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Clinical Cancer Research Vol. 12, 6043-6048, October 15, 2006
© 2006 American Association for Cancer Research

Imaging, Diagnosis, Prognosis

Hepatoma-Derived Growth Factor Is a Novel Prognostic Factor for Patients with Pancreatic Cancer

Hirokazu Uyama1, Yasuhiko Tomita2, Hideji Nakamura5, Shoji Nakamori3, Binglin Zhang2, Yoshihiko Hoshida2, Hirayuki Enomoto1, Yorihide Okuda1, Masato Sakon3, Katsuyuki Aozasa2, Ichiro Kawase4, Norio Hayashi1 and Morito Monden3

Authors' Affiliations: Departments of 1 Gastroenterology and Hepatology, 2 Pathology, 3 Surgery, and 4 Molecular Medicine, Osaka University Graduate School of Medicine, Yamada-oka, Suita, Osaka, Japan and 5 Division of Hepatobiliary and Pancreatic Medicine, Department of Internal Medicine, Hyogo College of Medicine, Mukogawa-cho, Nishinomiya, Hyogo, Japan

Requests for reprints: Hideji Nakamura, Division of Hepatobiliary and Pancreatic Medicine, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya 663-8501, Hyogo, Japan. Phone: 81-798-45-6472; E-mail: nakamura{at}hyo-med.ac.jp.


   Abstract
Top
 Abstract
Patients and Methods
 Results
 Discussion
 References
 
Purpose: Hepatoma-derived growth factor (HDGF) is a nucleus-targeted growth factor playing an important role in the development and progression of cancers. This study investigated the correlation of HDGF expression and prognosis in patients with pancreatic ductal carcinoma.

Patients and Methods: HDGF expression in pancreatic cancer cell lines was analyzed by Western blotting. HDGF expression was analyzed by immunohistochemistry for 50 patients with primary ductal carcinoma of the pancreas (33 male and 17 female) ranging in age from 48 to 80 years (median, 65 years) receiving surgical treatment. Cancer cells showing stronger staining than the noncancerous ducts were regarded as positive. Cases showing positive staining in <90% and >90% of tumor cells were regarded as HDGF labeling index (LI) levels 1 and 2, respectively. HDGF LI was determined separately for the nucleus and the cytoplasm.

Results: Western blotting showed HDGF expression in pancreatic cancer cells similar to that of hepatic cell lines. Twenty-three (46%) and 27 (54%) cases and 22 (44%) and 28 (56%) cases showed HDGF LI levels 1 and 2 for the nucleus and the cytoplasm, respectively. Patients with nuclear HDGF LI level 1 showed a significantly better 5-year survival rate (37.0%) than those with level 2 (6.8%; P = 0.023). No significant difference was observed in the cytoplasmic HDGF LI classification. Multivariate analysis revealed nuclear HDGF LI to be an independent prognosticator.

Conclusions: These findings suggest that HDGF could be a novel prognostic factor for pancreatic ductal carcinoma.

Pancreatic ductal carcinoma is one of the most fatal cancers showing a high potential for invasive activity and early recurrence with high recurrence rates (1–5). Even with the introduction of recent advances in imaging diagnostics such as computed tomographic scan and ultrasonography, most patients with pancreatic ductal carcinoma are diagnosed at an advanced stage, such as stage III or IV, and the tumors are usually unresectable (1). Even in resectable cases, the prognosis of patients with pancreatic ductal carcinoma is very poor (3–5). We are thus trying to identify useful prognostic factors other than the disease stage to predict the risk of disease recurrence more accurately.

Hepatoma-derived growth factor (HDGF) is a heparin-binding growth factor originally purified from media conditioned with the human hepatoma cell line HuH7 (6, 7). HDGF stimulates the proliferation of fibroblasts, endothelial cells, smooth muscle cells, and neuronal cells after translocation to the nucleus by use of the bipartite nuclear localization sequence in the COOH-terminal portion (8–11). HDGF is highly expressed in the early stage of organ development such as liver, lung, kidney, cardiovascular system, and gut, and thus considered to closely relate to the ontogeny (11–15). As for neoplasm, accumulating findings suggest that HDGF is closely related to the aggressive biological potential of cancer cells (16–19). These findings suggest that HDGF expression in cancer cells could be used as one of the prognosticators of cancer. Recently, a significant correlation has been reported between HDGF expression and the prognosis of patients with hepatocellular, gastric, and lung cancer (20–24).

