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MAP2K4/MKK4 Expression in Pancreatic Cancer: Genetic Validation of Immunohistochemistry and Relationship to Disease Course

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MAP2K4/MKK4 Expression in Pancreatic Cancer: Genetic Validation of Immunohistochemistry and Relationship to Disease Course 8516 Vol. 10, 8516–8520, December 15, 2004 Clinical Cancer Research MAP2K4/MKK4 Expression in Pancreatic Cancer: Genetic Validation of Immunohistochemistry and Relationship to Disease Course Wei Xin,1 Ki J. Yun,4 Francesca Ricci,2 ing patterns were also evaluated among unresectable pri- Marianna Zahurak,3 Wanglong Qiu,4 mary and metastatic cancer tissues from autopsy specimens, Gloria H. Su,4 Charles J. Yeo,5,6 indicating intact Mkk4 immunolabeling in 88.8% of the unresectable primary carcinomas as compared with 63.3% Ralph H. Hruban,4,5 Scott E. Kern,4,5 and 4,5 of distant metastases (P < 0.001). Our data indicate that the Christine A. Iacobuzio-Donahue loss of Mkk4 protein expression in pancreatic carcinomas 1 Department of Pathology, The University of Michigan Medical may be more frequent than suggested by the rates of genetic Center, Ann Arbor, Michigan; 2Department of Pathology, University La Sapienza, Rome, Italy; and the Departments of 3Biostatistics, inactivation alone and that MKK4 loss may contribute to 4Pathology, 5Oncology, and 6Surgery, The Johns Hopkins University disease progression. The correlation of MKK4 genetic status Hospital, Baltimore, Maryland with immunolabeling patterns validate this approach for the evaluation of MKK4 status in routine histologic sections and ABSTRACT may provide useful information regarding patient prognosis. MKK4 (MAP2K4/SEK1) is a member of the mitogen- activated protein kinase family, originally identified as a INTRODUCTION kinase involved in the stress-activated protein kinase path- The mitogen-activated protein (MAP) kinase cascades are way by directly phosphorylating c-Jun NH -terminal kinase. 2 multifunctional signaling pathways that are evolutionally well MKK4 genetic inactivation has been observed in a subset of conserved in all of the eukaryotic cells. One of the biological pancreatic carcinomas, implicating deregulation of the responses mediated through the MAP kinase pathways seems to stress-activated protein kinase pathway in pancreatic carci- be the decision of cell fate in response to stress-activation nogenesis. We evaluated Mkk4 protein expression patterns resulting in apoptosis. Specifically, three parallel MAP kinase by immunohistochemical labeling in a series of 60 resected cascades have been described that converge on extracellular primary infiltrating pancreatic adenocarcinomas (24 cases signal-regulated kinases, c-Jun NH -terminal kinases, or p38 with known MKK4 genetic status), and 14 different tissue 2 MAP kinases, and each consists of three classes of serine/ arrays representing the primary carcinoma and all of the threonine kinases, the MAP kinase, the MAP kinase kinase gross metastases from 26 patients that died of metastatic pancreatic cancer. Among the surgically resected carcino- (MAPKK, also known as MEK), and the MAPKK kinase mas, focal or diffuse-positive immunolabeling for Mkk4 pro- (MAPKKK). MAPKKK phosphorylates and thereby activates tein was found in 52 of 60 cases (86.7%). Among the eight MAPKK, and activated MAPKK in turn phosphorylates and carcinomas with negative Mkk4 immunolabeling, three har- activates MAP kinase (1–3). bored a homozygous deletion or intragenic mutation of the MKK4 (MAP2K4/SEK1) is a member of the MAP kinase MKK4 gene, in contrast to none of the 52 cases with positive family specifically involved in the stress-activated protein ki- Mkk4 immunolabeling (P < 0.01). Loss of Mkk4 immuno- nase (SAPK) pathway by directly phosphorylating the c-Jun labeling showed a trend toward shorter survival, with NH2-terminal kinase in response to Ask1 activation (4, 5). Mkk4-positive carcinomas having half the risk of death than Genetic inactivation of the MKK4 gene on chromosome 17p has Mkk4 immunolabel- been reported in a subset of pancreatic, biliary, and breast .(0.09 ؍ Mkk4-negative carcinomas (P carcinomas, suggesting dysregulation of the SAPK pathway may be selected for in carcinogenesis of these organs (6–8). In prostatic and ovarian carcinomas, Mkk4 is thought to function as a metastasis-suppressor gene in that loss of Mkk4 expression, Received 5/5/04; revised 6/29/04; accepted 8/2/04. but not genetic inactivation, is associated with metastasis for- Grant support: The Joseph C. Monastra Fund for Pancreas Cancer Research, the Jeff Zgonina Fund for Pancreas Cancer Research, and a mation in these organs (9, 10). Soongsan Fellowship from Wonkwang University, Wonkwang, Korea The purpose of this study was to perform a survey of Mkk4 (K. Yun). expression among a set of genetically well-characterized