Characterization of latently infected EBV+ antibody-secreting B cells isolated from ovarian tumors and malignant ascites.

Introduction: Intra-tumoral B cells mediate a plethora of immune effector mechanisms with key roles in anti-tumor immunity and serve as positive prognostic indicators in a variety of solid tumor types, including epithelial ovarian cancer (EOC). Several aspects of intra-tumoral B cells remain unclear, such as their state of activation, antigenic repertoires, and capacity to mature into plasma cells. Methods: B lymphocytes were isolated from primary EOC tissue and malignant ascites and were maintained in cell culture medium. The stably maintained cell lines were profiled with flow cytometry and B cell receptor sequencing. Secreted antibodies were tested with a human proteome array comprising more than 21,000 proteins, followed by ELISA for validation. Originating tumor samples were used for spatial profiling with chip cytometry. Results: Antibody-secreting B lymphocytes were isolated from the ovarian tumor microenvironment (TME) of four different EOC patients. The highly clonal cell populations underwent spontaneous immortalization in vitro, were stably maintained in an antibody-secreting state, and showed presence of Epstein-Barr viral (EBV) proteins. All originating tumors had high frequency of tumor-infiltrating B cells, present as lymphoid aggregates, or tertiary lymphoid structures. The antigens recognized by three of the four cell lines are coil-coil domain containing protein 155 (CCDC155), growth factor receptor-bound protein 2 (GRB2), and pyruvate dehydrogenase phosphatase2 (PDP2), respectively. Anti-CCDC155 circulating IgG antibodies were detected in 9 of 20 (45%) of EOC patients' sera. Tissue analyses with multiparameter chip cytometry shows that the antibodies secreted by these novel human B cell lines engage their cognate antigens on tumor cells. Discussion: These studies demonstrate that within the tumor-infiltrating lymphocyte population in EOC resides a low frequency population of antibody-secreting B cells that have been naturally exposed to EBV. Once stably maintained, these novel cell lines offer unique opportunities for future studies on intratumor B cell biology and new target antigen recognition, and for studies on EBV latency and/or viral reactivation in the TME of non-EBV related solid tumors such as the EOC.

Longitudinal Modulation of Loco-Regional Immunity in Ovarian Cancer Patients Receiving Intraperitoneal Chemotherapy.

The immune tumor microenvironment (TME) of epithelial ovarian cancer (EOC) carries both effector and suppressive functions. To define immune correlates of chemotherapy-induced tumor involution, we performed longitudinal evaluation of biomarker expression on serial biological specimens collected during intraperitoneal (IP) platinum-based chemotherapy. Serial biological samples were collected at several time points during IP chemotherapy. RNA from IP fluid cells and tumor tissue was analyzed via NanoString. Meso Scale Discovery (MSD) multiplex assay and ELISA for MUC1 antibodies were performed on plasma and IP fluid. Differentially expressed genes in IP fluid demonstrate an upregulation of B cell function and activation of Th2 immune response along with dampening of Th1 immunity during chemotherapy. MSD analysis of IP fluid and gene expression analysis of tumor tissue revealed activation of Th2 immunity and the complement system. Anti-MUC1 antibodies were detected in IP fluid samples. IP fluid analysis in a secondary cohort also identified chemotherapy-induced B cell function genes. This study shows that serial IP fluid sampling is an effective method to capture changes in the immune TME during chemotherapy and reveals treatment induced changes in B cell function and Th2 immunity.

Phase I Trial Combining Chemokine-Targeting with Loco-Regional Chemoimmunotherapy for Recurrent, Platinum-Sensitive Ovarian Cancer Shows Induction of CXCR3 Ligands and Markers of Type 1 Immunity.

