Cell lines
The human cell lines NHEK, HK-168, SiHa, CaSki, and HeLa were used. They were kindly provided by Dr. Federico De Marco (Regina Elena Cancer Institute, Rome, Italy). NHEK is a HPV negative human keratinocyte diploid cell line; HK-168 is a non tumorigenic cell line derived from primary human keratinocytes transfected with HPV16 and containing about 1–2 viral copies/aploid genome [14]. SiHa, CaSki and HeLa are cervical carcinoma-derived cell lines (the SiHa and CaSki from squamous cancer and the HeLa from an adenocarcinoma) containing HPV sequences (SiHa: 1 HPV16 genome copy per cell; CaSki: 500–600 HPV16 genome copies per cell; HeLa: 10–50 HPV18 genome copies per cell).
NHEK and HK-168 were cultured in a K-SMC (Gibco, Invitrogen, Milan, Italy) definite medium while SiHa, CaSki and HeLa were cultured in DMEM supplemented with 10% of fetal bovine serum and antibiotics (penicillin 100 U/mL, and streptomycin 100 μg/mL).
Western blotting
Analysis with rabbit polyclonal antibody against claspin (Bethyl Laboratories, Montgomery, TX, USA) was performed according to a published protocol [5]. Briefly, the samples were boiled in sample buffer and separated by SDS-PAGE. The proteins were, then, transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Freiburg, Germany) and incubated with the primary antibody. After removing the unbound primary antibody, the membranes were incubated with a secondary antibody-peroxidase conjugate (Sigma Chemical Co., MO, USA), processed for detection by chemiluminescence (Amersham), and imaged on Biomax film (Kodak, Milan, Italy). A rabbit polyclonal antibody against Actin (Santa Cruz Biotechnology, CA, USA) was used as a loading control.
Histological samples
This study was reviewed by the Ethics Committee of the Regina Elena Cancer Institute. We selected 80 cases from the Regina Elena Cancer Institute files, which had been collected between June 2006 and July 2009, submitted to a colposcopy-guided cervical biopsy immediately after a cervico-vaginal sampling for HPV testing. All histological slides were independently reviewed by two experienced pathologists and an adjudicated final diagnosis was established. Of the 80 formalin fixed biopsies, 9 had no relevant lesions (herein referred to as WNL: within normal limits), 19 were diagnosed as Cervical Intraepithelial Neoplasia (CIN) 1, 21 as CIN2, 16 as CIN3 and 15 as invasive Squamous Cell Carcinomas (SCC). For the purpose of our study, the WNL and CIN1 diagnoses were referred to as CIN2-, while CIN2, CIN3 and SCC were referred to as CIN2+. Therefore, on the basis of this classification, we analyzed 28 CIN2- and 52 CIN2+ cases. This sample size was adequate to detect a significant difference at 1% level with a statistical power of 90%, according to an a priori power calculation based on data from our laboratory regarding Ki67 reactivity in cervical pathology (data not shown). In fact, given that Ki67 is a well known proliferation marker which is widely applied in solid tumors [15], we expected a similar distribution of immunoreactivity for claspin. The sample size calculation identified a minimum number of 54 patients, 27 per CIN2- group and 27 per CIN2+ group.
Cytological samples
Of the 80 cervico-vaginal specimens collected in PreservCyt (Hologic, Rome, Italy) for HPV testing, 19 had a residual sample which was sufficient to obtain thin layer slides which were adequate for both morphological and immunocytochemical analyses. The thin layer slides were prepared using the ThinPrep 2000 System (Hologic), following the manufacturer’s instructions. Papanicolau slides were interpreted independently from all other findings, and classified according to the Bethesda 2001 guidelines [16], by two experienced cytopathologists. An adjudicated final report was established and reported as 3 Negative for Intraepithelial Lesion or Malignancy (NILM), 7 Low-Grade Intraepithelial Lesions (L-SIL) and 9 High-Grade Intraepithelial Lesions (H-SIL).
Claspin immunostaining
The immunohistochemistry (IHC) performed to detect claspin expression was carried out on 3 μm thick sections cut from formalin-fixed paraffin embedded blocks. We used a monoclonal antibody which was kindly provided by Professor T. Halazonetis (University of Geneva Switzerland), and directed against the amino-acidic residues 785–1056 of the full-length protein as previously described [7]. Antigen retrieval was carried out pretreating dewaxed and rehydrated slides in a water bath at 96 °C for 40 minutes in ethylenediamine tetracetic acid buffer (EDTA, pH 8.0). Immunoreactivity was revealed by means of a super sensitive streptavidin-biotin immunoperoxidase system (Novocastra, Menarini, Florence, Italy), using 3-amino-9-ethyl-carbazole as a chromogenic substrate. Nuclear staining, independent of intensity, was considered positive, excluding the basal layer in which proliferating cells are physiologically present. For each sample, we counted the positive nuclei in four to six selected High Power field (HPF, 400X magnification) representative of the lesion using an image analyzer (Eureka Interface, Menarini). A maximum of 200 immunoreactive nuclei/HPF were counted. For each sample, we calculated the mean number of the positive nuclei counted in all selected fields, thus obtaining a single value reported as nuclei/HPF value. For the purpose of the study, on the basis of the nuclei/HPF value, we distinguished 4 different categories of claspin reactivity: negative (0 or less than 1 immunoreactive nuclei/HPF), low-positive (1 to <20 immunoreactive nuclei/HPF), moderate-positive (20 to 80 immunoreactive nuclei/HPF), high-positive (more than 80 immunoreactive nuclei/HPF).
The immunocytochemical analysis of the 19 available cervico-vaginal samples was performed following the above described procedure, after fixing Thin-Prep slides in 10% buffered formalin for 20 minutes followed by water rinsing. Immunostaining was considered positive when at least one cell showed nuclear reactivity either among the superficial and intermediate typical cells or among the clearly atypical cells, independently of all other findings.
HPV testing
The HR-HPV DNA detection was performed on cytological samples by the HR-HPV Hybrid Capture 2 (HC2) test (Qiagen, Milan, Italy), following the manufacturer’s recommendations. Before the HC2 test, 2 mL of each sample were processed using the HC2 Sample Conversion Kit (Qiagen). The HR-HPV HC2 assay detects the most common 13 HR-HPV types in cervical cancer: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68. The HPV genotyping test was performed by the PCR based Linear Array HPV Genotyping kit (Roche Diagnostics, Italy), utilizing 250 μL of the residual liquid sample and following the manufacturer’s instructions. This assay is able to detect 37 high, intermediate and low risk HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39 and CP6108). Only samples that were found positive to at least one of the 13 genotypes recognized by the HC2 test were considered to be HPV DNA positive by PCR. Moreover, because of the higher oncogenic potential displayed by HPV 16 and 18 types [13], we divided the positive results into two categories: 1) presence of HPV 16 and/or 18 sequences with or without other genotypes (16/18 positive), 2) detection of HR-HPV genotypes other than 16 and 18, as single or multiple infections (HR-HPV positive).
Statistical analyses
Data were analyzed with SPSS statistical software version 17.0. (SPSS Inc., Chicago IL, USA). The associations between variables of interest were performed by the non-parametric Pearson Chi-Square test and the Kruskall-Wallis test, when appropriate. Analyses for trend were also carried out by using the Chi-square test for trend. A p-value < 0.05 was considered to be statistically significant. To evaluate the sensitivity and specificity for the presence of a CIN2+ lesion, the cases showing negative or low claspin expression were considered as negative whereas the cases with moderate or high claspin immunoreactivity were regarded to as positive.