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Introduction: The immunomodulatory effects of dexamethasone and prednisone have been a staple of chemotherapy regimens since Mustargen, Oncovin, procarbazine, prednisone (MOPP) was used to treat lymphomas in 1963. Since then steroids have become ubiquitous in chemotherapy and were transitioned to an anti-nausea medication in solid tumor therapies and now anti-inflammatory therapies for common side effects of immune checkpoint inhibitors. While some modern immunomodulator therapies like rituximab and mycophenolate mofetil work through specific mechanisms, steroids have a much more non-specific effect working through a wide variety of pathways. Traditionally, chemotherapies such as paclitaxel, doxorubicin, and cyclophosphamide have been thought to induce cell death purely through cell-autonomous mechanisms such as DNA damage or interference with accurate chromosome segregation during mitosis. More recently, the strong correlation between tumor-infiltrating lymphocytes (TILs) and pathological complete response (pCR) to chemotherapy has been demonstrated. We hypothesized that dexamethasone use may adversely affect the efficacy of chemotherapy due to down-regulation of TILs. Therefore, we investigated the effects of dexamethasone exposure levels on response rates to neoadjuvant therapy of triple negative breast cancer (TNBC) with doxorubicin, cyclophosphamide and paclitaxel. We evaluated TNBC due to its high sensitivity to chemotherapeutic agents in the neoadjuvant setting and because pCR following neoadjuvant chemotherapy is an indicator of better long-term outcomes in TNBC patients. Dexamethasone use as an anti-emetic in the neo-adjuvant setting is highly variable, often being substituted for with olanzapine. Methods: All patients with TNBC who received neo-adjuvant chemotherapy with doxorubicin and cyclophosphamide (AC) between January 1st, 2012 and November 31st, 2018 at The James Comprehensive Cancer Center at The Ohio State University were included in this retrospective study, which totaled 174 patients. We omitted patients who received carboplatin with the paclitaxel, or received other experimental therapies during the neoadjuvant period. The primary exposure was dexamethasone dose by chemotherapy cycle, and the primary outcome was pCR. We used logistic regression to perform an intent-to-treat analysis, and defined P Results: We have found that there is no statistical or apparent difference in pCR by average dexamethasone dose per neoadjuvant chemotherapy cycle (P = 0.51), including when adjusting for diabetes status, metformin prescription and age (P=0.85). We found that there was sufficient variation in dexamethasone dose per cycle with the main mass of observations between 10 and 60 mg/cycle. Conclusions: We did not detect a difference in dexamethasone dose per neoadjuvant chemotherapy cycle between patients who did or did not achieve pCR. Thus, based on this retrospective analysis, the use of dexamethasone as an anti-emetic for triple negative breast cancer may not be harmful or beneficial in terms of the pathologic response to chemotherapy. This suggests that its use as an agent to mitigate side effects from chemotherapy is reasonable. Citation Format: Daniel Goldstein, Mahmoud Kassem, Dionisia Quiroga, Abdul Miah, Craig Vargo, Namrata Vilas Shinde, Michael Berger, Nicole Williams, Daniel Stover, Sagar Sardesai, Maryam Lustberg, Bhuvaneswari Ramaswamy, Gary Tozbikian, Patrick Schnell, Mathew Cherian. Immunomodulation with dexamethasone in neoadjuvant chemotherapy for triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS13-46.