PURPOSE: Increased prevalence of cytotoxic T lymphocytes (CTL) in the tumor microenvironment (TME) predicts positive outcomes in patients with epithelial ovarian cancer (EOC), whereas the regulatory T cells (Treg) predict poor outcomes. Guided by the synergistic activity of TLR3 ligands, IFNα, and COX-2 blockers in selectively enhancing CTL-attractants but suppressing Treg-attractants, we tested a novel intraperitoneal chemoimmunotherapy combination (CITC), to assess its tolerability and TME-modulatory impact in patients with recurrent EOC. PATIENTS AND METHODS: Twelve patients were enrolled in phase I portion of the trial NCT02432378, and treated with intraperitoneal cisplatin, intraperitoneal rintatolimod (dsRNA, TLR3 ligand), and oral celecoxib (COX-2 blocker). Patients in cohorts 2, 3, and 4 also received intraperitoneal IFNα at 2, 6, and 18 million units (MU), respectively. Primary objectives were to evaluate safety, identify phase 2 recommended dose (P2RD), and characterize changes in the immune TME. Peritoneal resident cells and intraperitoneal wash fluid were profiled via NanoString and Meso Scale Discovery (MSD) multiplex assay, respectively. RESULTS: The P2RD of IFNα was 6 MU. Median progression-free survival and overall survival were 8.4 and 30 months, respectively. Longitudinal sampling of the peritoneal cavity via intraperitoneal washes demonstrated local upregulation of IFN-stimulated genes (ISG), including CTL-attracting chemokines (CXCL-9, -10, -11), MHC I/II, perforin, and granzymes. These changes were present 2 days after chemokine modulation and subsided within 1 week. CONCLUSIONS: The chemokine-modulating intraperitoneal-CITC is safe, tolerable, and associated with ISG changes that favor CTL chemoattraction and function. This combination (plus DC vaccine) will be tested in a phase II trial. See related commentary by Aranda et al., p. 1993.

Multiplex profiling identifies distinct local and systemic alterations during intraperitoneal chemotherapy for ovarian cancer: An NRG Oncology/Gynecologic Oncology Group Study.

OBJECTIVES: Ovarian cancer leads to abdominal carcinomatosis and late stage (III/IV) diagnosis in 75% of patients. Three randomized phase III trials have demonstrated that intraperitoneal (IP) chemotherapy improves outcomes in epithelial ovarian cancer. While IP treatment is validated by clinical trials, there is a poor understanding of the mechanism(s) leading to the survival advantage other than the increased concentration of cytotoxic drugs within the tumor microenvironment. A better understanding of this process through analysis of dynamic biomarkers should promote novel approaches that may enhance tumor clearance. We propose this pilot study to confirm the feasibility of collecting serial peritoneal samples from implanted catheters in women receiving IP chemotherapy. We believe these specimens may be used for multiplex analysis to reveal unique biomarker fluctuations when compared to peripheral blood. METHODS: From 13 women participating on GOG 252, 30 whole blood, 12 peritoneal fluid (PF), and 20 peritoneal wash (PW) with 30mL saline were obtained. Samples were requested prior to the first three chemotherapy cycles. Samples were assessed for volume, cell populations, protein, RNA, and miRNA content changes. RESULTS: Median volume for PF was 1.6mL and 3.1mL for PW. PW is a dilution of PF capable of capturing measurable biomarkers. Peritoneal aspirates contain a unique profile of biomarkers distinct from blood. miRNA undergo earlier alteration with chemotherapy than genes. Flow cytometry does not adequately capture biomarker fluctuations. CONCLUSIONS: As a proof of principle study, this trial provides evidence that sampling the peritoneal cavity can be adapted for biomarker analysis.

Proteomic Analysis of Matched Formalin-Fixed, Paraffin-Embedded Specimens in Patients with Advanced Serous Ovarian Carcinoma.

OBJECTIVE: The biology of high grade serous ovarian carcinoma (HGSOC) is poorly understood. Little has been reported on intratumoral homogeneity or heterogeneity of primary HGSOC tumors and their metastases. We evaluated the global protein expression profiles of paired primary and metastatic HGSOC from formalin-fixed, paraffin-embedded (FFPE) tissue samples. METHODS: After IRB approval, six patients with advanced HGSOC were identified with tumor in both ovaries at initial surgery. Laser capture microdissection (LCM) was used to extract tumor for protein digestion. Peptides were extracted and analyzed by reversed-phase liquid chromatography coupled to a linear ion trap mass spectrometer. Tandem mass spectra were searched against the UniProt human protein database. Differences in protein abundance between samples were assessed and analyzed by Ingenuity Pathway Analysis software. Immunohistochemistry (IHC) for select proteins from the original and an additional validation set of five patients was performed. RESULTS: Unsupervised clustering of the abundance profiles placed the paired specimens adjacent to each other. IHC H-score analysis of the validation set revealed a strong correlation between paired samples for all proteins. For the similarly expressed proteins, the estimated correlation coefficients in two of three experimental samples and all validation samples were statistically significant (p < 0.05). The estimated correlation coefficients in the experimental sample proteins classified as differentially expressed were not statistically significant. CONCLUSION: A global proteomic screen of primary HGSOC tumors and their metastatic lesions identifies tumoral homogeneity and heterogeneity and provides preliminary insight into these protein profiles and the cellular pathways they constitute.

Comparison of ERCC1/XPF genetic variation, mRNA and protein levels in women with advanced stage ovarian cancer treated with intraperitoneal platinum.

