The use of study specimens for analyses was approved by the Research Ethics Committee of Nanjing Medical University. Informed written consent was obtained from all the patients.

From June 1997 to February 2000, 80 ESCC and 15 adjacent non-cancerous paraffin-embedded samples were obtained from the tumor center of Nanjing First Hospital affiliated to Nanjing Medical University. There were 52 male and 28 female patients with a mean age of 60 years (range: 41-82). The patients were given preoperative examination including biopsy for diagnosis, barium X-ray, CT and ultrasonic endoscopy for clinical staging, and no treatment was given before operation. Radical resection was performed in each patient, and all the samples underwent postoperative pathological examination. There were 54 cases of stage I-II and 26 cases of stage III-IV cancer according to the American Joint Committee on Cancer staging manual (AJCC, 2002)[16]. With regard to postoperative histological results, 16 were well-differentiated, 40 moderately differentiated and 24 poorly differentiated. Another 70 ESCC and 10 non-cancerous paraffin-embedded samples were enlisted from January 2002 to December 2003 in the same institution. There were 48 male and 22 female patients with a mean age of 61 years (range: 38-89). All the patients were assessed for physiological ability and endoscopy and CT scan were performed for clinical staging prior to routine surgery for ESCC. The postoperative pathological examination found 56 cases of stage I-II and 14 cases of III-IV cancer according to AJCC (2002) pTNM standards[16]. Clinical follow-up after surgery and diagnosis was based on periodic visits (every 3 mo during the first year, every 6 mo the second year, and then yearly until relapse).

Real-time quantitative PCR was performed on paraffin-embedded sections from 80 ESCC patients and 15 adjacent non-cancerous samples. Briefly, total RNA was extracted by Recover All Total Nucleic Acid Isolation kit (Ambion), and 10 mg of DNase-treated total RNA was used for reverse transcription with Superscript III (Invitrogen, Carlsbad, CA, USA). An aliquot representing 100 ng input RNA was amplified by quantitative real-time PCR using the TaqMan PCR reagent kit and assay-on-demand gene expression products (FAM/Sybr, Foster City, CA, USA). RNA extracted from a non-cancerous lesion in one patient was used as a standard. After reverse transcription, standard cDNA was serially diluted to obtain five standard solutions for use in PCR to generate the reference curve. Sequences of the Bmi1 bidirectional primers were designed using Primer 5.0 rotor-gene 6.0 (Corbett Research) as follows: Bmi1 sense 5'-GTATTCCCTCCACCTCTTCTTG-3', Bmi1 antisense 5'-TGCTGATGACCCATTTACTGAT-3'. House-keeping gene: β-actin sense 5'-CCTGTACGCCAACACAGTGC-3', antisense 5'-ATACTCCTGCTTGCTGATCC-3'. Quantitative real-time PCR was carried out in a Rotor-Gene 3000 PCR kit (Corbett Research) with 10000 × Syber Green (Molecular Probes). After reverse transcription, standard cDNA was serially diluted to obtain five standard solutions for use in PCR to generate the reference curve. The relative amount of cDNA in each sample was measured by interpolation using the standard curve (Figure 1), and then the relative ratio of Bmi1 to β-actin (housekeeping gene) expression was calculated for each ESCC sample.

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Figure 1 Amplification curve (A) and standard curve (B) of quantitative real-time PCR for Bmi1 mRNA. Total RNA was extracted for subsequent reverse transcription, and standard cDNA was serially diluted to obtain five standard solutions (1 × 10^-1, 1 × 10^-2, 1 × 10^-3, 1 × 10^-4, 1 × 10^-5) for use in PCR to generate the reference curve, slope rate of the straight line (a) = -3.42089, intercept (b) = 5.97517, correlation coefficient (r) = 0.9996. The strength of Bmi1 and β-actin was directly generated by the machine.

Histopathological evaluation was performed on 4-&mgr;m slides stained with hematoxylin and eosin (HE) (Figure 2). Commercially available rabbit monoclonal antibodies against Bmi-1 (1:100, Santa Cruz Biotechnology) were used as primary antibodies. A paraffin section of the ESCC sample was deparaffinized and rehydrated in graded alcohol to water. Antigenic enhancement was performed by submerging in citrate buffer (pH 6.0) and microwaving. Endogenous peroxide activity was quenched by applying 0.3% hydrogen peroxide for 10 min, followed by incubation with 1% BSA to block the non-specific binding. The primary monoclonal anti-Bmi1 antibody was incubated for 60 min at 37°C. After washing, the tissue section was reacted with the biotinylated anti-rabbit IgG, and visualized using a Dako Envision System horseradish peroxidase for monoclonal antibodies. The slides were immersed in the prepared diaminobenzidine solution, which produces a brown precipitate at the level of the antigen-primary antibody. Slides were then counterstained with hematoxylin, dehydrated through alcohols of increasing concentration, placed in xylene, coverslipped using Permount, and analyzed under light microscopy. Each section was evaluated by at least two independent professional pathologists, the distribution of Bmi-1 was scored on a semi-quantitative scale, the percentage of positive tumor cells was recorded and divided as follows: negative (< 10% of tumor cells positive), locally positive (10%-50% of tumor cells positive), and diffusely positive (> 50% of tumor cells positive).