Introduction: Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer associated with poor prognosis and limited treatment options. Validated prognostic and predictive biomarkers are needed to guide treatment decisions and prognostication. Areas covered: In this review, we discuss established and developing prognostic and predictive biomarkers in TNBC and associated emerging and approved therapies. Biomarkers reviewed include epidermal growth factor receptor (EGFR), vascular endothelial growth factors (VEGF), fibroblast growth factor receptor (FGFR), human epidermal growth factor receptor 2 (HER2), androgen receptor, NOTCH signaling, oxidative stress/redox signaling, microRNAs, TP53 mutation, breast cancer susceptibility gene 1 or 2 (BRCA1/2) mutation/homologous recombination deficiency (HRD), NTRK gene fusion, PI3K/AKT/mTOR, immune biomarkers (programmed death-ligand 1 (PDL1), tumor-infiltrating lymphocytes (TILs), tumor mutational burden (TMB), neoantigens, defects in DNA mismatch repair proteins (dMMR)/microsatellite instability-high (MSI-H)), circulating tumor cells/cell-free DNA, novel targets of antibody-drug conjugates, and residual disease. Expert opinion: Biomarker-driven care in the management of TNBC is increasing and has helped expand options for patients diagnosed with this subtype of breast cancer. Research efforts are ongoing to identify additional biomarkers and targeted treatment options with the ultimate goal of improving clinical outcomes and survivorship.

INTRODUCTION: Since the advent of anti-HER2 therapies, evidence surrounding adjuvant treatment of small (T1mic, T1a, and T1b), node-negative, HER2-positive breast cancer (HER2+BC) has remained limited. Practices vary widely between institutions with little known regarding the added benefit of systemic therapy, including cytotoxic chemotherapy and HER2-directed treatments. Our group has set out to perform an extensive review of available literature on this topic. AREAS COVERED: In this review, we examined HER2 biology, anti-HER therapies, outcome definitions, and available prospective and retrospective data surrounding the use of adjuvant therapy in those with small, node-negative, HER2+BC. For outcomes, we primarily explored breast cancer-specific survival (BCSS), invasive disease-free survival (iDFS), and overall survival (OS). We also investigated the incidence of adverse events with a particular focus on symptomatic and asymptomatic declines in left ventricular ejection fraction. EXPERT OPINION: Retrospective data will likely be the main driver for future treatment decisions. Given what we know, high risk T1b and T1c subgroups derive measurable added benefit from HER2-guided combination therapies but it’s not clear whether these benefits outweigh known risks associated with this combination therapy. For tumors ≤0.5cm (T1mic and T1a), treatment remains highly controversial with limited evidence available through retrospective analysis that suggest over-treatment may be occurring.

Background Sarcomas constitute a heterogeneous group of tumors with different clinical behaviors and variable responses to systemic therapies. Recent immunotherapy studies with PD1 inhibitors (PD1i) show promising results with use in certain soft-tissue sarcomas; however, the clinical and molecular features that best predict response to PD1i remain unclear. Methods Demographic, imaging, histologic, and genetic sequencing data was collected for sarcoma patients who received nivolumab or pembrolizumab (PD1i) treatment at our institution between January 1st 2015 and April 23rd 2018. The primary objective was to determine progression-free survival (PFS) in patients with advanced sarcomas receiving PD1i. Secondary objectives included determining overall survival (OS) and assessment of characteristics associated with response to PD1i. Fifty-six patients who were treated with PD1i therapy met inclusion criteria for this study. Results Partial response towards PD1i treatment was seen in 3 in 26 evaluable patients, but no complete responses were observed (overall response rate 11.5%). Within this group of patients, the 90 day PFS was found to be 48.8%. In patients in whom PD1 expression was known, there was a statistically significant positive correlation between expression of PD1 and longer PFS and OS rates. Patients that were treated with more than four cycles of PD1i therapy were also more likely to have a greater OS. Conclusions This study suggests activity of PD1i in a pretreated cohort of advanced sarcoma patients, particularly for the subset of patients with PD1 positive tumors. Our results highlight the importance of further research to better target the optimal patient population and markers of response.

