The study is a retrospective correlation analysis to test the degree of CD31 expression among three (3) groups of diagnosed cervical lesions; cervicitis, CIN and SCC cervix. Inclusion criterion is confirmed cases with the afore mentioned three diagnoses. The exclusion criterion is any diagnosis other than the three.
Selected cases were reviewed with histomorphological exam to verify degree of dysplasia or neoplasia and paraffin blocks were examined for integrity prior to inclusion into sample population. These blocks were then sectioned and stained for IHC examination to assess CD31 expression and MVD. Results obtained were then subjected to statistical analysis.
A series of 150 patients with cervical lesions ranging from cervicitis (45), CIN (65) to carcinoma (40) were retrospectively studied. Specimens were obtained either, via routine Pap smear prior to surgical treatment or from hysterectomy specimens. Age of the patients ranged from 23 to 86 years (mean 46.5 years). All cases were selected from the archives of the Department of Pathology of HTJ Seremban, derived from the period from March 2007 to March 2015, based on availability of representative paraffin blocks and H&E slides.
An experienced pathologist verified all histological diagnoses. All lesions were classified using the histopathological criteria of the World Health Organization (WHO) classification, and staging was made according to the American Joint Committee on Cancer system. Normal tonsil samples were used as a control group for IHC staining.
The consent was obtained from the Director of HTJ, Seremban to access and utilize the cervical tissue samples in HTJ. The study was approved by the Ethics Committee of IMU, BMS-I1-2015(06), and is registered under the National Medical Research Registry (NMRR) and is Medical Research & Ethics Committee approved, NMRR-15-1253-25215.
Paraffin-embedded blocks were cut serially into 4-micron sections using a microtome (Leica 1205, USA) and mounted onto silanized glass slides. All IHC methods done in this study followed a standard protocol by the manufacturers with minor optimisation to suit local laboratory conditions.
Monoclonal CD31 antibody (clone: mouse, DAKO, USA) was used at a dilution of 1:50 for 30 min at room temperature. Tonsil tissue was used as a positive control. The CD31 positivity was indicated by the presence of cytoplasmic or membranous brown staining. Microvessel counting was performed in areas with maximal neovascularization within the tumors, outside any areas of artifact, necrosis, or inflammation, and without prior knowledge of the patient outcome. It was performed in a blind fashion with patient’s diagnosis and outcome withheld.
Sections were scanned at low and medium magnifications to identify the areas of greatest density of stained microvessels in the cervical stroma ‘‘hot spots’’. These were found to be below the basement membrane in cervicitis (Fig. 1), adjacent to the greatest depth of invasion in CIN (Fig. 2) and anywhere in the tumor masses of SCC (Fig. 3).
Within these areas of high vascularization, the MVD was determined in three randomly chosen fields, at a magnification of 400. Any brown-staining endothelial cell or a separate cell cluster was considered one single countable microvessel. Vessel lumens and red blood cells were not required for counting as a microvessel. Unstained lumens were considered artifacts even if they contained blood or tumor cells. At 100x magnification, counts were made of all distinct brown staining endothelial cells over three fields in each slide. The microvessel density (MVD) was defined as the average value of the three readings.
The data were analysed with SPSS-PC package (version 11.0, SPSS, Chicago, IL). Statistical analysis included the analysis of variance (ANOVA) and Tukey post-hoc tests for evaluation of MVD counts between cervicitis, CIN (CIN1–3), and SCC groups.
The means of MVD counts from various groups were evaluated by independent sample t-test. IHC scoring was made based on the Allred and methods used by other IHC studies on breast, urinary bladder and prostate cancer. Scanning at 40x, 3 brightest spots with the densest staining were identified for each slide, and the number of visibly identifiable microvessels was recorded. The mean of these three readings was then obtained to be used as the MVD score for that slide. This was to ensure a representative evaluation of the entire section.