1. Top of page
  2. Abstract

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.


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  2. Abstract

Patients and tissue samples

Two hundred and three patients with HCC undergoing partial hepatectomy (segmental or lobar resection) between 1979 and 1997 were included in our retrospective study.

No patient received pre-operative or adjuvant chemo- or radiotherapy. All patients were operated curatively (R0 resections). Patients who received orthotopic liver transplantation were excluded.

Each tumor was re-evaluated with regard to typing, staging, and Edmondson grading.

Tumor typing and staging was performed using WHO (Ishak, 1994) and UICC (Sobin and Wittekind, 1997) criteria. Maximum tumor diameter was measured macroscopically in fresh specimens. In addition, every tumor was examined macro- and microscopically for the presence of vascular invasion, capsule formation, satellites, multiplicity, inflammatory reaction, necrosis, bile production, giant cells, dysplasia within the surrounding liver, and co-existing cirrhosis. Multiplicity includes multiple nodules representing multiple, independent primary tumors as well as intrahepatic metastasis from a single primary hepatic carcinoma. Vascular invasion includes gross as well as histological involvement.

Tumor samples, free of necrosis or hemorrhage, were obtained from all patients. By macroscopic inspection, the periphery of the given tumor was preferred, to obtain 4 pieces of tissue in each case. For all analyses, the material was routinely fixed in 4% formaldehyde solution and embedded in paraffin. Sections (4 μm thick) were cut, dewaxed in xylene, and then rehydrated. In all cases, slides from 4 different paraffin blocks, sampled from different tumor areas, were examined.

For histopathological data see Tables 1 and 2.

 Number of patients1-Year survival rate (%) (95% CI)Median survival time in days (95% CI)p
   I12  (6%)66  (25–86)996  (581–1,410)<0.05 (stage I/II vs. stage III/IV)
   II37  (18%)58  (37–78)728  (102–2,043)
   IIIA82  (40%)47  (36–58)340  (144–535)
   IIIB18  (9%)24  (4–44)103  (27–179)
   IVA54  (27%)40  (27–53)272  (144–400)
pT category   
   pT112  (6%)66  (25–95)996  (581–1,410)n.s.
   pT246  (23%)50  (34–65)260  (141–305)
   pT391  (45%)46  (36–56)260  (110–410)
   pT454  (27%)40  (27–53)272  (144–400)
Tumor diameter    
   ≤6 cm94  (46%)45  (32–54)344  (193–495)n.s.
   >6 cm109  (54%)46  (36–56)260  (72–448)
   Present120  (59%)42  (32–52)224  (155–291)n.s.
   Absent83  (41%)52  (39–64)355  (110–598)
Edmondson grading    
   G171  (35%)48  (36–60)336  (112–580)<0.01 (G1/G2 vs. G3/G4)
   G266  (33%)57  (44–70)381  (48–714)
   G352  (26%)28  (15–41)127  (54–200)
   G414  (7%)26  (12–36)123  (52–195)
Age (years)    
   ≤60100  (49%)57  (50–62)595  (278–911)<0.01
   >60103  (51%)34  (29–38)206  (116–295)
p27 expression    
   ≤50%100  (49%)34  (29–41)207  (133–280)0.019
   >50%103  (51%)58  (46–62)661  (352–969)
ParameterTotal (%)LI ≤ 50LI > 50p
N (%)203  (100%) 100 (49%) 103 (51%) 
   I12  (6%)57<0.001 (stage I–IIIA vs. stage IIIB–IV)
   II37  (18%)1027
   IIIA82  (40%)3349
   IIIB18  (9%)108
   IVA54  (27%)4212
pT category    
   pT112  (6%)57<0.001 (pT1–3 vs. pT4)
   pT246  (23%)1531
   pT391  (45%)3853
   pT454  (27%)4212
Tumor diameter   
   <6 cm94  (46%)3064<0.05
   >6 cm109  (54%)7039
pN category    
   pN0176  (87%)8691n.s.
   pN126  (13%)1412
Edmondson grading    
   G171  (35%)3437n.s.
   G266  (33%)3630
   G352  (26%)2428
   G414  (7%)68
   Present120  (59%)7050<0.02
   Absent83  (41%)3053
   Present110  (54%)5258n.s.
   Absent93  (46%)4845
   Median (SD, range)(±11.48, 20–84)(±11.83, 24–84)(±11.17, 20–81)n.s.

