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 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/, 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 tumor suppressor genes (3–7). Although the lung, colon, and breast (1). 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

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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 formalin- Results fixed, paraffin-embedded archival tissue samples of 66 specimens from patients with stage II PDAs who had undergone pancreaticoduo- Thirty of 66 (46%) of stage II PDAs were MAP4K4-H. Among denectomy at The University of Texas M. D. Anderson Cancer Center these 66 cases, the paired benign pancreatic tissue samples from between 1990 and 2005 and had not received any preoperative 62cases were available. Twelve (19%) of them (11 of 48 chemotherapy and/or radiation therapy. Patients who received preop- chronic pancreatitis samples and 1 of 14 normal pancreatic erative chemotherapy and/or radiation and who died from postoper- tissue sample) were MAP4K4-H, which was significantly lower ative complications were excluded. Our study group consisted of than that in PDAs (P = 0.002; Table 1). PDAs and the 44 male patients and 22 female patients with a median age at time of surgery of 63.7 years (range, 39.8-79.9 years). This study was approved proliferating pancreatic ductules in chronic pancreatitis showed by the Institutional Review Board of The University of Texas M. D. cytoplasmic staining for MAP4K4 (Fig. 1). The acinar cells or Anderson Cancer Center. islet cells were negative for MAP4K4. Tissue microarrays. To construct the tissue microarrays used in this Clinicopathologic characteristics of the study population are study, formalin-fixed, paraffin-embedded archival tissue blocks and summarized in Table 2. According to the WHO classification their matching H&E-stained slides were retrieved, reviewed, and standards, 10 (15%) cases were well-differentiated, 41 (62%) screened for representative tumor regions and nonneoplastic pancreatic parenchyma by a gastrointestinal pathologist (H.W.). For each patient, two cores of tumor and two cores of paired benign pancreatic tissue Table 1. Expression of MAP4K4 in stage II PDAs were sampled from representative areas using a 1.0 mm punch. The and paired benign pancreatic tissue samples tissue microarrays were constructed with a tissue microarrayer (Beecher Instruments) as described previously (11). n MAP4K4-L MAP4K4-H Immunohistochemical analysis for MAP4K4. Immunohistochemical staining for MAP4K4 was done on 4 Am unstained sections from the Normal pancreas 14 13 1 tissue microarray blocks using a rabbit polyclonal antibody against Chronic pancreatitis 4837 11 MAP4K4 (1:250 dilution), which has been characterized previously PDA 66 36 30 (12). To retrieve the antigenicity, the tissue sections were treated at 100jC in a steamer containing 10 mmol citrate buffer (pH 6.0) for NOTE: P = 0.002. 60 min. The sections were then immersed in methanol containing 0.3%

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Fig. 1. Representative micrographs showing no staining for MAP4K4 in normal pancreas (A), weak cytoplasmic staining for MAP4K4 in pancreatic ductules in chronic pancreatitis (B), strong cytoplasmic staining for MAP4K4 in PDA (C), and a PDA that was negative for MAP4K4 (D). Original magnification, Â100. moderately differentiated, and 15 (23%) poorly differentiated lymph nodes examined between these two groups (20.0 F 9.2 adenocarcinoma. Of the 66 cases, 52(79%) had R0 resection in MAP4K4-H group versus 19.4 F 7.3 for MAP4K4-L group; (defined as all surgical margins negative microscopically), 14 P = 0.80). No association was found between MAP4K4 patients had R1 resection, and no patients had a grossly positive expression and other clinicopathologic features (Table 3). margin of resection. Fifty-two (79%) patients underwent After pancreaticoduodenectomy, the median and mean fol- postoperative therapy due to recurrence or metastasis. At the low-up of the patients in this study were 22.9 and 38.0 months, time of last follow-up, 48 patients had died of disease and 18 respectively. No patients were lost to follow-up. The patients patients were alive. whose tumors were MAP4K4-H had a worse prognosis. The There were significant differences in tumor size and the median overall survival was 19.5 F 2.9 months for patients frequencies of recurrence/distant metastasis after resection with MAP4K4-H PDAs compared with 65.2 F 19.4 months for between MAP4K4-H and MAP4K4-L groups (Table 2). Recur- patients whose tumors were MAP4K4-L (P = 0.02, log-rank test; rence/distant metastasis was present in 83% (25 of 30) of the Fig. 2). The median recurrence-free survival were 9.3 F 1.8 patients with MAP4K4-H tumors compared with 58% (21 of and 28.8 F 18.0 months for patients with MAP4K4-H and 36) of patients with MAP4K4-L PDAs (P = 0.03). High levels of MAP4K4-L PDAs, respectively (P = 0.0005, log-rank test; Fig. 2). MAP4K4 expression were also associated with larger tumor size High levels of MAP4K4 expression were associated with (P = 0.01). Although there was no significant difference in the poor overall and recurrence-free survival in univariate analysis frequencies of lymph node metastasis between MAP4K4-H and (P = 0.025 and 0.001, respectively; Table 2). In multivariate MAP4K4-L tumors, the average number of positive lymph analysis, MAP4K4 expression was a predictor of overall and nodes in pancreaticoduodenectomy specimens was 3.9 F 4.3 recurrence-free survival (P = 0.025 and 0.004) independent of in MAP4K4-H group compared with 1.5 F 1.8 in MAP4K4-L age, tumor size, differentiation, and lymph node status/stage (P = 0.003). There was no difference in the total number of (Table 4).

