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The objective of this study was to analyze the expression and prognostic role of the gap junction protein claudin-10 in high-grade serous carcinoma (HGSC). Claudin-10 protein expression by immunohistochemistry was analyzed in 588 HGSC (414 effusions, 174 surgical specimens). Expression in mesotheliomas (n = 97; 47 effusions, 50 surgical specimens) was studied for comparative purposes. CLDN10 mRNA expression by quantitative RT-PCR (qRT-PCR) was analyzed in 40 HGSC effusions. Claudin-10 protein expression was found in 360/588 (61%) HGSC vs. 19/97 (20%) mesotheliomas (p CLDN10 mRNA in HGSC effusions. High (> 25%) claudin-10 expression in HGSC effusions was significantly associated with shorter overall survival (OS; p = 0.036) and progression-free survival (PFS; p = 0.045) in univariate analysis, and was an independent prognosticator of OS in multivariate analysis (p = 0.045). In conclusion, claudin-10 protein expression is higher in HGSC compared to mesothelioma, although the diagnostic power of this marker appear to be lesser than other claudin family members. Claudin-10 expression in HGSC effusions is marker of more aggressive disease.

Gene promoters identified by PBX1 ChIP-on-chip.

Supplementary Table 1 from Gene Expression Signatures Differentiate Ovarian/Peritoneal Serous Carcinoma from Diffuse Malignant Peritoneal Mesothelioma

Including Supplementary Figures and Methods

Supplemental Figure 1

Supplementary Table 2 from Gene Expression Signatures Differentiate Ovarian/Peritoneal Serous Carcinoma from Diffuse Malignant Peritoneal Mesothelioma

Table S2

Purpose:Undifferentiated uterine sarcomas (UUS) are rare, extremely deadly, sarcomas with no effective treatment. The goal of this study was to identify novel intrinsic molecular UUS subtypes using integrated clinical, histopathologic, and molecular evaluation of a large, fully annotated, patient cohort.Experimental Design:Fifty cases of UUS with full clinicopathologic annotation were analyzed for gene expression (n = 50), copy-number variation (CNV, n = 40), cell morphometry (n = 39), and protein expression (n = 22). Gene ontology and network enrichment analysis were used to relate over- and underexpressed genes to pathways and further to clinicopathologic and phenotypic findings.Results:Gene expression identified four distinct groups of tumors, which varied in their clinicopathologic parameters. Gene ontology analysis revealed differential activation of pathways related to genital tract development, extracellular matrix (ECM), muscle function, and proliferation. A multivariable, adjusted Cox proportional hazard model demonstrated that RNA group, mitotic index, and hormone receptor expression influence patient overall survival (OS). CNV arrays revealed characteristic chromosomal changes for each group. Morphometry demonstrated that the ECM group, the most aggressive, exhibited a decreased cell density and increased nuclear area. A cell density cutoff of 4,300 tumor cells per mm2 could separate ECM tumors from the remaining cases with a sensitivity of 83% and a specificity of 94%. IHC staining of MMP-14, Collagens 1 and 6, and Fibronectin proteins revealed differential expression of these ECM-related proteins, identifying potential new biomarkers for this aggressive sarcoma subgroup.Conclusions:Molecular evaluation of UUS provides novel insights into the biology, prognosis, phenotype, and possible treatment of these tumors.

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.

PDF file, 1202KB, Supplementary Figure 1. Kaplan-Meier Survival Curves. Supplementary Figure 2. Kaplan-Meier Survival Curves. Supplementary Table 1. Univariate analysis of clinical covariates.

