A retrospective study including 300 cases of vulvar squamous cell carcinoma was performed. These patients underwent resection at The Norwegian Radium Hospital from 1977 to 2006. The median age at diagnosis was 74 years (range 35-96 years). Pre-surgery treatment was given to 9 patients, of which 6 received radiotherapy, whereas the other 3 were treated with radiotherapy/chemotherapy. Two hundred and one (67%) patients received radical vulvectomy. Postoperative treatment including irradiation, chemotherapy and irradiation/chemotherapy were performed on 63 (21%), 3 (1%), and 4 (1%) of the patients, respectively. Relapse was observed in 107 (36%) patients. All patients were followed up since confirmed diagnosis until death or 31. December, 2006. One hundred and twenty (40%) patients died of vulvar cancer. The median follow-up time for patients still alive was 131 months (range 11 to 346 months). All tumors were staged based on the International Federation of Gynecology and the Obstetrics (FIGO) classification [16]. Approval of the study has been given by The Regional Committee for Medical Research Ethics South of Norway (S-06012), The Social- and Health Directorate (04/2639 and 06/1478) and The Data Inspectorate (04/01043).

Histological specimens were reviewed by J.M.N, one of the co-author, who was concealed from all clinical information. Classification was performed according to World Health Organization recommendations [17]. Two hundred and eighty-two (94%) tumors were keratinizing/nonkeratinizing, 14 (5%) were basaloid and 4 (1%) were veruccoid. Previously, we have examined the expression of 14-3-3σ and human papillomavirus (HPV) infection in primary vulvar carcinomas [18,19], which was compared with the expression of CDC25s from the present study. Ten samples of normal vulva form patients undergoing surgery for benign gynecological diseases were included as control.

Two human vulvar squamous cell carcinoma cell lines, SW-954 (ATCC, Manassas, VA, USA) and CAL-39 (DSMZ, Germany), were cultured in RPMI 1640 medium (BioWhit-taker Europe, Verviers, Belgium) supplemented with 5% fetal bovine serum (FBS) (Biochrom KG, Berlin, Germany). For Western blot analysis and immunohistochemistry, monolayer cells were harvested by 0.01 M EDTA and thereafter washed in PBS.

Four-μm sections made from formalin-fixed, paraffin-embedded tissues and cell lines were immunostained using the Advance™ HRP System (K4068, Dako Corporation, CA, USA). After deparaffinization, sections for CDC25A staining were microwaved in 10 mM Tris-1 mM EDTA, pH 9.0, sections for CDC25B and phospho-CDC25C (Ser 216) staining were microwaved in 1 mM EDTA, pH 8.0 and sections for CDC25C staining were microwaved in 10 mM citrate buffer, pH 6.0 to regain the epitopes blocked by formalin fixation. To block endogeneous peroxidase the sections were treated with 0.3% hydrogen peroxide (H₂O₂) for 5 min. Sections were incubated overnight at 4°C with monoclonal antibodies, including CDC25A (clone DCS-120+DCS-121, 1:500, 0.4 μg IgG_2a/ml), CDC25B (clone 25B03, 1:150, 1.3 μg IgG₁/ml), CDC25C (clone 25C07, 1:100, 2 μg IgG₁/ml), all from NeoMarkers, CA, USA, and phospho-CDC25C (Ser 216) (clone 63F9, 1:500), from Cell Signaling, MA, USA. The specimens were then given a sequential 30 min incubation with Advance™ HRP link and Adance HRP enzyme, followed by treatment with 3'3-diaminobenzidine tetrahydrochloride (DAB) for 10 min, counterstained with hematoxylin, dehydrated and mounted in Diatex.

Sections from tonsil with known CDC25A, CDC25B and phospho-CDC25C (Ser 216) expression and from breast carcinoma with known CDC25C expression were used as positive control. Negative control included i) substitution of the monoclonal antibody with mouse myceloma protein of the same subclass and concentration as the monoclonal antibody, ii) incubation of sections with phospho-CDC25C (Ser 216) absorbed with phospho-CDC25C (Ser 216) peptide (Cell Signaling, MA, USA) as recommended by the supplier.

