data not shown). The CD44s protein abundance in tumor tissue from patients with N1 status was greater than that in patients with N0 status (P ⬍ .05). A significant correlation did exist between OPN and CD44s expression (rho ⫽ 0.3324, P ⬍ .001). No significant correlation existed between the expression of CD44s and overall survival or patient characteristics, such as age, gender, etiology of liver disease, and CTP classification (data not shown). DISCUSSION
Our study revealed that OPN was overexpressed in HCC. The expression tended to be higher in undifferentiated tumors. In addition, this expression was associated with higher tumor grade and was significantly greater in tumor tissues from patients with a positive lymph node status compared with patients with a negative lymph node status. These results are in line with a previous study demonstrating OPN overexpression in HCC with capsular infiltration and OPN-positive cancer cells often dispersed in the periphery of cancer nodules adjacent to stromal cells.9 OPNspecific antibodies effectively blocked HCC cell invasion in vitro and inhibited pulmonary metastasis in an animal model.5 Recently, it has been shown that an OPN fragment derived from an alternative splicing event may be essential for HCC cellular invasion. Its presence appeared to correlate with metastatic potential.10 Our results indicated a close correlation between OPN mRNA expression and OPN protein abundance, thus hinting at only minor posttranscriptional modifications in the production of OPN by HCC. Our study revealed a significant correlation between the expression of OPN and CD44s in HCC. The CD44s expression was much higher in HCC than in adjacent nontumor hepatic parenchyma, suggesting aberrant induction of CD44s protein synthesis in HCC. We observed greater expression of CD44s in HCC from patients with positive lymph node status as compared to patients with negative lymph node status. Activated forms of Ras induced the expression of both OPN and its receptor CD44 in cancer cells in vitro, thus suggesting the existence of an autocrine loop that synergistically stimulates mitogenactivated protein kinase and foci formation.11 The OPN binding to CD44 cell-surface molecules has been shown to promote tumor cell spreading and migration.12 OPN binding to alpha(v)beta(3)-integrin increased CD44v6 expression and augmented cell adhesion in hepatoma cells in vitro.13
BECKEBAUM, CHEN, SOTIROPOULOS ET AL
Although it has been suggested that up-regulation of CD44s and its isoforms is associated with poorly differentiated HCC,14 our findings and the results of others15,16 did not indicate a correlation between CD44s expression and HCC differentiation. In line with findings from Terris et al,15 we did not observe a correlation between patient survival and the extent of CD44s expression in HCC. In summary, our study revealed that OPN and CD44s were both overexpressed in HCC irrespective of the underlying liver disease. Expression of both proteins was associated with advanced HCC, and particularly with lymphatic tumor spread. Combined analysis of these two biomarkers may add to the prognostic information yielded by clinicopathological parameters alone, and may facilitate therapeutic management of HCC patients. REFERENCES 1. Parkin DM, Bray F, Ferlay J, et al: Global cancer statistics, 2002. CA Cancer J Clin 55:74, 2005 2. Llovet JM, Burroughs A, Bruix J: Hepatocellular carcinoma. Lancet 362:1907, 2003 3. Rittling SR, Chambers AF: Role of osteopontin in tumour progression. Br J Cancer 90:1877, 2004 4. Kim J, Ki SS, Lee SD, et al: Elevated plasma osteopontin levels in patients with hepatocellular carcinoma. Am J Gastroenterol 101:2051, 2006 5. Ye QH, Qin LX, Forgues M, et al: Predicting hepatitis B viruspositive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning. Nat Med 9:416, 2003 6. Furger KA, Menon RK, Tuck AB, et al: