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Human Cancer Biology

Gene Expression Signatures Differentiate Ovarian/Peritoneal Serous Carcinoma from Diffuse Malignant Peritoneal Mesothelioma

Ben Davidson, Zhen Zhang, Lilach Kleinberg, Mei Li, Vivi Ann Flørenes, Tian-Li Wang and Ie-Ming Shih
DOI: 10.1158/1078-0432.CCR-06-1059 Published October 2006

Abstract

Purpose: Ovarian/primary peritoneal serous carcinoma (OC/PPC) and diffuse peritoneal malignant mesothelioma (DMPM) are highly aggressive tumors that are closely related morphologically and histogenetically. It remains unclear whether both tumors are molecularly distinct neoplasms. The current study compared global gene expression patterns in OC/PPC and DMPM.

Experimental Design: Ten OC/PPC and five DMPM effusions were analyzed for gene expression profiles using the Affymetrix U133 Plus 2 arrays and the dCHIP analysis program. Differentially expressed candidate genes were validated using quantitative real-time PCR and immunohistochemistry.

Results: Unsupervised hierarchical clustering using all 54,675 genes in the array classified the samples into two groups: DMPM specimens versus OC/PPC specimens. A total of 189 genes that were differentially expressed in these two groups were selected based on statistical significance. Genes overexpressed in DMPM (n = 68) included calretinin, vitronectin, claudin 15, α4 laminin, hyaluronan synthase 1, cadherin 11, RAB7, v-maf, and the epidermal growth factor–containing fibulin-like extracellular matrix protein 1. Genes overexpressed in OC/PPC (n = 121) included insulin-like growth factor II (IGF-II); IGF-II binding protein 3; cyclin E1; folate receptors 1 and 3; RAB25; MUC4; endothelin-1; CD24; kallikreins 6, 7, and 8; claudins 3, 4, and 6; Notch3; and MMP-7. Quantitative real-time PCR validated the differential expression of 13 genes, and immunohistochemistry confirmed the differences for four gene products.

Conclusions: Expression profiling separates OC/PPC from DMPM and identifies a number of genes that are differentially expressed in these tumors. The molecular signatures unique to OC/PPC and DMPM should provide a molecular basis to study both tumors and new potential markers for facilitating their differential diagnosis.

  • Ovarian carcinoma
  • Malignant mesothelioma
  • Effusions

Diffuse malignant peritoneal mesothelioma (DMPM) is an aggressive cancer that originates from the native mesothelial cells of the peritoneal cavity. DMPM is less common than its pleural counterpart (9:1 ratio), differs in its gender predilection (roughly similar incidence in women and men), and has a weaker etiologic link to asbestos exposure (1, 2). The prognosis of DMPM patients has been extremely poor in earlier series, with median survival of 10 to 12 months (2). However, recent studies have shown improved survival (26-92 months) when DMPM patients are treated aggressively with combined debulking and preheated i.p. chemotherapy consisting of cisplatin, doxorubicin, and paclitaxel as major agents (2–4). Recently, a response rate of 25% was achieved following pemetrexed treatment (5). The majority of DMPM are of the epithelioid type (6).

Ovarian cancer (OC) is the most lethal gynecologic malignancy in the Western world and the fourth most frequent cause of cancer-related death in women (7). As DMPM, OC and the closely related and morphologically indistinguishable primary peritoneal carcinoma (PPC) are thought to develop from the peritoneal mesothelium or its closely linked variant, the ovarian surface epithelium. Similarly to DMPM, OC/PPC diffusely involves the peritoneal surface in the majority of cases, with cancer cells displaying the ability to form solid nodules on the peritoneal or pelvic wall and survive in the ascites fluid. Platinum compounds and taxanes are the mainstay of chemotherapy for OC. However, despite an initial response rate of 75% to surgery followed by chemotherapy, half of the advanced-stage patients develop recurrence within the first 2 to 3 years, resulting in a dismal 5-year survival rate of 35% (8).

In addition to their similar clinical presentation, OC/PPC of the serous type (the most common one) and DMPM can be morphologically indistinguishable. Furthermore, both tumor types are characterized by closely similar immunostaining profiles in routine pathology practice, including the differentiation markers Wilms' tumor-1 antigen and mesothelin, reflecting their common histogenesis (9–12).

