Olga Efimova

Ncf1 (p47phox) is essential for direct regulatory T cell mediated suppression of CD4+ effector T cells.

Background Multiple mechanisms have been advanced to account for CD4+FOXP3+ regulatory T cell (Treg)-mediated suppression of CD4+ effector T cells (Teffs) but none appear to completely explain suppression. Previous data indicates that Tregs may affect the microenvironment redox state. Given the inherent redox sensitivity of T cells, we tested the hypothesis that oxidants may mediate the direct suppression of Teffs by Tregs. Methodology/Principal Findings Tregs and Teffs were isolated from the spleens of wild type (WT) C57BL/6 mice or Ncf1(p47phox)-deficient C57BL/6 mice which lack NADPH oxidase function. Teffs were labeled with CFSE and co-cultured with unlabeled Tregs at varying Treg:Teff ratios in the presence of anti-CD3/CD28 coated beads for 3 days in suppression assays. Treg-mediated suppression was quantified by flow cytometric analysis of CFSE dilution in Teffs. The presence of the antioxidants n-acetylcysteine (NAC) or 2-mercaptoethanol or inhibitors of NADPH oxidase (diphenyleneiodonium and VAS-2870) resulted in reduced WT Treg-mediated suppression. The observed suppression was in part dependent upon TGFβ as it was partially blocked with neutralizing antibodies. The suppression of Teff proliferation induced by exogenous TGFβ treatment could be overcome with NAC. Ncf1-deficient Teff were slightly but significantly less sensitive than WT Teff to suppression by exogenous TGFβ. Ncf1-deficient Tregs suppressed Ncf1-deficient Teff very poorly compared to wild type controls. There was partial but incomplete reconstitution of suppression in assays with WT Tregs and Ncf1-deficient Teff. Conclusions/Significance We present evidence that NADPH oxidase derived ROS plays a role in the direct Treg mediated suppression of CD4+ effector T cells in a process that is blocked by thiol-containing antioxidants, NADPH oxidase inhibitors or a lack of Ncf1 expression in Tregs and Teffs. Oxidants may represent a potential new target for therapeutic modulation of Treg function.

Induction of granzyme B expression in T-cell receptor/CD28-stimulated human regulatory T cells is suppressed by inhibitors of the PI3K-mTOR pathway

BackgroundRegulatory T cells (Tregs) can employ a cell contact- and granzyme B-dependent mechanism to mediate suppression of bystander T and B cells. Murine studies indicate that granzyme B is involved in the Treg-mediated suppression of anti-tumor immunity in the tumor microenvironment and in the Treg-mediated maintenance of allograft survival. In spite of its central importance, a detailed study of granzyme B expression patterns in human Tregs has not been performed.ResultsOur data demonstrated that natural Tregs freshly isolated from the peripheral blood of normal adults lacked granzyme B expression. Tregs subjected to prolonged TCR and CD28 triggering, in the presence of IL-2, expressed high levels of granzyme B but CD3 stimulation alone or IL-2 treatment alone failed to induce granzyme B. Treatment of Tregs with the mammalian target of rapamycin (mTOR) inhibitor, rapamycin or the PI3 kinase (PI3K) inhibitor LY294002 markedly suppressed granzyme B expression. However, neither rapamycin, as previously reported by others, nor LY294002 inhibited Treg proliferation or induced significant cell death in TCR/CD28/IL-2 stimulated cells. The proliferation rate of Tregs was markedly higher than that of CD4+ conventional T cells in the setting of rapamycin treatment. Tregs expanded by CD3/CD28/IL-2 stimulation without rapamycin demonstrated increased in vitro cytotoxic activity compared to Tregs expanded in the presence of rapamycin in both short term (6 hours) and long term (48 hours) cytotoxicity assays.ConclusionTCR/CD28 mediated activation of the PI3K-mTOR pathway is important for granyzme B expression but not proliferation in regulatory T cells. These findings may indicate that suppressive mechanisms other than granzyme B are utilized by rapamycin-expanded Tregs.

