Racial differences in the pathophysiology of atherothrombosis are poorly understood. We explored the function and transcriptome of platelets in healthy black (n = 70) and white (n = 84) subjects. PAR4 thrombin receptor induced platelet aggregation and calcium mobilization were significantly greater in black subjects. Numerous differentially expressed (DE) RNAs were associated with both race and PAR4 reactivity, including phosphatidylcholine transfer protein (PCTP), and platelets from blacks expressed higher levels of PC-TP protein. PC-TP inhibition or depletion blocked activation of platelets or megakaryocytic cell lines through PAR4 but not PAR1. MiR-376c levels were DE by race and PAR4 reactivity, and were inversely correlated with PCTP mRNA levels, PC-TP protein levels and PAR4 reactivity. MiR-376c regulated expression of PC-TP in human megakaryocytes. A disproportionately high number of miRNAs DE by race and PAR4 reactivity, including miR-376c, are encoded in the DLK1-DIO3 locus, and were lower in platelets from blacks. These results support PC-TP as a regulator of the racial difference in PAR4-mediated platelet activation, indicate a genomic contribution to platelet function that differs by race, and emphasize a need to consider race effects when developing anti-thrombotic drugs.

Thymic-independent peripheral expansion of CD8+ cells derived from the graft in the initial stage of post-HSCT immune recovery is a well-known physiological event. Nevertheless, the description of symptomatic LGL leukemias and aggressive malignant cases in this setting may generate uncertainty, mostly in those cases in which the cytotoxic T lymphocyte expansion CTLe persists beyond the early transplantation period. We aimed to assess the nature of CTLe in adults during the post-alloHSCT period in a series of 154 patients with a long term surveillance. We studied the longitudinal kinetics of those expansions, their relation to clinical events, and their phenotypic and molecular features, including recently reported CTL leukemia-STAT3 mutations. In our study, trying to adhere to the WHO annotation of T-LGL, we considered two definitions for a CTL expansion: an absolute increase (≥ 2000 x109/L), and a relative expansion (a CD8/CD4 ratio ≥ 1.5), persisting more than six months in both cases. Persistent relative CTLe cases are frequent (49%) and related with timoglobulin prophylaxis (p≤0.001), acute graft versus host disease (GVHD, p=0.02), reduced intensity conditioning (p=0.04) and fungal and viral infections in the early post-HSCT. No differences in the number of serious infectious events from day 180 was found. Absolute CTLe are scarce (9%), related with chronic GVHD and absence of relapses. TCR rearrangement was reported as clonal and oligoclonal in the majority of patients with CTLe. We studied in a cross sectional manner with an extended immunophenotypic panel 17 patients: 5 patients with an absolute CTLe and 12 cases with a relative CTLe. A similar cytotoxic T αβ-effector phenotype was observed in all cases, with slight differences in the expression of CD25, CD16 and 1a. One patient with a relative CTLe expressed CD56 intensely: his ratio normalized at day 730 and no immune-related events were recorded. DNA stored during the post-alloHSCT setting was available from 68/75 relative CTLe patients (14/14 absolute CTLe cases). All of them went through molecular TCR rearrangement and STAT3 exon 21 mutations determination. In the relative CTLe cohort, TCR rearrangement was described as clonal, oligoclonal or polyclonal in 77%, 16% and 7%, respectively. Regarding absolute CTLe patients, TCR rearrangement was described as clonal in all the patients (n=14) of this subset. To increase the sensibility of the Sanger PCR, it was performed on DNA from CD3+ sorted cells in 54 out of 68 cases. No STAT3 mutation could be found in the CD3+ sorted fraction of relative or absolute defined CTLe. Not using an absolute threshold would establish a diagnosis of a persistent CTL expansion in 49% of our cohort of allo-transplanted patients. Additional diagnostic tools, as an effector phenotype, the presence of a NK marker or a monoclonal TCR rearrangement would not reduce significantly that percentage: CD57 was invariably expressed in CTLe cases, and 80% of our patients with expansions showed a TCR monoclonal pattern. STAT3 mutations resulting in persistent proliferation of CTL clones are a frequent event in large granular lymphocytic leukemia, and those clones have also been described in autoimmunity-driven disorders as acquired aplastic anemia and hypocellular myelodysplastic syndromes. We establish in this study the absence of exon 21 STAT3 mutations in the persistent CTL expansions found in a large series of patients with a long-term post-alloHSCT surveillance. The absence of STAT3 mutations and the CD8/CD4 declining longitudinal kinetics in the late period, supports its benign nature, expressed clinically by the null detrimental impact of these expansions on post-transplant outcome and/or serious infectious events. Figure 1. Relative and absolute CTLe kinetics. A) Linear representation over time of the CD8/CD4 ratio in the 75 