Interconnected lineage trajectories link conventional and natural killer (NK)-like exhausted CD8+ T cells beneficial in type 1 diabetes.

Distinct Natural Killer (NK)-like CD57+ and PD-1+ CD8+ exhausted-like T cell populations (Tex) have both been linked to beneficial immunotherapy response in autoimmune type 1 diabetes (T1D) patients. The origins and relationships between these cell types are poorly understood. Here we show that while PD-1+ and CD57+ Tex populations are epigenetically similar, CD57+ Tex cells display unique increased chromatin accessibility of inhibitory Killer Cell Immunoglobulin-like Receptor (iKIR) and other NK cell genes. PD-1+ and CD57+ Tex also show reciprocal expression of Inhibitory Receptors (IRs) and iKIRs accompanied by chromatin accessibility of Tcf1 and Tbet transcription factor target sites, respectively. CD57+ Tex show unappreciated gene expression heterogeneity and share clonal relationships with PD-1+ Tex, with these cells differentiating along four interconnected lineage trajectories: Tex-PD-1+, Tex-CD57+, Tex-Branching, and Tex-Fluid. Our findings demonstrate new relationships between Tex-like populations in human autoimmune disease and suggest that modulating common precursor populations may enhance response to autoimmune disease treatment.

Responders to low-dose ATG induce CD4+ T cell exhaustion in type 1 diabetes.

BACKGROUNDLow-dose anti-thymocyte globulin (ATG) transiently preserves C-peptide and lowers HbA1c in individuals with recent-onset type 1 diabetes (T1D); however, the mechanisms of action and features of the response remain unclear. Here, we characterized the post hoc immunological outcomes of ATG administration and their potential use as biomarkers of metabolic response to therapy (i.e., improved preservation of endogenous insulin production).METHODSWe assessed gene and protein expression, targeted gene methylation, and cytokine concentrations in peripheral blood following treatment with ATG (n = 29), ATG plus granulocyte colony-stimulating factor (ATG/G-CSF, n = 28), or placebo (n = 31).RESULTSTreatment with low-dose ATG preserved regulatory T cells (Tregs), as measured by stable methylation of FOXP3 Treg-specific demethylation region (TSDR) and increased proportions of CD4+FOXP3+ Tregs (P < 0.001) identified by flow cytometry. While treatment effects were consistent across participants, not all maintained C-peptide. Responders exhibited a transient rise in IL-6, IP-10, and TNF-α (P < 0.05 for all) 2 weeks after treatment and a durable CD4+ exhaustion phenotype (increased PD-1+KLRG1+CD57- on CD4+ T cells [P = 0.011] and PD1+CD4+ Temra MFI [P < 0.001] at 12 weeks, following ATG and ATG/G-CSF, respectively). ATG nonresponders displayed higher proportions of senescent T cells (at baseline and after treatment) and increased methylation of EOMES (i.e., less expression of this exhaustion marker).CONCLUSIONAltogether in these exploratory analyses, Th1 inflammation-associated serum and CD4+ exhaustion transcript and cellular phenotyping profiles may be useful for identifying signatures of clinical response to ATG in T1D.TRIAL REGISTRATIONClinicalTrials.gov NCT02215200.FUNDINGThe Leona M. and Harry B. Helmsley Charitable Trust (2019PG-T1D011), the NIH (R01 DK106191 Supplement, K08 DK128628), NIH TrialNet (U01 DK085461), and the NIH NIAID (P01 AI042288).

IL-6-Driven pSTAT1 Response Is Linked to T Cell Features Implicated in Early Immune Dysregulation.