Background: Breast cancer treatment includes neoadjuvant chemotherapy (NAC), offered to patients with locally advanced breast cancer and who may benefit from down-staging before conservation therapy. NAC allows for evaluation of treatment response with pathologic complete response (pCR) acting as a marker of survival. Black women receive NAC more frequently as they often present with more advanced stage tumors and the triple negative subtype. Furthermore, Black women without pCR following NAC are at greater risk of mortality. Obesity is a prognostic factor for breast cancer. Non-Hispanic Black women have the greatest prevalence of obesity in most states. Patients with higher Body Mass Index (BMI) have previously been shown to have lower rates of chemotherapy response. Data on racial and ethnic differences in pCR rates are limited and whether obesity is a confounding factor requires investigation. Methods: Retrospective review of patients diagnosed with non-metastatic breast cancer who completed NAC and had surgery at Ohio State University James Comprehensive Cancer Center between January 1, 2005, and December 31, 2019, were analyzed. Clinical stage was calculated based on tumor size and nodal status. Operative treatment received was recorded to determine pathologic stage and chemotherapy response. The study endpoint, pCR, was assessed after definitive surgery. BMI categories were based on World Health Organization classification and obese defined as 30kg/m2. For the data analysis, we included self-reported Black and White women, excluding patients classified as “Other” race. Preliminary analyses included the distribution of sample descriptive characteristics. Differences by race and demographic characteristics were compared using Pearson’s chi-square test for categorical variables and t-test or Wilcoxon rank-sum test for continuous variables. Univariate analysis and multivariable logistic regression for pCR by age, race, BMI, menopausal status, insurance status and employment status were performed. Results: A final sample of 882 met criteria (11.7% Black and 88.3% white women, 1% Hispanic ethnicity). Median age of diagnosis is 51, with median 147.4 months of follow-up. 64% of the sample had clinical stage 2 disease, 22% were triple negative, 62% Her-2 positive subtypes. For tumor characteristics 67% of Black women and 59% white women had high grade tumor. Black women also had more triple negative disease (30% vs. 21%), more advanced stage at presentation (27% vs. 21%). More white women were employed and had private insurance compared to Black women, who predominantly had public insurance. The median BMI was higher among Black women (31.5) than white women (28.6). 52% of white women vs. 47% Black had mastectomy over lumpectomy. 67% of white women had radiation vs. 61% of Black women. Overall, 33% of Black and white women had pCR, with 67% having no pCR. Race and BMI were not significant predictors of PCR rates on univariate or multivariable analysis. Age < 40 is the only variable associated with pCR (OR 1.645, [95 CI 1.117-2.420] p-value: 0.012). Conclusions: BMI was not a significant predictor of pCR in this limited retrospective review. However, further exploration with a larger sample evaluating differences in pCR by BMI can lead to a better understanding of the association between obesity and pCR. Though race was not significant in predicting pCR, there is also room for further research considering socioeconomic disparities and obesity rates by race. Table 1. Table 1. Predictors for pCR. Table 1. Multivariable logistic regression for pCR adjusted by age, race, BMI, menopause status, insurance status, employment status. Univariate analysis for predictors of pCR performed for race and BMI. Citation Format: Ruvarashe Rumano, Michael Grimm, Marilly Palettas, Julie Stephens, Nicole Williams, Sagar Sardesai, Dionisia Quiroga, Bhuvaneswari Ramaswamy, Electra Paskett, Bridget Oppong. Differences in breast tumor response to neoadjuvant chemotherapy by race- Is obesity the key? [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-01-06.