surgi- The costs of publication of this article were defrayed in part by the cally resected primary infiltrating pancreatic cancers as well as payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to among unresectable metastatic pancreatic cancers with an im- indicate this fact. munohistochemical approach. In doing so, we hoped to deter- Requests for reprints: Christine A. Iacobuzio-Donahue, The Johns mine the specificity and sensitivity of this assay for the MKK4 Hopkins Hospital, Division of Gastrointestinal/Liver Pathology, 720 gene status, the relative rates of MKK4 loss among primary Rutland Street, Ross Building, Room 632, Baltimore, MD 21205. Phone: (410) 955-3511; Fax: (410) 614-0671; E-mail: [email protected]. infiltrating and metastatic pancreatic cancers, and the relation- ©2004 American Association for Cancer Research. ship of MKK4 to long-term prognosis. Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 8517 MATERIALS AND METHODS Determinations of Loss of Heterozygosity and Sequenc- Tissues and Cell Lines. Paraffin-embedded blocks of 60 ing of MKK4. The genetic status of MKK4 was available for surgically resected primary infiltrating pancreatic adenocarcino- 24 of the resected primary infiltrating pancreatic adenocarcino- mas resected between 1992 and 1996 were collected from the mas analyzed in the current study. These genetic analyses have Surgical Pathology Files of The Johns Hopkins Hospital. Clin- been reported previously (7). ical and pathological data were also obtained from the Surgical Statistical Analysis. The frequencies of Mkk4 immuno- Pathology Files, including age, gender, race, tumor size, tumor labeling among cancer samples with known genetic status were ␹2 location, lymph node status, histologic subtype of invasive analyzed by the test with modification by the Fisher’s exact Ͻ carcinoma, and patient survival. The mean patient age was 63.3 test to account for frequency values 5. For purposes of statis- years old and included 34 males and 26 females. The H&E- tical analysis, focal and diffuse-positive labeling carcinomas were combined for comparison to negative labeling cancers. For stained slides from each case of primary infiltrating carcinoma determinations of overall survival in relation to immunohisto- were screened by light microscopy, and representative sections chemical labeling of Mkk4 protein, event time distributions containing infiltrating adenocarcinoma were selected for immu- were estimated with the Kaplan-Meier method (11) and com- nolabeling. pared with the log-rank statistic (12) or the proportional hazards Construction of Metastatic Pancreatic Cancer Tissue regression model (13). Other factors tested for prognostic value Microarrays. The paraffin-embedded tissues from 26 patients included age, race, gender, size of tumor, nodal status, and who died of histologically confirmed metastatic pancreatic can- tumor differentiation. Hazard ratios were expressed relative to a cer between 1972 to 1993 were collected from the Autopsy baseline reference category. Computations were done with the Pathology Files of The Johns Hopkins Hospital and used to Statistical Analysis System or EGRET (14). All Ps reported are construct 14 different tissue arrays representing the primary two-sided. For all of the statistical analyses, PsofՅ0.05 were carcinoma and all of the gross metastases from these patients. considered significant. Patient ages ranged from 35 to 89 years with a mean of 62.8. Fifteen patients were male and 11 were female. A total of 20 RESULTS different target organs were represented with a mean number of Mkk4 Protein Expression in Normal Pancreas and Pan- metastatic sites per patient of 3.4. The three major metastatic creatic Cancers. Mkk4 protein expression was noted in 52 of sites were liver, lung, and lymph node. For tissue microarray 60 (86.7%) infiltrating pancreatic ductal adenocarcinomas (Fig. construction, representative paraffin-embedded sections con- 1, A and B). In all of the cases, Mkk4 labeling had a cytoplasmic taining primary infiltrating or metastatic pancreatic ductal ade- distribution with scattered nuclear labeling. Of these 52 adeno- nocarcinoma were circled on the glass slides and used as a carcinomas with positive immunolabeling, 45 (87%) showed template. The tissue microarray was constructed with a manual diffuse labeling and 7 (13%) showed focal labeling. Adjacent Tissue Puncher/Arrayer (Beecher Instruments, Silver Spring, normal pancreatic tissue was also available for study within the MD). For each individual primary or metastatic sample, up to 4 same sections of infiltrating carcinoma. Immunolabeling for 1.4-mm cores were punched from the donor block to account for Mkk4 was detected within normal acini, islets, and duct epithe- tissue heterogeneity (depending on the size of the lesion). A lium (Fig. 1C).
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