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  • Veliparib br Statistical analysis br Correlations between the clinicopathological parameters and


    2.7. Statistical analysis
    Correlations between the clinicopathological parameters and claspin Veliparib were analyzed using the Fisher exact test. Kaplan-Meier survival curves were constructed for clas-pin-positive and claspin-negative patients, and survival rates of the 2 groups were compared. Differences between the sur-vival curves were tested for statistical significance using the
    Claspin expression in GC
    A C E
    Fig. 3 Immunohistochemical analysis of the correlation between claspin expression and various molecules including CSC markers in consec-utive tumor sections of GC. The expression of claspin was coexpressed with CD44, HER2, and p53. A, C, and E, The nuclear expression of claspin (A, C, and E, anticlaspin antibody immunohistochemical staining, original magnifications ×200, ×200, and ×200, respectively). B, The membrane expression of CD44 (anti-CD44 antibody immunohistochemical staining, ×400). D, The membrane expression of HER2 (anti-HER2 antibody im-munohistochemical staining, ×200) and HER2 gene amplification (HER2/CEP17 ratio of 3.88, ×1000). F, The nuclear expression of p53 (anti-p53 antibody immunohistochemical staining, ×400).
    log-rank test. Univariate and multivariate Cox regression anal-yses were used to evaluate the associations between clinical covariates and survival as described previously [20]. A P value of less than .05 was considered to indicate statistical signifi-cance. The SPSS software program (SPSS, Chicago, IL) was used Veliparib for all statistical analyses. 
    MKN-74 and TMK-1 (Fig. 1B). Moreover, we analyzed CLSPN expression in 14 GC tissue samples and 14 corre-sponding nonneoplastic mucosa samples by quantitative RT-PCR. We calculated the ratio of mRNA expression levels be-tween GC tissue (T) and corresponding nonneoplastic mucosa
    3. Results
    3.1. Messenger RNA expression of CLSPN in the spheroid body–forming GC cells, systemic normal or-gans, GC cell lines, and GC tissue
    To confirm up-regulation of the CLSPN gene in the spher-oid body–forming cells, the expression of CLSPN messenger RNA (mRNA) was measured by quantitative RT-PCR in the MKN-45 and MKN-74 cell lines. CLSPN mRNA expression was more than 2-fold higher in the spheroid body–forming cells than in the parental cells in both the MKN-45 and MKN-74 cells (Fig. 1A). Next, to confirm whether the CLSPN gene is cancer specific, quantitative RT-PCR was per-formed in 5 GC cell lines and in 14 types of normal tissue. CLSPN expression was detected at low levels or to even a lesser extent in various normal organs. However, high CLSPN expression was observed in GC cell lines MKN-45, 
    3.2. Immunohistochemical analysis of claspin in GC
    To analyze the tissue localization, pattern of distribu-tion, and relationship between clinicopathological charac-teristics and claspin in GC, we performed IHC in the 203 human GC samples. Claspin expression was detected in 94 (47%) of the 203 GCs, and it showed nuclear staining in tumor cells irrespective of the histology (Fig. 2A and B). In the nonneoplastic gastric mucosa, the staining of claspin was either weak or absent in epithelial and stromal cells (Fig. 2C). Next, we analyzed the relationship between claspin expression and various clinicopathological characteristics. Claspin expression was associated with higher T grade (P =
    G. Kobayashi et al.
    Table 2 Relationship between claspin expression and various molecules including CSC markers in 123 of the GC cases
    Claspin expression
    Positive, n (%) Negative
    NOTE. P values were calculated using the Fisher exact test.
    Abbreviation: NS, not significant.
    3.3. Relationship between claspin expression and prognosis in GC
    We performed a Kaplan-Meier analysis to investigate the association between claspin expression and patient prognosis to further elucidate the clinical impact of claspin on GC in 187 of our patients. Claspin expression was significantly asso-ciated with a poorer prognosis (P = .0468, log-rank test; Fig. 2D). We also performed univariate and multivariate Cox pro-portional hazards analyses but did not find claspin expression to be an independent prognostic predictor (data not shown).