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Expression of MAP4K4 Is Associated with Worse Prognosis in Patients with Stage II Pancreatic Ductal Adenocarcinoma John J

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Expression of MAP4K4 Is Associated with Worse Prognosis in Patients with Stage II Pancreatic Ductal Adenocarcinoma John J Imaging, Diagnosis, Prognosis Expression of MAP4K4 Is Associated with Worse Prognosis in Patients with Stage II Pancreatic Ductal Adenocarcinoma John J. Liang,1, 5 Hua Wang,2 Asif Rashid,1Ts e-Hu a Tan, 4 Rosa F. Hwang,2 Stanley R. Hamilton,1 James L. Abbruzzese,2 Douglas B. Evans,3 and Huamin Wang1 Abstract Purpose: Mitogen-activated protein 4 kinase 4 (MAP4K4) is a serine/threonine kinase and belongs to the mammalian STE20/MAP4K family. Recent studies have shown that MAP4K4 is overexpressed in many types of human cancer and cancer cell lines. MAP4K4 plays an important role in transformation, invasiveness, adhesion, and cell migration. However, the expression of MAP4K4 and its significance in pancreatic ductal adenocarcinoma (PDA) has not been studied. Experimental Design: We examined the expression of MAP4K4 by immunohistochemistry using tissue microarrays consisting of 66 stage IIPDA and their paired benign pancreatic tissue. The staining results were categorized as MAP4K4-H or MAP4K4-L. The results were correlated with clinicopathologic features and patient survival. Results: MAP4K4 was overexpressed (MAP4K4-H) in 30 of 66 (46%) PDAs and was higher than the paired benign pancreatic tissue samples (19%; P = 0.002).The median overall and recur- rence-free survival for patients with MAP4K4-H PDAs were 19.5 and 9.3 months, respectively, compared with 65.2 and 28.8 months for patients with MAP4K4-L tumor (P = 0.02 and 0.0005, log-rank test).MAP4K4 expression was associated with poor overall and recurrence-free survival in univariate analysis (P = 0.02 and 0.001). In multivariate analysis, MAP4K4 expression significantly correlated with overall and recurrence-free survival (P = 0.025 and 0.004) indepen- dent of age, tumor size, differentiation, and stage. MAP4K4 expression was also associated with higher frequency of recurrence/metastasis, larger tumor size, and increased number of positive lymph nodes (P < 0.05). Conclusion: MAP4K4 was overexpressed in about half of PDAs. Overexpression of MAP4K4 was associated with worse prognosis and is a prognostic marker for stage IIPDAs. Pancreatic ductal adenocarcinoma (PDA) is the fourth leading oncogene by point mutation and inactivation of p16, SMAD4/ cause of cancer death in the United States, preceded only by DPC4, and p53 tumor suppressor genes (3–7). Although the lung, colon, and breast cancers (1). Pancreatic cancer has one of genetic profile for pancreatic cancer is emerging, the role of the highest mortality rates, and virtually all patients with PDA specific genetic alterations involved in the aggressive growth die from it (2). Even for early stages of resectable PDAs, and metastasis of PDA is unclear. the tumor commonly recurs, with the most common sites of Mitogen-activated protein 4 kinase 4 (MAP4K4; also called recurrence being the liver, lung, peritoneal cavity, and pan- hepatocyte progenitor kinase-like/germinal center kinase-like creatic surgery bed. Studies have shown that multiple genes are kinase) is a serine/threonine kinase and belongs to the mam- frequently altered in PDAs, including activation of the K-ras malian STE20/MAP4K family. Recent studies have shown that MAP4K4 is overexpressed in many types of human cancer cell lines and tumors compared with normal tissue (8). Dominant- Authors’ Affiliations: Departments of 1Pathology, 2Gastrointestinal Medical negative mutant of MAP4K4 suppresses the Ras-induced trans- Oncology, and 3Surgical Oncology, The University of Texas M. D. Anderson formation in NIH3T3 cells and rat intestinal epithelial cells Cancer; 4Department of Immunology, Baylor College of Medicine, Houston,Texas (8). In addition, expression of inactive mutant MAP4K4 5 and Division of Anatomic Pathology, Penn State Milton S. Hershey Medical inhibits the anchorage-independent cell growth and hepatocyte Center, Hershey, Pennsylvania Received 2/14/08; revised 4/25/08; accepted 4/29/08. growth factor-stimulated epithelial cell invasion (8). Further- Grant support: AACR-PanCAN Career Development Award in Pancreatic Cancer more, knockdown of MAP4K4 mRNA by small interfering RNA Research and Institutional Research Grant atThe University ofTexas M. D. Anderson inhibits the tumor cell migration and invasion of ovarian Cancer Center. cancer, breast cancer, prostate cancer, and malignant melano- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance ma (9). These studies show that MAP4K4 plays an important with 18 U.S.C. Section 1734 solely to indicate this fact. role in transformation, cell migration, and invasiveness of Requests for