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

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.

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 adenocarcinomas 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). total of 99 cores each were included on each of the 14 recipient Relationship of Mkk4 Protein Expression to MKK4 Ge- blocks, representing the primary infiltrating pancreatic cancers, netic Status. The status of the MKK4 gene was previously the matched metastases, and a variety of normal control tissue determined for 24 of the 60 pancreatic cancers that were also cores from each patient. immunolabeled (7), providing the opportunity to correlate Mkk4 Immunohistochemical Labeling for Mkk4 Protein. immunolabeling patterns to the genetic status for each case Unstained 5-micron sections were cut from the paraffin blocks (Table 1). Among these 24 pancreatic adenocarcinomas, 23 selected for each case or the tissue microarrays and deparaf- showed loss of heterozygosity at 17p. MKK4 was genetically finized by routine techniques. Slides were treated with 1ϫ sodium citrate buffer (diluted from 10ϫ heat-induced epitope retrieval buffer; Ventana-Bio Tek Solutions, Tucson, AZ) before steaming for 20 minutes at 80°C. Slides were then cooled Table 1 Immunohistolabeling of MKK4 in pancreatic cancers with 5 minutes before incubating with antihuman Mkk4 monoclonal known genetic status antibody [NCL-MKK4 (1:80 dilution), Novacastra, Newcastle, Mkk4 IHC- Mkk4 IHC- United Kingdom] with a Dako automated stainer. Finally, Mkk4 MKK4 genetic status * positive negative primary antibody was detected by adding secondary antibody Ј Mutant † 0 3 ‡ followed by avidin-biotin complex and 3,3 -diaminobenzidine LOH with wild-type allele 18 2 chromagens. Sections were counterstained with hematoxylin. No LOH 0 1 Immunohistochemical labeling of Mkk4 was evaluated by two Total 18 6 of the authors (Wei Xin and Christine A. Iacobuzio-Donahue) Abbreviations: IHC, immunohistochemistry; LOH, loss of het- with agreement in all of the cases examined. The labeling of erozygosity. each carcinoma was recorded as diffusely positive if Ͼ50% of * Reported by Su et al. (7). † Codon exons were sequenced in all of the cases with LOH, and the neoplastic cells labeled for Mkk4, focally positive if 1 to mutations confirmed by repeat sequencing of an independently derived 50% of the neoplastic cells labeled, and negative (loss of ex- PCR product. pression) if no immunolabeling of Mkk4 protein was observed. ‡ P Ͻ 0.01.

Relationship of Mkk4 Expression to Patient Survival. Long-term, follow-up information was available for 45 of the 60 patients for which Mkk4 immunolabeling was done on the resected primary carcinoma. Thirty-seven of these 45 patients (82%) showed positive Mkk4 immunolabeling, and eight patients (18%) were negative for Mkk4. No significant difference in age, gender distribution, tumor size, tumor location, differentiation, or lymph node status among the Mkk4 immunonega- tive and immunopositive groups were found (Table 2). The only factor marginally associated with survival was Mkk4 immunolabeling status (Table 2; Fig. 2). Patients with positive MKK4 had half the risk of death compared with MKK4 negative patients, Hazard Ratio ϭ 0.5 (95% confidence interval: 0.22, 1.12), P ϭ 0.09. Mkk4 Protein Expression in Metastatic Pancreatic Cancer. Loss of Mkk4 expression has been reported in association with metastatic ability (9, 10). Therefore, we also determined the Mkk4 immunohistochemical labeling patterns among a variety of metastatic pancreatic adenocarcinoma tissues from archival autopsy material of 26 different patients with wide- spread disease, 18 for which the primary pancreatic carcinoma was also available for study. Intact Mkk4 immunolabeling was noted within the neoplastic epithelium of 16 of 18 (88%) primary pancreatic carcinomas analyzed, similar to the rate of Mkk4 immunolabeling found for surgically resected pancreatic cancers. In contrast, among 460 different metastases from these 26 patients, only 291 (63.3%) showed intact Mkk4 immunolabeling (P Ͻ 0.001). Mkk4 immunolabeling patterns of the metastatic lesions were also evaluated with respect to the matched primary carcinomas also available for study. For the 16 patients whose primary carcinoma showed intact Mkk4 immunolabeling, the matched metastatic disease for 15 of these patients showed heterogeneity of Mkk4 expression in that metastases with both positive and

