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Clinical Cancer Research Vol. 6, 4096-4100, October 2000
© 2000 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

Overexpression of Retinoblastoma Protein Predicts Decreased Survival and Correlates with Loss of p16INK4 Protein in Gallbladder Carcinomas1

Ya-Zhou Shi, Ai-Min Hui2, Xin Li, Tadatoshi Takayama and Masatoshi Makuuchi

Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study was designed to determine whether the level of retinoblastoma protein (pRb) expression predicts tumor progression and prognosis in gallbladder carcinomas (GBCs) and the relationship between pRb and p16INK4 protein expression. The expression of these two proteins was evaluated immunohistochemically in 37 tumors from 36 patients with GBC. pRb loss and overexpression were observed in 5 (13.5%) and 18 (48.6%) of the 37 tumors, respectively. Both pRb loss and overexpression were significantly correlated with advanced TNM stage, lymph node metastasis, and tumor perineural invasion. Moreover, pRb overexpression was significantly associated with decreased overall survival (P = 0.001; log-rank test). Further analysis indicated that the influence of pRb overexpression on survival was independent of TNM stage and lymph node metastasis. Loss of p16INK4 protein was observed in 28 of the 37 GBCs (75.7%), but was not significantly associated with any clinicopathological factors or survival. pRb overexpression was significantly associated with the loss of p16INK4 protein (P < 0.0001). These results suggest that pRb overexpression significantly predicts decreased survival in GBCs.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inactivation of the tumor suppressor gene products pRb3 and p16INK4 protein are common events in human cancers. The progression of cells from the G1 to the S phase is regulated via pRb phosphorylation by cyclin D complexed with cdks, which are in turn regulated by cdk inhibitors such as the p16INK4 protein. pRb is underphosphorylated throughout the G1 phase and phosphorylated just before the S phase. Hypophosphorylated pRb arrests cells in the G1 phase, and phosphorylation relieves this inhibition resulting in S phase entry (1) . p16INK4 associates with the cyclin D-cdk4 complex, preventing pRb phosphorylation and, consequently, S phase entry (2 , 3) . Dysregulation of the p16INK4/pRb pathway has been reported in numerous tumor types (4) . Li et al. (5) demonstrated that transcription of p16INK4 is repressed by pRb in cultured cells, and recently Fang et al. (6) showed that pRb expression is also transcriptionally repressed by p16INK4. These observations suggest that a bidirectional feedback may exist between pRb and p16INK4, and that the two proteins would be strictly and precisely regulated in this manner (6) .

The molecular events involved in tumor pathogenesis and progression of GBCs remain poorly characterized. p53 inactivation and K-ras gene mutation have been shown to contribute to the early stages of carcinogenesis of the gallbladder (7) and have no effect on tumor progression and clinical outcome (8 , 9) . Little is known about the role of pRb and p16INK4 in GBC. The purpose of this study was to determine whether the level of pRb expression predicts tumor progression and prognosis of GBC and the relationship between pRb and p16INK4 protein expression in GBC.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients and Specimens.
Resected specimens of 37 primary GBCs from 36 patients were obtained from our department between January 1990 and April 1999. One patient had double cancers of the gallbladder. The patients consisted of 17 men and 19 women with a median age of 65 years (range, 45–84 years). The clinicopathological variables were evaluated according to the General Rules for Surgical and Pathological Studies on Cancer of the Biliary Tract of the Japanese Society of Biliary Surgery (10) . The histological types included 12 papillary adenocarcinomas, 21 tubular adenocarcinomas, one mucinous adenocarcinoma, as well as one signet-ring cell, one adenosquamous, and one undifferentiated carcinoma. Thirty-seven tumors were classified into 11 stage I, 11 stage II, 7 stage III, and 8 stage IV GBCs according to the TNM classification system (11) . Survival was analyzed in 32 patients; 4 patients had tumor remnants after operation due to their advanced stage and were excluded from survival analysis. Seven specimens of normal gallbladder epithelia obtained from healthy persons who had undergone cholecystectomy when donating partial livers for transplantation were used as controls. Twenty-three specimens of noncancerous gallbladder epithelia from GBC patients were also available for pRb and p16INK4 evaluation.