Semiquantitative classes were used to describe the intensity (absent, 0; weak, 1; moderate, 2; strong, 3) and extent of staining (percent of positive tumor cells: absent, 0; < 10%, 1; 10-50%, 2; > 50%, 3). By multiplying intensity score with extent score, product scores for both cytoplasm staining and nucleus staining were produced which ranging from 0 to 9. By taking product scores from cytoplasm and nucleus into account at the same time, a composed score was given for each section. Based on staining pattern observed in normal vulvar epithelium, cutoff values in cytoplasm and/or nucleus were set. High CDC25A and CDC25B immunostaining in the nucleus was classified with a score > 6, and low with a score ≤ 6, whereas, high CDC25C and phospho-CDC25C (Ser 216) immunostaining in cytoplasm was classified with a score > 3 and low with a score ≤ 3. In addition, high phospho-CDC25C (Ser 216) immunostaining in nucleus was classified with a score > 0 and low with a score 0. Examination of immunostaining was performed by two independent observers (Z.W. and R.H.) with no knowledge of patient outcome. All discordant scores were reviewed until final agreement was obtained.

Protein extraction was performed as described previously [20]. Cells were lysed in ice-cold NP-40 lysis buffer [1% NP-40, 10% glycerol, 20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 100 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.02 mg/ml each of aprotinin, leupeptin, pepstatin, and 10 μl/ml phosphatase inhibitor cocktail I (Sigma-Aldrich, St. Louis, MO)]. Lysates were sonicated and clarified by centrifugation. Protein quantitation was done by Bradford analysis. Twenty-five μg protein/lane was resolved by 12% SDS polyacrylamide gel electrophoresis (PAGE) and then Transferred to PVDF membranes.

The PVDF membranes with protein extract from cell lines or 1 μg CDC25A, CDC25B and CDC25C antigens (Upstate, NY, USA) were blocked with 5% nonfat dry milk in tris-buffered saline-Tween (TBST) and subsequently hybridized with antibodies against CDC25s [CDC25A, 1:200; CDC25B, 1:200, (Santa Cruz Biotechnology, CA, USA); CDC25C, 1:200 and phospho-CDC25C (Ser 216), 1:500] overnight at 4°C, respectively. Membranes were then washed in TBST for 3 times, with 10 mins each, and further hybridized with corresponding anti-mouse or anti-rabbit IgG conjugated with Horseradish Peroxidase (HRP Labelled, 1:5000, 0.2 μg IgG₁/ml dilution) for 1 hour at room temperature. After 3 times rince in TBST for 10 mins each, membranes were finally treated by Western Lightning Enhanced Chemiluminescence (ECL) reagent (Perkin Elmer, MA, USA).

The NE-REP nuclear and cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, USA) was used to separate cytoplasm and nuclear proteins. Western blot analysis on each fraction was performed as described above. To confirm the pure separation of nuclear and cytoplasmic fractions, Lamin B, a nuclear protein exclusive recognizing antibody (Pierce Biotechnology), and Tubulin, a cytoplasmic protein exclusive recognizing antibody (Oncogene, San Diego, USA), were used, respectively.

Proteins extracted from CAL-39 were dephosphorylated by Calf intestinal alkaline phosphatase (CIAP) (Promega, Madison, WI, USA). Proteins were treated by CIAP in a 50 μl reaction volume (protein 5 μg, CIAP 20 units, 10 × buffer 5 μl and H₂O 42.5 μl). Following treatment for 30 min at 37°C, the reaction mixture was added an extra 20 units of CIAP and left for an additional 30 min at 37°C. Untreated and CIAP treated proteins were separated by SDS-PAGE and hybridized with CDC25C, phospho-CDC25C (Ser 216), CDC25A and CDC25B antibodies, respectively.

