- Top of page
- MATERIAL AND METHODS
Reduced expression of the cyclin-dependent kinase inhibitor p27 has previously been correlated with fatal clinical outcome in some tumors, including gastric, breast, and prostate cancers. For hepatocellular carcinoma, the findings are equivocal. In situ hybridization and immunohistochemistry were performed on a series of 203 curatively (R0) resected hepatocellular carcinomas and in corresponding non-cancerous liver tissue to detect p27. Patients receiving liver transplantation were excluded. The results were correlated with histopathological stage according to the UICC system, Edmondson grade, several other histopathological factors of possible prognostic significance, and finally patient survival. Whereas p27 mRNA was expressed homogeneously in all carcinomas examined, the p27 protein was found in various amounts. The labeling index of p27 protein was significantly lower in advanced stages of the disease (P < 0.001, χ2 = 28.1). We observed decreased p27 protein in higher pT categories (P < 0.001, χ2 = 24.7) and in multiple tumor nodules (P < 0.001, χ2 = 9.3). Multivariate Cox survival analysis identified age, co-existing cirrhosis, and Edmondson grade as independent prognostic factors. We conclude that evaluation of p27 in hepatocellular carcinoma is useful to predict stage of disease and may have clinical significance, e.g., in predicting optimal therapeutic regimes. Int. J. Cancer 89:350–355, 2000. © 2000 Wiley-Liss, Inc.
Identification of multiple clinical and pathological prognostic factors in hepatocellular carcinomas (HCCs) has permitted a reasonable degree of risk stratification (Wittekind, 1995). However, clinical and pathological data fail to predict the actual virulence of a tumor concerning relapse or spread to distant organs in individual cases. Cell-cycle progression is regulated by a family of cyclin-dependent kinases (cdks) and inhibited by cdk inhibitors. Cyclins, cdks, and cdk inhibitors are frequently deregulated in cancer (Nurse, 1997). Members of the kinase inhibitor protein (KIP) family, currently composed of p21Cip1/Waf1/Sdi1, p27Kip1, and p57Kip2, bind and inhibit cyclin E–cdk2 and cyclin A–cdk2 complexes (Ladha et al.,1998). p27Kip1 is an inhibitor in cells arrested by TGF-β and regulated by growth-inhibitory cytokines and by contact inhibition (Santoni-Rugiu et al.,1999). p27 is strongly expressed in non-proliferating cells and plays important roles in the regulation of both quiescence and G1 progression (Glaise et al.,1998). Levels of p27 decrease as cells re-enter the cell cycle, mostly due to ubiquitin-proteasome–dependent degradation (Loda et al.,1997). Although mutations of the p27Kip1 gene are rare in human tumors (Kawamata et al.,1995; Spirin et al.,1996), decreased p27 protein levels have been found in aggressive breast (Fredersdorf et al.,1997; Tan et al.,1997), lung (Esposito et al.,1997; Catzavelos et al.,1999), gastric (Mori et al.,1997; Ohtani et al.,1999), and colon (Thomas et al.,1998) cancers, suggesting that its loss may both reflect and participate in the process of tumor progression (Tsihlias et al.,1999).
The relationship between p27Kip1 expression and prognosis or histopathological parameters has not been examined in a large series of HCCs. To date, all reported studies have been based on small numbers of patients (Ito et al.,1999), have employed different techniques for p27 assessment, and have not assessed predictive value. The latter is an important concept since it is becoming clear that identification of specific molecular defects may be used to predict not only prognosis but also optimal treatment regimes. Additionally, potential prognostic indicators have almost exclusively been investigated in Asian (Qin et al.,1998; Ito et al.,1999) rather than Western medical centers. Given the differences in patient populations and etiology of HCC, it is reasonable to question whether the relative importance of various new prognostic factors differs as well.
We therefore assessed p27 mRNA and protein expression in a series of surgically removed primary HCCs, to establish whether there is a correlation between degree of expression in the primary tumor and histopathological data including tumor grade, stage, and lymph node involvement.
- Top of page
- MATERIAL AND METHODS
In the present study, p27 expression was examined in a series of 203 HCCs, to verify whether it is related to histopathological factors or prognosis. We used a double approach to examine p27 expression, performing mRNA in situ hybridization and immunohistochemistry on each specimen. Despite detecting normal p27 mRNA transcripts in all tumors examined, the amount of p27 protein varied considerably. Statistical evaluation revealed that p27 protein was strongly and inversely correlated with stage of disease at the time of surgery. Reduced p27 was significantly related to advanced locoregional extent of the primary tumor and to tumor size.
