Block, Matthew S.

Abstract Both genetic and epigenetic factors influence the development and progression of epithelial ovarian cancer (EOC). However, there is an incomplete understanding of the interrelationship between these factors and whether they interact to impact disease risk. Given that the development of ovarian cancer is complex, we aimed to identify DNA methylation marks that are candidates for mediating ovarian cancer genetic risk. We used 214 cases and 214 age-matched controls from the Mayo Clinic Ovarian Cancer Study. Pretreatment, blood-derived DNA was profiled for genome-wide methylation (Illumina Infinium HumanMethylation27 BeadArray) and single nucleotide polymorphisms (SNPs, Illumina Infinium HD Human610-Quad BeadArray). We employed a three-step filtering procedure, followed by the Causal Inference Test (CIT), to distinguish CpG sites that mediate genetic risk, from those that are consequential or independently acted on by genotype. Controlling for the estimated distribution of immune cells and other key covariates, we identified 1,993 out of 25,926 (7.7%) CpGs that were significantly differentially methylated between cases and controls (FDR, q < 0.05). The relationship between methylation and case-control status among these 1,993 CpGs was found to be highly consistent with the results of an independent study. Implementation of the CIT test revealed 13 CpG/SNP pairs, comprising 13 unique CpGs and 17 unique SNPs, which represent potential methylation-mediated relationships between genotype and EOC risk. These findings provide additional insight into EOC etiology and may serve as novel biomarkers for EOC susceptibility. Citation Format: Devin C. Koestler, Prabhakar Chalise, Mine S. Cicek, Julie M. Cunningham, Sebastian Armasu, Melissa C. Larson, Jeremy Chien, Matthew Block, Kimberly R. Kalli, Thomas A. Sellers, Ellen L. Goode, Brooke L. Fridley. Integrative genomic analysis identifies epigenetic marks that mediate genetic risk for epithelial ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 260. doi:10.1158/1538-7445.AM2014-260

Abstract Background: Ovarian cancer (OC) is the most common cause of death among gynecologic cancers. A key reason for the high lethality of OC is that early detection of OC is uncommon, and the majority of OC patients have advanced stage disease at diagnosis. Current non-invasive tests do not adequately distinguish benign from malignant adnexal masses. Diagnostic tests that reliably distinguish early stage OC from benign conditions may lead to earlier diagnosis of OC and improved survival. Methods: We designed a cohort study of plasma biomarkers in ovarian cancer patients. Specimens were analyzed from 100 patients with advanced OC (AJCC Stage III and IV), 50 patients with early stage OC (Stage I and II), and 50 patients with benign surgical conditions from the Mayo Ovarian SPORE Biospecimens Core. Presurgical plasma samples were assayed for multiple toll-like receptor agonists, cytokines, and vascular growth factors by ELISA and electrochemiluminescence. Biomarkers that were reliably detected in plasma were analyzed for association with OC. Differences in plasma biomarker levels between benign, early, and advanced OC patient groups were assessed using plate-adjusted logistic regression models. Results: Out of 23 biomarkers tested, 7 were excluded due to unreliable plasma detection. Of the remaining 16 biomarkers, 6_including interferon gamma (IFNγ), interleukin 6 (IL-6), IL-8, IL-10, tumor necrosis factor alpha (TNFα), and placental growth factor (PlGF)_were univariately associated with OC (all p<0.005), and one_IL-6_was associated with early stage OC (p < 0.0001). Heat shock protein 90kDa beta member 1 (HSP90B1, gp96) was associated with OC and early stage OC with borderline statistical significance (p = 0.039 and p = 0.026, respectively). However, when adjusted for cancer antigen 125 (CA-125), only HSP90B1 independently predicted OC (p = 0.008), as well as early stage OC (p = 0.014). Conclusions: The plasma cytokines IFNγ, IL-6, IL-8, IL-10, TNFα, and PlGF are associated with OC. However, after adjusting for CA-125, only HSP90B1 independently predicts OC, including early stage OC. These data warrant further investigation to determine whether measuring plasma HSP90B1 can aid in patient evaluation. Citation Format: Matthew S. Block, Matthew J. Maurer, Krista Goergen, Kimberly R. Kalli, Courtney L. Erskine, Marshall D. Behrens, Keith L. Knutson. Plasma heat shock protein 90kDa beta member 1 levels predict both early stage and advanced stage ovarian cancer independently from cancer antigen 125 in patients with an indeterminate adnexal mass. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1873. doi:10.1158/1538-7445.AM2014-1873