The functional and clinical roles of osteopontin in cancer and metastasis. Curr Mol Med 1:621, 2001 7. Goodison S, Urquidi V, Tarin D: CD44 cell adhesion molecules. Mol Pathol 52:189, 1999 8. Giulietti A, Overbergh L, Valckx D, et al: An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25:386, 2001 9. Gotoh M, Sakamoto M, Kanetaka K, et al: Overexpression of osteopontin in hepatocellular carcinoma. Pathol Int 52:19, 2002 10. Takafuji V, Forgues M, Unsworth E, et al: An osteopontin fragment is essential for tumor cell invasion in hepatocellular carcinoma. Oncogene 26:6361, 2007 11. Teramoto H, Castellone MD, Malek RL, et al: Autocrine activation of an osteopontin-CD44-Rac pathway enhances invasion and transformation by H-RasV12. Oncogene 24:489, 2005 12. Weber GF, Ashkar S, Cantor H: Interaction between CD44 and osteopontin as a potential basis for metastasis formation. Proc Assoc Am Physicians 109:1, 1997 13. Gao C, Guo H, Downey L, et al: Osteopontin-dependent CD44v6 expression and cell adhesion in HepG2 cells. Carcinogenesis 24:1871, 2003 14. Endo K, Terada T: Protein expression of CD44 (standard and variant isoforms) in hepatocellular carcinoma: relationships with tumor grade, clinicopathologic parameters, p53 expression, and patient survival. J Hepatol 32:78, 2000 15. Terris B, Laurent-Puig P, Belghitti J, et al: Prognostic influence of clinicopathologic features, DNA-ploidy, CD44H and p53 expression in a large series of resected hepatocellular carcinomas in France. Int J Cancer 74:614, 1997 16. Mathew J, Hines JE, Obafunwa JO, et al: CD44 is expressed in hepatocellular carcinomas showing vascular invasion. J Pathol 179:74, 1996
Role of Osteopontin and CD44s Expression for Patients With Hepatocellular Carcinoma Undergoing Liver Transplantation or Resection S. Beckebaum, X. Chen, G.C. Sotiropoulos, A. Radtke, M. Daoudaki, H.A. Baba, J. Wohlschlaeger, C.E. Broelsch, G. Gerken, and V.R. Cicinnati ABSTRACT Background. Detection of new biomarkers for hepatocellular carcinoma (HCC) is needed to estimate prognosis after liver transplantation (OLT) or hepatic resection. Osteopontin (OPN) is a secreted, calcium-binding, phosphorylated, acidic glycoprotein that is overexpressed in various cancers. Cluster differentiation 44 standard isoform (CD44s) is one of the primary receptors of OPN; it may contribute to metastatic tumor spread. Materials and Methods. Tumor tissue and surrounding hepatic parenchyma were obtained from 53 HCC patients who underwent liver resection. Their RNA was extracted from nitrogen-frozen tissues, and OPN mRNA levels were estimated by quantitative reverse transcription–polymerase chain reactions. Formalin-fixed, paraffin-embedded tissues were obtained from the same patients, and additionally from 60 OLT HCC patients to perform expression analysis for OPN and CD44s by standard avidin-biotin immunostaining methods. Results. Expression of OPN and CD44s was significantly higher among HCC compared with adjacent nontumor tissue. The OPN mRNA expression and protein abundance correlated positively; OPN overexpression was associated with high tumor grade. A positive correlation existed between OPN and CD44s expression; both proteins were significantly overexpressed in HCC lesions with positive lymph nodes. No significant correlation existed between patient survival and OPN and CD44s expression. Conclusion. Expression of both OPN and CD44s in HCC is associated with advanced tumor stage, thus possibly contributing prognostic information when evaluated together with classical clinicopathological parameters.
From the Department of Gastroenterology and Hepatology (S.B., G.G., V.R.C.), Department of General, Visceral, and Transplantation Surgery (S.B., X.C., G.C.S., A.R., M.D., C.E.B., V.R.C.), and Institute of Pathology and Neuropathology (H.A.B., J.W.), University Hospital Essen, University of Duisburg, Essen, Germany.