Background: Hormone receptor (HR) low (1-10%) HER2-negative breast cancer (BC) is emerging as a distinct subtype with similarities in clinical outcomes to triple-negative BC. However, there is a lack of consensus on treatment recommendations for chemo-immunotherapy and endocrine therapy in this subset. Here, we present results from a US National Cancer Database (NCDB) analyses of patients with HER2-negative BC evaluating response to neoadjuvant chemotherapy (NAC) and patterns of care by HR expression. Methods: Patients with stage I-III HER2-negative BC diagnosed in 2018 were identified in NCDB, a nationwide oncology outcomes database in the US. Quantitative HR expression was unavailable prior to 2018. Data were categorized into four groups by estrogen receptor (ER) and progesterone receptor (PR) expression: ER< 1% & PR< 1% (HR-Neg), ER 1-10% and/or PR 1-10% (HR-Low), ER >11-30% and/or PR>11-30% (HR-Int), ER> 30% and/or PR > 30% (HR-High). Those with undocumented HR status (3%) or without curative intent surgery (5%) were excluded. The primary outcome was pathologic complete response (pCR) by HR expression in those undergoing neoadjuvant chemotherapy. Key secondary objectives included assessment of clinicopathologic characteristics and practice patterns. The categorical variables were compared between the four groups using a Chi-square test. Age was compared using a Kruskal-Wallis test. Results: Out of 104,205 incident cases, 2541 (2.4%) were HR-Low and 1241 (1.2%) were HR-Int. Significant differences were found between HR groups with higher grade, clinical stage, and Ki-67 in HR-Low vs. HR-Int or HR-High groups (Table 1). Patients with HR-Low and HR-Int BC were more likely to receive chemotherapy than HR-High (74%, 70% vs. 20%; p < 0.001) and the use of adjuvant endocrine therapy correlated with HR expression. Only half of patients in the HR-Low group received any endocrine therapy compared to higher rates in the HR-Int and HR-High groups (52% vs. 74%, 92%; p< 0.001). pCR rates in those receiving neoadjuvant chemotherapy were significantly different by HR status, with higher pCR rates in HR-Low vs. HR-High groups (p< 0.001) (Table 2). NAC utilization significantly differed between groups. A higher proportion of patients with HR-Low BC received NAC than other HR-positive groups (p < 0.001). Additionally, there was an increased use of NAC in patients with HR-Low BC treated at academic vs. community cancer centers (p< 0.001). Conclusions: This is one of the largest real-world analyses comparing key differences in biology and practice patterns of HR-Low, HER2-negative BC. Consistent with prior studies, we report HR-Low BC to be a rare and distinct subtype with higher pCR rates compared to HR-High BC. Practice patterns show wide variability in utilization of neoadjuvant chemotherapy and endocrine therapy for these patients. Future studies should address this disparity and enhance representation of patients with HR-Low BC in clinical trials to improve long-term outcomes. Table 1: Patient demographics Table 2: Neoadjuvant chemotherapy response amongst differing HR expression levels Citation Format: Dionisia Quiroga, Michael Grimm, Julie Stephens, Kai Johnson, Nicole Williams, Preeti K. Sudheendra, Mathew A. Cherian, Daniel Stover, Ashley C. Pariser, Margaret Gatti-Mays, Robert Wesolowski, Jose G. Bazan, Sasha Beyer, Ko Un Park, Bridget Oppong, Bhuvaneswari Ramaswamy, Julia White, Sachin R. Jhawar, Sagar Sardesai. Impact of low hormone receptor expression on neoadjuvant chemotherapy response and patterns of care in early-stage HER2-negative breast cancer: a US National Cancer Database analysis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-02-17.

Background Adipose tissue has emerged as an important window into cancer pathophysiology, revealing potential targets for novel therapeutic interventions. The goal of this study was to compare the breast adipose tissue (BrAT) immune milieu surrounding breast carcinoma and contralateral unaffected breast tissue obtained from the same patient. Materials and methods Patients undergoing bilateral mastectomy for unilateral breast cancer were enrolled for bilateral BrAT collection at the time of operation. After BrAT was processed, adipocyte and stromal vascular fraction (SVF) gene expression was quantified by PCR. SVF cells were also processed for flow cytometric immune cell characterization. Results Twelve patients underwent bilateral mastectomy for unilateral ductal carcinoma. BrAT adipocyte CXCL2 gene expression trended higher in the tumor-affected breast as compared to the unaffected breast. Macrophage MCP-1 and PPARγ gene expression also tended to be higher in the tumor-affected breasts. T cell gene expression of FOXP3 (p = 0.0370) were significantly greater in tumor-affected breasts than unaffected breasts. Affected BrAT contained higher numbers of Th2 CD4+ cells (p = 0.0165) and eosinophils (p = 0.0095) while trending towards increased macrophage and lower Th1 CD4+ cells infiltration than tumor-affected BrAT. Conclusion This preliminary study aimed to identify the immunologic environment present within BrAT and is the first to directly compare this in individual patients’ tumor-associated and unaffected BrAT. These findings suggest that cancer-affected BrAT had increased levels of T cell specific FOXP3 and higher levels of anti-inflammatory/regulatory cells compared to the contralateral BrAT.

