The material studied comprised formalin-fixed paraffin embedded tissue samples from 31 previously untreated patients with squamous cell carcinoma of the oral cavity. Tumor sites included: 4 of the retromolar area, 10 of the floor of mouth, 3 of the lower gingival area and 14 of the oral tongue. All patients received local resection and elective lymph node neck dissection (unilateral or bilateral) at Service of Head and Neck Surgery, Hospital Heliopolis, Sao Paulo – Brazil. None of these patients had received any drugs therapy before surgery. All patients had histologically confirmed squamous cell carcinoma. Fourteen patients received adjuvant postsurgical radiotherapy following the clinical protocol of the Hospital. These patients were submitted to resection margins, positive lymph nodes and/or perineural invasion. Additionally, all patients have had at least 24 months of clinical follow up. The group was constituted by patients with a median age of 50 years (range, 38 – 70 years) and included 29 men and 2 women. Of these, 29 had a prolonged smoking history and 26 had chronic alcohol consumption.
The distribution of patients was made according to the post-surgery TNM classification based on the American Joint Committee on Cancer (AJCC) and the Union Internationale Contre le Cancer (UICC) and the clinical classification was based on AJCC-UICC; additionally, the T, N, and M characteristics are combined to produce a "stage" of the cancer, from I to IVB .
D2-40 Immunohistochemical procedure
Immunohistochemistry was carried out using the avidin-biotin-peroxidase complex assay, with the monoclonal antibody D2-40 (DAKO Corporation, Carpinteria, CA, USA). Briefly, deparaffinized and re-hydrated sections were immersed in 0.01 M citrate buffer (pH 6.0) and heated at 98°C for 20 min.; then slides were incubated with 0.3 % hydrogen peroxide in methanol for 30 min., followed by incubation with Normal Horse Serum (Vector Laboratories Inc., CA, USA) for 20 min., at room temperature, before incubating with the primary antibody diluted 1:100, overnight at 4°C. Sections were sequentially washed in PBS 1× and incubated with Biotinylated Universal Secondary antibody (Vector Laboratories Inc., CA, USA) for 30 min., Vectastain® Elite ABC reagent (Vector Laboratories Inc., CA, USA) for 45 min. at 37°C, and developed with 3,3'-diamino-benzidine (DAKO Corporation, Carpinteria, CA, USA) for 10 min. Negative controls were performed by omitting the primary antibody and, as positive control, tonsil tissue was used.
CD31 Immunohistochemical procedure
For CD31, the immunohistochemistry was carried out with the streptavidin-biotin-peroxidase complex technique (Ultravision Detection System Anti-polyvalent, HRP, Lab Vision Corporation, Fremont, CA, USA), using specific primary antibody raised against CD31/PECAM-1 (rabbit monoclonal antibody, clone Ab-1/JC/70A, Neomarkers, Freemont, CA, USA) diluted 1:50. Briefly, deparaffinized and rehydrated sections were heated up to 98°C for 20 min. in 0.01 M citrate buffer (pH 6.0). Endogenous peroxidases were inactivated with 3 % hydrogen peroxide in methanol for 10 min., followed by washing in PBS/Tween. Tissue sections were incubated with blocking solution for 10 min. and incubated with the primary antibody for 60 min. at room temperature. Sections were then sequentially washed in PBS/Tween and incubated with biotinylated goat anti-polyvalent antibody for 10 min., streptavidin peroxidase for 10 min., and developed with 3,3'-diamino-benzidine (DAB Substrate System; Lab Vision Corporation, Fremont, CA) for 10 min. Appropriated positive and negative controls were included in each run. Negative controls were performed by omission of the primary antibody and angiosarcoma was used as positive control. The slides were counterstained with haematoxylin and mounted with Synthetic Mountant Entellan (Merck, Darmstadt, Germany).
The immunohistochemical positive reaction of D2-40 and CD31 antibodies was evaluated considering its expression in the cytoplasm of lymphatic and blood endothelial cells, respectively. The evaluation was performed blindly and both LVD and BVD were assessed as postulated by Weidner et al , with slightly modifications. Microvessel was defined as a single endothelial cell or a cluster of endothelial cells positive for D2-40 or CD31, respectively, sitting around a visible lumen clearly separate from adjacent microvessels and from other connective tissue components. Additionally, as lymphatic vessels could generally appear as distorted and overlapped structures in cancer setting, the packed vessels were assumed as one lymphatic unit. In contrary, blood vessels commonly do not display distorted and packaged appearance. The number of vessels was quantified at ×200 (×20 objective lens and ×10 ocular lens) magnification. A median of 10 hot spot fields was defined as vessel density. The examination of each hotspot corresponds to a number of vessels confined to an area of 0.15 mm2. Both D2-40 and CD31 immunohistochemical positive reactions were independently counted in lymphatic and blood vessels from intratumoral and peritumoral areas. Intratumoral area was defined as the stromal tissue within two or more neoplastic aggregates, and peritumoral area was defined as the stroma tissue surrounding these neoplastic mass. D2-40 and CD31 positivity in tumor cells was classified, considering immunoreaction extension as negative (negative or weak immunoreaction) and positive (moderate to strong immunoreaction). For evaluation of lymphatic and blood vessels invasion, only D2-40 and CD31 positive vessels occupied by neoplastic cells, respectively, were considered. Seven cases were excluded for CD31 evaluation due to technical limitations.
Data were stored and analyzed using the SPSS statistical software (for Windows, version 14.0, Chicago, IL). The Shapiro-Wilk test was applied to assess normality of the results. Data was examined for statistical significance using the T-Student, the One-Way ANOVA, the Mann-Whitney U, the Kruskal-Wallis and the Pearson's chi-square (χ2) tests, as appropriate, being threshold for significance p values < 0.05.