In the present study, HDGF expression in pancreatic cancers was examined, and the correlation between the immunohistochemical index and recurrence or survival in patients with pancreatic ductal carcinoma was evaluated.


   Patients and Methods
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 Abstract
 Patients and Methods
Results
 Discussion
 References
 
Fifty patients who underwent resections for primary ductal carcinoma of the pancreas at the Gastroenterological Surgery Division, Osaka University Hospital from May 1995 to May 2003 were analyzed for this study. There were 33 males and 17 females of ages ranging from 48 to 80 years (median, 65 years). Preoperative diagnostic imaging examinations, ultrasonography, computed tomographic scan, magnetic resonance imaging study, and angiography were done for all patients. Forty-six patients initially received curative resection of the tumor whereas four patients received noncurative surgery.

Surgically resected specimens were fixed in 10% formalin, macroscopically examined, and then sliced at 5-mm intervals. The section containing the largest volume of tumor was processed for paraffin embedding. Four to five blocks per case were obtained.

Histologic sections, cut 4 µm thick, were stained with H&E and observed by three of the authors (Y.T., B.Z., and Y.H.) to determine the following categories: tumor histology, differentiation of tumor cells, vascular invasion, lymph node metastasis, and positivity of surgical margins. The surgical margin was identified as positive when cancer cells were present at the edge. Tumor stages were defined based on the pathologic tumor-node-metastasis (pTNM) classification (25). One representative slide per case was used for HDGF immunohistochemistry.

After resection, all patients were followed up by monitoring serum carcinoembryonic antigen and/or CA19-9, ultrasonography, computed tomographic scan, magnetic resonance imaging study, and chest X-ray every 1 and 3 months. The follow-up periods for survivors ranged from 3 to 72 months (median, 23 months) after surgery.

Antihuman HDGF antibody and Western blotting. Rabbit polyclonal antibody was raised against COOH-terminal amino acids (amino acids 231-240) of the human HDGF. The specificity and sensitivity of the antibody have previously been described (12, 16). Protein samples were extracted from human pancreatic cell lines with CelLytic-M Mammalian Cell Lysis Extraction Reagent (Sigma, St. Louis, MO). Ten micrograms of cell lysates, along with recombinant HDGF (10 ng) produced by Escherichia coli as a positive control, were electrophoresed in SDS-polyacrylamide gel and transblotted onto polyvinylidene transfer membranes (Millipore, Bedford, MA). The blotted membranes were reacted with an affinity-purified polyclonal anti–COOH terminus of HDGF antibody at a dilution of 1:10,000 and then visualized with an electrochemiluminescence detection system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom).

Immunohistochemical analysis. Immunohistochemical staining was done on formalin-fixed, paraffin-embedded sections (4 µm thick) with the avidin-biotin complex method. Antigen retrieval was done with microwave treatment (5 minutes, thrice) in 10 mmol/L citrate buffer (pH 6.0). The sections were immersed in methanol containing 0.3% hydrogen peroxidase for 20 minutes to block the endogenous peroxidase activity and incubated in 2.5% blocking serum for 30 minutes to reduce nonspecific binding. Anti-HDGF antibody was used as the primary antibody at a dilution of 1:5,000. Biotinylated antirabbit immunoglobulin G (Vector Laboratories, Burlingame, CA) was used as the secondary antibody. Sections were lightly counterstained with methyl green.

Stained sections were evaluated in a blind manner without prior knowledge of clinical information. Epithelial cells of the pancreatic ducts in the nonneoplastic lesion constantly showed weakly positive HDGF staining both for the nucleus and cytoplasm, then considered as an internal positive control. Cancer cells showing stronger staining than the noncancerous ducts were regarded as positive. The proportion of HDGF positive cells among all the cancer cells in the slide was estimated for both the nucleus and the cytoplasm. Finally, the HDGF labeling index (LI) was determined as follows: samples with <90% of cancer cells showing positive staining were regarded as HDGF LI level 1, and those with >90% as level 2. HDGF LI was determined separately for the nucleus and the cytoplasm.