Nicotinic Receptor Alpha7 Expression Identifies a Novel Hematopoietic Progenitor Lineage

How inflammatory responses are mechanistically modulated by nicotinic acetylcholine receptors (nAChR), especially by receptors composed of alpha7 (α7) subunits, is poorly defined. This includes a precise definition of cells that express α7 and how these impact on innate inflammatory responses. To this aim we used mice generated through homologous recombination that express an Ires-Cre-recombinase bi-cistronic extension of the endogenous α7 gene that when crossed with a reporter mouse expressing Rosa26-LoxP (yellow fluorescent protein (YFP)) marks in the offspring those cells of the α7 cell lineage (α7lin+). In the adult, on average 20–25 percent of the total CD45+ myeloid and lymphoid cells of the bone marrow (BM), blood, spleen, lymph nodes, and Peyers patches are α7lin+, although variability between litter mates in this value is observed. This hematopoietic α7lin+ subpopulation is also found in Sca1+cKit+ BM cells suggesting the α7 lineage is established early during hematopoiesis and the ratio remains stable in the individual thereafter as measured for at least 18 months. Both α7lin+ and α7lin– BM cells can reconstitute the immune system of naïve irradiated recipient mice and the α7lin+:α7lin– beginning ratio is stable in the recipient after reconstitution. Functionally the α7lin+:α7lin– lineages differ in response to LPS challenge. Most notable is the response to LPS as demonstrated by an enhanced production of IL-12/23(p40) by the α7lin+ cells. These studies demonstrate that α7lin+ identifies a novel subpopulation of bone marrow cells that include hematopoietic progenitor cells that can re-populate an animal’s inflammatory/immune system. These findings suggest that α7 exhibits a pleiotropic role in the hematopoietic system that includes both the direct modulation of pro-inflammatory cell composition and later in the adult the role of modulating pro-inflammatory responses that would impact upon an individual’s lifelong response to inflammation and infection.

Combined testing for CCAAT/enhancer-binding protein alpha (CEBPA) mutations and promoter methylation in acute myeloid leukemia demonstrates shared phenotypic features

Loss of function mutations in CCAAT/enhancer binding protein alpha (CEBPA) have been identified in acute myeloid leukemia (AML) and bi-allelic (double) CEBPA mutations are associated with improved prognosis in cases of cytogenetically normal-AML. In a subset of AML patients lacking CEBPA mutations, core promotor methylation of CEBPA has been described and is associated with a gene expression profile similar to the mutated cases including the expression of T cell associated genes such as CD7. However, the overall incidence and pattern of CEBPA mutations and core promoter methylation has not been thoroughly explored in a larger subset of AML with expression of CD7. Here we describe a simple and clinically deployable CEBPA promoter methylation test and the results of combined testing for CEBPA mutations and promoter methylation in 102 cases of AML, including 43 CD7+ cases. Overall, there were 5 methylated cases, 6 cases with double mutations, and 3 cases with single mutations. Significantly, 10 of 43 CD7+ cases (23%) had either methylated or double-mutated CEBPA. The CD7+ subset included all 5 methylated cases and 5 of the 6 cases with double mutations. All 3 cases with single mutations were CD7-. No case exhibited both hypermethylation and mutations. We find that promoter methylation accounts for half of those CD7+ cases with CEBPA dysregulating abnormalities. Furthermore, methylated cases and those with bi-allelic CEBPA mutations have similar phenotypic features including expression of CD7 and lack of co-incident NPM1 mutations. Our study suggests that methylation testing may be as important as mutation testing for identifying AML cases with CEBPA dysregulation and may be indicated in the routine prognostic workup of AML.