The biology of DMPM is poorly understood, and the molecular differences separating this tumor from OC/PPC have not been studied. Identification of new markers may aid in improved diagnosis of these tumors and may contribute to our knowledge regarding the expression of potential therapeutic targets on both cancers. In an attempt to elucidate molecular differences between DMPM and OC/PPC, we did a mRNA analysis of a small series of specimens, with the aim of comparing ascitic cells from both cancer types. Our findings show that despite their histogenetic and morphologic similarities, DMPM and OC/PPC show extensive differences in gene expression that may aid in understanding the biology of these tumors and provide new diagnostic markers.

Materials and Methods

Patients and material. The clinical material consisted of 15 peritoneal effusions (6 OC, 4 PPC, and 5 DMPM) submitted to the Department of Pathology at the Rikshospitalet-Radiumhospitalet Medical Center, Oslo, Norway, during 1999 to 2004. All OC/PPC were of high-grade serous type, and all DMPM specimens were of the epithelioid type. Effusions were centrifuged within minutes after aspiration from patients, aliquoted, and frozen in RPMI supplemented with 20% FCS and 20% DMSO. The diagnoses of all specimens were validated by pathologists, using both morphologic criteria and immunocytochemistry with a broad antibody panel that recognizes carcinoma or mesothelial cells (9). Based on these analyses, only cases with >50% tumor cells were selected for the study. This study was approved by the Regional Committee for Medical Research Ethics in Norway.

Microarray expression and GeneChip analysis. RNA was prepared from tumor samples using a Qiagen RNAeasy kit. Affymetrix U133 Plus 2 arrays were used to analyze gene expression in both OC/PPC and DMPM. Probe labeling, hybridization, and scanning for the arrays were done using the standard protocols in the JHMI Microarray Core (http://www.microarray.jhmi.edu). The dCHIP program was used to analyze the array data by performing the unsupervised tree-view clustering on 54,675 genes for all 15 array data to produce a dendogram. Genes with the high differential expression pattern between OC/PPC and DMPM were selected based on the following criteria: (a) E/B > 100 or B/E > 100; (b) E/B ≥ 6 or B/E ≥ 6. E and B correspond to the average expression value in the OC/PPC and DMPM groups, respectively (E = experiment, B = baseline).

Quantitative real-time reverse transcription-PCR. Quantitative real-time PCR was done to determine gene expression levels in all OC/PPC and DMPM using the protocol previously described (13). Primers were designed for 13 genes to test the performance in quantitative real-time PCR, and those containing robust and specific PCR products without detectable primer dimers were selected in analysis (Table 1 ). Approximately 16 to 100 ng of cDNA was included in the real-time PCR, which was done using an iCycler, and threshold cycle numbers were obtained using the iCycler Optical system interface software (Bio-Rad Lab, Hercules, CA). Averages in the threshold cycle number (Ct) of duplicate measurements were obtained. The results were expressed as the difference between the Ct of the gene of interest and the Ct of a control gene, beta-amyloid precursor protein (APP) for which expression is relatively constant among previously analyzed SAGE libraries. In cases where no gene expression was observed, a cutoff Ct value of 45 cycles was used.

View this table:
Table 1.

Primers used in quantitative PCR analysis

Immunocytochemistry. Protein expression of cyclin E, claudin-3, MUC4, and calretinin was analyzed in 30 DMPM (9 effusions and 21 solid tumors) and tissue arrays containing one hundred thirty 2-mm cores of OC/PPC specimens (effusions, primary tumors, and solid metastases) from 30 patients. Immunohistochemistry was done using the EnVision+ peroxidase system (DakoCytomation, Glostrup, Denmark). The cyclin E monoclonal antibody was purchased from Novocastra (Newcastle upon Tyne, United Kingdom) and used in 1:100 dilution using antigen retrieval in EDTA buffer. A polyclonal claudin-3 antibody was purchased from Zymed (San Francisco, CA) and used in 1:100 dilution using antigen retrieval in citrate buffer. Staining for MUC4 was done using a monoclonal antibody developed by Moniaux et al. (14) using a 1:1,500 dilution and antigen retrieval in EDTA buffer. Calretinin immunostaining was done using a monoclonal antibody from DakoCytomation at 1:20 dilution using antigen retrieval in EDTA. Appropriate positive and negative controls were used.