Statistics. Statistical analysis was done with JMP software (SAS Institute, Inc., Cary, NC). Overall survival was measured from the date of surgery. The correlation between HDGF LI and clinicopathologic variables was evaluated by {chi}2 test and Fisher's exact probability test. The Kaplan-Meier method was used to calculate the overall patient survival rate and the difference in survival curves was evaluated with a log-rank test (26). Cox proportional hazards regression model was used in a stepwise manner to analyze the independent prognostic factors (27). P < 0.05 was considered to be statistically significant.


   Results
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 Abstract
 Patients and Methods
 Results
Discussion
 References
 
HDGF expression in pancreatic ductal carcinoma. HDGF was highly expressed in pancreatic cancer cell lines as well as hepatic cancer cells by Western blot analysis (Fig. 1 ). Immunohistochemically, 27 (54%) cases showed strong staining in the nucleus of >90% of tumor cells and were regarded as nuclear HDGF LI level 2; 28 (56%) cases with strong cytoplasmic staining in >90% of tumor cells were classified as cytoplasmic HDGF LI level 2 (Fig. 2 ).


Figure 1
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  		Fig. 1. Western blotting of HDGF. All the four cell lines of pancreatic ductal carcinoma (MIA PaCa-2, PANC-1, PL45, and KP-4) showed the strong band sized 39 kDa similar to hepatic cell lines (HuH7 and Hep G3). R, recombinant HDGF.

 

Figure 2
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  		Fig. 2. HDGF immunohistochemistry. A and B, pancreatic ductal carcinoma with nuclear HDGF LI level 1. Less than 90% of tumor cells show stronger staining than the endothelial cells. C and D, pancreatic ductal carcinoma with nuclear HDGF LI level 2. More than 90% of tumor cells show stronger staining than the endothelial cells. Bar, 200 µm.

 
Correlation between HDGF expression and clinicopathologic features in pancreatic ductal carcinoma. The histologic findings of pancreatic ductal carcinomas were as follows: 47 cases were tubular adenocarcinoma, 14 being well differentiated, 27 moderately differentiated, and 6 poorly differentiated. Of the remaining cases, two were papillary adenocarcinoma and one was spindle cell carcinoma. The pTNM classification of the tumor was stage II in 6 cases, stage III in 18, and stage IV in 26 (Table 1 ). The relationship between HDGF expression and the clinicopathologic features is presented in Table 1. There was no significant relationship between HDGF expression and any clinicopathologic variable and between nuclear and cytoplasmic HDGF expression.


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  		Table 1. Correlation between HDGF expression and clinicopathologic factors in 50 patients with pancreatic ductal carcinoma

 
Prognostic significance of HDGF expression in pancreatic ductal adenocarcinoma. Four patients who could not initially receive curative resection of the tumor, two with papillary adenocarcinoma, and one with spindle cell carcinoma were excluded from the prognostic analysis. The 5-year overall survival rate of the 43 patients receiving curative surgery for pancreatic carcinoma was 22.3%. Tumor recurrence was found in 35 (81.4%) patients, local recurrence in 11 (31.4%) patients, and metastasis to other organs in 24 (55.8%) patients. Twenty-eight patients died due to tumor, 11 died due to other cause (gastrointestinal hemorrhage, 2; heart failure, 1; pneumonia, 1; rupture of superior mesenteric aneurysm, 1; hepatorenal failure, 2; liver abscess, 1; cerebral infarction and hemorrhage, 2; respiratory failure, 1), and 7 were alive at the end of the observation period.

Patients with nuclear HDGF LI level 1 showed a significantly better 5-year survival rate (37.0%) than those with level 2 (6.8%; P = 0.023; Fig. 3 ). No significant difference was observed in the cytoplasmic HDGF LI classification. Hazard ratio and 95% confidence interval of the clinicopathologic factors are shown in Table 2 . T factor of the pTNM classification (pT2 versus pT3-4), histologic differentiation (poorly differentiated adenocarcinoma versus others), and nuclear HDGF LI were significant factors for overall survival. Multivariate analysis revealed nuclear HDGF LI to be an independent prognosticator (Table 3 ).


Figure 3
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  		Fig. 3. Overall survival curves of patients with pancreatic ductal carcinoma showing nuclear HDGF LI levels 1 and 2. Significant difference was observed between the two groups (P = 0.023).

