Cervical carcinoma samples were retrieved from the archives of the Department of Pathology, Makerere University, Kampala, Uganda, and were diagnosed during the period 1968-1990. The study protocol has been approved of Higher Degrees Research & Ethics Committee at Makerere University, Uganda.
HPV testing
The cervical carcinoma samples sections selected for HPV testing were digested with proteinase K and the resulting extract was used for PCR. SPF10 PCR was performed using 10 μl of a 1:10 dilution of the DNA extract in a final reaction volume of 50 μl. The amplified PCR products were tested using probe hybridization with a cocktail of conservative probes recognizing at least 54 mucosal HPV genotypes in a microtiter plate format for the detection of HPV DNA. Optical densities (OD450) were read on a microtiter plate reader. HPV DNA positive samples were subsequently analysed by HPV SPF10-LIPA25 (version 1: produced at Labo Biomedical Products, Rijswijk, The Netherlands) [10, 11], a reverse hybridization technique that detects 25 high-risk and low-risk HPV types (6, 11, 16, 18, 31, 33, 34, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 54, 56, 58, 59, 66, 68, 70, 74). The sequence variation within the SPF10 primers allows the recognition of these different HPV genotypes, except for the types 68 and 73 as their interprimer regions are identical and cannot be distinguished on this test. After PCR, 10 μl of the amplimers was used to perform reverse hybridization for HPV genotype identification. The positive hybridization on the strips is visualized as a purple band by means of a precipitating colour substrate on the probe site. All SPF10-LIPA25 PCR detection and typing was performed at the facilities of DDL Diagnostic Laboratories (DDL, Voorburg, Netherlands) and at Institut Català d'Oncologia (ICO, Barcelona). Detailed HPV results were published elsewhere [12].
Tissue Microarrays (TMA)
Three punches (gauge 1 mm) of pre-existing paraffin embedded tissues were obtained from each block and then re-embedded in an arrayed master block using the manual tissue microarrayer (Beecher Instruments, Silver Spring MD). The punch specimens were performed in the most representative areas of the tumours, discarding necrosis or artefacts. Four micron paraffin sections were cut from the TMA blocks and deparaffinised through alcohols and xylene before immunostaining.
Immunohistochemistry (IHC)
Monoclonal antibodies included: CEA (clone B0194-11M-P, BioGenex), p16 INK4a, p16 (clone JC8, Biocare Medical), Vimentin (clone V9, Dako), estrogen receptor alpha, ER (clone 1D5, Dako), progesterone receptor, PR (clone PgR 636, Dako).
Heat Induced Epitope Retrieval (HIER) was done using a pressure cooker as a heating device. Retrieval solutions that were used were: Citrate buffer for Vimentin, PR, ER; EDTA 10% for p16 and Saponin for CEA. Incubation was done at room temperature for 30 min for all the antibodies.
The Dako Autostainer universal staining system was used with the EnVision+ Dual Link System-HRP, a two-step IHC staining technique. This system is based on an HRP labelled polymer which is conjugated with secondary antibodies.
The final reaction was done with diaminobenzydine and slides were counterstained with hematoxylin. The immunohistochemical stains were interpreted by one of the authors (BL) and the results were reported as positive or negative using the following criteria for each antibody. ER and PR were considered positive when more than 10% of the tumour cells displayed nuclear positive staining. p16 was considered positive/overexpressed if more than 75% of the tumour cells showed cytoplasmic and/or nuclear staining, CEA was considered as positive if more than 10% of the neoplastic cells showed cytoplasmic positive staining, and vimentin was considered positive only when more than 20% tumour cells displayed cytoplasmic staining. The cut-offs for most antibodies were those used at laboratory in Barcelona, while the 75% for p16 was chosen because this cut-off is both sensitive and specific for HPV related cervical adenocarcinoma [13].
Statistical analysis
The chi-square test was used to determine significant differences between HPV positive and HPV negative adenocarcinomas and expression of, p16, CEA, ER, PR and vimentin.