TPX2 expression is associated with poor survival in gastric cancer

Between January 2003 and December 2008, 290 patients underwent gastrectomy in the Department of Gastric Surgery of Tokyo Medical and Dental University. All patients were given sufficient explanation of the study, and written informed consent was obtained. This study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Board of Tokyo Medical and Dental University.

Each tumor was classified according to the tumor-node-metastasis classification system recommended by the Union for International Cancer Control. Follow-up surveillance was performed every 3 to 6 months with computed tomography, abdominal ultrasonography, and tumor marker analysis. Patients with distant metastatic or recurrent disease received chemotherapy with S-1 (oral fluoropyrimidine consisting of tegafur, gimeracil, and oteracil potassium; Taiho Co, Tokyo, Japan) alone or combined chemotherapy. The median follow-up period was 62 months (range, 2–111 months). During follow-up, a total of 99 patients (34%) died as a result of their disease, 84 (29%) had recurrent disease, and 15 (5%) died of other causes.

Immediately after surgery, small samples of gastric cancer tissue and adjacent normal tissue were collected, individually placed in RNA stabilization reagent (RNAlater; Qiagen, Valencia, CA, USA) and stored at −80 °C until analysis. Total RNA was extracted from each sample using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. The concentration of total RNA was determined by absorption measurements at 260 and 280 nm using a UV spectrophotometer (Beckman Counter; Beckman Coulter, Brea, CA, USA). For complementary DNA (cDNA) synthesis, 10 μg of total RNA from each sample was reverse-transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol.

Expression levels of TPX2 and the gene for β-actin, which served as the endogenous control, were determined by real-time quantitative reverse transcription PCR (qRT-PCR) using the 7300 Real-Time PCR System (Applied Biosystems). TaqMan gene expression assays were purchased from Applied Biosystems (Hs00201616_m1). The PCR reaction was carried out using TaqMan Universal Master Mix II (Applied Biosystems) with 1 μl of cDNA in a 24-μl final reaction volume. Thermal cycling conditions were as follows: 2 min at 50 °C, 10 min at 95 °C, 40 cycles each of 15-s denaturation at 95 °C, and 1 min of annealing at 60 °C. The cDNA synthesized by the gastric cancer cell line MNK 45 was used for calibration. Each sample was run in duplicate for both the target gene and the endogenous control gene. The amount of TPX2, normalized to the endogenous control and relative to the calibration samples, was calculated by the comparative threshold cycle (Ct) method using Relative Quantification Study Software version 1.4 on the 7300 Sequence Detection System (Applied Biosystems).

Immunostaining was carried out with a peroxidase-labeled polymer conjugated to secondary antibodies (Histofine Simple Stain Max PO (MULTI), Nichirei Co., Tokyo, Japan). A monoclonal mouse antibody against TPX2 (sc-376812) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA.). All of the original hematoxylin and eosin-stained slides of the surgical specimens were reviewed if available, and representative paraffin blocks for each case were selected for immunohistochemistry. Four-micrometer-thick sections were cut from formalin-fixed, paraffin-embedded tissue blocks. After deparaffinization and rehydration, antigen retrieval treatment was conducted for 25 min in 1 × TE (1 × Tris-EDTA, pH 8.0) at 98 °C in a microwave processor (MI-77; AZUMAYA, Tokyo, Japan). Endogenous peroxidase activity was quenched by a 15-min incubation in a solution of 3% hydrogen peroxide in 100% methanol. After washing with phosphate-buffered saline (PBS), the slides were incubated with the primary monoclonal antibody against TPX2 (dilution 1:50) for 30 min at room temperature and then overnight (16 h) at 4 °C. The slides were washed three times with 0.1% Tween 20/PBS and were then incubated with Simple Stain Max PO (MULTI) for 30 min at room temperature. After three additional washes, 3,3’-diaminobenzidine tetrahydrochloride solution (Histofine Simple Stain DAB Solution; Nichirei Co., Tokyo, Japan) was applied to the slides, which were then counterstained with Mayer’s hematoxylin (Wako, Tokyo, Japan). Gastric cancer tissue from participants in this study that were strongly positive for TPX2 served as positive controls.