PURPOSE: Adjuvant ovarian function suppression (OFS) in premenopausal hormone receptor (HR) positive breast cancer (BC) improves survival. Adherence to adjuvant gonadotropin-releasing hormone analogs (GnRHa) remains a challenge and is associated with toxicities and inconvenient parenteral administration. The goal of this study was to describe real-world adherence patterns and patient preferences surrounding adjuvant GnRHa. METHODS: We analyzed the medical records of premenopausal women with non-metastatic HR positive BC from January 2000 to December 2017; participants received adjuvant monthly goserelin or leuprolide at The Ohio State University. Data collected included demographics, clinicopathologic characteristics, and OFS adherence/side effects. We defined non-adherence as discontinuation of GnRHa within 3 years for a reason other than switching to an alternate OFS, delay > 7 days from a dose, or a missed dose. Chi-square tests assessed associations between clinical characteristics and outcomes. RESULTS: A total of 325 patients met eligibility. Of these, 119 (37%) patients were non-adherent to GnRHa; 137 (42%) underwent elective bilateral salpingo-oophorectomy after initial GnRHa. Those opting for surgery reported significantly more hot flashes (74% vs 48%, p < 0.001), arthralgias (46% vs 30%, p = 0.003), and vaginal dryness (37% vs 21%, p = 0.001) compared with patients remaining on GnRHa. CONCLUSION: Non-adherence to adjuvant GnRHa occurred in over a third of patients and almost half the patients initiating GnRHa underwent subsequent surgical ablation. These high frequencies highlight real-world patterns of OFS. Additionally, treatment toxicities may impact personal preference of OFS modality. Personalized practices to target predictors of adjuvant GnRHa non-adherence are critical to optimize symptoms, adherence, and survivorship.

Tenosynovial giant cell tumor (TGCT) is a rare benign tumor that involves the synovium, bursa, and tendon sheath, resulting in reduced mobility of the affected joint or limb. The current standard of care for TGCT is surgical resection. However, some patients have tumor recurrence, present with unresectable tumors, or have tumors that are in locations where resection could result in amputations or significant debility. Therefore, the development of systemic agents with activity against TGCT to expand treatment options is a highly unmet medical need. Pathologically, TGCT is characterized by the overexpression of colony -stimulating factor 1 (CSF-1), which leads to the recruitment of colony -stimulating factor-1 receptor (CSF-1R) expressing macrophages that make up the primary cell type within these giant cell tumors. The binding of CSF-1 and CSF-1R controls cell survival and proliferation of monocytes and the switch from a monocytic to macrophage phenotype contributing to the growth and inflammation within these tumors. Therefore, molecules that target CSF-1/CSF-1R have emerged as potential systemic agents for the treatment of TGCT. Given the role of macrophages in regulating tumorigenesis, CSF1/CSF1R-targeting agents have emerged as attractive therapeutic targets for solid tumors. Pexidartinib is an orally bioavailable and potent inhibitor of CSF-1R which is one of the most clinically used agents. In this review, we discuss the biology of TGCT and review the pre-clinical and clinical development of pexidartinib which ultimately led to the FDA approval of this agent for the treatment of TGCT as well as ongoing clinical studies utilizing pexidartinib in the setting of cancer.