Staining extent was scored by an experienced cytopathologist (B.D.) using a scale of 0 to 4, corresponding to percentage of immunoreactive tumor cells of 0%, 1% to 5%, 6% to 25%, 26% to 75%, and 76% to 100%, respectively. No DMPM specimen or OC/PPC core contained <100 tumor cells. Differences in protein expression between DMPM and OC/PPC cases and between solid tumors and effusions were analyzed using the two-sided χ2 test (SPSS version 12.0, Chicago IL).

Results

Unsupervised hierarchical clustering classified the samples into two distinct groups: one consisting of all DMPM specimens and the other consisting of all the OC/PPC specimens (Fig. 1 ). Supervised analysis using the dCHIP program was done to identify genes with highest difference in expression between DMPM and OC/PPC groups. Using a cutoff ratio of at least 6-fold difference between these two tumor types, we identified 189 genes that were differentially expressed (Fig. 2 ). Sixty-eight genes were significantly overexpressed in DMPM, including those coding for calretinin, vitronectin, claudin-15, α4 laminin, hyaluronan synthase 1, cadherin 11, RAB7, the v-maf musculoaponeurotic fibrosarcoma oncogene homologue, and the epidermal growth factor–containing fibulin-like extracellular matrix protein 1. The remaining 121 genes were overexpressed in OC/PPC and included insulin-like growth factor II (IGF-II); IGF-II binding protein 3; cyclin E1; folate receptors 1 and 3; RAB25; MUC4; endothelin-1; CD24 (small cell lung carcinoma cluster 4 antigen); kallikreins 6, 7, and 8; claudins 3, 4, and 6; Notch3; and MMP-7 (matrilysin). The full gene list is available in Supplementary Tables S1 and S2.

Fig. 1.
Fig. 1.

Unsupervised cluster analysis of gene expression profiling of OC/PPC and DMPM. Based on the analysis of all genes available on the Affymetrix chip U133 Plus 2, all five DMPM cluster to each other and are distant from the OC/PPC group.

Fig. 2.
Fig. 2.

Cluster analysis shows two distinct groups of samples (horizontal tree) corresponding to DMPM (specimens 1-5) and OC/PPC (specimens 6-15). The dendrogram of the vertical tree shows a total of 189 genes that are preferentially expressed in either DMPM or OC/PPC group. The expression levels are expressed by the increased gradient of red intensity.

Validation experiments. Among the 189 differentially expressed genes, we selected 13 for validation using quantitative real-time PCR. These consisted of genes that were overexpressed in OC/PPC (Notch 3, claudins 3 and 4, cyclin E1, MUC4, RAB25, folate receptor 1, IGF-II, and tumor-associated calcium signal transducer 1) and those overexpressed in DMPM (calretinin, vitronectin, epidermal growth factor–containing fibulin-like extracellular matrix protein 1, and v-maf musculoaponeurotic fibrosarcoma oncogene homologue). Expression levels of these 13 transcripts were analyzed using hierarchical clustering in the 10 OC/PPC and 5 DMPM specimens. As shown in Fig. 3 , the levels of OC/PPC markers were significantly higher in OC/PPC samples than in DMPM samples (P < 0.001). Similarly, the levels of DMPM markers were higher in DMPM specimens than in OC/PPC specimens (P < 0.001).

Fig. 3.
Fig. 3.

Gene expression analysis of 13 differentially expressed genes. Quantitative real-time PCR was done for 13 differentially expressed genes identified by the dCHIP program. The expression level of each gene in individual specimen is shown as a pseudo-color gradient based on the relative expression level of a given gene to the average value derived from the control gene APP. The first nine genes are more highly expressed in OC/PPC and the remaining four in DMPM.

