Knutson, Keith L.

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 Patients with HER2+ breast cancer treated with trastuzumab and chemotherapy have superior survival compared with patients treated with chemotherapy alone. Polymorphisms within FCGR2A and FCGR3A are associated with binding affinity of natural killer cells to the IgG1 portion of trastuzumab, and a polymorphism in FCGR2B (I232T) is associated with impaired regulatory activity. The association of these polymorphisms with clinical response among trastuzumab-treated patients is equivocal, with both positive and negative associations. We performed genotyping analysis on the FCGR3A V158F, FCGR2A R131H, and FCGR2B I232T polymorphisms in 1,325 patients from the N9831 clinical trial. Patients in arm A (N = 419) received chemotherapy only. Patients in arms B (N = 469) and C (N = 437) were treated with chemotherapy and trastuzumab (sequentially in arm B and concurrently in arm C). Using log-rank test and Cox proportional hazard models, we compared disease-free survival (DFS) among genotypic groups within pooled arms B/C. We found no differences in DFS between trastuzumab-treated patients who had the FCGR3A 158 V/V and/or FCGR2A 131 H/H high-affinity genotypes and patients without those genotypes. Furthermore, there was no significant interaction between FCGR3A and FCGR2A and treatment. However, there was a difference in DFS for FCGR2B I232T, with I/I patients deriving benefit from trastuzumab (P < 0.001), compared with the T carriers who did not (P = 0.81). The interaction between FCGR2B genotype and treatment was statistically significant (P = 0.03). Our analysis did not reveal an association between FcγR high-affinity genotypes and outcomes. However, it seems that the FCGR2B inhibitory gene may be predictive of adjuvant trastuzumab benefit. Cancer Immunol Res; 2(10); 962–9. ©2014 AACR.

Abstract Tumors evade both natural and pharmacologically induced (e.g., vaccines) immunity by a variety of mechanisms, including induction of tolerance and immunoediting. Immunoediting results in reshaping the immunogenicity of the tumor, which can be accompanied by loss of Ag expression and MHC molecules. In this study, we evaluated immunoediting in the neu-transgenic mouse model of breast cancer. A tumor cell line that retained expression of rat neu was generated from a spontaneous tumor of the neu-transgenic mouse and, when injected into the non-transgenic parental FVB/N mouse, resulted in the development of a strong immune response, initial rejection, and ultimately the emergence of neu Ag-loss variants. Morphologic and microarray data revealed that the immunoedited tumor cells underwent epithelial to mesenchymal transition accompanied by an up-regulation of invasion factors and increased invasiveness characteristic of mesenchymal tumor cells. These results suggest that immunoediting of tumor results in cellular reprogramming may be accompanied by alterations in tumor characteristics including increased invasive potential. Understanding the mechanisms by which tumors are immunoedited will likely lead to a better understanding of how tumors evade immune detection.

Abstract Studies in cancer patients have suggested that breast tumors recruit regulatory T cells (Tregs) into the tumor microenvironment. The extent to which local Tregs suppress antitumor immunity in breast cancer is unknown. We questioned whether inhibiting systemic Tregs with an IL-2 immunotoxin in a model of neu-mediated breast cancer, the neu-transgenic mouse, could impact disease progression and survival. As in human breast cancer, cancers that develop in these mice attract Tregs into the tumor microenvironment to levels of ∼10–25% of the total CD4+ T cells. To examine the role of Tregs in blocking immune-mediated rejection of tumor, we depleted CD4+CD25+ T cells with an IL-2 immunotoxin. The treatment depleted Tregs without concomitant lymphopenia and markedly inhibited tumor growth. Depletion of Tregs resulted in a persistent antitumor response that was maintained over a month after the last treatment. The clinical response was immune-mediated because adoptive transfer of Tregs led to a complete abrogation of the therapeutic effects of immunotoxin treatment. Further, Treg down-modulation was accompanied by increased Ag-specific immunity against the neu protein, a self Ag. These results suggest that Tregs play a major role in preventing an effective endogenous immune response against breast cancer and that depletion of Tregs, without any additional immunotherapy, may mediate a significant antitumor response.

Abstract Dendritic cells (DC) are important regulators of T cell immunity. The degree of stimulation, the pattern of costimulatory molecules expressed, and the cytokines secreted by DC dictate the nature of the effector and memory cells generated, particularly with respect to their Th1 or Th2 phenotypes. In this study, we demonstrate that the addition of activated DC to spleen cultures containing established Th2-polarized CD4+ T cells was sufficient to suppress Th2 and induce Th1 cytokines in a recall response, a phenomenon referred to as phenotype reversal. The ability of activated DC to induce phenotype reversal displayed exquisite Ag specificity. The DC activator B7-DC cross-linking Ab (XAb) was >10,000-fold more efficient at inducing phenotype reversal than the TLR agonists CpG-oligodeoxynucleotide and Gardiquimod. Characterization of the mechanisms governing phenotype reversal revealed the requirement for cognate interaction between the TCR:peptide-MHC complex, the expression of the costimulation/adhesion molecule ICAM-1, and secretion of IL-12 and IFN-γ by the activated DC. The requirement for the costimulation/adhesion molecule SLAM (signaling lymphocytic activation molecule) was found to be quantitative. Thus, activation of DC, particularly by crosslinking B7-DC, can modulate well-established Th2 T cell responses in an Ag-specific manner. Because the regulation of mouse and human DC by B7-DC XAb overlaps in several significant ways, immune modulation with B7-DC XAb is a potential strategy for treating Th2-mediated diseases.

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 Background: Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer-related death in females and leading gynecologic cause of cancer-related death. Despite the identification of a number of serum biomarkers, methods to identify early-stage disease and predict prognosis remain scarce. We have evaluated two biologically connected serum biomarkers, serum leukocyte protease inhibitor (SLPI) and progranulin (PGRN). Methods: Two-hundred frozen plasma samples were acquired from the Mayo Clinic Biospecimen Repository for Ovarian Cancer Research. Samples were obtained from 50 patients with benign conditions, 50 with American Joint Committee on Cancer (AJCC) stage I and II EOC, and 100 with AJCC stage III and IV EOC. Samples were obtained before surgical resection of a mass and were analyzed for absolute levels of SLPI and PGRN using ELISA assays. Receiver-operator characteristic curves were generated for SLPI and PGRN. Median follow-up was 48 months. Results: Absolute levels of SLPI were significantly elevated in patients with EOC compared with benign disease and predicted the presence of EOC (AUC of 0.812; P = 0.04); SLPI remained elevated in the subset of patients with normal CA-125. PGRN levels were not significantly increased in patients with early-stage or late-stage EOC as a whole, but an increase in PGRN levels was associated with decreased overall survival in advanced EOC. Conclusions: SLPI levels are elevated in EOC, and SLPI shows promise as a diagnostic biomarker for patients with both elevated and normal CA-125 levels. An increase in PGRN is associated with decreased overall survival. Impact: SLPI is elevated in EOC and warrants investigation in a screening study in women at risk for EOC. Cancer Epidemiol Biomarkers Prev; 22(10); 1730–5. ©2013 AACR.