While combinations of immune checkpoint (ICP) inhibitors and neo-adjuvant chemotherapy (NAC) have begun to be tested in patients with breast cancer (BC), chemotherapeutic effects on ICP expression in circulating T cells are still unclear. This information could help design future clinical trials including the selection of the best ICP inhibitors to be incorporated into NAC and finding predictive/prognostic biomarkers. Peripheral blood samples and/or tumor specimens before and after NAC were obtained from twenty-four women with operable BC. Using flow cytometry, the expression of CTLA4, PD-1, Lag3, OX40, and Tim3 on circulating T lymphocytes before and at the end of NAC were measured. Differences in the percentage of CD4+ and CD8+ T cells expressing various checkpoint receptors pre- and post-NAC were determined by a paired t-test. This data showed decreased ICP expression after NAC by circulating CD4+ T cells, including significant decreases in CTLA4 (p Citation Format: Dionisia M. Quiroga, Andrew Stiff, Christopher McQuinn, Zaibo Li, Hiroaki Nitta, Himanshu Savardekar, Brooke Benner, Bhuvaneswari Ramaswamy, Maryam Lustberg, Rachel Layman, Erin Macrae, Mahmoud Kassem, Nicole Williams, Sagar Sardesai, Jeffrey VanDeusen, Daniel Stover, Mathew Cherian, Thomas Mace, Lianbo Yu, Megan Duggan, William E. Carson, Robert Wesolowski. Analysis of immune checkpoint receptor expression by circulating T cells and tumor specimens in patients pre- and post-neoadjuvant chemotherapy for operable breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5514.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) gene polymorphisms have been linked to several autoimmune diseases; however, the molecular mechanisms underlying these associations are not well understood. Recently, we demonstrated that ERAP1 regulates key aspects of the innate immune response. Previous studies show ERAP1 to be endoplasmic reticulum-localized and secreted during inflammation. Herein, we investigate the possible roles that ERAP1 polymorphic variants may have in modulating the innate immune responses of human peripheral blood mononuclear cells (hPBMCs) using two experimental methods: extracellular exposure of hPBMCs to ERAP1 variants and adenovirus (Ad)-based ERAP1 expression. We found that exposure of hPBMCs to ERAP1 variant proteins as well as ERAP1 overexpression by Ad5 vectors increased inflammatory cytokine and chemokine production, and enhanced immune cell activation. Investigating the molecular mechanisms behind these responses revealed that ERAP1 is able to activate innate immunity via multiple pathways, including the NLRP3 (NOD-like receptor, pyrin domain-containing 3) inflammasome. Importantly, these responses varied if autoimmune disease-associated variants of ERAP1 were examined in the assay systems. Unexpectedly, blocking ERAP1 cellular internalization augmented IL-1β production. To our knowledge, this is the first report identifying ERAP1 as being involved in modulating innate responses of human immune cells, a finding that may explain why ERAP1 has been genetically associated with several autoimmune diseases.

Recombinant adenovirus serotype 5 (Ad5) vectors are promising vaccine candidates due to their intrinsic immunogenicity and potent transgene expression; however, widespread preexisting Ad5 immunity has been considered a developmental impediment to the use of traditional, or conventional, E1 and E3 gene-deleted Ad5 (Ad5[E1−]) vaccines. Even in the presence of anti-Ad5 immunity, recent murine and human studies have confirmed E2b gene-deleted Ad5 (Ad5[E1−,E2b−]) vaccines to be highly efficacious inducers of transgene-specific memory responses and significantly less toxic options than Ad5[E1−] vaccines. While these findings have been substantially confirmed, the molecular mechanisms underlying the different reactions to these vaccine platforms are unknown. Using cultures of human peripheral blood mononuclear cells (hPBMCs) derived from multiple human donors, we found that Ad5[E1−,E2b−] vaccines trigger higher levels of hPBMC proinflammatory cytokine secretion than Ad5[E1−] vaccines. Interestingly, these responses were generated regardless of the donors' preexisting anti-Ad5 humoral and cell-mediated immune response status. In vitro hPBMC infection with the Ad5[E1−,E2b−] vaccine also provoked greater Th1-dominant gene responses yet smaller amounts of Ad-derived gene expression than Ad5[E1−] vaccines. These results suggest that Ad5[E1−,E2b−] vaccines, in contrast to Ad5[E1−] vaccines, do not promote activities that suppress innate immune signaling, thereby allowing for improved vaccine efficacy and a superior safety profile independently of previous Ad5 immunity.