Supplementary Tables A and B from Proteomic Analysis of Malignant Ovarian Cancer Effusions as a Tool for Biologic and Prognostic Profiling

Purpose: This study assesses the ability of multidrug resistance (MDR)–associated gene expression patterns to predict survival in patients with newly diagnosed carcinoma of the ovary. The scope of this research differs substantially from that of previous reports, as a very large set of genes was evaluated whose expression has been shown to affect response to chemotherapy.Experimental Design: We applied a customized TaqMan low density array, a highly sensitive and specific assay, to study the expression profiles of 380 MDR-linked genes in 80 tumor specimens collected at initial surgery to debulk primary serous carcinoma. The RNA expression profiles of these drug resistance genes were correlated with clinical outcomes.Results: Leave-one-out cross-validation was used to estimate the ability of MDR gene expression to predict survival. Although gene expression alone does not predict overall survival (OS; P = 0.06), four covariates (age, stage, CA125 level, and surgical debulking) do (P = 0.03). When gene expression was added to the covariates, we found an 11-gene signature that provides a major improvement in OS prediction (log-rank statistic P < 0.003). The predictive power of this 11-gene signature was confirmed by dividing high- and low-risk patient groups, as defined by their clinical covariates, into four specific risk groups on the basis of expression levels.Conclusion: This study reveals an 11-gene signature that allows a more precise prognosis for patients with serous cancer of the ovary treated with carboplatin- and paclitaxel-based therapy. These 11 new targets offer opportunities for new therapies to improve clinical outcome in ovarian cancer. Clin Cancer Res; 18(11); 3197–206. ©2012 AACR.

The evolution of chemoresistance is a fundamental characteristic of cancer that ultimately hampers its clinical management. However, it may be possible to improve patient outcomes significantly by a better understanding of resistance mechanisms, which cancers rely upon during the evolution to an untreatable state. Here we report an essential role of the stem cell reprogramming factor, PBX1, in mediating chemoresistance in ovarian carcinomas. In the clinical setting, high levels of PBX1 expression correlated with shorter survival in post-chemotherapy ovarian cancer patients. In tumor cells with low endogenous levels of PBX1, its enforced expression promoted cancer stem cell-like phenotypes, including most notably an increase in resistance to platinum-based therapy used most commonly for treating this disease. Conversely, silencing PBX1 in platinum-resistant cells that overexpressed PBX1 sensitized them to platinum treatment and reduced their stem-like properties. An analysis of published genome-wide chromatin immunoprecipitation data indicated that PBX1 binds directly to promoters of genes involved in stem cell maintenance and the response to tissue injury. We confirmed direct regulation of one of these genes, STAT3, demonstrating that the PBX1 binding motif at its promoter acted to positively regulate STAT3 transcription. We further demonstrated that a STAT3/JAK2 inhibitor could potently sensitize platinum-resistant cells to carboplatin and suppress their growth in vivo. Our findings offer a mechanistic rationale to target the PBX1/STAT3 axis to antagonize a key mechanism of chemoresistance in ovarian cancers and possibly other human cancers. Cancer Res; 76(21); 6351–61. ©2016 AACR.

Purpose: Malignant epithelial ovarian cancer effusions are important in disease dissemination and clinical outcome. The identification of biochemical events active in effusions may improve our identification and application of targeted therapeutics.Experimental Design: Archival effusion samples for which outcome information was known were studied. Clinical variables were comparable between these groups. Two cohorts of patients with malignant effusion were assessed: those with effusion at presentation (Tap1) or at first recurrence (Tap2). Expression and activated fraction of selected signaling proteins were quantitated on serial protein microarrays using validated antibodies. Proteomic results and clinical variables were analyzed by univariate analysis followed by Cox proportional hazards model analysis.Results: Malignant effusions (>80% malignant cells) were distinguished from benign effusions by higher expression of AKT, activated extracellular signal-regulated kinase, activated (P ≤ 0.001) and total cAMP-responsive element binding protein (P = 0.01), and JNK (P = 0.03). Malignant pleural effusions could not be differentiated from ascites by signaling profiles. Both had signal expression clusters for survival, proliferation and metastasis, and injury pathways. Cox proportional hazards model analysis revealed high p38 and pEGFR/EGFR ratio as jointly associated with poor survival in Tap1 cases (both P ≤ 0.002). Phospho-JNK quantity was associated with worse outcome in Tap2 patients (P = 0.004), when taking other factors into consideration.Conclusions: Proliferation, survival, and apoptosis signaling dysregulation can be identified in ovarian cancer effusion samples. Biochemical characterization of clinical effusions may provide either predictive and/or correlative information on patient outcome from which to further understand the mechanisms of effusion development and target clinical intervention.