Abstract Edmonston vaccine strains of measles virus (MV) have significant antitumor activity in mouse xenograft models of ovarian cancer. MV engineered to express the sodium iodide symporter gene (MV-NIS) facilitates localization of viral gene expression and offers a tool for tumor radiovirotherapy. Here, we report results from a clinical evaluation of MV-NIS in patients with taxol- and platinum-resistant ovarian cancer. MV-NIS was given intraperitoneally every 4 weeks for up to 6 cycles. Treatment was well tolerated and associated with promising median overall survival in these patients with heavily pretreated ovarian cancer; no dose-limiting toxicity was observed in 16 patients treated at high-dose levels (108–109 TCID50), and their median overall survival of 26.5 months compared favorably with other contemporary series. MV receptor CD46 and nectin-4 expression was confirmed by immunohistochemistry in patient tumors. Sodium iodide symporter expression in patient tumors after treatment was confirmed in three patients by 123I uptake on SPECT/CTs and was associated with long progression-free survival. Immune monitoring posttreatment showed an increase in effector T cells recognizing the tumor antigens IGFBP2 and FRα, indicating that MV-NIS treatment triggered cellular immunity against the patients' tumor and suggesting that an immune mechanism mediating the observed antitumor effect. Our findings support further clinical evaluation of MV-NIS as an effective immunovirotherapy. Cancer Res; 75(1); 22–30. ©2014 AACR.

Abstract Background: Ovarian cancer (OC) is the leading cause of gynecologic cancer death in the United States. Survival in advanced OC is highly variable and is impacted by the endogenous anti-tumor immune response. Toll-like receptors (TLR) on tumor-infiltrating leukocytes can modulate anti-OC immunity. Heparan sulfate (HS) is an oligosaccharide component of the extracellular matrix that, when released, binds and triggers TLR-4 to induce production of multiple cytokines; HS is liberated from the extracellular matrix by heparanase, which is overexpressed by many ovarian cancers. We therefore explored the association between plasma levels of HS, heparanase, proinflammatory and immunosuppressive cytokines, and survival in patients with advanced OC. Methods: We designed a two stage cohort study of plasma biomarkers in ovarian cancer patients. For the pilot stage, we enrolled 100 patients with advanced OC (AJCC Stage III and IV), 50 patients with early stage OC (Stage I and II), and 50 patients with benign surgical conditions from the Mayo Ovarian SPORE Biospecimens Core. For the expansion stage, 257 patients with advanced OC were enrolled. Presurgical plasma samples were assayed for HS, heparanase, and multiple cytokines by ELISA and electrochemiluminescence. Differences in plasma levels between benign, early, and advanced OC patient groups were assessed using plate-adjusted linear models. Associations between plasma levels of HS and heparanase with overall survival in advanced OC patients were assessed by Cox proportional hazards models using a log2 transformation; total effects across stages were estimated using a meta-analysis approach. Results: In the pilot cohort, soluble HS levels were similar in control (mean +/ STD: 88.26 +/- 32.23), early stage (93.91 +/- 37.77), and advanced OC (93.55 +/- 40.44), with similar values observed in the expansion cohort (129.29 +/- 95.64). There was no correlation between heparanase and HS levels. Higher levels of HS were associated with improved overall survival (log2 HR = 0.62, p = 0.042; HR = 0.84, p = 0.014; HR = 0.82, 95% CI: 0.71-0.93, p = 0.003 for the pilot, expansion, and combined sets, respectively). There was no association between heparanase levels and overall survival (all p > 0.45). Conclusions: Neither soluble HS nor heparanase were elevated in presurgical OC patients relative to controls; however, higher levels of HS were associated with improved survival in patients with advanced OC. These results suggest that HS may play a key role in modulating the innate immune response to OC. Citation Format: Matthew S. Block, Matthew J. Maurer, Krista M. Goergen, Courtney L. Erskine, Marshall D. Behrens, Kimberly R. Kalli, Keith L. Knutson. Higher plasma levels of heparan sulfate are associated with improved survival of patients with advanced ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3476. doi:10.1158/1538-7445.AM2013-3476