Fig. 1 Mkk4 immunolabeling patterns in normal and neoplastic pancreatic tissues (A). Diffuse-positive Mkk4 immunolabeling in an infil- Table 2 Univariate Cox regression hazard ratios for survival trating pancreatic adenocarcinoma. Mkk4 immunolabeling is noted in a cytoplasmic distribution, with nuclear labeling also present. Scattered Hazard Confidence positive labeling of surrounding stromal cells can also be seen (B). Clinicopathologic variable * ratio interval P Mkk4 immunolabeling in an infiltrating pancreatic adenocarcinoma MKK4 immunolabeling pattern with a homozygous deletion of the MKK4 gene. Loss of Mkk4 protein Negative (n ϭ 8) 1.00 (0.22, 1.12) 0.09 expression is seen, in contrast to the surrounding stromal tissue that Positive (n ϭ 37) 0.50 retains Mkk4 immunopositivity (C). Normal pancreas. Positive Mkk4 Age (y) immunolabeling is present within normal ductal epithelium and sur- 66.6 Ϯ 3.5 1.00 (0.97, 1.02) 0.77 rounding acinar cells (ϫ200). Race Other (n ϭ 6) 1.00 (0.22, 1.29) 0.16 White (n ϭ 39) 0.53 Gender inactivated in three of these 23 cancers with 17p loss of het- Female (n ϭ 17) 1.00 (0.32, 1.20) 0.16 ϭ erozygosity. Two of the 23 carcinomas contained a homozygous Male (n 28) 0.62 Nodal status deletion of the MKK4 gene, and one carcinoma with allelic loss Negative (n ϭ 9) 1.00 (0.49, 2.57) 0.79 contained a missense mutation (G12S) of the remaining allele. Positive (n ϭ 36) 1.12 Immunohistochemical labeling of these 24 cancers indicated Tumor size Ͻ ϭ that six were negative and 18 were positive for Mkk4 protein. Of 3cm(n 15) 1.00 (0.35, 1.34) 0.28 Ն3cm(n ϭ 30) 0.69 the six negative cancers, two contained a homozygous deletion, Tumor differentiation one contained a missense mutation, and three were wild-type for Well/Moderately (n ϭ 32) 1.00 (0.57, 2.35) 0.69 MKK4. In contrast, all 18 of 18 carcinomas with positive Mkk4 Poor (n ϭ 13) 1.16 immunolabeling had wild-type MKK4 genetic status (P Ͻ 0.01, * Values shown are based on 45 patients for which complete Fisher’s exact test). clinicopathologic information was available.

rate of Mkk4 loss in pancreatic cancers to be higher than reflected by genetic alterations alone [13% in the current study versus 5% reported by Su et al. (7)]. Epigenetic inactivation of tumor suppressor genes has been reported for other genes known to play a role in pancreatic carcinogenesis, such as CDKN2A/p16 (15). CDKN2A/p16 has been shown to be inactivated by a variety of mechanisms in pancreatic cancer, including homozygous deletion, allelic loss coupled with genetic alterations of the remaining allele, and methylation of the gene promoter (15, 16). Thus, we cannot rule out that mechanisms such as hypermethylation of the MKK4 gene may also occur in pancreatic cancers. Our data also indicate a possible relationship among loss of Mkk4 expression and the poor prognosis often associated with pancreatic cancers. We found a trend toward worse survival for those patients with carcinomas that showed a loss of Mkk4 expression as compared with those with carcinomas with intact Mkk4 expression (P ϭ 0.09). Thus, the evaluation of Mkk4 Fig. 2 Kaplan-Meier survival curves in relation to Mkk4 immunola- immunolabeling status may have prognostic value for patients beling status. Negative immunolabeling shows a trend toward shorter with pancreatic cancer as has been shown for MADH4 (DPC4), survival among patients with surgically resectable pancreatic cancer. another marker of genetic status in patients with pancreatic cancer (17). Immunohistochemical labeling for Mkk4 also indicate that negative immunolabeling patterns noted. In one of the 16 pa- Mkk4 loss may correlate with metastasis formation in target tients with positive Mkk4 labeling of the primary tumor, all of organs. Loss of Mkk4 immunolabeling was significantly more the matched metastases showed loss of Mkk4 expression (Fig. common in distant metastases (36.7% of cases negative) than in 3). In the remaining two patients with negative labeling of the the primary carcinomas in these same patients (P Ͻ 0.001). primary tumor, all of the matched metastases were also Mkk4 These findings were not related to differences in immunogenic- negative. ity among surgical and autopsy materials or the number of metastases per patient, as identical patterns and rates of immu- DISCUSSION nolabeling of primary pancreatic cancers were found among The ability to correlate Mkk4 immunohistochemical label- surgically resected or autopsy material. The MKK4 gene has ing patterns to MKK4 genetic status previously determined for been reported to function as a metastasis-suppressor gene in these same pancreatic cancers provides a unique opportunity to breast and prostatic carcinomas, in that the loss of MKK gene directly correlate gene status, gene expression, and morphology. expression facilitates metastasis formation without affecting pri- This is particularly advantageous in studies of pancreatic cancer, mary tumor growth (9, 10). Our findings support the concept of a tumor type well known for its low neoplastic cellularity that Mkk4 as playing a role in metastatic behavior and suggest that often hinders genetic analyses of this tumor type. We now MKK4 may have metastasis-suppressor gene properties in pan- present data that relate Mkk4 immunolabeling to the genetic creatic cancer as well. status previously determined for those same cases, indicating the In summary, we propose that immunolabeling for the MKK4

Fig. 3 Mkk4 immunolabeling patterns in a primary pancreatic cancer (A) and a matched liver metastasis (B). Diffuse-positive immunolabeling of the cyto- plasm and nucleus is seen within the neoplastic cells of the primary tumor. In contrast, loss of Mkk4 immunolabeling was found in all of the matched metastases from this patient (ϫ200).

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Wei Xin, Ki J. Yun, Francesca Ricci, et al.

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