Elevated levels and enhanced sensing of the pro-inflammatory cytokine interleukin-6 (IL-6) are key features of many autoimmune and inflammatory diseases. To better understand how IL-6 signaling may influence human T cell fate, we investigated the relationships between levels of components of the IL-6R complex, pSTAT responses, and transcriptomic and translational changes in CD4+ and CD8+ T cell subsets from healthy individuals after exposure to IL-6. Our findings highlight the striking heterogeneity in mbIL-6R and gp130 expression and IL-6-driven pSTAT1/3 responses across T cell subsets. Increased mbIL-6R expression correlated with enhanced signaling via pSTAT1 with less impact on pSTAT3, most strikingly in CD4+ naïve T cells. Additionally, IL-6 rapidly induced expression of transcription factors and surface receptors expressed by T follicular helper cells and altered expression of markers of apoptosis. Importantly, many of the features associated with the level of mbIL-6R expression on T cells were recapitulated both in the setting of tocilizumab therapy and when comparing donor CD4+ T cells harboring the genetic variant, IL6R Asp358Ala (rs2228145), known to alter mbIL-6R expression on T cells. Collectively, these findings should be taken into account as we consider the role of IL-6 in disease pathogenesis and translating IL-6 biology into effective therapies for T cell-mediated autoimmune disease.

Guidelines for standardizing T-cell cytometry assays to link biomarkers, mechanisms, and disease outcomes in type 1 diabetes.

Cytometric immunophenotyping is a powerful tool to discover and implement T-cell biomarkers of type 1 diabetes (T1D) progression and response to clinical therapy. Although many discovery-based T-cell biomarkers have been described, to date, no such markers have been widely adopted in standard practice. The heterogeneous nature of T1D and lack of standardized assays and experimental design across studies is a major barrier to the broader adoption of T-cell immunophenotyping assays. There is an unmet need to harmonize the design of immunophenotyping assays, including those that measure antigen-agnostic cell populations, such that data collected from different clinical trial sites and T1D cohorts are comparable, yet account for cohort-specific features and different drug mechanisms of action. In these Guidelines, we aim to provide expert advice on how to unify aspects of study design and practice. We provide recommendations for defining cohorts, method implementation, as well as tools for data analysis and reporting by highlighting and building on selected successes. Harmonization of cytometry-based T-cell assays will allow researchers to better integrate findings across trials, ultimately enabling the identification and validation of biomarkers of disease progression and treatment response in T1D.

Exhausted-like CD8+ T cell phenotypes linked to C-peptide preservation in alefacept-treated T1D subjects.

Clinical trials of biologic therapies in type 1 diabetes (T1D) aim to mitigate autoimmune destruction of pancreatic ß cells through immune perturbation and serve as resources to elucidate immunological mechanisms in health and disease. In the T1DAL trial of alefacept (LFA3-Ig) in recent-onset T1D, endogenous insulin production was preserved in 30% of subjects for 2 years after therapy. Given our previous findings linking exhausted-like CD8+ T cells to beneficial response in T1D trials, we applied unbiased analyses to sorted CD8+ T cells to evaluate their potential role in T1DAL. Using RNA sequencing, we found that greater insulin C-peptide preservation was associated with a module of activation- and exhaustion-associated genes. This signature was dissected into 2 CD8 memory phenotypes through correlation with cytometry data. These cells were hypoproliferative, shared expanded rearranged TCR junctions, and expressed exhaustion-associated markers including TIGIT and KLRG1. The 2 phenotypes could be distinguished by reciprocal expression of CD8+ T and NK cell markers (GZMB, CD57, and inhibitory killer cell immunoglobulin-like receptor [iKIR] genes), versus T cell activation and differentiation markers (PD-1 and CD28). These findings support previous evidence linking exhausted-like CD8+ T cells to successful immune interventions for T1D, while suggesting that multiple inhibitory mechanisms can promote this beneficial cell state.

Unsupervised machine learning reveals risk stratifying glioblastoma tumor cells.

A goal of cancer research is to reveal cell subsets linked to continuous clinical outcomes to generate new therapeutic and biomarker hypotheses. We introduce a machine learning algorithm, Risk Assessment Population IDentification (RAPID), that is unsupervised and automated, identifies phenotypically distinct cell populations, and determines whether these populations stratify patient survival. With a pilot mass cytometry dataset of 2 million cells from 28 glioblastomas, RAPID identified tumor cells whose abundance independently and continuously stratified patient survival. Statistical validation within the workflow included repeated runs of stochastic steps and cell subsampling. Biological validation used an orthogonal platform, immunohistochemistry, and a larger cohort of 73 glioblastoma patients to confirm the findings from the pilot cohort. RAPID was also validated to find known risk stratifying cells and features using published data from blood cancer. Thus, RAPID provides an automated, unsupervised approach for finding statistically and biologically significant cells using cytometry data from patient samples.

