Kalli, Kimberly R.

Abstract Background: Ovarian cancer (OC) is commonly associated with ascites and predominantly remains in the peritoneal cavity. Soluble factors present in ascites such as growth factors and cytokines likely play a key role in local tumor behavior including stage, invasiveness and resectability and survival. Members of IL-12 family (IL-12p70, IL-23, IL-27 and IL-35) play a critical role in the regulation of inflammatory response. However, their role as local factors in ovarian cancer growth has not been well studied. We hypothesized that that the level of these important immunomodulatory and pro-inflammatory cytokines are associated with the clinical behavior of OC. Methods: Ascites fluid was collected and analyzed from women undergoing maximal debulking surgery for presumed OC, and relevant clinical endpoints were abstracted. Concentrations of IL-12 related cytokines (IL-23, IL-27, IL-35, and IL-12p70) were assessed by ELISA. Cytokines were also assessed on matching pre-surgery plasma samples. Cytokine concentrations between matched ascites and plasma samples were examined using scatter plots and Spearman correlations. Associations between ascites concentrations and clinical characteristics were assessed using Wilcoxon rank-sum tests. Associations between ascites levels with overall survival in newly diagnosed invasive OC patients were assessed by Cox proportional hazards models using a log2 transformation. Results: Ascites fluid was analyzed from 20 patients with primary epithelial OC including 18 serous histology, 19 patients with stage III or IV disease: 16 (80%) were optimally debulked. We observed that higher levels of IL-12p70 were associated with locally invasive disease including higher rates of sub-optimal debulking (p=0.0074) and stage IV disease (p=0.012). For other IL-12 family members we observed similar associations between higher cytokine levels and suboptimal disease and stage IV disease including IL-23 (p=0.73, p=0.17), IL-27 (p=0.041, p=0.018), and IL35 (p=0.12, p=0.11), respectively. In addition, increase in IL-23 was associated with worse overall survival (log2 HR = 1.85, p=0.031). Conclusions: Higher levels of IL-12 family of cytokines in ascitic fluid are associated with several surrogates of biological aggressiveness in OC including stage, resectability and survival. Given the importance of local tumor environment on clinical behavior, a detailed understanding of IL-12 pathway may help in developing novel therapeutic strategies to treat OC. Citation Format: Eati Basal, Mathew S. Block, Matthew J. Maurer, Krista Goergen, Courtney L. Erskine, Marshall Behrens, Kimberly R. Kalli, Keith Knutson, William Cliby. Levels of IL-12 family cytokines in ascitic fluid are associated with extent and resectability of ovarian cancer and correlate with overall 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 1434. doi:10.1158/1538-7445.AM2013-1434

Abstract Purpose: The therapeutic potential of PARP inhibitors is predicted to extend beyond BRCA mutant phenotypes to homologous recombination (HR)-deficient cancers. Response of individual ovarian cancers to niraparib was characterized using treatment naïve, patient-derived tumorgraft models and correlated with predicted HR status and gene expression profile. Methods: Utilizing a living tumor bank of patient-derived ovarian xenografts (PDXs), niraparib (MK-4827) was evaluated in five high-grade ovarian cancer tumorgrafts (PDX077, PDX087, PDX039, PDX080 and PDX95) as monotherapy, combination with carboplatin ± paclitaxel and maintenance therapy. Intraperitoneal tumorgrafts were monitored for tumor growth with twice-weekly transabdominal ultrasound imaging. In vivo response to niraparib was correlated to massively parallel sequencing data (BROCA) for HR genes and with each source patient's platinum response. Tumorgrafts were accordingly classified into predicted HR-proficient and HR-deficient phenotypes. PARP1 and poly(ADP-ribose) polymer (pADPr) expression as well as gene expression by DNA microarray were compared between treatment and control tumor replicates, and HR-deficient vs. HR-proficient tumors. Results: PDX077 (verified to harbor a somatic BRCA2 mutation (predicted HR deficient)) and PDX039 without any HR mutations detected by BROCA (predicted HR proficient) both responded to niraparib monotherapy and combination therapy in vivo (p=0.002 and 0.009 for treated vs. control groups, respectively). Significant growth attenuation, but not regression, in response to single agent treatment was demonstrated in PDX080 (CDK12 mutation; predicted HR-deficient; p=0.02 vs. control). In comparison, a BRCA2-mutant model (PDX095) failed to respond to niraparib monotherapy (p=0.97). As predicted, niraparib was not active as a single agent or in combination with chemotherapy in a BRCA wild-type, predicted HR-proficient tumorgraft (PDX087; p=not significant). Niraparib enhanced tumor regression with carboplatin but not with paclitaxel/carboplatin in an HR-deficient tumorgraft (PDX077), and maintenance niraparib therapy was effective in prolonging recurrence-free survival. pADPr inhibition was demonstrated in all niraparib-treated tumorgrafts, while PARP1 degrada-tion was evident in tumorgrafts responsive to single agent niraparib. Differential gene expression was seen in niraparib treated vs. control tumors in HR-deficient tumorgrafts in comparison to HR-proficient tumors at the end of 4 weeks of treatment. Clustering of 6 most/least differentially expressed genes was predictive of response to niraparib. Furthermore, RAD51C and PTEN gene expression were diminished in PDX039 (HR proficient by BROCA; niraparib responsive), suggesting a possible mechanism for its sensitivity to niraparib. Conclusion: Response to niraparib was seen in both BRCA-mutant and BRCA-wildtype xenografts. Thus, BRCA or HR gene mutational status alone was not sufficient to predict response to niraparib. Low RAD51C, PTEN and loss of PARP1 expression following treatment were most characteristic of tumors responding to niraparib. Paclitaxel may be detrimental to the sensitizing effects of PARP inhibition on carboplatin, which may have clinical implications for the incorporation of PARP inhibitors in front-line treatment. Our data are supportive of the phase III study of niraparib as maintenance therapy in patients with platinum-sensitive ovarian cancer. This abstract is also presented as Poster A30. Citation Format: Mariam Al Hilli, S. John Weroha, Marc Becker, Xiaonan Hou, Sean Harrington, Sarah McKinstrey, Kimberly Kalli, Karin Goodman, Keith Wilcoxen, Elizabeth Swisher, Scott Kaufmann, Paul Haluska. In vivo antitumor activity of the PARP inhibitor niraparib (MK-4827) in homologous recombination deficient and proficient ovarian cancer. [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 PR05.