OBJECTIVE: Approximately 20% of patients receiving platinum-based chemotherapy for epithelial ovarian cancer (EOC) are refractory or develop early recurrence. Identifying these patients early could reduce treatment-associated morbidity and allow quicker transfer to more effective therapies. Much attention has focused on ERCC1 as a potential predictor of response to therapy because of its essential role in the repair of platinum-induced DNA damage. The purpose of this study was to accurately measure protein levels of ERCC1 and its essential binding partner XPF from patients with EOC treated with platinum-based therapy and determine if protein levels correlate with mRNA levels, patient genotypes or clinical outcomes. METHODS: ERCC1 and XPF mRNA and protein levels were measured in frozen EOC specimens from 41 patients receiving intraperitoneal platinum-based chemotherapy using reverse transcription polymerase chain reaction and western blots. Genotypes of common nucleotide polymorphisms were also analyzed. Patient outcomes included progression free (PFS) and overall survival (OS). RESULTS: Expression of ERCC1 and XPF were tightly correlated with one another at both the mRNA and protein level. However, the mRNA and protein levels of ERCC1 were not positively correlated. Likewise, none of the SNPs analyzed correlated with ERCC1 or XPF protein levels. There was an inverse correlation between mRNA levels and patient outcomes. CONCLUSION: Neither genotype nor mRNA levels are predictive of protein expression. Despite this, low ERCC1 mRNA significantly correlated with improved PFS and OS.

Differential analysis of ovarian and endometrial cancers identifies a methylator phenotype.

Despite improved outcomes in the past 30 years, less than half of all women diagnosed with epithelial ovarian cancer live five years beyond their diagnosis. Although typically treated as a single disease, epithelial ovarian cancer includes several distinct histological subtypes, such as papillary serous and endometrioid carcinomas. To address whether the morphological differences seen in these carcinomas represent distinct characteristics at the molecular level we analyzed DNA methylation patterns in 11 papillary serous tumors, 9 endometrioid ovarian tumors, 4 normal fallopian tube samples and 6 normal endometrial tissues, plus 8 normal fallopian tube and 4 serous samples from TCGA. For comparison within the endometrioid subtype we added 6 primary uterine endometrioid tumors and 5 endometrioid metastases from uterus to ovary. Data was obtained from 27,578 CpG dinucleotides occurring in or near promoter regions of 14,495 genes. We identified 36 locations with significant increases or decreases in methylation in comparisons of serous tumors and normal fallopian tube samples. Moreover, unsupervised clustering techniques applied to all samples showed three major profiles comprising mostly normal samples, serous tumors, and endometrioid tumors including ovarian, uterine and metastatic origins. The clustering analysis identified 60 differentially methylated sites between the serous group and the normal group. An unrelated set of 25 serous tumors validated the reproducibility of the methylation patterns. In contrast, >1,000 genes were differentially methylated between endometrioid tumors and normal samples. This finding is consistent with a generalized regulatory disruption caused by a methylator phenotype. Through DNA methylation analyses we have identified genes with known roles in ovarian carcinoma etiology, whereas pathway analyses provided biological insight to the role of novel genes. Our finding of differences between serous and endometrioid ovarian tumors indicates that intervention strategies could be developed to specifically address subtypes of epithelial ovarian cancer.

High-resolution methylation analysis of the BRCA1 promoter in ovarian tumors.

Both hereditary and sporadic ovarian tumors frequently have decreased BRCA1 expression. One mechanism of downregulating BRCA1 expression is hypermethylation of the BRCA1 promoter. Studies have shown that the BRCA1 promoter is aberrantly hypermethylated in a subset of ovarian tumors, although the proportion varies widely between reports. High-resolution analysis of the BRCA1 promoter in ovarian cancer may provide information regarding the extent and heterogeneity of methylation and guide future studies using methylation-specific polymerase chain reaction (MS-PCR). We screened 50 primary epithelial ovarian tumors for BRCA1 promoter hypermethylation using MS-PCR. The BRCA1 promoter was hypermethylated in 16% (8 of 50) of the tumors, including two stage IA tumors. Sequence analysis of the promoter revealed that methylation of the CpG island is both extensive and mosaic in the methylated samples. Two CpG dinucleotides in the BRCA1 promoter, within and adjacent to a Myb consensus binding site, were most frequently methylated in ovarian tumors. BRCA1 expression was significantly lower in methylated than in unmethylated samples. Our analysis of the BRCA1 promoter revealed preferential methylation of specific CpG sites in ovarian tumors. This finding could be exploited in the design of highly sensitive MS-PCR assays for direct assessment of tumor DNA and potentially for early detection of ovarian cancer in body fluids.