This discrepancy may result from different clinical samples, the heterogeneity of gene expression in tumors and different research method. the CRC tissues tested, 70.0% of which showed high expression. There was no MAGE-D4 protein detected in any paired adjacent non-tumor tissue. No MAGE-D4 expression was found in colorectal adenomas and normal colorectal tissues by either RT-PCR or immunohistochemistry. Patients with high MAGE-D4 protein expression had significantly shorter overall survival than those with low MAGE-D4 protein expression (median, 68.6 vs 122.2 months; P=0.030). Furthermore, multivariate analysis exhibited high GM 6001 MAGE-D4 protein expression had a trend toward an independent prognostic factor (hazard ratio: 6.124; value of 0.05 were entered into the final model. Results Manifestation of MAGE-D4 mRNA We investigated the manifestation of MAGE-D4 mRNA in 60 CRCs with combined adjacent non-tumor cells, 24 colorectal adenomas and 10 normal colorectal cells by RT-PCR. MAGE-D4 mRNA was recognized in 46 of Mouse monoclonal to SMAD5 60 (76.7%) CRCs and 9 of 60 (15.0%) of adjacent non-tumor cells (Number 1). Statistical analysis revealed that there was significant difference between CRCs and adjacent non-tumor cells (P=0.000). No positive manifestation was detected in any of both colorectal adenomas and normal colorectal cells (Number 1). Open in a separate window Number 1 RT-PCR analysis of MAGE-D4 mRNA manifestation in CRC cells (T) with combined adjacent non-tumor cells (Nt), colorectal adenomas (A) and normal colorectal cells (N). GAPDH was used as internal control for the parallel PCR analysis of the same sample. P, positive control (glioma); Ne, bad control (no cDNA template). Manifestation of MAGE-D4 protein MAGE-D4 protein manifestation was examined in 30 CRCs with combined adjacent non-tumor cells, 12 colorectal adenomas and 10 normal colorectal cells by IHC. Positive staining was observed with different staining intensity in all the CRC samples tested and primarily located in the cytoplasm. Large MAGE-D4 protein manifestation was shown in 21 of 30 individuals (70.0%, Number 2C) and low in the other 9 individuals (30.0%, Number 2A). Notably, a rather heterogeneous manifestation was present in a significant quantity of CRC samples, which assorted from individual positive cells, foci of stained cells to standard staining of tumor cells. None of combined adjacent non-tumor cells (Number 2B and ?and2D),2D), colorectal adenomas (Number 2E) and normal colorectal cells (Number 2F) was positive for MAGE-D4 protein immunostaining. Open in a separate window Number 2 Representative immunohistochemical staining of MAGE-D4 protein in CRC cells (A, C), combined adjacent non-tumor cells (B, D), colorectal adenomas (E) and normal colorectal cells (F). Low and high immunoreactivity of MAGE-D4 protein immunostaining were demonstrated in (A and C) respectively, using polyclonal MAGE-D4 antibody. No positive reactivity was observed in combined adjacent non-tumor cells (B, D), colorectal adenomas (E) and normal colorectal cells (F). Initial magnification, 100 (400 in the right down corner). Prognostic effect of high MAGE-D4 protein expression Prognostic effect of MAGE-D4 protein expression in individuals with CRC was analyzed as a continuous variable inside a regression analysis. The result showed that mean overall survival (68.6 vs 122.2 months) was significantly shorter in patients with GM 6001 high MAGE-D4 protein expression than in those with low expression (P=0.030, Figure 3). Furthermore, Univariate analysis recognized carcinoembryonic antigen (CEA) 5 ng/mL, distant metastasis and high MAGE-D4 protein manifestation as significant prognostic GM 6001 factors (Table 1). In multivariate analysis, although distant metastasis exhibited the only independent prognostic element, high MAGE-D4 protein manifestation tended to become as an independent prognostic element (hazard percentage: 6.124; P=0.050; Table 1). Open in a separate window Number 3 KaplanCMeier analysis of MAGE-D4 protein in 30 individuals with CRC, classified as having low and high manifestation of MAGE-D4. Individuals with high MAGE-D4 protein manifestation in CRC cells had significantly shorter overall survival than individuals with low MAGE-D4 protein expression. Table 1 Prognostic factors in 30 individuals with CRC valuevaluevaluevaluevalue /th th align=”center” rowspan=”1″ colspan=”1″ hr / /th th align=”center” rowspan=”1″ colspan=”1″ hr / /th th align=”center” rowspan=”1″ colspan=”1″ hr / /th th align=”center” rowspan=”1″ colspan=”1″ Positive/Total (%) /th th align=”center” rowspan=”1″ colspan=”1″ High-expression/Total (%) /th th align=”center” rowspan=”1″ colspan=”1″ Positive/Total (%) /th /thead Gender????Male34/42 (81.0)0.31913/17 (76.5)0.44310/47 (21.3)0.490????Female12/18 (66.7)8/13 (61.5)????2/17 (11.8)Age (year)???? 5622/28 (78.6)0.7448/12 (66.7)1.0003/32 (9.4)0.055????56????24/32 (75.0)13/18 (72.2)9/32 (28.1)Tumor location????Colon28/36 (77.8)0.80317/24 (70.8)1.0006/32 (18.8)1.000????Rectum18/24 (75.0)4/6 (66.7)6/32 (18.8)Tumor size (cm)???? 523/27 (85.2)0.15810/13 (76.9)0.6917/41 (17.1)0.742????523/33 (69.7)11/17 (64.7)5/23 (21.7)CEA (ng/mL)a ????525/31 (80.6)0.45110/13 (76.9)0.6914/25 (16.0)0.751???? 521/29 (72.4)11/17 (64.7)8/39 (20.5)Depth of tumor invasion????T1-T28/10 (80.0)1.0000/0 (0)-2/18 (11.1)0.483????T3-T438/50 (76.0)21/30 (70.0)10/46 (21.7)Lymph node metastasis????N027/35 (77.1)0.9184/7 (57.1)0.6408/43 (18.6)1.000????N1-319/25 (76.0)17/23 (73.9)4/21 (19.0)Distant metastasis????M036/48 (75.0)0.71311/18 (61.1)0.24910/57 (17.5)0.607????M110/12 (83.3)10/12 (83.3)2/7 (19.0)TNM stage????? + II27/35 (77.1)0.9184/7 (57.1)0.6408/43 (18.6)1.000????III + IV19/25 (76.0)17/23 (73.9)4/21 (19.0)Histological typea ????Non-mucin-producing41/54 (75.9)1.00020/27 (74.1)0.20710/54 (18.5)1.000????Mucin-producing5/6 (83.3)1/3 (33.3)2/10 (20.0)Histological gradeb ????G113/17 (76.5)0.8625/8 (62.5)0.4841/16 (6.2)0.259????G220/27 (74.1)????14/18 (77.8)6/28 GM 6001 (21.4)????G313/16 (81.2)2/4 (50.0)5/20 (25.0) Open in a separate window aNon-mucin-producing malignancy includes tubular and (or) papillary adenocarcinoma, Mucin-producing malignancy includes mucinous malignancy and signet-ring cell malignancy. bHistological grade (G). G1, well differentiated; G2, moderately differentiated; G3, poorly differentiated. Discussion One.