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Table 2. Clinicopathologic correlation of MAP4K4 expression in stage II PDAs

Characteristics n MAP4K4-L (n = 36) MAP4K4-H (n = 30) P

Age (y) <60 23 13 10 0.97 60-70 26 14 12 >70 17 9 8 Gender Female 22 10 12 0.31 Male 44 26 18 Tumor size (cm) V2.0 1816 2 0.01 >2.0 4820 28 Tumor differentiation Well 10 7 3 0.32 Moderate 41 23 18 Poor 15 6 9 Margin status Negative 52 29 23 0.77 Positive 14 7 7 Lymph node status (stage) Negative (IIA) 23 15 80.30 Positive (IIB) 43 21 22 Postoperative chemotherapy No 14 7 7 0.77 Yes 52 29 23 Postoperative radiotherapy No 188 10 0.41 Yes 4828 20 Recurrence/distant metastasis No 20 15 5 0.03 Yes 46 21 25 Vital status Alive 1810 8 1.00 Dead 4826 22

Table 3. Univariate analysis of overall and recurrence-free survival in patients with stage II PDAs

Overall survival Recurrence-free survival n Hazard ratio (95% confidence interval) P Hazard ratio (95% confidence interval) P

Age (y) <60 (reference) 23 1.00 1.00 60-70 26 1.74 (0.77-3.90) 0.181.41 (0.71-2.79) 0.32 >70 17 2.16 (0.89-5.23) 0.09 1.79 (0.82-3.88) 0.14 Gender Female (reference) 22 1.00 1.00 Male 44 0.65 (0.34-1.27) 0.21 0.69 (0.38-1.26) 0.22 Tumor size (cm) V2.0 (reference) 181.00 1.00 >2.0 481.62 (0.74-3.55) 0.23 2.02 (0.97-4.19) 0.06 Tumor differentiation Well (reference) 10 1.00 1.00 Moderate 41 2.56 (0.88-7.4) 0.08 2.71 (1.05-7.01) 0.04 Poor 15 2.72 (0.83-8.90) 0.10 2.22 (0.75-6.51) 0.15 Margin status Negative (reference) 52 1.00 1.00 Positive 14 1.33 (0.60-2.96) 0.49 1.12 (0.55-2.27) 0.76 Lymph node status (stage) Negative (IIA) 23 1.00 1.00 Positive (IIB) 43 1.59 (0.78-3.23) 0.20 2.22 (1.14-4.31) 0.02 Postoperative chemotherapy No (reference) 14 1.00 1.00 Yes 52 1.3 (0.50-3.36) 0.59 0.97 (0.45-2.10) 0.95 Postoperative radiotherapy No (reference) 181.00 1.00 Yes 481.3 (0.50-3.36) 0.59 0.68 (0.34-1.28) 0.24 MAP4K4 expression Low (reference) 36 1.00 1.00 High 30 2.16 (1.10-4.23) 0.025 2.83 (1.55-5.19) 0.01

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Fig. 2. Kaplan-Meier curves for overall survival by stage (A) and MAP4K4 expression (C) and recurrence-free survival by stage (B) and MAP4K4 expression (D)inpatients with stage IIPDAs.