Immunohistochemistry showed cyclin E protein expression in 111 of 116 (97%) OC/PPC cores (14 noninformative cores) and 10 of 30 (33%) DMPM specimens (P < 0.001; Fig. 4A-C ). MUC4 protein expression was found in 117 of 122 (96%) OC/PPC cores (8 noninformative cores) and 1 of 30 (3%) DMPM (P < 0.001; Fig. 4D-F). Claudin-3 protein was expressed in 89 of 130 (68%) OC/PPC cores and was absent in all 30 DMPM (P < 0.001; Fig. 4G-I). Calretinin was uniformly expressed in tumor cells in 30 of 30 (100%) DMPM specimens and was expressed in 20 of 112 (18%) OC/PPC cores (18 noninformative cores), predominantly in a focal manner (<5% of cells; P < 0.001; Fig. 4J-L).

Fig. 4.
Fig. 4.

Immunohistochemistry analysis using cyclin E, MUC4, claudin-3, and calretinin as markers for the differentiation of OC/PPC from DMPM. A to C, cyclin E. Expression of cyclin E (nuclear immunoreactivity) in the majority of tumor cells is shown in two OC/PPC effusions (A and B) with negative expression in solid DMPM. Staining is nuclear. D to F, MUC4. Similar MUC4 immunointensity (both membrane and cytoplasmic staining) is shown in solid tumor (D) and effusion (E) from patients diagnosed with OC/PPC, with negative expression in a DMPM effusion (F). Staining is cytoplasmic and membranous. G to I, claudin-3. Claudin-3 immunoreactivity (both membrane and cytoplasmic staining) is shown with diffuse immunoreactivity in a solid tumor (G) and effusion (H) from patients diagnosed with OC/PPC and negative expression in solid DMPM (I). J to L, calretinin. Tumor cells in an OC/PPC effusion are calretinin negative (only few reactive mesothelial cells are stained (J), whereas an effusion (K) and solid tumor (L) from patients diagnosed with DMPM are positive with both cytoplasmic and nuclear immunoreactivity.

Comparative analysis of protein expression in effusions and solid tumors was done separately for each tumor type. OC/PPC effusions showed significantly higher claudin-3 expression than solid tumors (P < 0.001) and no expression of calretinin (0 of 15 compared with 20 of 107 solid tumors), although the latter finding failed to reach statistical significance. In DMPM, we found a trend for lower cyclin E protein in effusions (0 of 9 versus 10 of 21 positive specimens, P = 0.093), with comparable or similar expression level of the remaining three proteins, claudin-3, MUC-4, and calretinin.

Discussion

Compared with OC/PPC, DMPM is a relatively rare cancer type, and its molecular pathogenesis is poorly understood. In view of the similarities in clinical and pathologic features between these two tumor types, we attempted to characterize molecular discriminators that may aid in diagnosis, treatment, and prognostication in this clinical setting. Our data on a small series of cases provide the first evidence of distinct molecular profiles that are unique to OC/PPC and DMPM.

Cyclin E is a nuclear protein regulating the G1 to S-phase transition in the cell cycle by associating with cyclin-dependent kinase-2. The cyclin E/cyclin-dependent kinase 2 complex then phosphorylates and inactivates the retinoblastoma protein (15). Cyclin E is one of the genes that have been shown to be frequently amplified in ovarian carcinoma (16, 17), and its expression has been shown to correlate with poor survival in several studies (18–20). To the best of our knowledge, Cyclin E expression has not been studied in DMPM to date. Our data on both mRNA and protein level show that cyclin E expression is significantly higher in OC/PPC than in DMPM, suggesting that its overexpression may not be a central mechanism in the biology of malignant mesothelioma. The difference was most pronounced in effusions, in which cyclin E was uniformly negative and positive in DMPM and OC/PPC, respectively. Cyclin E may be a useful new marker for the differentiation between OC/PPC and DMPM in both effusions and solid tumors, especially at the former site, and a potential therapeutic target in OC/PPC.