IRF5 genetic risk variants drive myeloid-specific IRF5 hyperactivation and presymptomatic SLE.

Genetic variants within or near the interferon regulatory factor 5 (IRF5) locus associate with systemic lupus erythematosus (SLE) across ancestral groups. The major IRF5-SLE risk haplotype is common across populations, yet immune functions for the risk haplotype are undefined. We characterized the global immune phenotype of healthy donors homozygous for the major risk and nonrisk haplotypes and identified cell lineage-specific alterations that mimic presymptomatic SLE. Contrary to previous studies in B lymphoblastoid cell lines and SLE immune cells, IRF5 genetic variants had little effect on IRF5 protein levels in healthy donors. Instead, we detected basal IRF5 hyperactivation in the myeloid compartment of risk donors that drives the SLE immune phenotype. Risk donors were anti-nuclear antibody positive with anti-Ro and -MPO specificity, had increased circulating plasma cells and plasmacytoid dendritic cells, and had enhanced spontaneous NETosis. The IRF5-SLE immune phenotype was conserved over time and probed mechanistically by ex vivo coculture, indicating that risk neutrophils are drivers of the global immune phenotype. RNA-Seq of risk neutrophils revealed increased IRF5 transcript expression, IFN pathway enrichment, and decreased expression of ROS pathway genes. Altogether, the data support that individuals carrying the IRF5-SLE risk haplotype are more susceptible to environmental/stochastic influences that trigger chronic immune activation, predisposing to the development of clinical SLE.

Human Germinal Center B Cells Differ from Naïve and Memory B Cells in CD40 Expression and CD40L-Induced Signaling Response.

CD40 expression is required for germinal center (GC) formation and function, but the kinetics and magnitude of signaling following CD40 engagement remain poorly characterized in human B cells undergoing GC reactions. Here, differences in CD40 expression and signaling responses were compared across differentiation stages of mature human tonsillar B cells. A combination of mass cytometry and phospho-specific flow cytometry was used to quantify protein expression and CD40L-induced signaling in primary human naïve, GC, and memory B cells. Protein expression signatures of cell subsets were quantified using viSNE and Marker Enrichment Modeling (MEM). This approach revealed enriched expression of CD40 protein in GC B cells, compared to naïve and memory B cells. Despite this, GC B cells responded to CD40L engagement with lower phosphorylation of NFκB p65 during the first 30 min following CD40L activation. Before CD40L stimulation, GC B cells expressed higher levels of suppressor protein IκBα than naïve and memory B cells. Following CD40 activation, IκBα was rapidly degraded and reached equivalently low levels in naïve, GC, and memory B cells at 30 min following CD40L. Quantifying CD40 signaling responses as a function of bound ligand revealed a correlation between bound CD40L and degree of induced NFκB p65 phosphorylation, whereas comparable IκBα degradation occurred at all measured levels of CD40L binding. These results characterize cell-intrinsic signaling differences that exist in mature human B cells undergoing GC reactions. © 2019 International Society for Advancement of Cytometry.

Machine learning reveals chronic graft-versus-host disease phenotypes and stratifies survival after stem cell transplant for hematologic malignancies.

The application of machine learning in medicine has been productive in multiple fields, but has not previously been applied to analyze the complexity of organ involvement by chronic graft-versus-host disease. Chronic graft-versus-host disease is classified by an overall composite score as mild, moderate or severe, which may overlook clinically relevant patterns in organ involvement. Here we applied a novel computational approach to chronic graft-versus-host disease with the goal of identifying phenotypic groups based on the subcomponents of the National Institutes of Health Consensus Criteria. Computational analysis revealed seven distinct groups of patients with contrasting clinical risks. The high-risk group had an inferior overall survival compared to the low-risk group (hazard ratio 2.24; 95% confidence interval: 1.36-3.68), an effect that was independent of graft-versus-host disease severity as measured by the National Institutes of Health criteria. To test clinical applicability, knowledge was translated into a simplified clinical prognostic decision tree. Groups identified by the decision tree also stratified outcomes and closely matched those from the original analysis. Patients in the high- and intermediate-risk decision-tree groups had significantly shorter overall survival than those in the low-risk group (hazard ratio 2.79; 95% confidence interval: 1.58-4.91 and hazard ratio 1.78; 95% confidence interval: 1.06-3.01, respectively). Machine learning and other computational analyses may better reveal biomarkers and stratify risk than the current approach based on cumulative severity. This approach could now be explored in other disease models with complex clinical phenotypes. External validation must be completed prior to clinical application. Ultimately, this approach has the potential to reveal distinct pathophysiological mechanisms that may underlie clusters. Clinicaltrials.gov identifier: NCT00637689.