Immunohistochemistry.
The procedure for staining pRb protein has been described by us elsewhere (12) . Briefly, following antigen retrieval, the sections were reacted with the mouse monoclonal anti-pRb antibody (clone G3-245; PharMingen, San Diego, CA; dilution, 1:500). The protocol for p16INK4 staining was similar, except that two different primary antibodies were used. One was a mouse monoclonal anti-p16INK4 antibody (clone G175-405, PharMingen) dilution, 1:1000; the other was a rabbit polyclonal anti-p16INK4 antibody (C-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA; dilution, 1:1000). The antigen site was detected by the avidin-biotin-peroxidase complex method using a commercial kit (Vector Laboratories, Inc., Burlingame, CA). 3,3'-Diaminobenzidine tetrahydrochloride was used as the color reagent, and hematoxylin was used as a counterstain. According to a previous report and our experience, p16INK4 staining should be performed within 2 weeks after sectioning because antigen reactivity is lost soon after preparation (13) . All sections were stained within 1 week after sectioning in our study.

A section of normal esophageal squamous epithelium, which had been shown to be pRb-positive (14) , was processed in each run as a positive control. Normal human liver tissue was used as a positive control for p16INK4 (15) . In addition, positive staining of the surrounding stromal cells was used as an internal positive control for both pRb and p16INK4 for each specimen. Negative controls were obtained by omitting primary antibodies. Only nuclear staining of target cells was considered to be positive pRb immunoreactivity or positive p16INK4 immunoreactivity. Cytoplasmic staining was common in most p16INK4-positive cases.

Statistical Analysis.
The {chi}2 test was used to examine the association of pRb and p16INK4 expression with clinicopathological parameters. Survival statistics were evaluated using Kaplan-Meier analysis with a log-rank test. A P <0.05 was judged to indicate statistical significance.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
pRb Expression.
pRb nuclear immunoreactivity was observed in all 7 normal gallbladder epithelia from healthy persons (Fig. 1Citation A) and in 23 noncancerous gallbladder epithelia from GBC patients; the percentage of positive cells was >90%. The intensity of nuclear immunoreactivity was graded as weak and strong. All of the seven normal gallbladder epithelia and a large portion of the noncancerous gallbladder epithelia showed homogeneously weak nuclear immunoreactivity, and strongly stained cells were observed in some noncancerous gallbladder epithelia (<50%).



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  		Fig. 1. Immunohistochemical staining of pRb (A–D) and p16INK4 (E–H) (x350). A, a normal gallbladder epithelium showing weak pRb nuclear immunoreactivity. B, a tumor showing no pRb nuclear reactivity (pRb 0). Some stromal cells were pRb-positive (arrow). C, a tumor showing normal pRb expression (pRb 1+). D, a tumor showing pRb overexpression (pRb 2+). E, a normal gallbladder epithelium showing no p16INK4 nuclear immunoreactivity. F, a p16INK4-negative tumor. The surrounding stromal cells were p16INK4-positive (arrow). A tumor that was p16INK4-positive by a monoclonal antibody (clone G175-405) (G) and by a polyclonal antibody (C-20) (H). D and F represent a same portion of a tumor.

