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ANTICANCER RESEARCH 30: 5239-5244 (2010)

Clinical Significance of Vimentin-positive Gastric Cancer Cells

YUHIKO FUYUHIRO1, MASAKAZU YASHIRO1,2, SATORU NODA1, SHINICHIRO KASHIWAGI1, JUNKO MATSUOKA1, YOSUKE DOI1, YUKIHIRO KATO1, NAOSHI KUBO1, MASAICHI OHIRA1 and KOSEI HIRAKAWA1

1Department of Surgical Oncology and 2Oncology Institute of Geriatrics and Medical Science, Osaka City University Graduate School of Medicine, Osaka, Japan

Abstract. Background/Aim: Vimentin expression in cells seem to acquire -like properties, with changes epithelial cells is reported to be associated with the in morphology, architecture, adhesion, and migration malignant phenotype of cancer cells in vitro. However, the capacity (4). The expression of vimentin in epithelial cells is clinical significance of vimentin expression in is essential to the successive EMT through the interaction with not well understood. The aim of this study was to clarify the and other intermediate filaments (5). Although the significance of vimentin-positive gastric cancer (GC). vimentin expression of cancer cells has been linked to a Patients and Methods: A total of 265 GCs were examined by more malignant potential in vitro, the clinical significance of immunofluorescent staining with antibodies against vimentin vimentin expression in carcinomas is still controversial (6, and . GCs were determined to be 7). The aberrant overexpression of vimentin has been linked vimentin-positive when cells were positive for both vimentin to a more aggressive status in various tumors including and carcinoembryonic antigen staining. Results: A total of (8), (9, 10), cervical (11), 86 (32%) of 265 GCs were vimentin positive. There was a renal cell carcinoma (12), and prostate carcinoma (13). On statistically significant correlation between vimentin-positive the other hand, some reports have shown that vimentin GCs and advanced clinical stage (p<0.001), macroscopic expression does not inversely predict patient survival (14). scirrhous-type (p<0.001), histological diffuse-type Since gastric carcinoma is one of the most frequent causes (p<0.001), lymph node metastasis (p=0.008) and lymphatic of cancer death due to its high metastatic ability (15), invasion (p=0.013). The prognosis of patients with vimentin- vimentin expression of cancer cells might be correlated with positive GCs was significantly (p<0.001) worse than that more malignant phenotype in gastric carcinoma. However, with vimentin-negative GCs. Conclusion: Vimentin there are few studies of the clinical significance of vimentin expression might contribute to the high invasive phenotype expression in gastric carcinoma. This study examined the of GC, and may be a useful biomarker to determine the significance of vimentin-positive cancer cells in gastric biological aggressiveness of GC. carcinoma.

Vimentin is a 57 kDa , which Patients and Methods forms a part of the (1). Vimentin is a characteristic component of mesenchymal cells and it is not Clinical materials. A total of 265 patients who had undergone usually expressed in epithelial cells (2). The epithelial– resection of a primary gastric tumor at the host institute were mesenchymal transition (EMT) is a process in which cells enrolled in this study. Tumor specimens were fixed in 10% formaldehyde solution and embedded in paraffin. The pathologic undergo a developmental switch from an epithelial to a diagnoses and classifications were made according to the Japanese mesenchymal phenotype (3). During the EMT, epithelial Classification of Gastric Carcinoma. ‘Early cancer’ indicates cancer invasion limited to the submucosa, and ‘advanced cancer’ indicates cancer invasion into the muscularis propria or serosa. The median follow-up time for all 265 patients was 58 months (range 1 to 177 Correspondence to: Masakazu Yashiro, MD, Ph.D., Department of months). The median follow-up time for the patients that died of the Surgical Oncology, Osaka City University Graduate School of disease was 25 months (n=88), compared with 75 months for Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. surviving patients (n=177). A total of 31 patients were lost from the Tel: +81 666453838, Fax: +81 666466450, e-mail: m9312510@ study after more than 60 months of follow-up. med.osaka-cu.ac.jp Antibodies and reagents. FITC mouse monoclonal vimentin (clone Key Words: Vimentin, gastric carcinoma cells, scirrhous type, lymph VIM 3B4, Cat.No.61413) was obtained from PROGEN Biotechnik node , prognosis. (Heidelberg, Germany). PE mouse anti-Human CD66 (carcino-