reprints:HuaminWang, Department of Pathology,The University of cancer cells (9, 10). The prognostic value of MAP4K4 Texas M. D. Anderson Cancer Center, Unit 085, 1515 Holcombe Boulevard, expression in PDA has, however, not been studied, because Houston, TX 77030. Phone: 713-563-1846; Fax: 713-563-1848; E-mail: majority of the patients with surgically resectable PDAs have [email protected]. F 2008 American Association for Cancer Research. stage II disease and little is known about the prognosis in this doi:10.1158/1078-0432.CCR-08-0381 group of patients. In this study, we examined the expression www.aacrjournals.org 7043 Clin Cancer Res 2008;14(21)November 1, 2008 Downloaded from clincancerres.aacrjournals.org on October 5, 2021. © 2008 American Association for Cancer Research. Imaging, Diagnosis, Prognosis hydrogen peroxidase for 20 min to block the endogenous peroxidase Translational Relevance activity and were incubated in 2.5% blocking serum to reduce nonspecific binding. Sections were incubated for 90 min at 37jC with MAP4K4 is a serine/threonine kinase and belongs to the primary anti-MAP4K4 at a 1:250 dilution. Standard avidin-biotin mammalian STE20/MAP4K family. Recent studies have immunohistochemical analysis of the sections was done according to shown that MAP4K4 is overexpressed in many types of the manufacturer’s recommendations (Vector Laboratories). Diamino- human cancer and cancer cell lines. MAP4K4 plays an benzidine was used as a chromogen, and hematoxylin was used for counterstaining. The staining results were evaluated independently by important role in transformation, invasiveness, adhesion, two gastrointestinal pathologists (J.J.L. and H.W.) to determine the and cell migration. However, the expression of MAP4K4 average percentage of positive cells in two cores. Using the median and its significance in PDA has not been studied. In this value of 10% as a cutoff, high expression of MAP4K4 (MAP4K4-H) was study, we examined the expression of MAP4K4 in 66 stage defined as cytoplasmic staining of z10% of the tumor cells and low II PDAs and found that MAP4K4 was overexpressed in expression of MAP4K4 (MAP4K4-L) was defined as cytoplasmic 46% (30 of 66) of stage IIPDAs. In addition, we showed staining of <10% of the tumor cells or no cytoplasmic staining as that MAP4K4 overexpression was associated with higher reported previously (13). frequency of distant metastasis/recurrence after pancreati- Statistical analysis. The Fisher’s exact tests were used to compare coduodenectomy, larger tumor size, and increased number categorical data. Overall survival and recurrence-free probability curves of positive lymph nodes in patients with stage II PDAs. were constructed using the Kaplan-Meier method, and the log-rank test was used to evaluate the statistical significance of differences. The Furthermore, MAP4K4 expression was associated with patients’ follow-up information through September 2006 was extracted poor overall and recurrence-free survival and was a prog- from the prospectively maintained institutional pancreatic cancer nostic factor for patients with stage II PDA in multivariate database managed in the Department of Surgical Oncology and, if analysis. These findings support the role of MAP4K4 in necessary, updated by review of the U.S. Social Security Index. The the invasion/metastasis of pancreatic cancer. The ability recurrence information was updated every time a patient came to the to inhibit MAP4K4 and its downstreamsignaling pathways clinic/institution for a follow-up visit. Recurrence-free survival was may offer new potential avenues for the development of calculated as the time from the date of surgery to the date of first novel therapeutic strategies. recurrence after surgery (in patients with recurrence) or to the date of last follow-up (in patients without recurrence). The overall survival was calculated as the time from the date of diagnosis to the date of death or the date of last follow-up (if death did not occur). of MAP4K4 in 66 pancreaticoduodenectomy specimens of stage The prognostic significance of clinical and pathologic characteristics II PDA and their paired nonneoplastic pancreatic ductal was determined using univariate Cox regression analysis. Cox propor- epithelial cells. Using univariate and multivariate analysis, we tional hazards models were fitted for multivariate analysis. After correlated the expression level of MAP4K4 with survival and interactions between variables were examined, a backward stepwise procedure was used to derive the best-fitting model. Statistical analysis other clinicopathologic features in patients with stage II PDA. was done using Statistical Package for Social Sciences software (for Windows 12.0; SPSS). We used a two-sided significance level of 0.05 Materials and Methods for all statistical analyses. Case selection. We constructed tissue microarrays using
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