 
On the basis of the preliminary observations on normal and noncancerous gallbladder epithelia, GBCs were classified into three categories: (a) pRb 0 (loss), few or no tumor cell showing pRb immunoreactivity (Fig. 1Citation B); (b) pRb 1+ (normal), more than 1% of the tumor cells demonstrating pRb reactivity, but the strongly stained cells <50% of the total cells examined (Fig. 1Citation C); and (c) pRb 2+ (overexpression), strongly stained cells >=50% of the total cells examined (Fig. 1Citation D). In the 37 GBCs, pRb 0 was found in 5 (13.5%) tumors, pRb 1+ in 14 (37.8%) tumors, and pRb 2+ in 18 (48.6%) tumors (Table 1)Citation . Both pRb 0 and pRb 2+ were significantly correlated with late-stage (stage III or IV) disease (pRb 0 versus pRb 1+, P = 0.0015; pRb 2+ versus pRb 1+, P = 0.004; Table 1Citation ). Moreover, pRb 0 and pRb 2+ expression were often accompanied by lymph node metastasis, whereas no pRb 1+ tumors had lymph node metastasis (pRb 0 versus pRb 1+, P = 0.012; pRb 2+ versus pRb 1+, P = 0.0008; Table 1Citation ). In addition, both pRb 0 and pRb 2+ were significantly related to tumor perineural invasion (pRb 0 versus pRb 1+, P = 0.01; pRb 2+ versus pRb 1+, P = 0.0095; Table 1Citation ). The pRb expression level was not correlated with any other factors analyzed (Table 1)Citation .


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  		Table 1 %Relationship between pRb expression and clinicopathological features in GBCs

 
For the 32 patients who underwent radical surgery, the 5-year overall survival rates in patients with pRb 1+, pRb 0, and pRb 2+ tumors were 90%, 60%, and 21.5%, respectively. Kaplan-Meier curves showed that overall survival was shorter in the pRb 0 group as compared with pRb 1+ group, and even shorter in pRb 2+ group (Fig. 2Citation ; pRb 0 versus pRb 1+, P = 0.19; pRb 2+ versus pRb 1+, P = 0.001; pRb 2+ versus pRb 0, P = 0.28; log-rank test). In the group of 22 patients with stage I/II disease, patients with pRb 2+ tumors (8 patients) showed a significantly decreased overall survival compared with patients with pRb 1+ tumors (13 patients; P = 0.01); the 5-year overall survival rates were 18.8% for patients with pRb 2+ tumors and 90% for those with pRb 1+ tumors. Moreover, in the group of 25 patients without lymph node metastasis, pRb 2+ (n = 8) also significantly correlated with decreased survival (pRb 2+ versus pRb 1+, P = 0.01); the 5-year overall survival rates were 18.8% and 90% for patients with pRb 2+ tumors and pRb 1+ tumors, respectively. The number of pRb 0 patients was too small in both the stage I/II group (one tumor) and the node-negative group (three tumors) to allow a possible survival analysis. For all of the 36 patients, including 4 patients who underwent nonradical surgery, the 5-year overall survival rates were 90% in pRb 1+ group, 60% in pRb 0 group, and 18% in pRb 2+ group (pRb 0 versus pRb 1+, P = 0.19; pRb 2+ versus pRb 1+, P = 0.0003; pRb 2+ versus pRb 0, P = 0.17; log-rank test).



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  		Fig. 2. Kaplan-Meier curves for overall survival according to pRb status in 32 patients with resectable GBCs (pRb 0 versus pRb 1+, P = 0.19; pRb 2+ versus pRb 1+, P = 0.001; pRb 2+ versus pRb 0, P = 0.28; log-rank test).

 
p16INK4 Expression.
p16INK4 immunoreactivity was not observed in any normal gallbladder epithelia from the 7 healthy persons (Fig. 1Citation E) and the 23 noncancerous epithelia samples from GBC patients. Thirty-seven GBCs demonstrated varied p16INK4 immunoreactivity with the percentage of positive tumor cells ranging from 0–90%, and the tumors were classified into two categories: (a) p16INK4 -, few or no tumor cells demonstrating p16INK4 immunoreactivity (Fig. 1Citation F); and (b) p16INK4 +, >=1% of the tumor cells showing p16INK4 immunoreactivity (Fig. 1G and H)Citation . Two different primary antibodies used to detect p16INK4 showed similar patterns of expression (Fig. 1G and H)Citation . In the 37 GBCs, p16INK4 was negative (p16INK4 -) in 28 tumors (75.7%) and positive (p16INK4 +) in 9 tumors (24.3%) with the percentage of positive tumor cells ranging from 5–90%. The levels of p16INK4 expression were not associated with any clinicopathological parameter analyzed, including sex, age, histological status, tumor venous involvement, lymphatic or perineural invasion, node status, or TNM stage. The 5-year survival rate for patients with p16INK4-positive tumors (77.8%) was higher than that for patients with p16INK4-negative tumors (40.3%), and this difference approached significance (P = 0.07).