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 Survival in epithelial ovarian cancer (EOC) is influenced by the host immune response, yet the key genetic determinants of inflammation and immunity that affect prognosis are not known. The nuclear factor-κB (NF-κB) transcription factor family plays an important role in many immune and inflammatory responses, including the response to cancer. We studied common inherited variation in 210 genes in the NF-κB family in 10,084 patients with invasive EOC (5,248 high-grade serous, 1,452 endometrioid, 795 clear cell, and 661 mucinous) from the Ovarian Cancer Association Consortium. Associations between genotype and overall survival were assessed using Cox regression for all patients and by major histology, adjusting for known prognostic factors and correcting for multiple testing (threshold for statistical significance, P < 2.5 × 10−5). Results were statistically significant when assessed for patients of a single histology. Key associations were with caspase recruitment domain family, member 11 (CARD11) rs41324349 in patients with mucinous EOC [HR, 1.82; 95% confidence interval (CI), 1.41–2.35; P = 4.13 × 10−6] and tumor necrosis factor receptor superfamily, member 13B (TNFRSF13B) rs7501462 in patients with endometrioid EOC (HR, 0.68; 95% CI, 0.56–0.82; P = 2.33 × 10−5). Other associations of note included TNF receptor–associated factor 2 (TRAF2) rs17250239 in patients with high-grade serous EOC (HR, 0.84; 95% CI, 0.77–0.92; P = 6.49 × 10−5) and phospholipase C, gamma 1 (PLCG1) rs11696662 in patients with clear cell EOC (HR, 0.43; 95% CI, 0.26–0.73; P = 4.56 × 10−4). These associations highlight the potential importance of genes associated with host inflammation and immunity in modulating clinical outcomes in distinct EOC histologies. Cancer Epidemiol Biomarkers Prev; 23(7); 1421–7. ©2014 AACR.

Abstract To reveal biologic mechanisms underlying clinical outcome of high-grade serous (HGS) epithelial ovarian carcinomas (EOC), we evaluated the association between tumor epigenetic changes and time to recurrence (TTR). We assessed methylation at approximately 450,000 genome-wide CpGs in tumors of 337 Mayo Clinic (Rochester, MN) patients. Semi-supervised clustering of discovery (n = 168) and validation (n = 169) sets was used to determine clinically relevant methylation classes. Clustering identified two methylation classes based on 60 informative CpGs, which differed in TTR in the validation set [R vs. L class, P = 2.9 × 10−3, HR = 0.52; 95% confidence interval (CI), 0.34–0.80]. Follow-up analyses considered genome-wide tumor mRNA expression (n = 104) and CD8 T-cell infiltration (n = 89) in patient subsets. Hypomethylation of CpGs located in 6p21.3 in the R class associated with cis upregulation of genes enriched in immune response processes (TAP1, PSMB8, PSMB9, HLA-DQB1, HLA-DQB2, HLA-DMA, and HLA-DOA), increased CD8 T-cell tumor infiltration (P = 7.6 × 10−5), and trans-regulation of genes in immune-related pathways (P = 1.6 × 10−32). This is the most comprehensive assessment of clinical outcomes with regard to epithelial ovarian carcinoma tumor methylation to date. Collectively, these results suggest that an epigenetically mediated immune response is a predictor of recurrence and, possibly, treatment response for HGS EOC. Cancer Res; 74(11); 3084–91. ©2014 AACR.