Abstract Background: Current options to prevent ovarian and breast cancer recurrence are limited. However, signs of anti-tumor immunity are associated with improved survival in patients who have experienced a number of different malignancies. After identifying folate receptor alpha (FR)-derived class II-binding peptides recognized by lymphocytes from patients who previously had breast or ovarian cancer, we developed a multi-peptide vaccine with five peptides, each of which was recognized by more than 25% of the patients. We combined this vaccine with metronomic cyclophosphamide in an attempt to selectively reduce T regulatory cells in the periphery and the tumor microenvironment. Methods: We obtained an Investigational New Drug approval from the Food and Drug Administration to perform a phase I study in 22 patients who were currently in remission from their breast or ovarian cancer. Participants were required to be at least 90 days past any treatment for their disease (other than hormonal treatment, if applicable) and show no current evidence of disease. After 28 days of metronomic cyclophosphamide, six monthly intradermal injections of the vaccine were given. The primary endpoints of the trial are safety and immunogenicity. Results: To date, 15 of 22 patients have enrolled (7 breast and 8 ovarian cancer patients). 4 breast and 2 ovarian cancer patients are available for toxicity evaluation. No serious major adverse events have been observed, with the expected injection site reactions being the primary toxicity experienced by most participants; grade 2 decreased white blood cell counts were observed in 3 patients at completion of cyclophosphamide. Two patients went off study prior to completing all six vaccinations, one for vision changes, and one for disease progression. Immune monitoring is underway, with major assays being ELISAs for high-affinity IgG targeting the FR and FR-peptide specific ELIspots. These data will be presented at the meeting. Conclusions: Metronomic cyclophosphamide followed by intradermal vaccinations with a multi-peptide vaccine targeting the tumor antigen FR appears to be a tolerable regimen at this preliminary point of the study. With the exception of vision changes in one patient, no serious safety concerns have arisen. Pursuit of this strategy in further clinical study will require demonstration of FR-specific immune responses, an acceptable toxicity profile, and demonstration that T regulatory cells are altered in the periphery. Citation Format: Kimberly R. Kalli, Courtney L. Erskine, James W. Krempski, Linda M. Benson, Vera J. Suman, Douglas J. Padley, Allan B. Dietz, Timothy J. Hobday, Amy C. Degnim, Lynn C. Hartmann, Matthew S. Block, Keith L. Knutson. Metronomic cyclophosphamide followed by a multi-peptide folate receptor vaccine for ovarian and breast cancer immunotherapy in the setting of minimal residual disease - a feasibility study. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A82.

Abstract Under optimal Ag stimulation, CTL become functional effector and memory T cells. Professional APCs (pAPC) are considered essential for the activation of CTL, due to their unique capacity to provide costimulation and present exogenous Ags through MHC class I molecules. In this study, we report a novel means by which Th lymphocytes acquire and present MHC class I determinants to naive CTL. Although previous studies have looked at T cell Ag presentation to activated T cells, this study presents the first example of Ag presentation by Th cells to naive CTL. We report that activated Th cells can function as effective pAPC for CTL. Our results show that: 1) In addition to acquisition of cell surface molecules, including MHC class I/peptide complexes, from pAPC, Th cells can acquire and present MHC class I-binding peptides through TCR-MHC class II interactions with pAPC; 2) the acquired Ag can be functionally presented to CTL; and 3) Ag presentation by Th cells induces naive CTL to proliferate and preferentially differentiate into cells that phenotypically and functionally resemble central memory T cells. These findings suggest a novel role of Th cells as pAPC for the development of memory immune responses.