Computational Immune Monitoring Reveals Abnormal Double-Negative T Cells Present across Human Tumor Types.

Advances in single-cell biology have enabled measurements of >40 protein features on millions of immune cells within clinical samples. However, the data analysis steps following cell population identification are susceptible to bias, time-consuming, and challenging to compare across studies. Here, an ensemble of unsupervised tools was developed to evaluate four essential types of immune cell information, incorporate changes over time, and address diverse immune monitoring challenges. The four complementary properties characterized were (i) systemic plasticity, (ii) change in population abundance, (iii) change in signature population features, and (iv) novelty of cellular phenotype. Three systems immune monitoring studies were selected to challenge this ensemble approach. In serial biopsies of melanoma tumors undergoing targeted therapy, the ensemble approach revealed enrichment of double-negative (DN) T cells. Melanoma tumor-resident DN T cells were abnormal and phenotypically distinct from those found in nonmalignant lymphoid tissues, but similar to those found in glioblastoma and renal cell carcinoma. Overall, ensemble systems immune monitoring provided a robust, quantitative view of changes in both the system and cell subsets, allowed for transparent review by human experts, and revealed abnormal immune cells present across multiple human tumor types.

Frequency and phenotype consequence of APOC3 rare variants in patients with very low triglyceride levels.

BACKGROUND: High levels of triglycerides (TG ≥200 mg/dL) are an emerging risk factor for cardiovascular disease. Conversely, very low levels of TG are associated with decreased risk for cardiovascular disease. Precision medicine aims to capitalize on recent findings that rare variants such as APOC3 R19X (rs76353203) are associated with risk of disease, but it is unclear how population-based associations can be best translated in clinical settings at the individual-patient level. METHODS: To explore the potential usefulness of screening for genetic predictors of cardiovascular disease, we surveyed BioVU, the Vanderbilt University Medical Center's biorepository linked to de-identified electronic health records (EHRs), for APOC3 19X mutations among adult European American patients (> 45 and > 55 years of age for men and women, respectively) with the lowest percentile of TG levels. The initial search identified 262 patients with the lowest TG levels in the biorepository; among these, 184 patients with sufficient DNA and the lowest TG levels were chosen for Illumina ExomeChip genotyping. RESULTS: A total of two patients were identified as heterozygotes of APOC3 R19X for a minor allele frequency (MAF) of 0.55% in this patient population. Both heterozygous patients had only a single mention of TG in the EHR (31 and 35 mg/dL, respectively), and one patient had evidence of previous cardiovascular disease. CONCLUSIONS: In this patient population, we identified two patients who were carriers of the APOC3 19X null variant, but only one lacked evidence of disease in the EHR highlighting the challenges of inclusion of functional or previously associated genetic variation in clinical risk assessment.

BRAF and MEK inhibitor therapy eliminates Nestin-expressing melanoma cells in human tumors.

Little is known about the in vivo impacts of targeted therapy on melanoma cell abundance and protein expression. Here, 21 antibodies were added to an established melanoma mass cytometry panel to measure 32 cellular features, distinguish malignant cells, and characterize dabrafenib and trametinib responses in BRAFV600mut melanoma. Tumor cells were biopsied before neoadjuvant therapy and compared to cells surgically resected from the same site after 4 weeks of therapy. Approximately 50,000 cells per tumor were characterized by mass cytometry and computational tools t-SNE/viSNE, FlowSOM, and MEM. The resulting single-cell view of melanoma treatment response revealed initially heterogeneous melanoma tumors were consistently cleared of Nestin-expressing melanoma cells. Melanoma cell subsets that persisted to week 4 were heterogeneous but expressed SOX2 or SOX10 proteins and specifically lacked surface expression of MHC I proteins by MEM analysis. Traditional histology imaging of tissue microarrays from the same tumors confirmed mass cytometry results, including persistence of NES- SOX10+ S100ß+ melanoma cells. This quantitative single-cell view of melanoma treatment response revealed protein features of malignant cells that are not eliminated by targeted therapy.