In situ hybridization

Two segments of p27 cDNA were cloned into XbaI and HindIII sites of the pSPT18 vector (Boehringer-Mannheim, Mannheim, Germany) for synthesis of the 2 probes to detect different parts of p27 mRNA. Probe I detects nt 24–150 and probe II, nt 307–536 of the human p27 coding region.

Constructs were confirmed by DNA sequencing. RNA probes were synthesized with the DIG RNA labeling kit from Boehringer-Mannheim, according to the instructions of the manufacturer. mRNA in situ hybridization was performed as described before (Tannapfel et al.,1999a,b) on deparaffinized and dried sections after pre-treatment with proteinase K (Boehringer-Mannheim), 10 mg/ml (in 5× TE), for 30 min at 37°C. Post-fixation was performed with paraformaldehyde (4%). After drying, pre-hybridization was carried out under stringent conditions, covering the slides with ECLTM gold hybridization buffer solution (Amersham, Aylesbury, UK) for 30 min. After denaturation of the probe, slides were covered with the hybridization solution containing DIG-labeled probe (in ECLTM gold hybridization buffer) for 16 hr at 42°C. After the hybridization reaction, slides were incubated with RNase A at 37°C, followed by stringent washing procedures (2 × SSC, 0.1% SDS at room temperature and at 50°C). After washing with Triton X and covering with anti-DIG conjugates, BCIP/NBT solution for detection was applied (Boehringer-Mannheim). Slides were exposed at room temperature for 48 hr. Finally, the reaction was stopped and slides were covered and mounted.

Hybridization signals were evaluated by 2 observers trained in HCC histology (AT, DG). For all probes, signals were evaluated relative to background signal and RNase-treated control slides hybridized with the same anti-sense probe. Sense probes detecting the non-coding strand were used as negative controls. Sense and anti-sense probes were applied to paired serial slides. As an additional negative control, one slide in each set was treated with ribonuclease A prior to hybridization, to deplete the sample of mRNA. No-probe slides were prepared as additional controls for every 5 slides.

Immunohistochemical analysis

Immunohistochemical analysis was performed as described before (Tannapfel et al.,1999a,b). The material was routinely fixed in 4% formaldehyde solution and embedded in paraffin. After slicing into 4-μm-thick sections, preparations were dewaxed in xylene and then rehydrated. Endogenous peroxidase activity was blocked by 3% hydrogen peroxide in methanol for 30 min. After a short rinse in PBS, sections were pre-incubated with avidin-biotin for 15 min, to reduce non-specific background staining. Preparations were covered with normal goat serum for 20 min and then incubated with the primary antibody (p27, mouse monoclonal antibody, dilution 1:1,000; Transduction Laboratories, Lexington, KY). Then, sections were washed with PBS, incubated with biotinylated goat anti-mouse IgG (BioGenex, Hamburg, Germany) for 30 min and covered with peroxidase-conjugated streptavidin (Dako, Copenhagen, Denmark). The peroxidase reaction was allowed to proceed for 8 min, with 0.05% 3,3-diaminobenzidine tetrahydrochloride solution as substrate. Slides were counterstained with hematoxylin.

Negative controls were also performed by replacing the primary antibody with mouse or goat ascites fluid (Sigma-Aldrich, St. Louis, MO).

Slides were examined and scored independently by 2 of us (AT, DG) without any clinical or pathological information. At least 20 randomly selected high-power fields with a minimum of 4,000 cells were evaluated and scored for degree of expression. The percentage of p27-positive cells was expressed as a ratio of positive cells to the number of cells counted. Based on prior reports of p27 expression, cases were classified as exhibiting high or low p27 expression. According to the literature, a labeling index (LI) as a percentage of p27-positive cell nuclei was calculated. Patients with low p27 expression exhibited 50% or fewer positively stained tumor cell nuclei in the specimen (LI ≤50), while high expressors had more than 50% p27-positive tumor cell nuclei (LI >50) (Esposito et al.,1997; Mori et al.,1997; Ito et al.,1999).