Supplementary Figure Legends 1-2 from Activation of NF-κB Signaling by Inhibitor of NF-κB Kinase β Increases Aggressiveness of Ovarian Cancer

Gene amplification is one of the common mechanisms that activate oncogenes. In this study, we used single nucleotide polymorphism array to analyze genome-wide DNA copy number alterations in 31 high-grade ovarian serous carcinomas, the most lethal gynecologic neoplastic disease in women. We identified an amplicon at 19p13.12 in 6 of 31 (19.5%) ovarian high-grade serous carcinomas. This amplification was validated by digital karyotyping, quantitative real-time PCR, and dual-color fluorescence in situ hybridization (FISH) analysis. Comprehensive mRNA expression analysis of all 34 genes within the minimal amplicon identified Notch3 as the gene that showed most significant overexpression in amplified tumors compared with nonamplified tumors. Furthermore, Notch3 DNA copy number is positively correlated with Notch3 protein expression based on parallel immunohistochemistry and FISH studies in 111 high-grade tumors. 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 but not in those with minimal amount of Notch3 expression. These results indicate that Notch3 is required for proliferation and survival of Notch3-amplified tumors and inactivation of Notch3 can be a potential therapeutic approach for ovarian carcinomas. (Cancer Res 2006; 66(12): 6312-8)

The NF-κB family of transcription factors has been implicated in the propagation of ovarian cancer, but the significance of constitutive NF-κB signaling in ovarian cancer is unknown. We hypothesized that constitutive NF-κB signaling defines a subset of ovarian cancer susceptible to therapeutic targeting of this pathway. We investigated the biological relevance of NF-κB in ovarian cancer using a small-molecule inhibitor of inhibitor of NF-κB kinase β (IKKβ) and confirmed with RNA interference toward IKKβ. We developed a gene expression signature of IKKβ signaling in ovarian cancer using both pharmacologic and genetic manipulation of IKKβ. The expression of IKKβ protein itself and the nine-gene ovarian cancer–specific IKKβ signature were related to poor outcome in independently collected sets of primary ovarian cancers (P = 0.02). IKKβ signaling in ovarian cancer regulated the transcription of genes involved in a wide range of cellular effects known to increase the aggressive nature of the cells. We functionally validated the effect of IKKβ signaling on proliferation, invasion, and adhesion. Downregulating IKKβ activity, either by a small-molecule kinase inhibitor or by short hairpin RNA depletion of IKKβ, blocked all of these cellular functions, reflecting the negative regulation of the target genes identified. The diversity of functions controlled by IKKβ in ovarian cancer suggests that therapeutic blockade of this pathway could be efficacious if specific IKKβ inhibitor therapy is focused to patients whose tumors express a molecular profile suggestive of dependence on IKKβ activity. Cancer Res; 70(10); 4005–14. ©2010 AACR.

Supplementary Figure 1 from Small Interfering RNA Molecules Targeting Endothelin-Converting Enzyme-1 Inhibit Endothelin-1 Synthesis and the Invasive Phenotype of Ovarian Carcinoma Cells

Supplementary Table 1 from Activation of NF-κB Signaling by Inhibitor of NF-κB Kinase β Increases Aggressiveness of Ovarian Cancer

PDF file - 81K, Verification of IKKe knock down

PDF file - 233K, Viability and invasion of additional ovarian cancer cell lines

Supplementary Figure 1 from Activation of NF-κB Signaling by Inhibitor of NF-κB Kinase β Increases Aggressiveness of Ovarian Cancer