This work was supported in part by grant program 107-05710/ IFORES (V.R.C.) and grant program 107-05470/IFORES (S.B.). Address reprint requests to Susanne Beckebaum, MD, Interdisciplinary Liver Transplant Unit, University Hospital Essen, OPZ 2, Ebene A1, Hufelandstr. 55, 45122 Essen, Germany. E-mail: [email protected]
0041-1345/08/$–see front matter doi:10.1016/j.transproceed.2008.08.034
© 2008 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
3182
Transplantation Proceedings, 40, 3182–3184 (2008)
OSTEOPONTIN AND CD44S EXPRESSION
H
EPATOCELLULAR carcinoma (HCC) represents the third most common cause of cancer death in the world; its incidence is continuously increasing.1 Recurrence of HCC after surgical therapy, either liver resection or transplantation, is a major factor limiting patient survival.2 Therefore, the identification of new biomarkers to predict tumor spread and survival remains a subject of fundamental importance. Osteopontin (OPN), a secreted, calcium-binding, phosphorylated, acidic glycoprotein, has been implicated in tumorigenesis; it is recognized as a potential biomarker in a variety of human cancers.3 In a subgroup of patients with HCC and underlying hepatits B virus (HBV) infections, higher OPN levels correlated significantly with tumor stage.4,5 However, the role of OPN as a biomarker for HCC warrants further investigation to confirm its utility in all patients with HCC. OPN can engage a number of receptors, including cluster differentiation 44 (CD44). Moreover, OPN binding to CD44 has been shown to promote tumor cell spread and migration.6 CD44, a glycoprotein widely expressed on the cell surface, is involved in cell– cell and cell–matrix interactions. The standard isoform (CD44s), the smallest form of CD44, has been implicated in lymphocyte homing, tissue regeneration, and tumor cell dissemination.7 The aim of this study was to investigate the role of OPN and CD44s expression among HCC patients undergoing liver transplantation or resection. MATERIALS AND METHODS Patients and Specimens Formalin-fixed, paraffin-embedded tissues were obtained to evaluate the expression of OPN and CD44s among 60 HCC patients who were 16 to 67 years old (median age, 55 years) and who had undergone liver transplantation between 1989 and 2004. Tumor tissue and surrounding hepatic parenchyma were obtained from 53 HCC patients who underwent liver resection (range, 30 –79 years; median, 64 years). The male-to-female ratio was 3:1 in both study groups. The follow-up period of 78 months included 106 of 113 patients (93.8%). The study was approved by the local ethics committee; all patients provided written informed consent.
Quantitative Reverse Transcription–Polymerase Chain Reaction (qRT–PCR) Tumor and adjacent hepatic tissue were snap-frozen in liquid nitrogen immediately after surgical resection. Total RNA was isolated using the TRIzol reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s protocol. The average amount and purity of RNA was determined by spectrophotometry. The qRT–PCR was carried out in ABI 7700 sequence detection system (SDS, Applied Biosystems, Foster City, Calif). Expression of OPN was assessed by relative quantification using the standard curve method.8 Beta-actin mRNA was used as an internal control, and mRNA extracted from HepG2 hepatoma cell line served as the calibrator. Sequence-specific PCR primers (Carl Roth GmbH ⫹ Co. KG, Karlsruhe, Germany) and fluorochrome-labeled internal oligonucleotide TaqMan probes (Qiagen, Hilden, Germany) were: OPN-forward primer, 5=-CGAGGAGTTGAAT GGTGCATAC-3=; OPN-reverse primer, 5=-TTTCAGCACTCTGGTCATCCA-3=; OPN-probe, 5=-ACGAGGACATCACCTCACA-3=; beta-actin-forward primer, 5=-ACCGAGG CCCCCCTG-3=; beta-actin-reverse primer, 5=-GGTCTCAAACATGAT-
3183 CTGGGTCA-3=; beta-actin-probe, 5=-ACCCCAAGGC CA ACCGCGA-3=.
Immunohistochemistry Sections of formalin-fixed, paraffin-embedded tissue samples deparaffinized in xylene were rehydrated in ethanol. Endogenous peroxidase and nonspecific background staining were blocked by incubating slides with 3% aqueous hydrogen peroxide for 30 minutes. The slides were washed with phosphate-buffered saline (PBS; pH 7.4) for 5 minutes. Tissue sections were heated in citrate buffer (10 mmol/L, pH 6.0) in a microwave oven (100°C) for 20 minutes. Slides were then incubated with anti-human OPN mouse monoclonal antibody (MAb, clone 10A16, Immuno-Biological Laboratories, Japan, Cat. No. 10011) and anti-human CD44s mouse MAb (clone DF1485, Novocastra Laboratories, Newcastle-uponTyne, United Kingdom, Cat. No. NCL-CD44-2) overnight at 4°C, followed by incubation with biotinylated secondary antibody for 20 minutes and staining with Cell and Tissue Staining Kit (R&D Systems,) according to the manufacturer’s protocol. The slides were analyzed independently by two observers using light microscopy. Staining results were classified into four groups, according to the percentage of positively stained tumor cells. The staining criteria were negative (⫺); weak (⫹), ⱕ25%; moderate (⫹⫹), 25%–50%; and strong (⫹⫹⫹), ⱖ50%.