0250-7005/2010 $2.00+.40 5239 ANTICANCER RESEARCH 30: 5239-5244 (2010) embryonic antigen, CEA; clone B1.1/CD66, Cat.No.551480) was between vimentin-positive cancer and clinicopathological obtained from BD bioscience (Heidelberg, Germany). DAPI features are shown in Table I. The occurrence of vimentin- solution was obtained from WAKO (Kumamoto Japan). positive gastric cancer was significantly frequent in advanced stage of T2-T4 (p<0.001), macroscopically scirrhous-type, Immunofluorescence microscopy. Triple-immunofluorescence labeling was performed to examine the presence of vimentin- diffuse-type (p<0.001), lymph node metastasis (p=0.008), positive gastric cancer cells. The slides were deparaffinized in lymphatic invasion (p<0.013). In contrast, there was no xylene and hydrated in decreasing concentrations of ethyl alcohol. statistically significant association between vimentin-positive The tissues were heated for 20 min at 105˚C and at 0.4 kg/cm2 by expression and peritoneal dissemination, cytology positive, autoclave in Target Retrieval Solution (Dako Co., Carpinteria, CA, and venous invasion. USA). The slides were washed with 0.01 M PBS, and then blocked with 3% BSA (diluted in PBS) for 30 min at room temperature. Survival. Figure 2 shows Kaplan-Meier overall survival Sections were further incubated with anti-Human CD66 (CEA) antibody (1:100) and vimentin (1:50) and DAPI (1:10000) for curves in relation to vimentin-positive gastric cancer 60 min at room temperature. The sections were washed with expression levels in gastric carcinomas. The survival curve 0.01 M PBS (three times) and mounted. Stained sections were was calculated from the date of surgery. The 5-year survival viewed under a fluorescence microscope Leica Digital Microscopy of the patients with vimentin-positive gastric cancer DMI 6000 (Leica Microsystems, Heidelberg, Germany). Images expression tumors was 56.9% in comparison to 78.5% for were captured with a Leica TCS SP5. All three fluorescent those patients with negative tumors (Figure 2A). The parameters were investigated in the same section by switching to prognosis of patients with vimentin-positive gastric cancer the appropriate wavelength filter for each of the markers. Stained sections were viewed with a DAPI filter (365 nm excitation), CEA was significantly worse (p=0.003) than that of patients with fluorescence with a PE filter (546 nm excitation), and antibody vimentin-negative gastric cancer. In 214 patients with distribution with a FITC filter (450-490 nm excitation). curative resection (R0), the prognosis of with those with vimentin-positive gastric cancer expression tumors was Immunofluorescence determination of vimentin. The tumor significantly worse (p<0.001) than that of those with specimens showed various patterns of immunofluorescent staining vimentin-negative gastric cancer expression tumors (Figure against vimentin antibody. Cells were defined as being gastric 2B). In univariate analysis (Table II), vimentin-positive cancer cells when they were positive for CD66 (CEA) staining. Vimentin expression level was analyzed semi-quantitatively cancer (p<0.001), advanced cancer (p<0.001), peritoneal according to the percentage of cancer cells showing vimentin dissemination (p<0.001), venous invasion (p=0.042), and positivity: 0, 0%; 1+, 1-9%; 2+, 10-20%; 3+, >20%. Vimentin lymph node metastasis (p<0.001) were correlated expression was considered positive when scores were ≥2+, and significantly with patient survival. In multivariate analysis negative when scores were ≤1+ (Figure 1). Four scratched fields (Table III), invasion depth, macroscopic type, and peritoneal were chosen randomly and the number of vimentin-positive cancer dissemination were found to be independent prognostic cell was counted from the images (×200). The slides were factors, but not vimentin expression. interpreted by two investigators without knowledge of the corresponding clinicopathological data. The relationships between various clinicopathological factors and vimentin-positive cancer Discussion cells were analyzed. Since a number of stromal cells, such as , express Statistical analysis. The χ2 test, Fisher’s exact test or Mann-Whitney vimentin, it is difficult to distinguish vimentin-positive U-test were used to determine the significance of the differences cancer cells from stromal cells by comparing two different between the covariates. Survival durations were calculated using the slides of hematoxylin and eosin staining and vimentin Kaplan-Meier method and were analyzed by the log-rank test to compare the cumulative survival durations in the patient groups. The staining. Therefore vimentin-positive cancer cells were Cox proportional hazards model was used to compute univariate and examined using triple-immunofluorescence labeling by CEA, multivariate hazards ratios for the study parameters. For all tests, a p- vimentin, and DAPI staining in a single slide. Through the value <0.05 was defined as statistically significant. The SPSS software use of immunofluorescent staining it was possible to program (SPSS ver.11 Tokyo, Japan) was used for the analyses. distinguish vimentin-positive cancer cells from stromal cells. In this study, the aberrant overexpression of vimentin was Results found in 85 (32%) out of 265 patients. The vimentin expression was significantly high in patients with advanced Correlation between clinicopathological factors and vimentin- gastric cancer, especially those with macroscopically positive cancer expression. Vimentin-positive expression was scirrhous-type gastric carcinoma. Moreover, vimentin- found in 86 (32%) out of 265 gastric carcinomas, in 20 (17%) positive cancer was significantly associated with out of 119 early gastric carcinomas and in 66 (45%) out of histologically diffuse-type, lymphatic invasion, lymph node 146 advanced gastric carcinomas. CD66 (CEA) staining was metastasis. Macroscopically scirrhous type gastric cancer positive for most of the gastric carcinomas. The relationship (type 4 carcinoma, diffusely infiltrating carcinoma, or linitis