Intra-observer error was calculated in a preliminary examination using the same material. It 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%.

To minimize inter-observer error, all counting was performed separately. In 8 cases, in which conflicting numbers were evaluated, recounting was performed, to obtain concordance of opinion.

As a second independent method to verify nuclear p27 staining scores, an image cytometric analysis was performed with the CYRES 3.0 system (Zeiss, Jena, Germany), a video-based interactive image cytometer (Tannapfel et al.,1999a,b), using the Quantitative Nuclear Antigen Program software (KS400, Zeiss). Total nuclear area, percentage positive nuclear area, and the sum of the optical densities were quantified for each case. A minimum total nuclear area of 10,000 μm2 was measured for each case.


Differences in frequencies between subgroups were analyzed with the Kruskal-Wallis test and the Mann-Whitney U-test for unpaired samples. Correlation coefficients were calculated according to Pearson, and χ2 statistics were used for contingency tables. Overall survival functions and probabilities were estimated by the Kaplan-Meier method. The log-rank test was used to detect differences between survival curves for stratified variables. Relevant prognostic factors were identified by univariate and multivariate Cox regression analyses. The significance level was defined as P < 0.05.

The median follow-up was 89 months (range 12 days to 196 months). No patient was lost during follow-up.

The medical records of all 203 patients were re-examined to assess the status of disease at the closing date of the study (31 December 1997). At this time, 2 patients were still alive. All of the 201 patients who died during the follow-up period presented with intrahepatic and metastatic disease at last contact with the oncological outpatient clinic. We concluded that death in these patients was related to HCC.

The overall observed 1-year survival rate for all patients was 46% [95% confidence interval (CI) 37–53%]. The median disease-free survival was 321 days (95% CI 217–424 days). The 5-year disease-related survival rate was 19.5% (95% CI 11–22.8%).


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  2. Abstract

p27 in situ hybridization

Hybridization with the anti-sense probe for p27 was informative in 82 cases. In 121 cases, a high degree of non-specific binding to any component of the tissue section was observed, after comparison of the signal to the background signal on RNase-treated control slides hybridized with the same anti-sense probe.

Transcripts for p27 were detected in tumor cells of all carcinomas examined (Fig. 1A,B). Transcripts were nearly homogeneously distributed in the tumor without any predominance (Fig. 1A). Specific hybridization with strong signals localized to the cytoplasm and nucleus was observed. Tumor stroma, endothelial cells, cholangiocytes, fibroblasts, and inflammatory cells were only occasionally positive for p27. Transcripts were not observed within mitotic or apoptotic cells. Cirrhotic, tumor-surrounding, non-neoplastic tissue exhibited strong p27 mRNA expression as well. In comparison to cirrhotic liver tissue, the amount of p27 mRNA transcripts was slightly reduced in non-cirrhotic, tumor-surrounding liver tissue.

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Figure 1. In situ hybridization of p27 mRNA. (A) p27 mRNA transcripts (brown reaction product) within HCC (original magnification ×40). (B) p27 mRNA expression within a small HCC nodule. Staining signal localized to the cytoplasm. Non-neoplastic surrounding inflammatory cells and fibrous tissue without transcripts (original magnification ×20).

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In situ hybridization clearly revealed neoplastic hepatocytes as the main site of p27 RNA transcription.

The extent of mRNA transcripts detected in a given tumor did not correlate with the protein amount detected by immunohistochemistry. In contrast to p27 protein, which was expressed heterogeneously within the tumor to various amounts, abundant mRNA transcripts were present within each tumor. As illustrated in Figures 1 and 2, a tumor of stage IIIB exhibited homogeneous expression of p27 mRNA (Fig. 1A), whereas the corresponding p27 protein LI was <10% (Fig. 2B).