Generating Quantitative Cell Identity Labels with Marker Enrichment Modeling (MEM).

Multiplexed single-cell experimental techniques like mass cytometry measure 40 or more features and enable deep characterization of well-known and novel cell populations. However, traditional data analysis techniques rely extensively on human experts or prior knowledge, and novel machine learning algorithms may generate unexpected population groupings. Marker enrichment modeling (MEM) creates quantitative identity labels based on features enriched in a population relative to a reference. While developed for cell type analysis, MEM labels can be generated for a wide range of multidimensional data types, and MEM works effectively with output from expert analysis and diverse machine learning algorithms. MEM is implemented as an R package and includes three steps: (1) calculation of MEM values that quantify each feature's relative enrichment in the population, (2) reporting of MEM labels as a heatmap or as a text label, and (3) quantification of MEM label similarity between populations. The protocols here show MEM analysis using datasets from immunology and oncology. These MEM implementations provide a way to characterize population identity and novelty in the context of computational and expert analyses. © 2018 by John Wiley & Sons, Inc.

Mass cytometry deep phenotyping of human mononuclear phagocytes and myeloid-derived suppressor cells from human blood and bone marrow.

The monocyte phagocyte system (MPS) includes numerous monocyte, macrophage, and dendritic cell (DC) populations that are heterogeneous, both phenotypically and functionally. In this study, we sought to characterize those diverse MPS phenotypes with mass cytometry (CyTOF). To identify a deep phenotype of monocytes, macrophages, and DCs, a panel was designed to measure 38 identity, activation, and polarization markers, including CD14, CD16, HLA-DR, CD163, CD206, CD33, CD36, CD32, CD64, CD13, CD11b, CD11c, CD86, and CD274. MPS diversity was characterized for 1) circulating monocytes from healthy donors, 2) monocyte-derived macrophages further polarized in vitro (i.e., M-CSF, GM-CSF, IL-4, IL-10, IFN-γ, or LPS long-term stimulations), 3) monocyte-derived DCs, and 4) myeloid-derived suppressor cells (MDSCs), generated in vitro from bone marrow and/or peripheral blood. Known monocyte subsets were detected in peripheral blood to validate the panel and analysis pipeline. Then, using various culture conditions and stimuli before CyTOF analysis, we constructed a multidimensional framework for the MPS compartment, which was registered against historical M1 or M2 macrophages, monocyte subsets, and DCs. Notably, MDSCs generated in vitro from bone marrow expressed more S100A9 than when generated from peripheral blood. Finally, to test the approach in vivo, peripheral blood from patients with melanoma (n = 5) was characterized and observed to be enriched for MDSCs with a phenotype of CD14+HLA-DRlowS100A9high (3% of PBMCs in healthy donors, 15.5% in patients with melanoma, P < 0.02). In summary, mass cytometry comprehensively characterized phenotypes of human monocyte, MDSC, macrophage, and DC subpopulations in both in vitro models and patients.

Characterizing cell subsets using marker enrichment modeling.

Learning cell identity from high-content single-cell data presently relies on human experts. We present marker enrichment modeling (MEM), an algorithm that objectively describes cells by quantifying contextual feature enrichment and reporting a human- and machine-readable text label. MEM outperforms traditional metrics in describing immune and cancer cell subsets from fluorescence and mass cytometry. MEM provides a quantitative language to communicate characteristics of new and established cytotypes observed in complex tissues.

Transcriptomic evaluation of the American oyster, Crassostrea virginica, deployed during the Deepwater Horizon oil spill: Evidence of an active hydrocarbon response pathway.

