Material and Method
Patients’ General Information and Tissue Specimens
In our study, laryngectomy materials examined between January 2009 and December 2015 were investigated retrospectively. 114 cases who were diagnosed with LSCC and have not previously received chemotherapy and radiotherapy were included in the study. Previous chemotherapy and/or radiotherapy, 8 patients were excluded. The clinical information of the patients was obtained from the automation system of our hospital. Recurrence and metastasis were determined by imaging methods, clinical examination epicrisis and examination of pathological materials sent to our department in the postoperative period. In order to determine the survival rate, recurrence and metastasis, the follow-up period for all 114 patients was determined as 5 years, and changes after 5 years were not taken into account. Pathology reports, slides and paraffin blocks of the patients were extracted from our archive. Paraffin blocks containing adjacent non-cancerous tissues of at least 1 cm in length were selected for the immunohistochemical study.
Immunohistochemistry
4-micron sections were taken from the blocks where the tumor was the densest and were put into the Roche Ventana automatic immunohistochemistry staining device (Ventana Roche, ABD) after the tissues placed on charged slides in a 70-degree drying oven for 15 minutes. The tissues in the device were treated with ULTRA Cell Conditioning Solution, hydrogen peroxide, HSPA-2 antibodies (Nova Castra, Leica, Newcastle, United Kingdom) after being subjected to deparaffinization and dehydration processes respectively.
The immunohistochemical stains were evaluated by two pathologists. As negative control group adjacent squamous epithelium not containing dysplasia were accepted. Nuclear and/or cytoplasmic staining was considered positive for HSPA-2. For HSPA-2, staining rate=0 (Grade-0), without staining 1-10% = 1 (Grade-1), 11-49% = 2 (Grade-2), ≥50% = 3 (Grade-3) were accepted. HSPA-2 was evaluated as follows; staining intensity: 0; no staining (Grade-0), 1+; weak cytoplasmic and nuclear staining (Grade-1), 2+; moderate cytoplasmic and nuclear staining (Grade-2), 3+; strong cytoplasmic and nuclear staining (Grade-3) (Figure 1). The immunoreactivity score (histoscore) was calculated using the method of multiplying the intensity and rate. The histoscore values for HSPA-2 0 were evaluated as follows; negative (Grade-0), 1-3; weak (Grade-1), 4-6; moderate (Grade-2), 7-9 strong (Grade-3) [15]. The connection between HSPA-2 staining rate, intensity, histoscore and age, gender and important prognostic parameters such as histological grade, the TNM stage, the primary tumor, lymph nodule metastasis, the presence of lymphovascular invasion, survival rate, recurrence and metastasis was examined.
Our study was approved by our local ethics committee (08-31/26.12.2019).