Low p 27 Expression Is an Independent Predictor of Survival for Patients with Either Hilar or Peripheral Intrahepatic Cholangiocarcinoma 1

Purpose and Experimental Design: The prognosis for intrahepatic cholangiocarcinoma (ICC) depends mainly on the feasibility of complete surgical resection. In the absence of demonstrated biological predictors of survival, we evaluated the prognostic value of the cyclin-dependent kinase inhibitor p27 by immunohistochemistry in a series of routine specimens from 47 ICC patients, 22 with the hilar and 25 with the peripheral subtype. Proliferation rate was also evaluated in the same cases by the MIB1 index. Tumors were scored as high, low, and negative p27 expressers (>50%, <50%, and no positive nuclei, respectively). Results: High, low, and negative p27 expression was recorded in 18 (38%), 17 (36%), and 12 (26%) cases, respectively. No significant correlation was found between p27 expression and gender, age, tumor grade, tumor location, vascular or perineural invasion, or proliferative index. Tumors with low or absent p27 expression were associated with poor survival compared with the high-expresser group. Kaplan-Meier curves comparing different p27 expression levels with survival showed highly significant separation (P < 0.0001, log-rank test). With univariate Cox proportional hazard regression, only p27 score among all of the parameters was found to influence survival (P 0.0003). Conclusion: We conclude that in ICC, low or absent p27 expression can predict poor survival, regardless of tumor location, pathological features, and tumor proliferation. Immunohistochemical detection of p27 on routine sections may provide the first biological prognostic marker for ICC, thus influencing the therapeutic strategies for these patients. INTRODUCTION The p27 cyclin-dependent kinase inhibitor Cdki is a negative cell-cycle regulator that belongs to the Cip/Kip group of cyclin-dependent kinase inhibitors and shares sequence homology with p21 and p57 (1). The p27 protein penetrates the nucleus, where it preferentially binds to cyclin A–E/Cdk2 complexes and inactivates them, thereby preventing the cell from entering the S-phase (2, 3). Expression of the p27 gene is posttranscriptionally regulated by proteolytic degradation via the ubiquitin-proteasome pathway (4). Although some effective point mutations have been described, the main control system for the function of p27 seems to be retention of the protein in the cytoplasm, where ubiquination takes place (4, 5). Low or absent nuclear immunohistochemical expression of p27 is an important negative prognostic parameter for many human epithelial tumors in the breast, colon, stomach, prostate, lung, liver, and elsewhere (6–20). Regarding tumors of biliary origin, a recent study revealed the existence of an inverse correlation between p27 and cyclin D1 expression that can significantly predict survival in patients with resectable carcinoma of the extrahepatic bile ducts (21). Moreover, loss of p27 expression in gallbladder carcinoma patients has been associated with poor prognosis and tumor progression (22). ICCs are commonly classified as PCs (arising form small bile ducts) and HCs (originating from the large bile ducts of the hepatic hilus), also known as Klatskin tumors (23, 24). The prognosis for ICC is generally poor, with long-term as well as short-term survival almost exclusively depending on the feasibility of surgical resection (25). None of the many proposed biological prognostic parameters, such as p53 mutations, cerbB-2, c-met, and Muc1 expression, has proven reliable enough to be systematically adopted in clinical practice (26–30). It should also be noted that many studies have lumped ICCs together with distal extra-hepatic biliary tumors and carcinoma of the gallbladder, thus causing confusion in a field where tumor location is itself a prognostic parameter. In the present study we analyzed the expression of p27 by IHC in 47 human resected ICC specimens (25 PC and 22 HC) and in the adjacent nonneoplastic tissues. Expression of p27 was analyzed with respect to the main pathological prognostic variables of ICC, proliferation index and overall survival. MATERIALS AND METHODS Patient Population. We performed a retrospective study on 47 patients (22 men and 25 women; mean age, 60.7 years) Received 6/1/01; revised 8/30/01; accepted 9/4/01. 