In the tissue samples stained for TPX2, slides were considered positively stained when tumor cell nuclei were stained a darker brown than the background level of staining, regardless of intensity. TPX2 staining was graded as high (≥5% of tumor cells stained) or low (<5% of tumor cells stained). We stained representative cross-sectional slices of tumor tissue and counted at least five fields within most stained sections. The stained slides were evaluated by two independent observers (CT and YT) who were blinded to each patient’s prognosis. Any disagreements between the two observers were resolved by reassessment and consensus.

The messenger RNA (mRNA) expression of TPX2 in gastric cancer tissues and matched adjacent normal tissues was compared using paired t tests. The χ ² test and the Mann-Whitney U test were used to analyze the association between TPX2 protein expression and clinicopathological factors. Kaplan-Meier curves were compared using a log-rank test for univariate analysis of disease-specific survival (DSS) and relapse-free interval (RFI). For the RFI analysis, 25 stage IV patients were excluded. In all analyses, IBM SPSS Statistics Software version 22 (IBM, Armonk, NY, USA) was used, and p values <0.05 were considered significant.

Initially, we assayed TPX2 mRNA levels in a retrospective cohort of 19 gastric cancer tissues and matched adjacent normal tissues using qRT-PCR. In this cohort, the median TPX2 mRNA level was significantly higher in gastric cancer tissues than in matched adjacent normal tissues (p = 0.004; Fig. 1).

Expression of the TPX2 protein was assessed by immunohistochemical staining of gastric cancer tissue samples from 290 patients. Of these samples, 123 (42.4%) were classified as having high TPX2 expression (≥5% of tumor cells stained). TPX2 expression was mainly observed in the nuclei of tumor cells, with no expression observed in the nuclei of cells in normal tissue (Fig. 2). Weak expression was observed in the cytoplasm of some tumor cells.

High expression of TPX2 was significantly associated with the patient’s age (<65 vs. ≥65, p < 0.001), type of histology (differentiated vs. undifferentiated, p = 0.002), depth of tumor (T1 vs. T2 vs. T3 vs. T4, <0.001), lymph node metastasis (N0 vs. N1 vs. N2 vs. N3, p < 0.001), stage (I vs. II vs. III vs. IV, p < 0.001), and remote metastasis or recurrence (presence vs. absence, p = 0.02; Table 1).

High expression of TPX2 was associated with poorer DSS (p = 0.004) and RFI (p = 0.013) by univariate analysis. In patients with positive expression of TPX2, the 5-year DSS was 65.9% and the RFI was 69.9%; in patients with negative expression of TPX2, these values were 78.1 and 82.8%, respectively (Figs. 3 and 4).

After adjustment for significant prognostic factors (age, histopathology, tumor location, depth of invasion, lymph node involvement), the multivariate analysis of clinicopathological features affecting DSS indicated that independent prognostic factors were depth of tumor invasion (hazard ratio (HR) 7.7, 95% CI 2.7 to 21.6, p < 0.001) and lymph node metastasis (HR 6.4, 95% CI 3.1 to 13.2, p < 0.001; Table 2). After adjustment for the same significant prognostic factors, multivariate analysis of clinicopathological features affecting RFS indicated that independent prognostic factors were tumor main location (HR 1.9, 95% CI 1.1 to 3.3, p = 0.02), depth of tumor invasion (HR 6.0, 95% CI 2.1 to 17.1, p = 0.001), and lymph node metastasis (HR 6.7, 95% CI 3.0 to 15.3, p < 0.001; Table 3). High TPX2 expression was not an independent prognostic factor for either DSS or RFS. Comparison of Kaplan-Meier curves of the DSS of patients with low and high TPX2 expression after recurrence by using the log-rank test showed no significant difference between the DSS of these two groups (p = 0.52; Fig. 5).