Discussion stage II PDAs. Furthermore, MAP4K4 expression was associated with poor overall and recurrence-free survival and was a In this study, we examined the MAP4K4 expression in 66 prognostic factor for patients with stage II PDA. stage II PDAs and their matched benign pancreatic tissue MAP4K4 belongs to the mammalian STE20/MAP4K family. samples. We found that MAP4K4 was overexpressed in 46% MAP4K4 mRNA has been shown to be overexpressed in several of the stage II PDAs compared with 19% of the matched benign human malignancies compared with normal control tissue pancreatic tissue samples. MAP4K4 overexpression was asso- (8). In addition, MAP4K4 has been shown to be involved in ciated with higher frequency of distant metastasis/recur- cell migration and invasion in a variety of tumor cell lines, rence after pancreaticoduodenectomy, larger tumor size, and including malignant melanoma, ovarian, breast, and prostate increased number of positive lymph nodes in patients with cancers (9). Knockdown of MAP4K4 mRNA using small

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Table 4. Multivariate analysis of overall and recurrence-free survival in patients with stage II PDAs

Overall survival Recurrence-free survival n Hazard ratio (95% confidence interval) P Hazard ratio (95% confidence interval) P

Age (y) <60 (reference) 23 1.00 1.00 60-70 26 1.96 (0.80-4.78) 0.14 1.44 (0.68-3.04) 0.33 >70 17 2.77 (1.02-7.47) 0.05 1.65 (0.72-3.79) 0.24 Tumor size (cm) V2.0 (reference) 18 1.00 >2.0 48NA 1.22 (0.53-2.81) 0.97 Tumor differentiation Well (reference) 10 1.00 1.00 Moderate 41 2.39 (0.82-6.94) 0.11 2.54 (0.98-6.60) 0.06 Poor 15 2.48(0.76-8.15) 0.14 1.67 (0.56-4.96) 0.35 Lymph node status (stage) Negative (IIA) 23 1.00 1.00 Positive (IIB) 43 1.33 (0.60-2.95) 0.48 1.93 (0.98-3.82) 0.06 MAP4K4 expression Negative (reference) 36 1.00 1.00 Positive 30 2.16 (1.10-4.23) 0.025 2.44 (1.33-4.50) 0.004

interfering RNA in these tumor cell lines inhibits the cancer cell related strongly with poor overall and recurrence-free sur- migration and invasion (9). In the presence of hepatocyte vival in univariate analysis and was a prognostic factor asso- growth factor, overexpression of MAP4K4 increases the invasive ciated with worse prognosis in multivariate analysis. Because ability of rat intestinal epithelial cells (8). The promigratory most patients with PDA present with locally advanced dis- effect of MAP4K4 is mediated through the c-Jun NH2-terminal ease, other secondary prognostic factors, particularly MAP4K4, kinase, which plays critical role in stress responses, cell may be of use. Previous studies have shown potential prog- proliferation, , and tumorigenesis (9, 14–18). nostic markers for PDAs, including loss of SMAD4 expres- Although these studies clearly show the role of MAP4K4 in sion (19, 20), copy number of 17 (21), activated cancer cell migration and invasion, the role of MAP4K4 in Akt and Erk levels (22). Tascilar et al. examined SMAD4 pro- pancreatic cancer has not been examined. Our study showed tein expression in 249 patients who underwent pancrea- that 46% of stage II PDAs overexpressed MAP4K4 protein. It ticoduodenectomy for PDA and found that SMAD4 protein was interesting that MAP4K4 was also overexpressed in 23% of expression is a prognostic factor independent of other pancreatic ductules in chronic pancreatitis but only in 7% of clinicopathologic parameters (20). It would be interesting to pancreatic ducts from normal pancreas, suggesting that compare the prognostic value of MAP4K4 with these reported MAP4K4 may play a role in chronic pancreatitis and benign markers in future studies for patient stratification in clin- pancreatic ductular proliferation. In our study, MAP4K4 over- ical trials. expression correlated with larger tumor size, increased number In conclusion, our results showed that MAP4K4 was overex- of positive lymph nodes, and recurrence/metastasis of PDAs. In pressed in stage II PDAs. High levels of MAP4K4 expression in addition, we found that high levels of MAP4K4 expression were stage II PDAs were associated with poor prognosis and were a present in 3 of 3 patients with stage IV PDAs but not in the two prognostic marker for stage II PDAs. Further studies are needed patients with stage I PDAs (data not shown). These findings to determine whether MAP4K4 expression may be served as a support the role of MAP4K4 in the invasion/metastasis of potential target for directed therapy in PDA. pancreatic cancer. The ability to inhibit MAP4K4 and its down- stream signaling pathways may offer new potential avenues for the development of novel therapeutic strategies. Disclosure of Potential Conflicts of Interest In this study, we showed that MAP4K4 expression in stage II PDAs correlates with poor outcome. MAP4K4 expression cor- No potential conflicts of interest were disclosed.

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John J. Liang, Hua Wang, Asif Rashid, et al.

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