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 This research was supported by Ministero della Università e della Ricerca Scientifica (MURST) e Università degli Studi di Bologna. 2 To whom requests for reprints should be addressed, at Istituto Oncologico “Addarii,” Viale Ercolani 4/2, 40138 Bologna, Italy. Phone: 39051-6364546; Fax: 39-051-6364403; E-mail: grigioni@med.unibo. it. 3 The abbreviations used are: IHC, immunohistochemistry; ICC, intrahepatic cholangiocarcinoma; PC and HC, peripheral-type and hilar-type cholangiocarcinoma, respectively; DAB, 3,3 -diaminobenzidine. 3994 Vol. 7, 3994–3999, December 2001 Clinical Cancer Research Research. on July 9, 2017. © 2001 American Association for Cancer clincancerres.aacrjournals. org Downloaded from who underwent liver resection for ICC between 1984 and 1998 in our hospital. Pathological, demographic, and survival data of the patients were retrieved from the files of the Pathology Unit at the “Addarii” Institute of Oncology and the Department of Surgery at the University of Bologna. Twenty-two tumors were HC, whereas 25 were PC. All tumors were histologically diagnosed, graded, and grouped as moderately differentiated (grade 2; n 29) or poorly differentiated (grade 3; n 18). Tumors were also grouped according to the presence (n 15) or absence (n 32) of neoplastic vascular thrombosis and the presence (n 16) or absence (n 31) of neoplastic perineural invasion (Table 1). The follow-up interval ranged from 1 to 60 months from the date of surgery (mean, 12.7 months). None of the patients received postoperative adjuvant therapy. After surgical resection, specimens from neoplastic and surrounding nonneoplastic tissue were fixed in 10% buffered formalin. Informed consent was obtained for all patients. IHC. Fourm tissue sections were cut, placed on silanepretreated slides, deparaffinized, and rehydrated through graded alcohols. Antigen retrieval was performed by microwave heating at high power (750 W) in 10 mM sodium citrate buffer (pH 6) for four cycles of 5 min each. Slides were then allowed to cool for 30 min prior to incubation with either the p27 monoclonal antibody (Transduction Laboratories, Lexington, KY) diluted 1:200 or the anti-Ki67 monoclonal antibody (clone MIB1; Biogenex, San Ramon, CA) diluted 1:50 for 1 h at room temperature. Staining was performed with the Envision Monoclonal System (Dako, Carpinteria, CA). The reaction was developed with DAB. Slides were counterstained with Mayer’s hematoxylin and mounted. Nonimmune mouse serum was used as negative control. Lymphocytes were used as the internal positive control for p27. Identical reaction times allowed accurate comparison of all samples. Immunohistochemical staining was evaluated independently by two pathologists (M. F. and A. D.). IHC for p27 was scored as the ratio of nuclei displaying clear-cut positivity with respect to the total number of cells, as described previously (20). Tumors were categorized into three groups: high p27 expressers ( 50% positive nuclei; score 2), low p27 expressers ( 50% positive nuclei; score 1), and p27negative tumors; score 0). The proliferation index was calculated as the number of MIB1-positive nuclei over the total number of neoplastic cells, and tumors were scored as low proliferating ( 10% positive neoplastic cells) or high proliferating ( 10% positive neoplastic cells; Table 1). At least 20 high-power fields were randomly chosen, and 2000 cells were always counted. Statistical Analysis. Associations between the p27 score and other clinicopathological variables were assessed by Pearson’s 2 test (31). Survival times were calculated from the date of initial diagnosis. Associations between the relative risk of case fatality and the various categories of single prognostic variables were evaluated according to Cox’s proportional hazard regression model for censored data after dummy code transformation of the predictor (32). Survival curves were computed using the Kaplan-Meier product-limit method and compared using the log-rank test (33, 34). Analyses were performed using StatView 5.0 statistical software (SAS Institute, Cary, NC).