Patients and tissue specimens
A total of 179 cervical cancer patients treated between January 2001 and December 2008 were included in the study. The material was retrieved from archival paraffin-embedded surgical samples at Sun Yat-Sen University Cancer Center. In addition, 75 pairs of snap-frozen cervical cancer and normal cervical samples from above patients were collected for Real-time PCR. None of the patients had received chemotherapy or radiotherapy before surgery. After surgery, the patients were treated with adjuvant chemotherapy according to the national guidelines. Platinum-based chemotherapy was initiated within two weeks after surgery and then repeated for four cycles at three-week intervals. In all cases, the diagnoses and grading were peer-reviewed according to the principles laid down in the latest International Federation of Gynecology and Obstetrics criteria . Prior written consent was obtained from all patients and this study was approved by the Research Ethics Committee of Sun Yat-Sen University Cancer Center.
RNA extraction and real-time PCR
Total RNA from CSCC and paired normal cervical tissues was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The extracted RNA was pretreated with RNase-free DNase, and 2 μg RNA from each sample was used for cDNA synthesis with random hexamers. Real-time PCR was performed using the Applied Biosystems 7500 Sequence Detection system. The primers used are as follows: Msi1, forward, 5’- GAGGGTTCGGGTTTGTCACG-3’, reverse, 5’-GGCGACATCACCTCCTTTGG-3’; ALDH1, forward, 5’-GTTAGCTGATGCCGACTT GG-3’ , reverse, 5’-CCCACT CTCAATGAGGTCAAG-3’; Sox2, forward, 5’- GCTGTATGGCTGCTGCACTTC-3’, reverse, 5’-GCACACGCACCCAGCACT GT-3’; CD49f, forward, 5’- ATGGAG GAAACCCTGTGGCT-3’, reverse, 5’- ACGAGAGCTTGG CTCTTG GA-3’; GAPDH, forward, 5’-AGAAGGCTGGGCTCATTTG-3’, reverse, 5’-AGGGGCCATCCA CAGTCTTC-3’. The expression level of CSC markers mRNA was calculated using a ratio of CSC markers mRNA to GAPDH mRNA.
Immunohistochemical (IHC) staining
Immunostaining was performed on paraffin-embedded 4 μm sections and mounted on poly-L-lysine-coated slides. The sections were baked at 65 °C for 30 minutes, then deparaffinized in xylene and rehydrated. Antigen retrieval was performed by submerging the sections into a 10 μmol/L citrate buffer solution (pH 6.0) for 10 minutes in a microwave oven. The slides were then treated with 3 % hydrogen peroxide in methanol to quench the endogenous peroxidase activity, followed by incubation with 1 % fish skin gelatin to block the nonspecific staining. Tissue sections were incubated overnight with monoclonal rabbit antibody against Msi1 (Abcam, Cambridge, USA; 1:200), monoclonal rabbit antibody against ALDH1 (Abcam, Cambridge, USA; 1:200), monoclonal mouse antibody against Sox2 (Abcam, Cambridge, USA; 1:200), and monoclonal rabbit antibody against CD49f (Abcam, Cambridge, USA; 1:200). After washing, the sections were incubated with prediluted secondary antibody (Abcam, Cambridge, USA), followed by further incubation with 3,3-diaminobenzidine tetrahydrochloride (DAB). Finally, the slides were counterstained with hematoxylin and mounted in an aqueous mounting medium.
For negative controls, primary antibodies were replaced with normal serum.
Immunostaining was separately evaluated by two independent pathologists who were blinded as to the patients. Expression of the four CCSC markers was analyzed by an individual labeling score considering percent and staining intensity of positive cells. Intensity of stained cells was graded semi-quantitatively into four levels: 0 (no staining); 1 (weak staining = light yellow); 2 (moderate staining = yellow brown) and 3 (strong staining = brown); and the percentage was scored as: 0, negative; 1, 10 % or less; 2, 11 % to 50 %; 3, 51 % to 80 %; or 4, 80 % or more positive cells. Intensity and fraction of positive cell scores were multiplied for each marker and thus the scoring system was defined as low expression for scores of 0–3, and as high expression for scores of 4–12.
All statistical analyses were carried out using SPSS (version 16.0, SPSS Inc, Chicago, USA) statistical software. The overall survival (OS) and recurrence-free survival (RFS) were calculated as the time from the date of primary surgery to the date of first death or recurrence. Survival curves were plotted using the method of Kaplan-Meier, and the log-rank test was used to determine statistical differences between life tables. The correlations between clinicopathologic characteristics and recurrence were analyzed using the χ2 test. Univariate and multivariate analysis were applied to assess the independent predictive significance of variables on RFS. P < 0.05 in all cases was considered statistically significant.