Kalli, Kimberly R.

Abstract Purpose: It is well known that the immune system impacts the clinical course of ovarian cancer suggesting that immune-based approaches may effective in treating the disease. This has led to several clinical trials of novel treatments such as antibody, vaccine, and adoptive T cell therapy. PD-1:PD-L1 axis is a major immune regulatory pathway that blunts immune effectors in the tumor microenvironment. It is well known that high surface expression of PD-1 on tumor infiltrating T cells is a sign of their exhaustion. However, the complete mechanism behind the PD-1 regulation of T cell exhaustion is yet to be explored. Recently we identified that PD-1 is also expressed on ovarian tumor-infiltrating myeloid dendritic cells (DCs) that exhibit an immunosuppressive phenotype. Our goal in this study was to understand the mechanism by which PD-1 mediates the paralysis of ovarian cancer infiltrating DCs. Experimental procedures: PD-1+ DCs obtained from ID8 mouse model of ovarian cancer were used in this study. Data from this preclinical model was confirmed by using myeloid DCs obtained from ovarian cancer patient’s samples. Using standard flow cytometry, immunoassays (ELISA, multiplexed cytokine assay), PCR array, western blot and confocal microscopy, PD-1 mediated paralysis of ovarian cancer DCs was determined. Results: Our data shows that blockade of surface PD-1 on ovarian cancer infiltrating DCs results in their activation by, enhancing their surface co-stimulatory molecules expression, increasing their motility, enhancing their antigen presentation capacity, and increasing their production of immunostimulatory cytokines. Also, our results suggest that PD-1 expressed on tumor DCs mediates the suppression of NFkB which occurs through SHP-2 and IKK dependent mechanisms. Conclusion: Our study reveals that blockade of PD-1 on ovarian cancer infiltrating DCs results in the reversal of their paralysis to such an extent that it overrides their B7-H1 mediated suppression of T cells and this occurs through the reversal of PD-1 mediated tonic suppression of NFkB. Citation Format: Lavakumar Karyampudi James Krempski, Purushottam Lamichhane, Kimberly R. Kalli, Marshall D. Behrens, Doris M. Vargas, Lynn C Hartmann, Karen E Hedin, Ellen L. Goode, Keith L. Knutson. PD-1 mediated paralysis of ovarian cancer infiltrating dendritic cells [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-THER-1418.

Abstract Background: Epithelial ovarian cancer (EOC) is composed of five major histologic types: 1) high-grade serous carcinoma (HGSC), accounting for most cases (∼70%); and the rarer 2) clear cell, 3) endometrioid, 4) mucinous, and 5) low- grade serous carcinoma (LGSC). As EOC risk factors are histology specific, as are the site of precursor lesions, accurate subtyping is critical to understanding EOC prognostic factors and etiology. Our aim was to compare two currently employed histology assignment strategies using gene expression, DNA methylation, and clinical outcome data Methods: Histology assignment based on: a) pathologist, and b) an integrated pathologist and IHC prediction algorithm (pathIHC) (using ARID1A, CDKN2A, DKK1, HNF1B, MDM2, PGR, TP53, TFF3, VIM, and WT1 staining patterns), was compared for tumors of all histologies at the Mayo Clinic, Rochester. The 500 most variable probes from Illumina Methylation450 BeadChips (N = 259) and Agilent 4×44K expression arrays (N = 245) were used to perform unsupervised hierarchical clustering. Fisher's Exact test was used to test the association of clusters with histology. Cox proportional hazards regression analysis was used to test the association of clusters and histology with time to progression (TTP) and time to death (TTD). Results: Eighty-two percent of tumors were concordant between histology assignment strategies. Clustering based on methylation data produced two distinct clusters. PathIHC produced more homogenous clusters (p-value = 2.8×10-16) than pathology alone (p = 1.3×10-9). Cluster M1, characterized by high levels of methylation across almost all probes, was enriched for the rarer EOC subtypes; cluster M2, characterized by moderate levels of methylation across 50%-75% of probes, was predominantly HGSC. These patterns were observed irrespective of histology assignment strategy; however, when using histology by pathologist, cluster M2 had more endometrioid and LGSC tumors interspersed with HGSC tumors. Clustering based on expression data also produced two clusters. PathIHC produced slightly more homogenous clusters (p = 9.0×10-11, pathology alone, p = 2.3×10-10). Cluster E1 was enriched for the rarer EOC subtypes; cluster E2 was primarily serous lineage tumours (HGSC and LGSC), particularly when using histology by pathIHC. Considering only clinical outcomes, histologies by pathology were more significantly different in terms of TTP (p = 1.1×10-6) and TTD (p = 1.7×10-3) than histologies by pathIHC (TTP, p = 5.4×10-6; TTD, p = 5.7×10-3). Conclusions: Histology by pathIHC produced more homogeneous clusters in both the methylation and expression data; thus, molecular data supports this strategy. Differences in clinical outcomes (TTP and TTD) between histology groups were more pronounced when using assignment by pathologist; thus, clinical data supports this strategy. Citation Format: M A. Earp, S J. Winham, S M. Armasu, B L. Fridley, M C. Larson, Z C. Fogarty, K R. Kalli, C Wang, G L. Keeney, J M. Cunningham, S Ramus, M Kobel, E L. Goode. Comparison of pathology versus IHC-based ovarian carcinoma histology assignment using gene expression, DNA methylation, and clinical outcome data. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4246. doi:10.1158/1538-7445.AM2015-4246