The need for novel, effective adjuvants that are capable of eliciting stronger cellular and humoral adaptive immune responses to antigenic targets is well understood in the vaccine development field. Unfortunately, many adjuvants investigated thus far are either too toxic for human application or too weak to induce a substantial response against difficult antigens, such as tumor-associated antigens (TAAs). In spite of this trend, clinical investigations of recombinant Eimeria antigen (rEA) have revealed this protein to be a non-toxic immunogenic agent with the ability to trigger a Th1-predominant response in both murine and human subjects. Our past studies have shown that injection of an rEA-encoding adenovirus (rAd5-rEA) alongside an HIV antigen-encoding adenovirus greatly improves the adaptive immune response against this pathogen-derived transgene. In this report, we investigated if rAd5-rEA could promote and/or alter cytotoxic memory responses towards carcinoembryonic antigen (CEA), a colorectal cancer-related TAA. We found that the addition of rAd5-rEA to an Ad-based CEA vaccine (rAd5-CEA) induced a dose-dependent increase in several anti-CEA T and B cell responses. Moreover, inclusion of rAd5-rEA increased the number of CEA-derived antigenic epitopes that elicited significant cell-mediated and IgG-mediated recognition. These enhanced anti-CEA immune responses also translated into superior CEA-targeted cell killing, as evaluated by an in vivo cytotoxic T lymphocyte assay. Overall, these results suggest that co-administration of rAd5-rEA with a tumor antigen vaccine can substantially boost and broaden the TAA-specific adaptive memory response, thereby validating the potential of rAd5-rEA to be a beneficial adjuvant during therapeutic cancer vaccination.

Natural killer (NK) cells are innate immune effector cells that play a crucial role in immune surveillance and the destruction of cancer cells. NK cells express a low-affinity receptor for the Fc or constant region of immunoglobulin G (FcγRIIIa) and multiple cytokine receptors that respond to antibody-coated targets and cytokines in the tumor microenvironment. In the present work, microarray gene expression analysis revealed that the IL-21 receptor (IL-21R) was strongly upregulated following FcR stimulation. The IL-21R was found to be upregulated on FcR-stimulated NK cells at the transcript level as determined by reverse transcription polymerase chain reaction (RT-PCR). Immunoblot analysis revealed that protein expression of the IL-21R peaked at 8 h post-stimulation of the FcR. Inhibition of the mitogen-activated protein kinase (MAPK) pathway downstream of the FcR blocked the induction of IL-21R expression. Increased expression of the IL-21R sensitized NK cells to IL-21 stimulation, as treatment of FcR-stimulated NK cells led to significantly increased phosphorylation of STAT1 and STAT3, as measured by intracellular flow cytometry and immunoblot analysis. Following FcR-stimulation, IL-21-activated NK cells were better able to mediate the lysis of trastuzumab-coated human epidermal growth factor receptor 2 (HER2+) SK-BR-3 tumor cells as compared to control-treated cells. Likewise, IL-21-induced NK cell secretion of IFNγ following exposure to antibody-coated tumor cells was enhanced following FcR-stimulation. The analysis of NK cells from patients receiving trastuzumab therapy for HER2+ cancer exhibited increased levels of the IL-21R following the administration of antibody suggesting that the presence of monoclonal antibody-coated tumor cells in vivo can stimulate the increased expression of IL-21R on NK cells.