Relationship Between pRb and p16INK4 Expression.
pRb expression was inversely associated with p16INK4 expression (Table 2)Citation . All 5 pRb 0 tumors were p16INK4-positive, whereas all 18 pRb 2+ tumors were p16INK4-negative. No tumor showed an absence of both Rb and p16INK4, and only four tumors were positive for both proteins. pRb 2+ expression was closely correlated with the loss of p16INK4 expression (P < 0.0001).


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  		Table 2 %Relationship between pRb and p16INK4 expression in GBCs

 
The 37 tumors were divided into two groups based on the combination of pRb and p16INK4 expression: group I (33 tumors, 89.2%), tumors showing either loss of p16INK4 or loss of pRb; and group II (4 tumors, 10.8%), tumors demonstrating both p16INK4 and pRb expression. The combined p16INK4/pRb expression was not significantly related to any of clinicopathological features examined. Patients in group I had a significantly decreased overall survival compared with group II patients (P = 0.04). The 5-year overall survival rates were 43% for group I patients and 100% for those in group II.

Surgical procedures included 23 cholecystectomies (single cholecystectomy or cholecystectomy with resection to <2 cm depth of the liver bed) and 13 extended operations (resection of adjacent organs in addition to the gallbladder). The patients who underwent extended operations showed significantly decreased survival compared with those treated by cholecystectomy (P = 0.007). This may be attributable to the fact that the patients who underwent extended operations had advanced diseases.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Loss of pRb has been demonstrated in a variety of cancers, including retinoblastoma, osteosarcoma, small cell lung carcinoma, and bladder cancer (16, 17, 18) . The present study is the first to examine pRb expression in GBC. Loss of pRb expression (pRb 0) was observed in 5 of 37 (13.5%) GBCs, and was significantly associated with advanced TNM stage, lymph node metastasis, and perineural invasion (Table 1)Citation . We failed to find a significant association between pRb loss and patient outcome. This may be due to the smaller size of the study and the low frequency of pRb loss (5 of 37 tumors). Of particular importance in this study was that pRb overexpression was frequently observed (48.6%, 18 of 37 tumors) and correlated with tumor progression to the same extent as pRb loss (Table 1)Citation . Moreover, pRb overexpression was significantly associated with decreased survival (Fig. 2)Citation . These results suggest that not only pRb loss, but also its overexpression, is involved in gallbladder carcinogenesis, and that the latter is an even more frequent event and influences the clinical status of GBC more significantly. Further analysis showed that pRb overexpression was significantly associated with decreased survival in both the stage I/II group and in the node-negative group, suggesting that the influence of pRb overexpression on clinical outcome is independent of disease stage. Moreover, pRb overexpression was significantly correlated to poor survival for all of the patients, including those who underwent nonradical surgery. This implies that pRb status influences prognosis for GBC patients independent of tumor resectability. We speculate that the overexpression of pRb indicates an inactive status in which tumor-suppressor function is lost. Taken together, either the loss of pRb expression or pRb overexpression is significantly correlated with tumor progression, and pRb overexpression significantly predicts an unfavorable survival.