Abstract Ovarian cancer is the most lethal gynecologic malignancy and the fifth leading cause of cancer deaths among women. The current standard of care post surgical cytoreduction is combination platinum/taxane chemotherapy. The initial response varies widely; subsets of carcinomas demonstrate resistance or sensitivity from the onset. The underlying cause of this response heterogeneity remains incompletely understood. Patient-derived xenografts (PDX) serve as useful in vivo models to study molecular response markers and test the efficacy of targeted therapies. Our group has demonstrated a high engraftment rate (>70%) of ovarian cancer PDXs (Avatars) by injecting treatment naïve patient tumor directly into the peritoneal cavity of an in immunocompromised mouse host in an effort to better mimic the anatomic context by which ovarian cancer naturally develops. We now have 328 engrafted Avatar models representing the wide range of ovarian cancer subtypes, including serous (65%), mixed epithelial (10%), endometrioid (7%), clear cell (4%), mucinous (3%), carcinosarcoma (1%) and other (10%). Using these models, we are performing a massively parallel sequencing strategy referred to as BROCA-HR to detect all mutation classes (e.g. gene rearrangements, copy number variations, etc.) and gene aberrations within the Fanconi Anemia-BRCA homologous recombination (HR), non-homologous end joining (NHEJ), PTEN, and DNA Mismatch repair pathways. To date, 148 Avatar models have undergone BROCA sequencing. Observed deleterious mutations included 12 BRCA1 (8%), 6 BRCA2 (4%), and 4 PIK3CA (3%); additional loss of function mutations were also evident in the following genes: ATM, RAD51C, FANCM, FANCD2, FANCA, CHEK2, PALB2, MHS6, CDK12 and GEN1. These models are representative of mutations seen in the TCGA mutation set. Models harboring said mutations are currently being tested with platinum agents and/or PARP inhibitors. Taken together, the Avatars represent a high throughput model system for pre-clinical testing in vivo that effectively recapitulates ovarian cancer (e.g. mutations in BRCA1, BRCA2 and other DNA damage response proteins). We have been able to establish avatars at a high rate, including those with mutations in BRCA1, BRCA2 and other DNA damage response genes. These models will be a unique resource for future studies of biomarkers and novel therapeutic approaches. This work is funded by the Ovarian Cancer Research Fund (750563), Mayo Ovarian SPORE (CA136393), National Cancer Institute (RO1 CA184502). Note: This abstract was not presented at the meeting. Citation Format: Kunal A. Lodhia, Marc A. Becker, Xiaonan Hou, Kimberly R. Kalli, Maria I. Harrell, Elizabeth M. Swisher, John S. Weroha, Paul Haluska. Characterization of 148 ovarian cancer tumografts (Avatars) using BROCA-HR deep sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1474. doi:10.1158/1538-7445.AM2015-1474

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 Tumor infiltrating regulatory T cells (Tregs) appear to play a role in survival in a number of solid tumors, including ovarian cancer. We assessed genetic variation via tag SNPs (N=749) in 25 Treg associated genes (CD28, CTLA4, FOXP3, IDO1, IL10, IL10RA, IL15, 1L17RA, IL23A, IL23R, IL2RA, IL6, IL6R, IL8, LGALS1, LGALS9, MAP3K8, STAT5A, STAT5B, TGFB1, TGFB2, TGFB3, TGFBR1, TGRBR2, and TGFBR3) in relation to ovarian cancer survival. SNPs were selected based on r2 ≥ 0.8, MAF ≥ 0.05, 5 kb upstream and downstream from the gene. We had SNP and outcome data for ovarian cancer from 10,084 combined ovarian cancer cases, including 5,248 high grade serous, 1,452 endometrioid, 661 mucinous, and 795 clear cell cases of European descent across multiple studies from the Ovarian Cancer Association Consortium (OCAC). Cox proportional hazards regression modeling was used to estimate per-allele hazard ratios (HRs) and 95% confidence intervals (CIs) for associations with overall survival, adjusting for age at diagnosis, population substructure PCs, study site, histology (for the all case analysis), tumor stage, tumor grade and oral contraceptive use. The strongest associations were found for endometrioid histologic subtype and IL2RA SNPs (maximum r2=0.67) rs11256497 [HR=1.42, 95% CI: 1.22-1.64; p=5.7 x 10−6], rs791587 [HR=1.36, 95% CI: 1.17-1.57; p=6.2 x 10−5], rs2476491 [HR=1.40, 95% CI: 1.19-1.64; p=5.6 x 10−5], and rs10795763 [HR=1.35, 95% CI: 1.17-1.57; p=7.9 x 10−5], as well as clear cell ovarian cancer and CTLA4 SNP rs231775 [HR=0.67, 95% CI: 0.54-0.82; p=9.3 x 10−5]. No other associations were observed at p<1 x 10−4. Overall, this study provided evidence that variation in genes related to Tregs, particularly IL2RA and CTLA4, influence ovarian cancer survival. Citation Format: Bridget Charbonneau, Keith Knutson, Robert A. Vierkant, Julie M. Cunningham, Zachary C. Fogarty, Kimberly R. Kalli, Matthew J. Maurer, Ann L. Oberg, Brooke L. Fridley, Claudia C. Preston, Paul D.P. Pharoah, Susan J. Ramus, Catherine M. Phelan, Kunle Odunsi, Kirsten Moysich, Ellen L. Goode. Polymorphisms in regulatory T cell related genes and ovarian cancer survival. [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 4844. doi:10.1158/1538-7445.AM2013-4844

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