Estuarine organisms were impacted by the Deepwater Horizon oil spill which released ∼5 million barrels of crude oil into the Gulf of Mexico in the spring and summer of 2010. Crassostrea virginica, the American oyster, is a keystone species in these coastal estuaries and is routinely used for environmental monitoring purposes. However, very little is known about their cellular and molecular responses to hydrocarbon exposure. In response to the spill, a monitoring program was initiated by deploying hatchery-reared oysters at three sites along the Alabama and Mississippi coast (Grand Bay, MS, Fort Morgan, AL, and Orange Beach, AL). Oysters were deployed for 2-month periods at five different time points from May 2010 to May 2011. Gill and digestive gland tissues were harvested for gene expression analysis and determination of aliphatic and polycyclic aromatic hydrocarbon (PAH) concentrations. To facilitate identification of stress response genes that may be involved in the hydrocarbon response, a nearly complete transcriptome was assembled using Roche 454 and Illumina high-throughput sequencing from RNA samples obtained from the gill and digestive gland tissues of deployed oysters. This effort resulted in the assembly and annotation of 27,227 transcripts comprised of a large assortment of stress response genes, including members of the aryl hydrocarbon receptor (AHR) pathway, Phase I and II biotransformation enzymes, antioxidant enzymes and xenobiotic transporters. From this assembly several potential biomarkers of hydrocarbon exposure were chosen for expression profiling, including the AHR, two cytochrome P450 1A genes (CYP1A-like 1 and CYP1A-like 2), Cu/Zn superoxide dismutase (CuZnSOD), glutathione S-transferase theta (GST theta) and multidrug resistance protein 3 (MRP3). Higher expression levels of GST theta and MRP3 were observed in gill tissues from all three sites during the summer to early fall 2010 deployments. Linear regression analysis indicated a statistically significant relationship between total PAH levels in digestive gland tissue samples with CYP1A-like 2, CuZnSOD, GST theta and MRP3 induction. These observations provide evidence of a potentially conserved AHR pathway in invertebrates and yield new insight into the development of novel biomarkers for use in environmental monitoring activities.

Deep phenotyping of Tregs identifies an immune signature for idiopathic aplastic anemia and predicts response to treatment.

Idiopathic aplastic anemia (AA) is an immune-mediated and serious form of bone marrow failure. Akin to other autoimmune diseases, we have previously shown that in AA regulatory T cells (Tregs) are reduced in number and function. The aim of this study was to further characterize Treg subpopulations in AA and investigate the potential correlation between specific Treg subsets and response to immunosuppressive therapy (IST) as well as their in vitro expandability for potential clinical use. Using mass cytometry and an unbiased multidimensional analytical approach, we identified 2 specific human Treg subpopulations (Treg A and Treg B) with distinct phenotypes, gene expression, expandability, and function. Treg B predominates in IST responder patients, has a memory/activated phenotype (with higher expression of CD95, CCR4, and CD45RO within FOXP3(hi), CD127(lo) Tregs), expresses the interleukin-2 (IL-2)/STAT5 pathway and cell-cycle commitment genes. Furthermore, in vitro-expanded Tregs become functional and take on the characteristics of Treg B. Collectively, this study identifies human Treg subpopulations that can be used as predictive biomarkers for response to IST in AA and potentially other autoimmune diseases. We also show that Tregs from AA patients are IL-2-sensitive and expandable in vitro, suggesting novel therapeutic approaches such as low-dose IL-2 therapy and/or expanded autologous Tregs and meriting further exploration.

High-Dimensional Analysis of Acute Myeloid Leukemia Reveals Phenotypic Changes in Persistent Cells during Induction Therapy.

The plasticity of AML drives poor clinical outcomes and confounds its longitudinal detection. However, the immediate impact of treatment on the leukemic and non-leukemic cells of the bone marrow and blood remains relatively understudied. Here, we conducted a pilot study of high dimensional longitudinal monitoring of immunophenotype in AML. To characterize changes in cell phenotype before, during, and immediately after induction treatment, we developed a 27-antibody panel for mass cytometry focused on surface diagnostic markers and applied it to 46 samples of blood or bone marrow tissue collected over time from 5 AML patients. Central goals were to determine whether changes in AML phenotype would be captured effectively by cytomic tools and to implement methods for describing the evolving phenotypes of AML cell subsets. Mass cytometry data were analyzed using established computational techniques. Within this pilot study, longitudinal immune monitoring with mass cytometry revealed fundamental changes in leukemia phenotypes that occurred over time during and after induction in the refractory disease setting. Persisting AML blasts became more phenotypically distinct from stem and progenitor cells due to expression of novel marker patterns that differed from pre-treatment AML cells and from all cell types observed in healthy bone marrow. This pilot study of single cell immune monitoring in AML represents a powerful tool for precision characterization and targeting of resistant disease.

