Prognostic Impact of Protein Overexpression of the Proto-oncogene PIM-1 in Gastric Cancer

Patients and Methods

Patients. A total of 117 consecutive patients with potentially curative resections for gastric cancer between 1996 and 2000, with a median age of 65 years (min. 32, max. 85), 70 men and 47 women, were included in the study. The clinical and pathological characteristics of the patients are summarized in Table I. Of these patients, 105 (89.7%) underwent gastrectomy with D2 lymphadenectomy with curative intent, and 12 (10.3%) underwent subtotal gastric resection with D2 lymphadenectomy. Tumor specimens were collected from all patients. A mean number of 39 lymph nodes were resected per patient. R0 resection was achieved in 106 (90.6%) patients. Lymph node metastases (N+) were found in 75 (64.1%) specimens, and distant metastases (M1) were seen in 24 (21%) patients. Follow-up of surviving patients was at least 5 years. Informed consent was obtained from each patient according to the local Ethics Committee.

Immunohistochemistry. PIM-1 protein was detected by the polyclonal antibody Anti-PIM-1 (Abgent, San Diego, CA, USA) raised against a synthetic peptide selected from C-terminal amino acids 298-313 of the protein.

Five μm sections of the paraffin-embedded tissues were cut and deparaffinized according to standard histological techniques. Subsequently, a high-sensitivity immunohistochemical staining was perfomed applying the DAKO EnVision System (DakoCytomation, Hamburg, Germany) according to the manufacturer's instructions. In brief, pretreatment for antigen retrieval was performed in a microwave using 10 mM citrate buffer, pH 6.0, for 3×5 min at 700 W. Endogenous peroxidase activity was blocked by immersing the slides in 0.03% hydrogen peroxide in methanol for 5 min Subsequently, sections were incubated with primary antibody anti-PIM-1 (polyclonal ab; Abgent, San Diego, USA), diluted 1:50 with 10 mM phosphate-buffered saline (PBS), pH 7.4, at 4°C overnight. After washing twice in Tris-buffered saline (TBS) sections were incubated with avidin-horse radish peroxidase-conjugated secondary antibody (goat anti-rabbit) of the Envision-kit for 30 min at room temperature. After washing (2×5 min in TBS) the sections were stained by the chromogen 3-amino-9-ethylcarbazol for 30 min and then rinsed with H₂O. Nuclei were counterstained with hematoxylin. The staining procedure without primary antibody was used to develop a negative control, a PIM-1 expressing gastric normal tissue specimen was applied as positive control. The staining results were monitored by concurrently treating negative and positive controls.

Evaluation of immunostaining and statistical analysis. The microscopic evaluation was performed at a magnification of ×400 independently and in a blind fashion by two pathologists (U.D.) and (S.E.B.)

Analysis of PIM-1 immunostaining was performed using a scoring system based on the fraction of positive cells and the staining intensity in the specimens. The whole tissue of each section was evaluated. PIM-1 status was defined as score 0: no expression <5%; score 1: low expression of 5-35%; score 2: medium expression of 35-65%; and score 3: strong expression >65% positive cells. Due to the limited number of patients, score 1 and 2 were grouped for graphic presentation. For statistical analyses, scores were grouped depending on the correlation analyzed, with foveolae or tumor parameter, respectively.

Associations between the degree of staining and the subgroups according to the clinical and pathological classifications were calculated by Chi² test. Related samples were compared using the Wilcoxon signed-rank test with two-sided assumption. Survival curves were calculated by the Kaplan-Meier method and the significant difference between PIM-1 status was evaluated by means of the log-rank test. P-values <0.05 were considered significant. All statistical calculations were performed using the statistical software package release 12.0 (SPSS Inc, Chicago, IL, USA).