5240 Fuyuhiro et al: Vimentin Expression in Gastric Carcinoma

Figure 1. Immunofluorescent staining of vimentin expression in gastric cancer. A: The expression of vimentin was positive for 43% of CEA-positive gastric cancer cells. B: In contrast, vimentin expression was low (8%) in gastric cancer cells.

Figure 2. A: The overall survival of 265 patients based on vimentin expression. B: The overall survival of 214 patients with curative resection (R0).

plastica-type) is characterized by cancer cell infiltration, acquisition of invasive properties of cancer cells (18, 19). reflecting frequent distant metastasis of cancer cells, and These findings suggest that vimentin expression might carries a worse prognosis among other types of gastric contribute to the highly invasive phenotype of scirrhous carcinomas (16, 17). The expression of vimentin in epithelial gastric carcinoma. Overexpression of transforming growth cells was essential to the EMT which is associated with the factor-β (TGF-β) was frequent in scirrhous gastric carcinoma

5241 ANTICANCER RESEARCH 30: 5239-5244 (2010)

Table I. Correlation between clinicopathological factors and vimentin- Table II. Univariate analysis with respect to overall survival in gastric positive cancer. cancer.

Clinicopathological factor Vimentin staining P-value Parameter Risk 95% P-value ratio Confidence Positive Negative interval n=86 (32%) n=179 (68%) Vimentin expression Tumor deptha Positive vs. Negative 2.292 1.453-3.616 <0.001 Early cancer (T1) 20 (17%) 99 (83%) Invasion depth Advanced cancer (T2-T4) 66 (45%) 80 (55%) <0.001 Early cancer vs. Advanced cancer 1.692 1.323-2.163 <0.001 Macroscopic typeb Macroscopic type Non-scirrhous-type 59 (27%) 163 (73%) Non-scirrhous-type vs. Scirrhous-type 3.366 2.389-4.742 <0.001 Scirrhous-type 27 (63%) 16 (37%) <0.001 Histological type Histologic type Diffuse vs. Intestinal 1.084 0.848-1.386 0.52 Intestinal type 19 (17%) 94 (83%) Peritoneal metastasis Diffuse type 67 (44%) 85 (56%) <0.001 Positive vs. Negative 6.462 3.926-10.638 <0.001 Peritoneal metastasis Cytology Positive 9 (43%) 12 (57%) Positive vs. Negative 4.417 3.002-6.501 <0.001 Negative 77 (32%) 167 (68%) 0.289 Venous invasion Venous invasion Positive vs. Negative 1.363 1.011-1.838 0.042 Positive 18 (32%) 38 (68%) Lymph node metastasis Negative 68 (33%) 141 (67%) 0.956 Positive vs. Negative 1.911 1.490-2.450 <0.001 Lymph node metastasis Lymphatic invasion Positive 46 (41%) 65 (59%) Positive vs. Negative 1.663 1.299-2.130 <0.001 Negative 40 (26%) 114 (74%) 0.008 Lymphatic invasion Positive 47 (41%) 69 (59%) Table III. Multivariate analysis with respect to overall survival in gastric Negative 39 (26%) 110 (74%) 0.013 cancer. Cytologyc Positive 12 (35%) 22 (65%) Parameter Risk 95% P-value Negative 74 (32%) 157 (68%) 0.705 ratio Confidence interval a‘Early cancer’ indicates cancer invasion limited to the submucosa, and ‘advanced cancer’ indicates cancer invasion into the muscularis propria Vimentin expression or serosa. T1: Tumor invades mucosa or submucosa, T2: tumor invades Positive vs. Negative 0.951 0.568-1.594 0.849 the muscularis propria or the subserosa, T3: tumor penetrates the serosa Invasion depth and exposed to abdominal cavity without invading the adjacent Early cancer vs. Advanced cancer 3.491 1.459-8.350 0.005 structures, T4: tumor invades the adjacent structures. bThe classification Macroscopic type according to the general rules for gastric cancer study of the Japanese Non-scirrhous-type vs. Scirrhous-type 4.424 2.162-9.051 <0.001 Research Society for Gastric Cancer. Type 1 is defined as a polypoid Histological type tumour, sharply demarcated from the surrounding mucosa and usually Diffuse vs. Intestinal 1.125 0.586-2.163 0.723 attached on a wide base. Type 2 is defined as polypoid tumour with Peritoneal metastasis ulceration and with sharply demarcated margins. Type 3 is defined as Positive vs. Negative 2.184 1.159-4.118 0.016 ulcerated carcinoma with cancer infiltration into the surrounding wall. Venous invasion Scirrhous-type (Type 4) is defined as diffusely infiltrating flat carcinoma Positive vs. Negative 1.521 0.853-2.712 0.156 in which ulceration is usually not a marked feature. c‘Cytology’ means Lymph node metastasis peritoneal lavage cytology at laparotomy as a standard method for the Positive vs. Negative 1.318 0.681-2.552 0.412 detection of free tumor cells.