TESTING POPULATION-SPECIFIC QUANTITATIVE TRAIT ASSOCIATIONS FOR CLINICAL OUTCOME RELEVANCE IN A BIOREPOSITORY LINKED TO ELECTRONIC HEALTH RECORDS: LPA AND MYOCARDIAL INFARCTION IN AFRICAN AMERICANS.

Previous candidate gene and genome-wide association studies have identified common genetic variants in LPA associated with the quantitative trait Lp(a), an emerging risk factor for cardiovascular disease. These associations are population-specific and many have not yet been tested for association with the clinical outcome of interest. To fill this gap in knowledge, we accessed the epidemiologic Third National Health and Nutrition Examination Surveys (NHANES III) and BioVU, the Vanderbilt University Medical Center biorepository linked to de-identified electronic health records (EHRs), including billing codes (ICD-9-CM) and clinical notes, to test population-specific Lp(a)-associated variants for an association with myocardial infarction (MI) among African Americans. We performed electronic phenotyping among African Americans in BioVU≥40 years of age using billing codes. At total of 93 cases and 522 controls were identified in NHANES III and 265 cases and 363 controls were identified in BioVU. We tested five known Lp(a)-associated genetic variants (rs1367211, rs41271028, rs6907156, rs10945682, and rs1652507) in both NHANES III and BioVU for association with myocardial infarction. We also tested LPA rs3798220 (I4399M), previously associated with increased levels of Lp(a), MI, and coronary artery disease in European Americans, in BioVU. After meta-analysis, tests of association using logistic regression assuming an additive genetic model revealed no significant associations (p<0.05) for any of the five LPA variants previously associated with Lp(a) levels in African Americans. Also, I4399M rs3798220 was not associated with MI in African Americans (odds ratio = 0.51; 95% confidence interval: 0.16 - 1.65; p=0.26) despite strong, replicated associations with MI and coronary artery disease in European American genome-wide association studies. These data highlight the challenges in translating quantitative trait associations to clinical outcomes in diverse populations using large epidemiologic and clinic-based collections as envisioned for the Precision Medicine Initiative.

In Vivo Autofluorescence Imaging of Tumor Heterogeneity in Response to Treatment.

Subpopulations of cells that escape anti-cancer treatment can cause relapse in cancer patients. Therefore, measurements of cellular-level tumor heterogeneity could enable improved anti-cancer treatment regimens. Cancer exhibits altered cellular metabolism, which affects the autofluorescence of metabolic cofactors NAD(P)H and FAD. The optical redox ratio (fluorescence intensity of NAD(P)H divided by FAD) reflects global cellular metabolism. The fluorescence lifetime (amount of time a fluorophore is in the excited state) is sensitive to microenvironment, particularly protein-binding. High-resolution imaging of the optical redox ratio and fluorescence lifetimes of NAD(P)H and FAD (optical metabolic imaging) enables single-cell analyses. In this study, mice with FaDu tumors were treated with the antibody therapy cetuximab or the chemotherapy cisplatin and imaged in vivo two days after treatment. Results indicate that fluorescence lifetimes of NAD(P)H and FAD are sensitive to early response (two days post-treatment, P<.05), compared with decreases in tumor size (nine days post-treatment, P<.05). Frequency histogram analysis of individual optical metabolic imaging parameters identifies subpopulations of cells, and a new heterogeneity index enables quantitative comparisons of cellular heterogeneity across treatment groups for individual variables. Additionally, a dimensionality reduction technique (viSNE) enables holistic visualization of multivariate optical measures of cellular heterogeneity. These analyses indicate increased heterogeneity in the cetuximab and cisplatin treatment groups compared with the control group. Overall, the combination of optical metabolic imaging and cellular-level analyses provide novel, quantitative insights into tumor heterogeneity.