 

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  		Table 2. Univariate analysis of clinicopathologic factors for overall survival of patients with pancreatic ductal adenocarcinoma

 

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  		Table 3. Multivariate analysis of clinicopathologic factors for overall survival of patients with pancreatic ductal adenocarcinoma

 

   Discussion
Top
 Abstract
 Patients and Methods
 Results
 Discussion
References
 
To establish effective therapeutic modalities for pancreatic ductal carcinoma, precise assessment of factors affecting tumor progression and patient prognosis is crucial. Although a conventional TNM staging system, based on tumor size, tumor progression, lymph node involvement, and distant metastasis, is useful for the stratification of patients with pancreatic ductal carcinoma, poor outcome of patients has been reported even for the low-stage (I and II) group (1, 2). Therefore, new prognosticators and the accumulation of statistical analyses are needed to develop an effective stratification system of pancreatic ductal carcinoma.

Previous studies in cellular biology have shown that HDGF, a member of the heparin-binding growth factor, has a wide range of biological functions, such as mitogenic activity and vascular development (7, 11, 17, 28). Overexpression of HDGF induces tumorigenesis in vivo through the combination of intrinsic angiogenic activity through induction of vascular endothelial growth factor (17, 28). We have shown that HDGF expression is increased in hepatocellular carcinoma compared with adjacent nontumorous areas in the mouse model (18). As for human cancers, a relationship of increased HDGF expression with poor outcome of patients has been reported for hepatocellular, gastric, and lung cancers (20–24). The present study tried to determine whether or not HDGF is related to the recurrence and prognosis of pancreatic ductal carcinoma.

We examined HDGF expression in pancreatic ductal carcinoma by immunohistochemical analysis using antibody to HDGF obtained by COOH-terminal peptide-conjugated Sepharose column, which shows prognostic significance based on the nuclear localization of HDGF. Prominent nuclear translocation of HDGF has been reported to be essential for increased DNA synthesis and proliferation, and growth-stimulating activity, which shows prognostic significance (8, 9). Pancreatic ductal carcinomas with higher HDGF LI in the nucleus might have shown a higher growth potential than those with lower HDGF LI, thus leading to poorer prognosis. In the present study, we divided HDGF-LI into four categories: <50%, 50% to 75%, 75% to 90%, and >90% positive staining in tumor cells. Among them, nuclear HDGF labeling was mostly significant when the cases were divided into those with staining in >90% of tumor cells and others, and then this cutoff level was employed. The actual number of patients was as follows: eight patients showed positive nuclear HDGF staining in <50% of tumor cells, eight patients in 50% to 75% of tumor cells, and seven patients in 75% to 90% of tumor cells. The last seven patients had values close to cutoff (>90%), and the former 16 patients showed truly low values.

Cytoplasmic localization of HDGF displayed no prognostic significance. Previous studies have shown the prognostic significance of cytoplasmic HDGF accumulation in hepatic and gastric cancers (20–22), although the precise role of cytoplasmic localization of HDGF is still unclear. Recently, the existence of a plasma membrane-located HDGF receptor has been reported (29). Further investigation is necessary to clarify the biological effect of cytoplasmic HDGF localization. The present analysis might suggest the importance of proliferation and growth activity induced by nuclear translocation of HDGF in the development of pancreatic ductal carcinoma.

Univariate and multivariate analyses showed nuclear HDGF expression to be an independent prognosticator for pancreatic ductal carcinoma. The main cause for the poor prognosis of pancreatic ductal carcinoma is local recurrence. Pleiotropic HDGF activities such as antiapoptosis, proliferation, invasiveness, and angiogenesis might enable the pancreatic carcinoma to display microinvasiveness at the resected margin, and thus promote the recurrence of cancer. These findings might explain why HDGF expression is a sign of poor prognosis in pancreatic ductal carcinoma.

In conclusion, HDGF expression as determined by immunohistochemistry could be used as a new prognosticator for pancreatic ductal carcinoma. Although further study is still needed to determine the precise role of HDGF in the malignant behavior of pancreatic ductal carcinoma, the results of the present study indicate that HDGF can serve as a potential target for anticancerous drug design.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 5/ 3/06; revised 7/12/06; accepted 7/25/06.


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 Patients and Methods
 Results
 Discussion
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Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
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