Clinical decisions surrounding genomic and proteomic testing among United States veterans treated for lung cancer within the Veterans Health Administration

BackgroundCurrent clinical guidelines recommend epidermal growth factor receptor (EGFR) mutational testing in patients with metastatic non-small cell lung cancer (NSCLC) to predict the benefit of the tyrosine kinase inhibitor erlotinib as first-line treatment. Proteomic (VeriStrat) testing is recommended for patients with EGFR negative or unknown status when erlotinib is being considered. Departure from this clinical algorithm can increase costs and may result in worse outcomes. We examined EGFR and proteomic testing among patients with NSCLC within the Department of Veterans Affairs (VA). We explored adherence to guidelines and the impact of test results on treatment decisions and cost of care.MethodsProteomic and EGFR test results from 2013 to 2015 were merged with VA electronic health records and pharmacy data. Chart reviews were conducted. Cases were categorized based on the appropriateness of testing and treatment.ResultsOf the 69 patients with NSCLC who underwent proteomic testing, 33 (48%) were EGFR-negative and 36 (52%) did not have documented EGFR status. We analyzed 138 clinical decisions surrounding EGFR/proteomic testing and erlotinib treatment. Most decisions (105, or 76%) were concordant with clinical practice guidelines. However, for 24 (17%) decisions documentation of testing or justification of treatment was inadequate, and 9 (7%) decisions represented clear departures from guidelines.ConclusionEGFR testing, the least expensive clinical intervention analyzed in this study, was significantly underutilized or undocumented. The records of more than half of the patients lacked information on EGFR status. Our analysis illustrated several clinical scenarios where the timing of proteomic testing and erlotinib diverged from the recommended algorithm, resulting in excessive costs of care with no documented improvements in health outcomes.

Neoplastic plasma cell aberrant antigen expression patterns and their association with genetic abnormalities

Abstract Little is known about aberrant antigen expression patterns and their association with cytogenetic aberrations in multiple myeloma (MM). We examined the correlation between flow cytometry and florescence in situ hybridization (FISH) in 167 marrow specimens with MM. Gene expression profiling of CD56, CD117, CD52 and CD20 mRNA in plasma cells (PCs) from patients treated on Total Therapy 2 and Total Therapy 3 trials were also evaluated. Higher expression of CD56 and CD117 was associated with hyperdiploidy. High CD52 mRNA expression was associated with c-MAF and FGFR3 subgroups. Higher expression of CD56 mRNA, but lower Kit expression, were noted in association with FGFR3. In contrast, the c-MAF subgroup showed high Kit expression but lacked NCAM mRNA expression. CKS1B amplification showed positive correlation with CD52 (p = 0.0065) but negative correlation with CD20 (p = 0.0207). These findings indicate that phenotypic differences in MM are associated with distinct genetic subgroups, which potentially has important diagnostic and prognostic value.

An initiative using informatics to facilitate clinical research planning and recruitment in the VA health care system

Background Randomized clinical trials are the gold standard for evaluating healthcare interventions and, more generally, add to the medical knowledge related to the treatment, diagnosis and prevention of diseases and conditions. Recent literature continues to identify health informatics methods that can help improve study efficiency throughout the life cycle of a clinical trial. Electronic medical record (EMR) data provides a mechanism to facilitate clinical trial research during the study planning and execution phases, and ultimately, can be utilized to enhance recruitment. The Department of Veterans Affairs (VA) has a strong history of clinical and epidemiological research with over four decades of data collected from Veterans it has served nationwide. The VA Informatics and Computing Infrastructure (VINCI) provides VA research investigators with a nationwide view of high-value VA patient data. Within VA, the Cooperative Studies Program (CSP) Network of Dedicated Enrollment Sites (NODES) is a consortium of nine sites that are part of an embedded clinical research infrastructure intended to provide systematic site-level solutions to issues that arise during the conduct of VA CSP clinical research. This paper describes the collaboration initiated by the Salt Lake City (SLC) node site to bring informatics and clinical trials together to enhance study planning and recruitment within the VA. Methods The SLC VA Medical Center physically houses both VINCI and a node site and the co-location of these two groups prompted a natural collaboration on both a local and national level. One of the functions of the SLC NODES is to enhance recruitment and promote the success of CSP projects. VINCI supports these efforts by providing VA researchers access to potential population pools. VINCI can provide 1) feasibility data during study planning, and 2) active patient lists during recruitment. The process for CSP study teams to utilize these services involves regulatory documentation, development of queries, revisions to the initial data request, and ongoing communications with several key study personnel including the requesting research team, study statisticians, and VINCI data managers. Results The early efforts of SLC NODES and VINCI aimed to provide patient lists exclusively to the SLC CSP study teams for the following purposes: 1) increasing recruitment for trials that were struggling to meet their respective enrollment goals, and 2) decreasing the time required by study coordinators to complete chart review activities. This effort was expanded to include multiple CSP sites and studies. To date, SLC NODES has facilitated the delivery of these VINCI services to nine active CSP studies. Conclusion The ability of clinical trial study teams to successfully plan and execute their respective trials is contingent upon their proficiency in obtaining data that will help them efficiently and effectively recruit and enroll eligible participants. This collaboration demonstrates that the utilization of a model that partners two distinct entities, with similar objectives, was effective in the provision of feasibility and patient lists to clinical trial study teams and facilitation of clinical trial research within a large, integrated healthcare system.