One μg antigen CDC25B was phosphorylated by CHK1 (Millipore, Billerica, MA, USA) in a 50 μl reaction volume (antigen CDC25B 1 μg, CHK1 20 ng/μl, ATP 200 μM/μl, 10 × kinase buffer 5 μl and H₂O 39 μl) for 30 min at room temperature. Untreated and CHK1 treated antigens were separated by SDS-PAGE, blotted onto PVDF membranes and hybridized with CDC25B and phospho-CDC25C (Ser 216) antibodies, respectively.

Pearson's chi-square (χ²) test was performed in order to evaluate associations between CDC25 protein expression and clinicopathologic variables. Kaplan and Meier estimate and the log-rank test were used to evaluate and compare survival data. Disease-specific survival was calculated from the date of diagnosis to vulvar cancer related death. A Cox proportional hazards regression model was used for both univariate and multivariate evaluation of survival rates. In the multivariate analysis, a backward stepwise regression was performed with a p = 0.05 as the inclusion criterion for variables in the univariate analysis. All calculations were processed using SPSS 15.0 statistical software package (SPSS, Chicago, IL) and statistical significance was considered as p ≤ 0.05.

The specifity of CDC25 antibodies were tested by Western blot analysis using the antigenes CDC25A, CDC25B and CDC25C. However, since anti-CDC25B used for immunohistochemistry was not recommended by the supplier to use for Western blot analysis, an alternative anti-CDC25B was utilized for Western blot analysis. Our results showed that anti-CDC25A detected CDC25A, but not CDC25B or CDC25C. Anti-CDC25B (only for Western blot analysis) identified CDC25B, but not CDC25A or CDC25C. Anti-CDC25C and anti-phospho-CDC25C (Ser 216) immunoblotted with CDC25C, but not with CDC25A or CDC25B (Figure 1a). These results indicate that anti-CDC25A, anti-CDC25B, anti-CDC25C and anti-phospho-CDC25C (Ser 216) specifically detect their own respective antigen without any crossreaction with other CDC25s isoform.

Anti-CDC25C, as well as anti-CDC25B, detected proteins treated or un-treated with CIAP (Figure 1b), which indicate that anti-CDC25C and anti-CDC25B recognize their respective protein in phosphorylated as well as dephosphorylated form. However, proteins treated with CIAP failed to be identified by anti-CDC25A or anti-phospho-CDC25C (Ser 216) (Figure 1b), which indicate that both CDC25A and phospho-CDC25C (Ser 216) antibody exclusively detect phosphorylated CDC25A and CDC25C, respectively.

Anti-CDC25B detected CDC25B antigen treated or un-treated with CHK1, but neither of them was identified by anti-phospho-CDC25C (Ser 216) (Figure 1c). Furthermore, no coexpression of CDC25B and phospho-CDC25C (Ser216) was seen in serial sections of vulvar carcinomas (Figure 1d-e). These results indicate that the phospho-CDC25C (Ser 216) antibody does not cross-react with phosphorylated CDC25B.

In normal vulvar squamous epithelium, nuclear membrane staining for CDC25A was identified in basal, parabasal, middle and top layers (10/10 cases with score 9), whereas nuclear staining for CDC25B was seen in basal, parabasal and middle layers (10/10 cases with score 6) (Figure 2a-b). Cytoplasmic staining for CDC25C was observed in basal, parabasal and middle layers (3/10 cases with score 3 and 7/10 cases with score 6), whereas phospho-CDC25C (Ser 216) was limited to the basal layer (7/10 cases negative and 3/10 cases with score 3) (Figure 2c-d). None of the normal cases showed nuclear staining for CDC25C and phospho-CDC25C (Ser 216).

The immunostaining results in vulvar carcinomas are summarized in Table 1. High CDC25A and CDC25B immunostaining (score > 6) in the nucleus was observed in 152/300 (51%) and 47/300 (16%) cases, respectively (Figure 2e-f). CDC25A expression was observed in the nuclear membrane. High CDC25C expression (score > 3) in the cytoplasm was seen in 188/300 (63%) cases (Figure 2g). CDC25C immunostaining in the nucleus was not observed in any cases. High phospho-CDC25C (Ser216) expression in cytoplasm (score > 3) and nucleus (score > 0) were detected in 151/300 (50%) and 211/300 (70%) cases, respectively. Taking both the cytoplasmic and nuclear immunostaining into account, we found that high phospho-CDC25C (Ser216) immunostaining (score > 3) appeared in 232/300 (77%) cases (Figure 2h).

