2. Methodology2.1. Patients and Tissue SpecimensSeventy-eight patients with histologically confirmed adenocarcinoma of the stomach operated on with curative intent (R0) entered selleck compound the study. The group of patients consisted of 54 males and 24 females with ages ranging between 37 and 84 years old (mean age: 62 years old). The stage of tumors was assessed according to the 5th ed. of TNM Classification of Malignant Tumors [14] (stage I: 25 patients (32%); II: 18 (23%); III: 17 (22%); IVM0: 18 (23.1%). According to Lauren’s classification: 19 (24%) cases were intestinal, 44 (56.%) diffused and 15 (19.%) were of a mixed type. All patients underwent elective total gastrectomy and D2 lymphadenectomy with curative intent (the mean number of dissected lymph nodes was 19).

The adjuvant chemotherapy was administered in 53 cases of tumors infiltrating beyond the muscularis propria or in patients with lymph node involvement. The followup was scheduled every 3 months for the first 2 years and then every 6 months. Chest X-ray, abdominal sonography, CT scan as well as clinical and endoscopic examinations were performed. The information about overall survival were obtained from Lower-Silesian Regional Cancer Registry. The data were collected in a retrospective manner.The tissue samples were fixed in 10% buffered formalin and embedded in paraffin. In each case, hematoxylin- and eosin-stained preparations were subjected to histopathological evaluation by two pathologists.2.2. ImmunohistochemistryFormalin-fixed, paraffin embedded tissue was freshly cut (4��m).

The sections were mounted on superfrost slides (Menzel Gl?ser, Germany), dewaxed with xylene, and gradually hydrated. The activity of endogenous peroxidase was blocked by 5min exposure to 3% H2O2. All the studied sections were Anacetrapib boiled for 15min at 250W in the Antigen Retrieval Solution (DakoCytomation, Denmark). Then, immunohistochemical reactions were performed using the rabbit antihuman antibody detecting HER-2 (optimally prediluted) (DakoCytomation, Denmark). The tested sections were incubated with antibodies for 1h at room temperature. The subsequent incubations involved biotinylated antibodies (15min, room temperature) and a streptavidin-biotinylated peroxidase complex (15min, room temperature) (LSAB+, HRP, DakoCytomation, Denmark). NovaRed (Vector Laboratories, UK) was used as a chromogen (10min, at room temperature). All the sections were counterstained with Meyer’s hematoxylin. In every case, control reactions were included, in which specific antibody was substituted by the Primary Mouse Negative Control (DakoCytomation, Denmark).2.3. Evaluation of Reaction IntensityThe intensity of immunohistochemical reactions was estimated independently by two pathologists.