MP86-16 A MULTI-CENTER ANALYSIS OF PROSTATE CANCER (PCA) TREATMENT AMONG VETERANS FOLLOWING INTRODUCTION OF THE 17-GENE GENOMIC PROSTATE SCORE (GPS) ASSAY

RESULTS: 627 men were eligible for the study. Age, ethnicity, primary language, education and Charlson comorbidity did not differ across PSA strata. Compared to the referent group PSA <10, those with PSA 50 were more likely to receive androgen deprivation therapy as their primary form of treatment (p <0.01). Patients with PSAs 10-19.9 were more likely to have sexual bother (b1⁄411.1, p<0.03) compared to the referent group. (See Table) There were no other differences in other HRQOL domains across PSA strata. CONCLUSIONS: In this population, we found no statistically significant difference in HRQOL outcomes by PSA level. The finding that patients with very elevated PSA levels having outcomes that were no worse than patients with less aggressive disease is important clinically because most quality of life detriments tend to be from treatment of localized disease. Further, these findings will inform physicians on patient symptomatology despite PSA level.

Clinical Impact of 21-Gene Recurrence Score Test Within the Veterans Health Administration: Utilization and Receipt of Guideline-Concordant Care

Introduction: Ensuring guideline‐concordant cancer care is a Department of Veterans Affairs (VA) priority, especially as the number of breast cancer patients at VA medical centers (VAMCs) grows. We assessed the utilization and clinical impact of the 21‐gene Recurrence Score test, which predicts 10‐year risk of breast cancer recurrence and the likelihood of chemotherapy benefit, on veterans newly diagnosed with breast cancer. Patients and Methods: We conducted a retrospective cohort study using 2011‐2012 VA Central Cancer Registry, chart review, and laboratory test data. Independent variables assessed included patient and site‐of‐care characteristics. The outcome of interest was whether newly diagnosed, eligible (node negative, hormone‐receptor positive, human epidermal growth factor receptor 2 [HER2] negative) veterans underwent the 21‐gene test. We performed descriptive statistics on all patients and multivariate logistic regression to determine associations. We correlated treatments received with test results. Results: Among 328 eligible veterans, 82 (25%) had the 21‐gene test; 100 eligible veterans (30%) sought care at a VAMC where no tests were ordered. Receiving care at a VAMC that had womens health services (odds ratio [OR], 1.84, 95% confidence interval [CI], 1.05‐3.22) and having tumor characteristics meeting the National Comprehensive Cancer Network 2010 test criteria (OR, 3.06, 95% CI, 1.69‐5.57) were positive predictors of testing; increasing age (OR, 0.93, 95% CI, 0.91‐0.96 per year) and fee‐based care (OR, 0.46, 95% CI, 0.26‐0.82) were negative predictors. The majority of tested patients received guideline‐concordant care. Conclusion: Site of care and tumor characteristics were important predictors of test uptake. Facilitating delivery of guideline‐concordant cancer care requires improved laboratory informatics and clinical decision support.