Supplementary Table 1, Figures 1-7 from Jagged-1 and Notch3 Juxtacrine Loop Regulates Ovarian Tumor Growth and Adhesion

PDF file - 67K

Notch3 gene amplification and pathway activation have been reported in ovarian serous carcinoma. However, the primary Notch3 ligand that initiates signal transduction in ovarian cancer remains unclear. In this report, we identify Jagged-1 as the highest expressed Notch ligand in ovarian tumor cells as well as in peritoneal mesothelial cells that are in direct contact with disseminated ovarian cancer cells. Cell-cell adhesion and cellular proliferation were reduced in Notch3-expressing ovarian cancer cells that were cocultured with Jagged-1 knockdown mesothelial and tumor feeder cells. Interaction of Notch3-expressing ovarian cancer cells with Jagged-1–expressing feeder cells activated the promoter activity of candidate Notch3 target genes, and this activity was attenuated by Notch3 siRNA. Constitutive expression of the Notch3 intracellular domain significantly suppressed the Jagged-1 shRNA–mediated growth inhibitory effect. In Notch3-expressing ovarian cancer cells, Jagged-1–stimulating peptides enhanced cellular proliferation, which was suppressed by γ-secretase inhibitor and Notch3 siRNA. Taken together, our results show that Jagged-1 is the primary Notch3 ligand in ovarian carcinoma and Jagged-1/Notch3 interaction constitutes a juxtacrine loop promoting proliferation and dissemination of ovarian cancer cells within the intraperitoneal cavity. [Cancer Res 2008;68(14):5716–23]

Supplementary Figure 2 from Activation of NF-κB Signaling by Inhibitor of NF-κB Kinase β Increases Aggressiveness of Ovarian Cancer

Supplementary Figure Legend from Small Interfering RNA Molecules Targeting Endothelin-Converting Enzyme-1 Inhibit Endothelin-1 Synthesis and the Invasive Phenotype of Ovarian Carcinoma Cells

PDF file - 1.8MB

Supplementary Table 1 from Notch3 Gene Amplification in Ovarian Cancer

PDF file - 23K, IKK isoform IHC scoring

PDF file - 172K, Validation of IKKe gene signature

The molecular characteristics of low-grade serous carcinoma (LGSC) in serous effusions have not been previously studied. The present study analyzed the molecular profile of LGSC at this anatomic site.Specimens consisted of a series of 17 serous effusions (15 peritoneal, 2 pleural) from 16 patients, of which 15 were LGSC and 2 serous borderline tumor (SBT) who later progressed to LGSC. For comparative purposes, 9 surgical specimens from 6 patients with LGSC were analyzed. Fresh-frozen cell pellets and surgical specimens underwent targeted next-generation sequencing covering 50 unique genes.Mutations were found in tumors from 14 of the 22 patients, of whom 4 had 2 different mutations and 10 had a single mutation. Overall, the most common mutations were in KRAS (n=3) and BRAF (n=3), followed by NRAS (n=2), CDK2NA (n=2), TP53 (n=2), ATM (n=2). Mutations in MET, STK11, ERBB2 and FLT3 were found in 1 case each. Patient-matched specimens had the same molecular profile. Both effusions with TP53 mutation had concomitant ATM mutation, and both stained immunohistochemically with wild-type pattern. Absence of mutations was associated with a trend for shorter overall survival in univariate analysis (p=0.072).The molecular alterations in LGSCs in serous effusions are consistent with the ones found in solid tumors, with frequent alterations in the MAPK pathway. Mutations in LGSC may be a marker of better outcome.