Ankylosing spondylitis (AS) is a chronic systemic arthritic disease that leads to significant disability and loss of quality of life in the ∼0.5% of the worldwide human population it affects. There is currently no cure for AS and mechanisms underlying its pathogenesis remain unclear. AS is highly genetic, with over 70% of the genetic risk being associated with the presence of HLA-B27 and endoplasmic reticulum aminopeptidase-1 (ERAP1) alleles. Furthermore, gene-gene interactions between HLA-B27 and ERAP1 AS risk alleles have recently been confirmed. Here, we demonstrate that various ERAP1 alleles can differentially mediate surface expression of antigens presented by HLA-B27 on human cells. Specifically, for all peptides tested, we found that an ERAP1 variant containing high AS risk SNPs reduced the amount of the peptide presented by HLA-B27, relative to low AS risk ERAP1 variants. These results were further validated using peptide catalysis assays in vitro, suggesting that high AS risk alleles have an enhanced catalytic activity that more rapidly destroys many HLA-B27-destined peptides, a result that correlated with decreased HLA-B27 presentation of the same peptides. These findings suggest that one mechanism underlying AS pathogenesis may involve an altered ability for AS patients harboring both HLA-B27 and high AS risk ERAP1 alleles to correctly display a variety of peptides to the adaptive arm of the immune system, potentially exposing such individuals to higher AS risk due to abnormal display of pathogen or self-derived peptides by the adaptive immune system.

Background Adenoviral based vectors remain promising vaccine platforms for use against numerous pathogens, including HIV. Recent vaccine trials utilizing Adenovirus based vaccines expressing HIV antigens confirmed induction of cellular immune responses, but these responses failed to prevent HIV infections in vaccinees. This illustrates the need to develop vaccine formulations capable of generating more potent T-cell responses to HIV antigens, such as HIV-Gag, since robust immune responses to this antigen correlate with improved outcomes in long-term non-progressor HIV infected individuals. Methodology/Principal Findings In this study we designed a novel vaccine strategy utilizing an Ad-based vector expressing a potent TLR agonist derived from Eimeria tenella as an adjuvant to improve immune responses from a [E1-]Ad-based HIV-Gag vaccine. Our results confirm that expression of rEA elicits significantly increased TLR mediated innate immune responses as measured by the influx of plasma cytokines and chemokines, and activation of innate immune responding cells. Furthermore, our data show that the quantity and quality of HIV-Gag specific CD8+ and CD8− T-cell responses were significantly improved when coupled with rEA expression. These responses also correlated with a significantly increased number of HIV-Gag derived epitopes being recognized by host T cells. Finally, functional assays confirmed that rEA expression significantly improved antigen specific CTL responses, in vivo. Moreover, we show that these improved responses were dependent upon improved TLR pathway interactions. Conclusion/Significance The data presented in this study illustrate the potential utility of Ad-based vectors expressing TLR agonists to improve clinical outcomes dependent upon induction of robust, antigen specific immune responses.

Despite recent advances in developing and licensing adjuvants, there is a great need for more potent formulations to enhance immunogenicity of vaccines. An Eimeria tenella derived antigen (rEA) augments immune responses against several pathogens in animal models and recently was confirmed to be safe for human use. In this study, we have analyzed the molecular mechanisms underlying rEA activity in mice, and confirmed that rEA activates multiple immune cell types, including DCs, macrophages, NK, B, and T cells. The rEA adjuvant also elicits the induction of pleiotropic pro-inflammatory cytokines, responses that completely depend upon the presence of the TLR adaptor protein MyD88. Surprisingly, we also found that the TRIF adaptor protein acts as a potent negative regulator of TLR agonist-triggered immune responses. For example, IL12 production and the induction of co-stimulatory molecule expression by DCs and IFNγ production by NK cells in vivo were significantly increased in rEA-treated TRIF-KO mice. Importantly, however, TRIF suppressive effects were not restricted to rEA-mediated responses, but were apparent in LPS- or ODN2006-activated DCs as well. Taken together, our findings confirm that rEA is a potent adjuvant, triggering robust activation of the innate immune system, in a manner that is augmented by MyD88 and inhibited by TRIF; thereby unveiling the potential complexities of modulating TLR activity to augment vaccine efficacy.