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Figure 2. Immunohistochemical demonstration of p27 protein. (A) Immunohistochemical demonstration of p27 protein. Strong expression of p27 within the carcinoma cells (brown reaction product within the tumor cell nuclei). LI <50% (original magnification ×20). (B) Immunohistochemical demonstration of p27 protein in HCC (UICC stage IIIB, same tumor as Fig. 1A). LI <50% (original magnification ×10).

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p27 immunohistochemistry

Nuclear p27 staining was consistently seen in normal hepatocytes and in tumor-infiltrating lymphocytes, which provided internal staining controls in most tumor slides. Cholangiocytes were occasionally positive as well. The average p27 LI in normal liver tissue was 15 (±12.8, range 0–30).

In HCC, tumor cells were significantly more often p27-positive. We observed an average LI of 49 (±32.5, range 0–95).

Correlation analysis between the results from light microscopy and those from automated image cytometric analysis revealed a significant positive correlation (r = 0.95, P < 0.01). The percentage of positive nuclear areas and the sum of the optical densities were highly correlated with the percentage of positive cells assessed by light microscopy in each tumor.

Among the 203 HCCs examined, 103 (51%) exhibited strong nuclear staining of at least 50% of the tumor cell nuclei (LI >50) (Fig. 2B). In contrast, 100 carcinomas (49%) showed weak staining, with 50% or fewer positive tumor cell nuclei (LI ≤50) (Fig. 2B).

When comparing p27 LI with the stage of disease at the time of surgery, a highly significant correlation was established. The grade of positivity was significantly lower in advanced stages of disease. Of 103 tumors that showed strong immunostaining (LI >50), 83 belonged to stage I, II, or IIIA, whereas only 20 were in advanced tumor stages IIIB or IVA (P < 0.001).

Since the pathological staging criteria are based on the pTNM classification and on multiplicity of the tumor and vascular invasion, we further examined the relationship between p27 expression and pT and N categories, multiplicity, and vascular invasion. pT category was significantly related to p27 LI. In the advanced pT category, significantly reduced p27 immunostaining was observed (P < 0.001). Of 54 patients with pT4 (multiple tumors in more than 1 lobe, involving a major branch of the portal or hepatic vein or with direct invasion of adjacent organs) at the time of surgery, 42 (78%) had weak immunostaining for p27 (LI ≤50), whereas only 12 (23%) exhibited strong staining (LI >50) (χ2 value 24.7, P < 0.001) (Table 2). A significant relationship was observed between multiplicity and p27 LI. Of 120 patients (58%) with multiple tumors, 70 exhibited reduced p27 staining (LI ≤50) compared to 30 of 83 patients (36%) with solitary tumors and LI ≤50 (χ2 value 9.27, P = 0.02). According to the tumor size, solitary tumors with a diameter >6 cm had significantly decreased p27 protein with LI ≤50 compared to those <6 cm (χ2 value 17.42, P < 0.001). We did not observe a significant relationship between p27 immunostaining and vascular invasion, Edmondson grading, concomitant cirrhosis and age.

Survival rate

The survival analysis was performed on 203 patients and took into account the following variables: p27 positivity (defined by immunohistochemistry), UICC tumor stage (Sobin and Wittekind, 1997), Edmondson grade, vascular invasion, capsule formation, multiplicity, satellites, dysplasia, inflammatory reaction, necrosis, bile production, presence of giant cells, co-existing cirrhosis and age.

Consistent with the published data, UICC stage, Edmondson grade, co-existing cirrhosis, and age were significant prognostic parameters. Survival was significantly shorter in patients older than 60 years and in those with co-existing cirrhosis. Univariate analysis showed p27 to be a strong predictor of survival (Fig. 3). Multiplicity, satellites, bile production, capsule formation, presence of giant cells, inflammatory infiltrate, and dysplasia within the non-tumurous liver lacked prognostic significance.

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Figure 3. Overall survival according to p27.

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Multivariate Cox regression analysis of the ungrouped variables UICC stage (Sobin and Wittekind, 1997), Edmondson grade, co-existing cirrhosis, age, and p27 identified age, cirrhosis, and Edmondson grade as independent prognostic factors. Due to the strong association of p27 with UICC stage, this factor was not identified as being of independent prognostic significance.


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  2. Abstract

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.


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  2. Abstract
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