(20), and TGF-β signals play an important role for the positive gastric cancer was significantly worse. Elevated metastatic spread of cancer cells (21-23) such as migration, vimentin expression level might be closely correlated with invasion, and EMT (4, 22, 24). These findings suggest that up-regulated migration and high invasiveness and poor TGF-β might up-regulate vimentin expression, and affect the prognosis of gastric carcinoma. On the other hand, highly metastatic ability of scirrhous gastric cancer cells. multivariate survival analysis for all patients showed that The prognosis of patients with vimentin-positive gastric vimentin expression did not influence the clinical outcome, cancer was significantly worse than those with vimentin- suggesting that vimentin-positive gastric cancer expression negative gastric cancer, and in patients who had undergone was not an independent prognostic factor. Although vimentin curative R0 resection, the prognosis of those with vimentin- failed to emerge as an independent prognostic factor, it was

5242 Fuyuhiro et al: Vimentin Expression in Gastric Carcinoma successful in identifying a subgroup of patients with disease 11 Gilles C, Polette M, Piette J, Delvigne AC, Thompson EW, of high malignancy. These findings suggested that vimentin Foidart JM and Birembaut P: Vimentin expression in cervical expression might be a useful biomarker to determine the carcinomas: association with invasive and migratory potential. J Pathol 180(2): 175-180, 1996. biological aggressiveness of gastric cancer. 12 Moch H, Schraml P, Bubendorf L, Mirlacher M, Kononen J, In conclusion, vimentin-positive gastric cancer was Gasser T, Mihatsch MJ, Kallioniemi OP and Sauter G: High- associated with the highly invasive phenotype of advanced throughput tissue microarray analysis to evaluate gastric cancer, especially with scirrhous type of gastric uncovered by cDNA microarray screening in renal cell carcinoma. Vimentin may play an important role in the carcinoma. Am J Pathol 154(4): 981-986, 1999. metastasis of gastric carcinoma. 13 Lang SH, Hyde C, Reid IN, Hitchcock IS, Hart CA, Bryden AA, Villette JM, Stower MJ and Maitland NJ: Enhanced expression Acknowledgements of vimentin in motile prostate cell lines and in poorly differentiated and metastatic prostate carcinoma. Prostate 52(4): 253-263, 2002. This study was supported in part by a Grant-in Aid for Scientific 14 Heatley MK, Ewings P, Odling Smee W, Maxwell P and Toner Research (C) 13671329 and (B) 13470260 from the Ministry of PG: Vimentin expression does not assist in predicting survival in Education, Science, Sports, Culture and Technology of Japan, and ductal carcinoma of the breast. Pathology 34(3): 230-232, 2002. by a Grant-in Aid for the Osaka City University Medical Research 15 Kiyasu Y, Kaneshima S and Koga S: Morphogenesis of Foundation. peritoneal metastasis in human gastric cancer. Cancer Res 41(3): 1236-1239, 1981. 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Eur J Cancer 30A(10): 1527-1534, 1994. 10 Hendrix MJ, Seftor EA, Seftor RE and Trevor KT: Experimental co-expression of vimentin and intermediate filaments in Received October 19, 2010 human breast cancer cells results in phenotypic interconversion and Revised November 15, 2010 increased invasive behavior. Am J Pathol 150(2): 483-495, 1997. Accepted November 16, 2010

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