The Notch family of receptors consists of four members. Notch receptors are transmembrane proteins that are cleaved into two subunits by furin-like convertases, with subsequent translocation of the intracellular domain to the nucleus and activation of downstream effectors that mediate cell proliferation and survival (21). Activation of Notch members has been described in acute lymphoblastic leukemia, neuroblastoma, and several carcinomas, including tumors of the lung, skin, prostate, and cervix (21). Notch3 was recently found to be up-regulated in ovarian carcinoma compared with the ovarian surface epithelium (22). Using SNP arrays, we recently identified an amplicon at 19p13.12 in high-grade ovarian serous carcinomas and showed that Notch3 was the gene that is most significantly overexpressed in amplified compared with nonamplified tumors. Notch3 DNA copy number correlated with Notch3 protein expression based on parallel immunohistochemistry and fluorescence in situ hybridization analyses in high-grade ovarian serous carcinomas. Inactivation of Notch3 by both γ-secretase inhibitor and Notch3-specific small interfering RNA suppressed cell proliferation and induced apoptosis in the cell lines that overexpressed Notch3 (23). In the present study, we showed that the level of Notch3 gene expression is significantly higher in OC/PPC than in DMPM, suggesting that activation of the Notch3 signaling pathway is common in OC/PPC but not in DMPM.

Tight junctions are areas of the cell surface that regulate ion flux and thereby cellular homeostasis and contain three major families of proteins: claudins, occludins, and junctional adhesion molecules. Claudins are a family of tight junction proteins that at present comprises 23 members (24). Despite the expectation that claudins are down-regulated in cancer, this finding holds true only for some members (e.g., claudin-1 in breast and colon cancer; ref. 24) because two proteins in this family (claudin-3 and claudin-4) have been shown to be overexpressed in ovarian carcinomas compared with normal ovarian tissue or benign ovarian tumors (22, 25, 26). Ovarian surface epithelial cells that constitutively express claudin-3 and claudin-4 have enhanced invasive ability and MMP-2 activation (27). In agreement with these studies, we observed significantly higher expression levels of claudin-3 and claudin-4, as well as claudin-6, in OC/PPC based on the array analysis. This finding was validated by quantitative PCR for claudin-3 and claudin-4 and immunohistochemistry for claudin-3 (Table 2 ). In contrast, claudin-15 was more highly expressed in DMPM, supporting the differential expression of claudins in different tumor types. Our finding that claudin-3 expression is significantly higher in OC/PPC effusions is currently being analyzed in a larger cohort.

View this table:
Table 2.

Imunnohistochemistry expression data for four proteins in DMPM and OC/PPC

MUC4 belongs to the mucin glycoprotein family, heavily glycosylated molecules that are produced by different types of epithelium for the purpose of protection. MUC4, a heterodimer with a mucin and protein backbone of 500 and 190 kDa, respectively, is normally expressed by most normal epithelia, as well as in carcinomas of the pancreas, lung, and breast (28). mRNA expression of MUC4, as determined by Northern blot analysis, is decreased with increasing tumor stage in ovarian carcinoma, and its expression is associated with a trend towards better survival (29). We found frequent expression of MUC4 on both mRNA and protein level in our specimens, the majority of which were diagnosed at advanced stage. Further research is, therefore, necessary to establish the clinical role of MUC4 in ovarian cancer. The significantly higher expression level of MUC4 in OC/PPC compared with DMPM is in agreement with the role of this molecule in differentiating lung adenocarcinoma from pleural mesothelioma (30). It is noteworthy that MUC4 is transactivated by PEA3 (31), a member of the Ets transcription factor family whose expression levels correlate with poor survival in patients with OC/PPC effusions (32).

Several other genes of interest that may prove to be therapeutic targets in ovarian cancer were found to be more highly expressed in OC/PPC, including IGF-II; IGF-II binding protein 3; folate receptors 1 and 3; RAB25; endothelin-1; CD24 (small cell lung carcinoma cluster 4 antigen); kallikreins 6, 7, and 8; and MMP-7. The IGF system is activated in an autocrine manner in ovarian carcinoma (33), and IGF-II induces proliferation in ovarian carcinoma cells (34). Blocking of IGF signaling may be of therapeutic value in OC/PPC (35). IGF-II has been shown to be a marker of poor survival in OC (36). Folate receptors were found to be more highly expressed in OC/PPC than in DMPM in our analysis, a finding which is different from previous reports showing that folate receptors are highly expressed in both OC and mesothelioma cells (37, 38). The gene for RAB25, a small GTPase involved in apical vesicle trafficking, is amplified at 1q22 in ovarian and breast cancer, and its mRNA is highly expressed in advanced-stage ovarian carcinoma (39). Engineered expression of RAB25 protects carcinoma cells against anoikis and apoptosis, including that induced by chemotherapy, and its expression correlates with poor survival in both tumor types (39). Endothelin-1 and its receptor constitute an autocrine growth factor pathway in ovarian cancer and mediate epithelial to mesenchymal transition (40, 41). CD24 is a small mucin-like membrane protein that functions as the receptor to P-selectin. CD24 is frequently expressed in ovarian carcinoma, and its expression has been shown to correlate with poor overall survival in this tumor (42). Its expression has not been reported in mesothelioma.

