Erskine, Courtney L.

Abstract Purpose: Folate receptor alpha (FR) is overexpressed in several cancers. Endogenous immunity to the FR has been demonstrated in patients and suggests the feasibility of targeting FR with vaccine or other immune therapies. CD4 helper T cells are central to the development of coordinated immunity, and prior work shows their importance in protecting against relapse. Our previous identification of degenerate HLA-class II epitopes from human FR led to the development of a broad coverage epitope pool potentially useful in augmenting antigen-specific immune responses in most patients. Patients and Methods: We conducted a phase I clinical trial testing safety and immunogenicity of this vaccine, enrolling patients with ovarian cancer or breast cancer who completed conventional treatment and who showed no evidence of disease. Patients were initially treated with low-dose cyclophosphamide and then vaccinated 6 times, monthly. Immunity and safety were examined during the vaccine period and up to 1 year later. Results: Vaccination was well tolerated in all patients. Vaccine elicited or augmented immunity in more than 90% of patients examined. Unlike recall immunity to tetanus toxoid (TT), FR T-cell responses developed slowly over the course of vaccination with a median time to maximal immunity in 5 months. Despite slow development of immunity, responsiveness appeared to persist for at least 12 months. Conclusions: The results demonstrate that it is safe to augment immunity to the FR tumor antigen, and the developed vaccine is testable for therapeutic activity in most patients whose tumors express FR, regardless of HLA genotype. Clin Cancer Res; 24(13); 3014–25. ©2018 AACR.

Tumor necrosis factor α antagonists are increasingly used to treat inflammatory and autoimmune disorders and are associated with increased risk of active tuberculosis. Diagnosis of active tuberculosis in patients taking tumor necrosis factor α antagonists can be challenging owing to increased incidence of extrapulmonary manifestations and false-negative results on current available diagnostic tests. We present a case of a young woman on infliximab for ulcerative colitis who presented with disseminated tuberculosis. As part of a research study, we performed flow cytometric immune profiling, which has previously not been reported in patients with active tuberculosis taking tumor necrosis α antagonists. The flow cytometry results were within the positive thresholds for tuberculosis infection. Flow cytometric immune profiling may be a valid diagnostic tool for patients taking tumor necrosis factor α antagonists.

Abstract The addition of trastuzumab to chemotherapy extends survival among patients with HER2+ breast cancer. Prior work showed that trastuzumab and chemotherapy augments HER2 extracellular domain (ECD)-specific antibodies. The current study investigated whether combination therapy induced immune responses beyond HER2-ECD and, importantly, whether those immune responses were associated with survival. Pretreatment and posttreatment sera were obtained from 48 women with metastatic HER2+ breast cancer on NCCTG (now Alliance for Clinical Trials in Oncology) studies, N0337 and N983252. IgG to HER2 intracellular domain (ICD), HER2-ECD, p53, IGFBP2, CEA, and tetanus toxoid were examined. Sera from 25 age-matched controls and 26 surgically resected HER2+ patients were also examined. Prior to therapy, some patients with metastatic disease had elevated antibodies to IGFBP2, p53, HER2-ICD, HER2-ECD, and CEA, but not to tetanus toxin, relative to controls and surgically resected patients. Treatment augmented antibody responses to HER2-ICD in 69% of metastatic patients, which was highly associated with improved progression-free survival (PFS; HR = 0.5, P = 0.0042) and overall survival (OS; HR = 0.7, P = 0.038). Augmented antibody responses to HER2-ICD also correlated (P = 0.03) with increased antibody responses to CEA, IGFBP2, and p53, indicating that treatment induces epitope spreading. Paradoxically, patients who already had high preexisting immunity to HER2-ICD did not respond to therapy with increased antibodies to HER2-ICD and demonstrated poorer PFS (HR = 1.6, P < 0.0001) and OS (HR = 1.4, P = 0.0006). Overall, the findings further demonstrate the importance of the adaptive immune system in the efficacy of trastuzumab-containing regimens. Cancer Res; 76(13); 3702–10. ©2016 AACR.

Abstract Rationale Most immunocompetent patients diagnosed with latent tuberculosis infection (LTBI) will not progress to tuberculosis (TB) reactivation. However, current diagnostic tools cannot reliably distinguish nonprogressing from progressing patients a priori, and thus LTBI therapy must be prescribed with suboptimal patient specificity. We hypothesized that LTBI diagnostics could be improved by generating immunomarker profiles capable of categorizing distinct patient subsets by a combinatorial immunoassay approach. Objectives A combinatorial immunoassay analysis was applied to identify potential immunomarker combinations that distinguish among unexposed subjects, untreated patients with LTBI, and treated patients with LTBI and to differentiate risk of reactivation. Methods IFN-γ release assay (IGRA) was combined with a flow cytometric assay that detects induction of CD25+CD134+ coexpression on TB antigen–stimulated T cells from peripheral blood. The combinatorial immunoassay analysis was based on receiver operating characteristic curves, technical cut-offs, 95% bivariate normal density ellipse prediction, and statistical analysis. Risk of reactivation was estimated with a prediction formula. Measurements and Main Results Sixty-five out of 150 subjects were included. The combinatorial immunoassay approach identified at least four different T-cell subsets. The representation of these immune phenotypes was more heterogeneous in untreated patients with LTBI than in treated patients with LTBI or unexposed groups. Patients with IGRA(+) CD4+CD25+CD134+ T-cell phenotypes had the highest estimated reactivation risk (4.11 ± 2.11%). Conclusions These findings suggest that immune phenotypes defined by combinatorial assays may potentially have a role in identifying those at risk of developing TB; this potential role is supported by risk of reactivation modeling. Prospective studies will be needed to test this novel approach.

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 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: 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 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.