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Figure 2 Staining of ESCC tissues. The tumor cells of cancerous tissues were stained as violet in the nucleus and pink in the cytoplasm (× 100).

Data were expressed as mean ± SD and analyzed using the Stata v9.0-CYGiSO bin (Computer Resource Center, USA). The significance of differences among groups was determined by Student’s t test and χ² test or Fisher’s exact test. The difference in free survival between groups was analyzed by the Kaplan-Meier method and log-rank test. The starting point for calculating free survival was the date of surgery, and the endpoint was the date of death. Statistical significance was assessed at the two sided 5% level, and P values less than 0.05 were considered statistically significant.

Clinical follow-up was made in 76 patients. The comparative expression levels were determined as a ratio between the Bmi1 and the housekeeping gene (β-actin) to correct for variation in the amounts of mRNA. The 5-year survival rate was 37.46%. The Bmi1 mRNA level was higher in the cancerous tissues than that in the non-cancerous tissues (2.181 ± 2.158 vs 0.931 ± 0.894, P = 0.0152). The Bmi1 expression level was unaffected by gender and age. The expression level of Bmi1 mRNA was much lower at the I-II stage than that at the III-IV stage, which varied inversely with the differentiation grade, and was lower in cases without metastatic lymph nodes than in those with metastatic lymph nodes (Table 1). Based on the detection of Bmi-1 median expression level (1.085), patients were divided into the down-expression group (Bmi1 mRNA level < 1.085) and the up-expression group (Bmi1 mRNA level > 1.085), and the accumulated survival rate was higher in the former than that in the latter (Figure 3).

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Figure 3 Effects of Bmi1 mRNA expression on prognosis of ESCC patients. Two patients did not complete the 5-year follow-up in each group (small circles). The survival rate was higher in the down-expression group (group 2) than in the up-expression group (group 1), χ² = 4.41, P = 0.0356.

Bmi1 oncoprotein expression was diffusely positive, focally positive and negative in 44, 16 and 10 of 70 ESCC cases, respectively. Compared with three, two and five of 10 adjacent non-cancerous cases, Bmi1 protein was significantly increased in ESCC samples (P = 0.027). Analysis of protein localization in ESCC cells was made, and the tumor cells with Bmi1 staining were divided into three categories, referring to both the nucleus and cytoplasm. In all 70 tested ESCC tissues, Bmi1 presented highly intense expression in both nucleus and cytoplasm with varied degrees, accompanied by less or even no expression in the nucleus, with significant differences (Figure 4). The positivity of the oncoprotein in samples of histological grade III was more frequent than that of grade II, but no significant differences were observed between other differentiated grades (P = 0.031). No relationship was found between the Bmi1 protein expression and lymph node metastases, pathological staging and cell differentiation (Table 2). Clinical follow-up was made in 67 patients. The 5-year survival rate was 40.01%. There was no statistical difference in survival rates between groups according to the Bmi1 expression (P = 0.1704).

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Figure 4 Immunohistochemical staining of ESCC using antibody to Bmi-1. A: Bmi1 protein expression in adjacent non-cancerous tissues (× 400); B: Cytoplasm staining of Bmi1 in ESCC cells (× 400); C: Intense Bmi-1 staining in the nucleus in ESCC cells (× 400). The brown staining under light microscopy indicates positivity.

Bmi1, a member of the polycomb group and a component of the polycomb repressive complex1, has been considered as a oncogene involved in many solid and hematological malignant tumors, and it may be of diagnostic and prognostic relevance. However, to date, no report about the role of Bmi1 in esophageal squamous cell carcinoma (ESCC) has been made. However, the up-regulation of c-Myc and the down-regulation of p53 and p16 in ESCC tissues make it plausible that Bmi1 may play an important role in the initiation and progression of ESCC.

This research, for the first time, investigated the expression of Bmi1 and its clinicopathological role in patients with ESCC.

A significant upregulation of Bmi1 was observed in cancerous tissues in contrast to adjacent non-cancerous paraffin-embedded esophageal specimens at the mRNA and the protein level by quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry. Furthermore, overexpression of Bmi1 positively correlated with lymph node metastases, pathological stage and differentiation grade at the mRNA level.

Bmi1 may act as a guide for the postoperative therapy and a differentiation marker of ESCC with high malignancy, and for prediction of the survival of ESCC patients.

The authors investigated the expression of Bmi1 at the mRNA and protein level in patients with ESCC in comparison with healthy adjacent tissue, by real-time PCR and immunohistochemistry. They observed a significant up-regulation of Bmi1 in tumor tissues in contrast to healthy control tissues. Up-regulation correlated positively with lymph node metastases, stage and histological grading at the mRNA level. However, the histological analysis showed no such correlation. The authors conclude that Bmi1 expression is common in ESCC and may serve as a marker to predict lymph node metastasis and survival in ESCC patients.