Abstract Immunosuppression in the tumor microenvironment blunts vaccine-induced immune effectors. PD-1/B7-H1 is an important inhibitory axis in the tumor microenvironment. Our goal in this study was to determine the effect of blocking this inhibitory axis during and following vaccination against breast cancer. We observed that using anti-PD-1 antibody and a multipeptide vaccine (consisting of immunogenic peptides derived from breast cancer antigens, neu, legumain, and β-catenin) as a combination therapy regimen for the treatment of breast cancer–bearing mice prolonged the vaccine-induced progression-free survival period. This prolonged survival was associated with increase in number of Tc1 and Tc2 CD8 T cells with memory precursor phenotype, CD27+IL-7RhiT-betlo, and decrease in number of PD-1+ dendritic cells (DC) in regressing tumors and enhanced antigen reactivity of tumor-infiltrating CD8 T cells. It was also observed that blockade of PD-1 on tumor DCs enhanced IL-7R expression on CD8 T cells. Taken together, our results suggest that PD-1 blockade enhances breast cancer vaccine efficacy by altering both CD8 T cell and DC components of the tumor microenvironment. Given the recent success of anti-PD-1 monotherapy, our results are encouraging for developing combination therapies for the treatment of patients with cancer in which anti-PD-1 monotherapy alone may be ineffective (i.e., PD-L1–negative tumors). Cancer Res; 74(11); 2974–85. ©2014 AACR.

Abstract The presence of regulatory T cells (Treg) in solid tumors is known to play a role in patient survival in ovarian cancer and other malignancies. We assessed inherited genetic variations via 749 tag single-nucleotide polymorphisms (SNP) 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. We analyzed genotype and overall survival in 10,084 women with invasive epithelial ovarian cancer, including 5,248 high-grade serous, 1,452 endometrioid, 795 clear cell, and 661 mucinous carcinoma cases of European descent across 28 studies from the Ovarian Cancer Association Consortium (OCAC). The strongest associations were found for endometrioid carcinoma and IL2RA SNPs rs11256497 [HR, 1.42; 95% confidence interval (CI), 1.22–1.64; P = 5.7 × 10−6], rs791587 (HR, 1.36; 95% CI, 1.17–1.57; P = 6.2 × 10−5), rs2476491 (HR, = 1.40; 95% CI, 1.19–1.64; P = 5.6 × 10−5), and rs10795763 (HR, 1.35; 95% CI, 1.17–1.57; P = 7.9 × 10−5), and for clear cell carcinoma and CTLA4 SNP rs231775 (HR, 0.67; 95% CI, 0.54–0.82; P = 9.3 × 10−5) after adjustment for age, study site, population stratification, stage, grade, and oral contraceptive use. The rs231775 allele associated with improved survival in our study also results in an amino acid change in CTLA4 and previously has been reported to be associated with autoimmune conditions. Thus, we found evidence that SNPs in genes related to Tregs seem to play a role in ovarian cancer survival, particularly in patients with clear cell and endometrioid epithelial ovarian cancer. Cancer Immunol Res; 2(4); 332–40. ©2014 AACR.

Abstract Purpose: Ovarian cancer has a high recurrence and mortality rate. A barrier to improved outcomes includes a lack of accurate models for preclinical testing of novel therapeutics. Experimental Design: Clinically relevant, patient-derived tumorgraft models were generated from sequential patients and the first 168 engrafted models are described. Fresh ovarian, primary peritoneal, and fallopian tube carcinomas were collected at the time of debulking surgery and injected intraperitoneally into severe combined immunodeficient mice. Results: Tumorgrafts demonstrated a 74% engraftment rate with microscopic fidelity of primary tumor characteristics. Low-passage tumorgrafts also showed comparable genomic aberrations with the corresponding primary tumor and exhibit gene set enrichment of multiple ovarian cancer molecular subtypes, similar to patient tumors. Importantly, each of these tumorgraft models is annotated with clinical data and for those that have been tested, response to platinum chemotherapy correlates with the source patient. Conclusions: Presented herein is the largest known living tumor bank of patient-derived, ovarian tumorgraft models that can be applied to the development of personalized cancer treatment. Clin Cancer Res; 20(5); 1288–97. ©2014 AACR.

Abstract Ovarian cancer is a clinically and molecularly heterogeneous disease. The driving forces behind this variability are unknown. Here, we report wide variation in the expression of the DNA cytosine deaminase APOBEC3B, with elevated expression in the majority of ovarian cancer cell lines (three SDs above the mean of normal ovarian surface epithelial cells) and high-grade primary ovarian cancers. APOBEC3B is active in the nucleus of several ovarian cancer cell lines and elicits a biochemical preference for deamination of cytosines in 5′-TC dinucleotides. Importantly, examination of whole-genome sequence from 16 ovarian cancers reveals that APOBEC3B expression correlates with total mutation load as well as elevated levels of transversion mutations. In particular, high APOBEC3B expression correlates with C-to-A and C-to-G transversion mutations within 5′-TC dinucleotide motifs in early-stage high-grade serous ovarian cancer genomes, suggesting that APOBEC3B-catalyzed genomic uracil lesions are further processed by downstream DNA "repair" enzymes including error-prone translesion polymerases. These data identify a potential role for APOBEC3B in serous ovarian cancer genomic instability. Cancer Res; 73(24); 7222–31. ©2013 AACR.