Genes that were preferentially expressed in DMPM included calretinin, vitronectin, claudin 15, RAB7, the α4 laminin chain, hyaluronan synthase 1, cadherin 11, the v-maf musculoaponeurotic fibrosarcoma oncogene homologue, and the epidermal growth factor–containing fibulin-like extracellular matrix protein 1. Among them, calretinin has been shown to be a useful marker for detecting benign and malignant mesothelial cells in routine effusion cytology (9). The higher calretinin mRNA and protein expression in DMPM can, therefore, serve as internal control for our analysis. Vitronectin is expressed by ovarian carcinoma cells (43) and enhances the expression of urokinase-type plasminogen activator in mesothelioma cells in vitro (44). Our data suggest that its expression levels in DMPM may be higher than in OC/PPC. Expression of the α4 laminin chain is in agreement with the reported ability of mesothelioma cells to synthesize laminin (45) and their strong expression of the α6 integrin subunit, part of the α6β1 and α6β4 laminin receptors (46). Hyaluronan synthase 1 is frequently expressed in benign and malignant mesothelial cells, although its expression is also seen in lung adenocarcinomas (47). Cadherin 11 expression has not been reported in mesotheliomas. The v-MAF oncogene is a homodimeric protein with a basic leucine zipper structure at its COOH terminus, which binds to palindromic DNA sequences termed MAREs and is able to induce cell transformation (48). Its expression has not been previously shown in mesothelioma. The epidermal growth factor–containing fibulin-like extracellular matrix protein 1, also known as fibulin 3, is an extracellular matrix protein that localizes to basement membranes and stroma and mediates cell-cell and cell-matrix communication. Fibulin 3 is involved in the pathogenesis of macular degeneration in Sorsby fundus dystrophy, a hereditary macular degenerative disease and binds tissue inhibitor of metalloproteinases-3 in this disease (49). Fibulin 3 inhibits angiogenesis, and its expression is reduced in different carcinomas, including tumors of the ovary, breast lung, kidney, and colon (50).

In conclusion, gene expression analysis of a limited series of OC/PPC and DMPM effusions reveals a large number of new genes that are differentially expressed in these tumors, with excellent correlation with the data in validation tests. The findings in this study provide the first evidence of extensive molecular differences in gene expression between these two histogenetically closely related tumors. Expansion of future series to a larger number of specimens, as done in the present study for four new markers, may prove valuable for characterization of these candidate genes as both diagnostic markers and therapeutic targets.

Acknowledgments

We gratefully acknowledge the excellent technical assistance of Ellen Hellesylt, Inger-Liv Nordli, and Martina Skrede at the Department of Pathology, Radiumhospitalet-Rikshospitalet Medical Center.

Footnotes

  • Grant support: National Cancer Institute grant CA103937.

  • 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 with 18 U.S.C. Section 1734 solely to indicate this fact.

  • Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

  • I. Shih (ishih{at}jhmi.edu) and B. Davidson (bend{at}ulrik.uio.no) share the correspondence of this study.

    • Accepted August 9, 2006.
    • Received May 1, 2006.
    • Revision received July 26, 2006.

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Clinical Cancer Research: 12 (20)
October 2006
Volume 12, Issue 20

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Gene Expression Signatures Differentiate Ovarian/Peritoneal Serous Carcinoma from Diffuse Malignant Peritoneal Mesothelioma
Ben Davidson, Zhen Zhang, Lilach Kleinberg, Mei Li, Vivi Ann Flørenes, Tian-Li Wang and Ie-Ming Shih
Clin Cancer Res October 15 2006 (12) (20) 5944-5950; DOI: 10.1158/1078-0432.CCR-06-1059
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