Loss of pRb expression is generally due to genetic alterations in the Rb gene (19 , 20) . Loss of heterozygosity at the Rb gene locus has been previously reported in 20% (3 of 15 tumors) of GBCs (21) , which is approximately comparable with the rate of pRb loss observed in our present study. Previous studies on pRb expression in cancers have focused on pRb loss. Consistent with our present study, pRb overexpression recently has been proved to be strongly correlated with tumor progression and disease outcome of bladder and hepatocellular carcinomas (12 , 22 , 23) . The actual mechanism by which overexpression of pRb blocks pRb function, however, remains unclear. There are at least two possibilities. It is possible that upstream genes involving pRb phosphorylation regulation are defective in such situations; p16INK4 can transcriptionally repress Rb expression, and its inactivation may lead to pRb overexpression through a feedback loop (6) . Simultaneously, inactivation of p16INK4 loses its inhibition on cdks, resulting in a disproportional level of the hyperphosphorylated pRb (inactive form). Supporting this speculation, in the present study, pRb overexpression was closely associated with loss of p16INK4 expression. The other possibility is that some mutations of the Rb gene or Rb protein binding to certain DNA viral oncoproteins may also lead to its functional loss while preserving its expression (19 , 24) . These dysfunctional pRbs may accumulate at a high level in tumor cells.

pRb controls the transition from the G1 to the S phase by interacting with the Early Region 2 of the Adenovirus 2 Genome (E2) Promoter Binding Factor (E2F) transcription factor. Recent studies indicated that phosphorylation of pRb by cyclin D-cdk4 disrupts its association with histone deacetylase, relieving repression of the cyclin E gene. The expression of cyclin E, and thus activation of cyclin E-cdk2, prevents pRb from binding and inhibiting E2F and enabling the transcription of genes required for passage through the G1 restriction point (25 , 26) . pRb phosphorylation is regulated by cdks, which are in turn positively regulated by cyclin D and cyclin E and negatively regulated by cdk inhibitors such as p16INK4, p21WAF1, p27Kip1, and p57Kip2 (2 , 27, 28, 29, 30, 31) . Of the factors regulating pRb phosphorylation, p16INK4 seems to be the most important; its inactivation leads to pRb hyperphosphorylation (2 , 3) . Therefore, we hypothesize that the inactivation of p16INK4 stimulates cells to increase pRb expression through a physiological feedback loop and simultaneously enhances the phosphorylation of pRb, resulting in an accumulation of hyperphosphorylated pRb (inactive form). In support of our hypothesis, a close association of pRb overexpression with p16INK4 protein loss has been found in bladder cancer (13) and in hepatocellular carcinoma (32) .

p16INK4 was not significantly associated with any clinicopathological features or survival. However, when grouping together tumors lacking p16INK4 and those lacking pRb, this subset of patients had a significantly poor survival compared with patients with tumors demonstrating both p16INK4 and pRb. This suggests that disruption of the p16INK4/pRb pathway plays an important role in GBC progression.

Data from this study suggest that: (a) pRb overexpression is associated with tumor progression to the same extent as pRb loss, and significantly predicts decreased survival in GBCs; and (b) pRb overexpression is closely correlated with the loss of p16INK4 protein in GBCs.


   ACKNOWLEDGMENTS
 
The authors are grateful to R. Miyazawa, Photographic Center, Tokyo University Hospital, for his help in photography.


   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.

1 Supported in part by a grant from the Ministry of Education, Science and Culture of Japan; a grant from the Japan-China Medical Association, Japan; and a grant from the Fujida Memorial Fund for Medical Research, a fund within Japan Society for the Promotion of Science (all to A-M. H.). Back

2 To whom requests for reprints should be addressed, at Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Phone: 81-3-3815-5411, extension 33321; Fax: 81-3-5684-3989; E-mail: amhui-tky{at}umin.ac.jp Back

3 The abbreviations used are: pRb, retinoblastoma protein; cdk, cyclin-dependent kinase; GBC, gallbladder carcinoma. Back

Received 2/26/00; revised 7/ 3/00; accepted 7/14/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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