Statistical Methods The OPN/beta-actin mRNA expressions in tumor versus adjacent hepatic tissue were compared using the Wilcoxon signed-rank tests. Multivariate correlation analysis was performed to determine the relationship between OPN Virgule/CD44s expression and clinicopathological features. Overall survival curves were calculated using the Kaplan-Meier method. Results are expressed as mean values ⫾ SEM unless otherwise specified. The level of critical significance was assigned at P ⬍ .05. All analyses were performed using SPSS 12.0 (SPSS, Chicago, Ill).
RESULTS
The qRT–PCR analysis revealed that OPN mRNA was overexpressed in 39 of 53 (74%) resected HCC patients. The average OPN/beta-actin ratio in tumor tissue was significantly higher compared with noncancerous tissue from the same patient (4.77 ⫾ 1.74 vs 0.07 ⫾ 0.03, P ⬍ .001). In adjacent nontumor hepatic tissue, the OPN/beta-actin ratio was less than 1.0 in 52 of 53 patients (98%). In contrast, in HCC, OPN/beta-actin ratios were lower than 1.0 in 33 of 53 subjects (62%), between 1.0 and 10.0 in 13 of 53 (25%), and higher than 10.0 in 7 of 53 (13%) patients. OPN was detected in cancer-cell cytoplasm by immunohistochemistry. Bile duct epithelia in adjacent nontumor tissue also stained intensely positive for OPN, whereas it was not detected in normal hepatocytes. We observed a significant correlation between OPN mRNA expression and OPN protein abundance (rho ⫽ 0.3497, P ⬍ .05). The OPN mRNA expression in HCC was significantly higher among patients older than 60 years than those younger than 60 years (P ⬍ .01). The OPN/beta-actin ratio in tumor tissue was less than 1 in 32 of 50 patients (64%) with high tumor differentiation, but greater than 1 in 3 of 3 patients (100%) with low tumor differentiation. The OPN
3184
protein abundance in tumor tissue of patients with N1 status was significantly greater than that in patients with N0 status (P ⬍ .05). Similarly, OPN/beta-actin ratio in tumor tissue was less than 1 in 32 of 48 patients (67%) with status N0, but more than 1 in 4 of 5 patients (80%) with status N1. No significant correlation was seen between the OPN expression and overall survival or patient characteristics, such as gender, etiology of liver disease, and Child-Turcotte-Pugh classification (data not shown). The CD44s protein abundance in tumor tissue from patients with N1 status was greater than that in patients with N0 status (P ⬍ .05). A significant correlation did exist between OPN and CD44s expression (rho ⫽ 0.3324, P ⬍ .001). No significant correlation existed between the expression of CD44s and overall survival or patient characteristics, such as age, gender, etiology of liver disease, and CTP classification (data not shown). DISCUSSION
Our study revealed that OPN was overexpressed in HCC. The expression tended to be higher in undifferentiated tumors. In addition, this expression was associated with higher tumor grade and was significantly greater in tumor tissues from patients with a positive lymph node status compared with patients with a negative lymph node status. These results are in line with a previous study demonstrating OPN overexpression in HCC with capsular infiltration and OPN-positive cancer cells often dispersed in the periphery of cancer nodules adjacent to stromal cells.9 OPNspecific antibodies effectively blocked HCC cell invasion in vitro and inhibited pulmonary metastasis in an animal model.5 Recently, it has been shown that an OPN fragment derived from an alternative splicing event may be essential for HCC cellular invasion. Its presence appeared to correlate with metastatic potential.10 Our results indicated a close correlation between OPN mRNA expression and OPN protein abundance, thus hinting at only minor posttranscriptional modifications in the production of OPN by HCC. Our study revealed a significant correlation between the expression of OPN and CD44s in HCC. The