patients with relative CTLe. B) Trend linear plot of 25 patients with a relative CTLe and a follow up of, at least, 1440 days from transplantation. Each line depicts a patientxs longitudinally measured CD8/CD4 ratio. Patients are grouped by similar pattern of ratio behaviour through follow up. The line "slope" depicts the magnitude of the change between time points. C) Linear representation over time of the CD3/CD8 count in PB in the 14 patients with an absolute CTLe. D) Trend linear plot illustrating the CD8/CD4 ratio behaviour of the 14 patients with an absolute CTLe. Figure 1. Relative and absolute CTLe kinetics. A) Linear representation over time of the CD8/CD4 ratio in the 75 patients with relative CTLe. B) Trend linear plot of 25 patients with a relative CTLe and a follow up of, at least, 1440 days from transplantation. Each line depicts a patientxs longitudinally measured CD8/CD4 ratio. Patients are grouped by similar pattern of ratio behaviour through follow up. The line "slope" depicts the magnitude of the change between time points. C) Linear representation over time of the CD3/CD8 count in PB in the 14 patients with an absolute CTLe. D) Trend linear plot illustrating the CD8/CD4 ratio behaviour of the 14 patients with an absolute CTLe. Disclosures No relevant conflicts of interest to declare.

3330 MicroRNAs (miRNAs) are important regulators of gene expression involved in virtually all physiological and pathological processes. Transcriptional profiling with subsequent bioinformatic analysis is increasingly used to identify miRNAs critical for hematopoiesis, normal blood cell function and hematological diseases. The biological variation observed in differential expression of these RNAs has been useful for better understanding of disease mechanism, and for identifying potential biomarkers and therapeutic targets. However, conclusions about the significance of measured biological variation is only legitimate when the RNA expression levels are normalized to internal controls, and most miRNA profiling data analyses are based on assumptions that have not been validated. For example, the expression level of U6 snRNA is often used to normalize expression across comparison tissues. However, it is not known whether U6 snRNA is expressed at equivalent levels among these cells or sample groups, and no reliable reference genes have been identified in cells from different hematopoietic lineages. The goal of this study was to identify reference miRNAs with the least variation for peripheral blood T-cells, B-cells, granulocytes and platelets. Subsets of cells were obtained from the peripheral blood of 5 healthy donors using density centrifugation followed by immunoselection. High purity (>97%) of the cells was verified by flow cytometry. Total RNA was extracted with Trizol and miRNA profiled by Nanostring technology (Nanostring Technologies, Denver, CO). Two different statistical algorithms - the NormFinder (NF) and Coefficient of variation (CV) methods - were used to identify miRNA reference genes with high stability within and among cell types. Geometric mean normalized raw data was analyzed by NF and CV, and candidate normalizer genes were selected based on three criteria: (1) stability measure of the variation in miRNA values (low values being more stable) (2) present in the top 10 candidate normalizer genes by both the NF and CV methods, and (3) a moderate expression value of 300–1000 counts (range = 50–12,000 counts for 95% of miRNAs). The last criterion is important because variation in a very low abundance reference gene would have an inappropriately large effect on normalization of the data. The reference miRNAs identified were hsa-miR-30b and hsa-miR-151-5p for B-cells, hsa-miR-484 and hsa-miR-425 for platelets, hsa-miR-301a, hsa-miR-30d and hsa-miR-424 for granulocytes, and hsa-miR-140-3p and hsa-miR-101 for T-cells. Hsa-let-7b and hsa-miR-423-3p were identified as common normalizers across T-cells, B-cells, platelets and granulocytes with stability values of 0.207 and 0.331 by NormFinder and 0.0599 and 0.0591 by CV method as shown in Fig. 1 and Fig. 2. Both hsa-let-7b and hsa-miR-423-3p were validated by RT-PCR to be stable normalizers (CV of Ct values were 9% and 12%, respectively) across T-cells, B-cells, platelets and granulocytes. Notably, hsa-let-7b showed lower variation across cell types than U6 snRNA, indicating hsa-let-7b is a more reliable reference gene for quantification of miRNA data from hematopoietic cells. In summary, we used a rigorous and formal approach to identify miRNAs in different hematopoietic lineages that can be used as appropriate reference transcripts for genome-wide profiling studies. Most ideally, this approach can be used for any new transcriptome data set. In addition, we provide ideal reference miRNAs for comparisons within or among different hematopoietic lineages. Normalization with the reference genes identified in this report, will allow more reliable and accurate determination of the biological variation involving differential expression of hematopoietic miRNAs than currently used normalizers. Disclosures: No relevant conflicts of interest to declare.