Phospholipase D (PLD) isoenzymes participate in a variety of cellular functions that are mostly attributed to phosphatidic acid (PA) synthesis. Dysregulation of PLD regulates tumor progression and metastasis, yet little is known about the underlying mechanism. We previously reported on the expression and clinical role of the PLD isoenzymes PLD1 and PLD2 in tubo-ovarian high-grade serous carcinoma (HGSC). In the present study, we investigated the biological function of PLD1 and PLD2 using the OVCAR-3 and OVCAR-8 HGSC cell lines. KO cell lines for both PLDs were generated using CRISPR/CAS9 technology and assayed for exosome secretion, spheroid formation, migration, invasion and expression of molecules involved in epithelial-mesenchymal transition (EMT) and intracellular signaling. Significant differences between PLD1 and PLD2 KO cells and controls were observed for all the above parameters, supporting an important role for PLD in regulating migration, invasion, metastasis and EMT.

The move toward consistent and comprehensive surgical pathology reports for cancer resection specimens has been a key development in supporting evidence-based patient management and consistent cancer staging. The International Collaboration on Cancer Reporting (ICCR) previously developed a data set for reporting of the ovarian, fallopian tube and primary peritoneal carcinomas which was published in 2015. In this paper, we provide an update on this data set, as a second edition, that reflects changes in the 2020 World Health Organization (WHO) Classification of Female Genital Tumours as well as some other minor modifications. The data set has been developed by a panel of internationally recognized expert pathologists and a clinician and consists of “core” and “noncore” elements to be included in surgical pathology reports, with detailed commentary to guide users, including references. This data set replaces the widely used first edition, and will facilitate consistent and accurate case reporting, data collection for quality assurance and research, and allow for comparison of epidemiological and pathologic parameters between different populations.

High-grade serous ovarian cancer (HGSOC) has poor prognosis and new treatment modalities are needed. Immunotherapy, with checkpoint inhibitors, have demonstrated limited impact. To evaluate the suitability for immunotherapeutics, contextualized preclinical models are required to secure meaningful clinical translation. Therefore, we developed and characterized humanized patient-derived xenograft (hu PDX) murine models of HGSOC, which were established by orthotopic implantation of tumor cell suspensions and intravenous injection of CD34+ cells isolated from umbilical cord blood samples. The developing human immune system in NSG and NSGS mice was followed longitudinally by flow cytometry and characterized by mass cytometry with a panel of 34 surface markers. Molecular imaging of tumor burden, survival analysis, and characterization of tumor-infiltrating immune cells was performed to assess the treatment response to anti-PD-1 (nivolumab) monotherapy. Successful generation of hu PDX models was achieved. Mice treated with nivolumab showed a decrease in tumor burden, however no significant survival benefit was identified when compared to untreated controls. No correlation was seen between PD-L1 expression and CD8 T cell infiltration and response parameters. As the characterization showed an immune infiltration of predominantly myeloid cells, similar to what is observed in HGSOC patients, the models may have the potential to evaluate the importance of myeloid cell immunomodulation as well. publishedVersion

Endometrial carcinomas (ECs) are histologically classified as endometrioid and nonendometrioid tumors, with each subgroup displaying different molecular profiles and clinical outcomes. Considerable biological and clinical heterogeneity exists within this scheme, however, reflecting its imperfection. We aimed to gather additional data that might help clarify the tumors’ pathogenesis and contribute toward a more meaningful classification scheme. In total, 33 ECs were examined for the presence of chromosomal aberrations, genomic imbalances, pathogenic variants, microsatellite instability, and expression profiles at both gene and miRNA levels. Chromosome 1 was the most frequently rearranged chromosome, showing a gain of all or part of the long arm. Pathogenic variants were found for PTEN (53%), PDGFRA (37%), PIK3CA (34%), and KIT (31%). High microsatellite instability was identified in 15 ECs. Comparing tumors and controls, we identified 23 differentially expressed genes of known importance in carcinogenesis, 15 genes involved in innate and adaptative immune responses, and altered expression of 7 miRNAs. miR-32-5p was the most upregulated. Our series showed a high degree of heterogeneity. Tumors were well-separated from controls, but there was no clear-cut separation between endometrioid and nonendometrioid ECs. Whether this means that the current phenotypic classification is of little relevance or if one still has not detected which genomic parameters to enter into correlation analyses remains unknown.