CD44s expression was much higher in HCC than in adjacent nontumor hepatic parenchyma, suggesting aberrant induction of CD44s protein synthesis in HCC. We observed greater expression of CD44s in HCC from patients with positive lymph node status as compared to patients with negative lymph node status. Activated forms of Ras induced the expression of both OPN and its receptor CD44 in cancer cells in vitro, thus suggesting the existence of an autocrine loop that synergistically stimulates mitogenactivated protein kinase and foci formation.11 The OPN binding to CD44 cell-surface molecules has been shown to promote tumor cell spreading and migration.12 OPN binding to alpha(v)beta(3)-integrin increased CD44v6 expression and augmented cell adhesion in hepatoma cells in vitro.13
BECKEBAUM, CHEN, SOTIROPOULOS ET AL
Although it has been suggested that up-regulation of CD44s and its isoforms is associated with poorly differentiated HCC,14 our findings and the results of others15,16 did not indicate a correlation between CD44s expression and HCC differentiation. In line with findings from Terris et al,15 we did not observe a correlation between patient survival and the extent of CD44s expression in HCC. In summary, our study revealed that OPN and CD44s were both overexpressed in HCC irrespective of the underlying liver disease. Expression of both proteins was associated with advanced HCC, and particularly with lymphatic tumor spread. Combined analysis of these two biomarkers may add to the prognostic information yielded by clinicopathological parameters alone, and may facilitate therapeutic management of HCC patients. REFERENCES 1. Parkin DM, Bray F, Ferlay J, et al: Global cancer statistics, 2002. CA Cancer J Clin 55:74, 2005 2. Llovet JM, Burroughs A, Bruix J: Hepatocellular carcinoma. Lancet 362:1907, 2003 3. Rittling SR, Chambers AF: Role of osteopontin in tumour progression. Br J Cancer 90:1877, 2004 4. Kim J, Ki SS, Lee SD, et al: Elevated plasma osteopontin levels in patients with hepatocellular carcinoma. Am J Gastroenterol 101:2051, 2006 5. Ye QH, Qin LX, Forgues M, et al: Predicting hepatitis B viruspositive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning. Nat Med 9:416, 2003 6. Furger KA, Menon RK, Tuck AB, et al: The functional and clinical roles of osteopontin in cancer and metastasis. Curr Mol Med 1:621, 2001 7. Goodison S, Urquidi V, Tarin D: CD44 cell adhesion molecules. Mol Pathol 52:189, 1999 8. Giulietti A, Overbergh L, Valckx D, et al: An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25:386, 2001 9. Gotoh M, Sakamoto M, Kanetaka K, et al: Overexpression of osteopontin in hepatocellular carcinoma. Pathol Int 52:19, 2002 10. Takafuji V, Forgues M, Unsworth E, et al: An osteopontin fragment is essential for tumor cell invasion in hepatocellular carcinoma. Oncogene 26:6361, 2007 11. Teramoto H, Castellone MD, Malek RL, et al: Autocrine activation of an osteopontin-CD44-Rac pathway enhances invasion and transformation by H-RasV12. Oncogene 24:489, 2005 12. Weber GF, Ashkar S, Cantor H: Interaction between CD44 and osteopontin as a potential basis for metastasis formation. Proc Assoc Am Physicians 109:1, 1997 13. Gao C, Guo H, Downey L, et al: Osteopontin-dependent CD44v6 expression and cell adhesion in HepG2 cells. Carcinogenesis 24:1871, 2003 14. Endo K, Terada T: Protein expression of CD44 (standard and variant isoforms) in hepatocellular carcinoma: relationships with tumor grade, clinicopathologic parameters, p53 expression, and patient survival. J Hepatol 32:78, 2000 15. Terris B, Laurent-Puig P, Belghitti J, et al: Prognostic influence of clinicopathologic features, DNA-ploidy, CD44H and p53 expression in a large series of resected hepatocellular carcinomas in France. Int J Cancer 74:614, 1997 16. Mathew J, Hines JE, Obafunwa JO, et al: CD44 is expressed in hepatocellular carcinomas showing vascular invasion. J Pathol 179:74, 1996