Reduced p27 protein has been associated with more aggressive tumor behavior in several other human gastro-intestinal cancers (Lloyd et al.,1999; Tsihlias et al.,1999). Studies of esophageal gastric carcinoma have shown that p27 protein expression is lower in more aggressive tumors (Anayama et al.,1998; Mori et al.,1997; Ohtani et al.,1999). In esophageal adenocarcinomas, low p27 protein expression correlated with higher histological grade, depth of invasion, presence of lymph node metastases, and survival (Singh et al.,1998; Fujieda et al.,1999). In colorectal cancer, p27 was a powerful negative prognostic marker, especially in stage II tumors (Loda et al.,1997; Fredersdorf et al.,1997). It was suggested that p27 may help in the selection of patients who would benefit from adjuvant therapy (Loda et al.,1997). However, p27 expression is not lost with gene mutation (Kawamata et al.,1995). More than 500 tumors have been examined for specific p27 gene mutations, and fewer than 5 of these have had specific mutations (Spirin et al.,1996).
For HCCs, our in situ hybridization results indicate that p27 mRNA was expressed at high levels, irrespective of tumor size or stage. In contrast, our protein data with low p27 protein in advanced HCCs suggested that loss of p27 resulted from post-translational mechanisms. Some studies have shown that p27 levels are regulated by alterations of protein stability at the post-transcriptional level by ubiquitin-proteasome–mediated proteolysis (Hengst and Reed, 1996; Catzavelos et al.,1999). In colorectal carcinomas, regulation of p27 by the ubiquitin-proteasome–mediated proteolysis was confirmed by Loda et al. (1997). Furthermore, in non-small-cell lung cancer, high p27 expression levels were associated with lower degradation activity compared to tumors with low or undetectable p27 expression, which presented high degradation activity (Esposito et al.,1997). In contrast to Barrett′s adenocarcinoma, where p27 protein was localized in the cytoplasm of tumor cells (Singh et al.,1998), we did not observe a cytoplasmic staining pattern, suggesting that this method of protein inactivation may not occur in liver cancer.
p27 acts as an inhibitor of CDK2 in regenerating liver, and the p27Kip gene is haplo-insufficient for tumor suppression (Fero et al.,1998). This implies that reduced expression of p27 protein may predispose to abnormal cell-cycle and tumor progression. The exact mechanisms used by aggressive tumors to eliminate p27 protein remain undefined. Several growth factors, including TGF-α, or activation of the ras/mitogen-activated protein kinase pathway can reduce p27 levels by decreasing translation and stability of the protein (Lloyd et al.,1999). As excessive over-expression of p27 induces apoptosis in normal and neoplastic cells (Wang et al.,1997), down-regulation of p27 may also represent a survival advantage for cancer cells, resulting in more aggressive tumors.
In contrast to the data reported for non-small-cell lung (Catzavelos et al.,1999), breast (Tan et al.,1997), prostate (Cote et al.,1998), and gastric (Ohtani et al.,1999) cancers, we failed to identify p27 as a significant independent prognostic factor in patients with HCC. In our 203 patients, all of whom received identical treatment, p27 expression was related to survival only in univariate analysis. In multivariate statistical survival analysis, however, concomitant liver cirrhosis, age, and Edmondson grade were of independent prognostic value. This was in contrast to the data reported by Ito et al. (1999), who identified p27 as a significant prognostic factor in 83 patients with HCC. Unfortunately, they did not report the exact pathological stage, grade, and modalities of treatment of their patients. Ito et al. (1999) performed p27 protein analysis using immunohistochemistry. They assessed their staining results by counting 500 tumor cells. Due to possible intra-tumor heterogeneity of staining and to minimize the intra-observer error, our staining results were evaluated twice in a preliminary examination using the same material. This showed that at least 900 tumor cell nuclei should be assessed to have the results fall within 5% of the estimated real mean, with a probability of 95%. Additionally, we performed image analysis to validate the visual scoring method, which can be more readily applied to routine immunohistological material. However, the average LIs we obtained for neoplastic and non-neoplastic liver tissue were comparable to those reported by others (Ito et al.,1999).
The clinical implications of our findings are evident as HCCs diagnosed at an early stage can usually be cured by radical surgical resection. Clearly, it would be advantageous to know prior to surgery which patients are likely to have advanced stage of disease or even multiple tumors. Therefore, our data could be used as an additional predictive test, e.g., at a liver biopsy for planning surgical treatment as pre-surgical prognostication. To avoid misinterpretation of p27 in a biopsy due to intra-tumor heterogeneity of expression, multiple biopsies from different tumor areas could be examined to obtain representative staining results. However, the practicable and valid method of p27 immunohistochemical assessment and the good correlation with clinicopathological data such as stage, tumor size, and multiplicity justify further prospective clinical studies with larger series of patients.