Abstract Peptide immunotherapy both activates and suppresses the T cell response against known peptide Ags. Although pretreatment with VP2121–130 peptide inhibits the development of antiviral CTL specific for the immunodominant Db:VP2121–130 epitope expressed during acute Theiler’s murine encephalomyelitis virus infection, i.v. injection of this same peptide or MHC tetramers containing the peptide during an ongoing antiviral CTL response results in a peptide-induced fatal syndrome (PIFS) within 48 h. Susceptibility to PIFS is dependent on peptide-specific CD8+ T cells, varies among inbred strains of mice, and is not mediated by traditionally defined mechanisms of shock. Analyses using bone marrow chimeras and mutant mice demonstrate that susceptibility to PIFS is determined by the genotype of bone marrow-derived cells and requires the expression of perforin. Animals responding to peptide treatment with PIFS develop classical stress responses in the brain. These findings raise important considerations for the development of peptide therapies for active diseases to modify immune responses involving expanded populations of T cells. In summary, treatment with peptides or MHC-tetramers during a peptide-specific immune response can result in a fatal shock-like syndrome. Susceptibility to the syndrome is genetically determined, is mediated by CD8+ T cells, and requires expression of perforin. These findings raise concerns about the use of peptides and MHC tetramers in therapeutic schemes.

Abstract Both CD8 and the TCR bind to MHC class I molecules during physiologic T cell activation. It has been shown that for optimal T cell activation to occur, CD8 must be able to bind the same class I molecule that is bound by the TCR. However, no direct evidence for the class I-dependent association of CD8 and the TCR has been demonstrated. Using fluorescence resonance energy transfer, we show directly that a single class I molecule causes TCR/CD8 interaction by serving as a docking molecule for both CD8 and the TCR. Furthermore, we show that CD3ε is brought into close proximity with CD8 upon TCR/CD8 association. These interactions are not dependent on the phosphorylation events characteristic of T cell activation. Thus, MHC class I molecules, by binding to both CD8 and the TCR, mediate the reorganization of T cell membrane components to promote cellular activation.

Abstract MHC class I molecules are highly polymorphic within populations. This diversity is thought to be the result of selective maintenance of new class I alleles formed by gene conversion. It has been proposed that rare alleles are maintained by their ability to confer resistance to common pathogens. Investigation has focused on differences in the presentation of foreign Ags by class I alleles, but the majority of peptides presented by class I molecules are self peptides used in shaping the naive T cell repertoire. We propose that the key substrate for the natural selection of class I gene conversion variants is the diversity in immune potential formed by new alleles. We show that T cells compete with each other for niches in the thymus and spleen during development, and that competition between different clones is dramatically affected by class I mutations. We also show that peripheral naive T cells proliferate preferentially in the presence of the class I variant that directed T cell development. The data argue that class I gene conversion mutations dramatically affect both the development and the maintenance of the naive CD8 T cell repertoire.

Abstract Natural selection drives diversification of MHC class I proteins, but the mechanism by which selection for polymorphism occurs is not known. New variant class I alleles differ from parental alleles both in the nature of the CD8 T cell repertoire formed and the ability to present pathogen-derived peptides. In the current study, we examined whether T cell repertoire differences, Ag presentation differences, or both account for differential viral resistance between mice bearing variant and parental alleles. We demonstrate that nonresponsive mice have inadequate presentation of viral Ag, but have T cell repertoires capable of mounting Ag-specific responses. Although previous work suggests a correlation between the ability to present an Ag and the ability to generate a repertoire responsive to that Ag, we show that the two functions of MHC class I are independent.