INTRODUCTION: Extracorporeal photoapheresis (ECP) is an immunomodulatory therapy for patients with acute or chronic graft-versus-host disease (aGVHD/cGVHD). So far, few studies have explored the molecular regulation of GVHD, and to date no studies have addressed how specific is miRNA expression change during ECP, and whether selected miRNA profiles might be predictive of clinical responses to ECP. OBJECTIVE: The aim of the study was to analyze the expression profile of miRNAs in plasma of patients with GVHD candidates for ECP, and their changes in responding and non-responding patients to this therapy. PATIENTS AND METHODS: Patients with GVHD underwent ECP therapy by off-line methods according to internal protocols. Peripheral blood samples were drawn pre-ECP and after 6 months of treatment. Data on patient characteristics, medical therapies and responses were obtained from medical records. We included the following study cohorts: 1) Initial cohort of 10 GVHD patients (7 cGVHD, 3 aGVHD) and 3 controls; 2) Internal validation cohort with 21 GVHD patients (14 cGVHD, 7 aGVHD) and 10 controls; and 3) External validation cohort (Policlinico S. Matteo, Pavia) composed of 24 GVHD patients (17 cGVHD, 7 aGVHD) and 12 controls. Additionally, samples from 12 patients undergoing ECP due to lung transplantation were also included. Plasma miRNAs were purified with NucleoSpin miRNA Plasma (Macherey-Nagel). In the initial cohort, we analyzed 178 miRNAs, using the Plasma focus miRNAs PCR array (Exiqon). In the validation cohorts we quantified, by qRT-PCR, candidate miRNAs using miRcury LNA RT miRNA PCR (Exiqon) and specific Exiqon primers. RESULTS: In the initial cohort, 4 miRNAs (miR-22-5p, miR-34a-5p, miR-148a-3p, and miR-505-3p) showed higher expression in patients with GVHD compared to controls (p<0.05), which significantly decreased to values similar to healthy individuals at 6 months post-ECP (Fig. 1A). In the validation study with the groups of both the internal and external cohorts, only miR-34-5p and miR-148a-3p were significantly increased in GVHD patients before ECP treatment compared to controls, and those levels significantly decreased with this therapy. This effect was not an intrinsic change associated with ECP, since this pattern was not observed in patients undergoing ECP for lung transplantation (data not shown). We explored the ability of these 2 miRNAs to predict clinical responses to ECP at 6 months: responders showed higher pre-ECP levels of miR-34a-5p and lower miR-148a-3p compared to non-responders (p=0.009, and p=0.003, respectively) (Fig. 1B). The ROC curves showed an area under the curve (AUC) of 0.82 for miR-34a-5p and 0.89 for miR-148a-3p. The ability to identify patients who respond to ECP was improved (p<0.01) by combining both markers (AUC=0.96) (Fig. 1C). CONCLUSION: This study identifies miR-34a-5p and miR-148a-3p as potential biomarkers to predict responses to ECP in patients with GVHD. The combinatorial function of these miRNAs can provide a more informative outlook on the pathophysiology of the disease by identifying potential target genes being inhibited and the pathways involved, both in the development of GVHD and following responses to ECP. Overall, this information would enable the implementation of more personalized patient treatment strategies, and likely lead to significant advances in the management of GVHD patients. Disclosures Lozano: Amgen: Consultancy; Terumo S.A.: Consultancy; Grifols S.A.: Consultancy; Novartis: Consultancy; Macopharma: Consultancy.