In the vulvar carcinoma cell lines SW-954 and CAL-39, immunohistochemistry identified CDC25A (score = 3), CDC25B (score = 9), CDC25C (score = 3) and phospho-CDC25C (Ser216) (score = 3) in the nucleus, whereas, CDC25B (score = 3), CDC25C (SW-954, score = 6 and CAL-39, score = 9), and phospho-CDC25C (Ser216) (score = 9) were observed in the cytoplasm (Figure 3a-d). Similar results were detected using Western blot analysis (Figure 3e). CDC25A was seen only in the nuclear fraction. CDC25B was weakly expressed in the cytoplasmic and strongly in the nuclear fraction. In contrast, CDC25C and phospho-CDC25C (Ser 216) were strongly expressed in the cytoplasmic fraction, but weakly in the nuclear fraction.

High expression of CDC25A in the nucleus was significantly correlated to low expression of 14-3-3σ protein in cytoplasm, nucleus and cytoplasm/nucleus (p = 0.03, p = 0.01 and p = 0.01, respectively) and to low expression of phospho-CDC25C (Ser216) in nucleus and cytoplasm/nucleus (p = 0.002 and p = 0.04, respectively). High expression of CDC25B in the nucleus was significantly correlated with poor differentiation (p = 0.004), vessel infiltration (p = 0.03), high expression of phospho-CDC25C (Ser216) in cytoplasm and cytoplasm/nucleus (p = 0.01, and p = 0.04 respectively) and HPV infected cases (p = 0.03). The levels of CDC25C and phospho-CDC25C (Ser216) immunostaining in relation to clinicopathological parameters are shown in Table 2. High expression of CDC25C in cytoplasm was significantly correlated with high FIGO substage (p = 0.004), presence of lymph node metastases (p = 0.04), large tumor diameter (p = 0.03), poor differentiation (p = 0.03), high expression of 14-3-3σ protein in cytoplasm and cytoplasm/nucleus (both p = 0.001) and high expression of phospho-CDC25C (Ser216) in cytoplasm, nucleus and cytoplasm/nucleus (all p < 0.001). High expression of phospho-CDC25C (Ser216) in cytoplasm, nucleus and cytoplasm/nucleus were significantly correlated with high FIGO substage (p = 0.05, p = 0.01 and p = 0.005, respectively), large tumor diameter (p < 0.001, p = 0.009 and p < 0.001, respectively), deep invasion (p = 0.01, p = 0.01 and p < 0.001, respectively), high 14-3-3σ protein levels in cytoplasm (p = 0.007, p < 0.001 and p < 0.001, respectively) and high 14-3-3σ protein expression in cytoplasm/nucleus (p = 0.005, p < 0.001 and p < 0.001, respectively). Factors showing significant correlation with high expression of phospho-CDC25C (Ser216) in cytoplasm and cytoplasm/nucleus were poor differentiation (p < 0.001 and p = 0.01, respectively). High nuclear expression of 14-3-3σ was significantly correlated with high expression of phospho-CDC25C (Ser216) in nucleus (p < 0.001) and cytoplasm/nucleus (p = 0.002). Cases negative for HPV was significantly correlation with high expression of phospho-CDC25C (Ser216) in nucleus (p < 0.001).

In the univariate analysis only high expression of phospho-CDC25C (Ser216) in cytoplasm/nucleus was associated with poor disease-specific survival (p = 0.04) (Figure 4). However, in multivariate analysis, when phospho-CDC25C (Ser216) cytoplasm/nucleus expression was added to the variables lymph node metastases, tumor diameter, infiltration of vessel, age and depth of invasion, only lymph node metastases, tumor diameter, vessel infiltration and age retained independent prognostic significance (Table 3).