Abstract Myelodysplastic syndromes (MDS) are hematologic malignancies characterized by hematopoietic stem cell dysfunction that leads to ineffective hematopoiesis and cytopenias. Even though a third of MDS cases transform to leukemia, most of the mortality in MDS is due to low blood counts that occur because of failure of hematopoiesis. Development of effective treatments for MDS have been impeded by a limited understanding of the molecular pathways that suppress hematopoietic stem cells. We previously demonstrated that the myelosuppressive cytokine TGF-β mediated SMAD2 activation can inhibit stem and progenitor cells in MDS. We demonstrated that SMAD7, a negative regulator of TGF-β receptor-I kinase, is markedly reduced in MDS, and can lead to ineffective hematopoiesis by overactivation of Smad2/3 mediated TGF-β signaling. To determine the cause of SMAD7 reduction in MDS, we analyzed the 3′UTR of the gene and found a highly conserved binding site for microRNA-21. Strikingly, we observed significantly elevated levels of miR-21 in MDS marrow samples when compared with age matched controls. miR-21 was shown to directly bind to the 3′UTR of SMAD7 and reduce its expression in hematopoietic cells. Next, we tested the role of miR-21 in regulating TGF-β signaling in a TGF-β overexpressing transgenic mouse model that develops progressive anemia and dysplasia and thus serves as a model of human bone marrow failure. Treatment with a chemically modified miR-21 inhibitor led to significant increases in hematocrit and an increase in SMAD7 expression in vivo. Inhibition of miR-21 also led to increase in erythroid colony formation from primary MDS bone marrow progenitors, demonstrating its ability in stimulating hematopoiesis in vitro. Even though miR-21 was found to be elevated in MDS, the mechanisms of its elevation in MDS as well as in other cancers are not well elucidated. The miR-21 gene locus has been shown to contain upstream STAT3 binding sites. Furthermore, STAT3 has also been shown to directly lead to miR-21 upregulation in myeloma and other immune cells. Genomic profiling of highly purified hematopoietic stem cells in MDS by us demonstrated that STAT3 is selectively upregulated in these cells. STAT3 overexpression was validated in an expanded set of 183 MDS CD34+ cells as well. These results suggest that a STAT3-miR-21 pathway leads to enhancement of TGF-β signaling, thus leading to ineffective hematopoiesis in MDS. Citation Format: Tushar D. Bhagat, Li Zhou, Lubomir Sokol, Ioannis Mantzaris, Sanchari Bhattacharyya, Shanisha A.K. Gordon, Yiting Yu, Krishna Gundabolu, Sangeeta Nischal, Rahul Polineni, Carolina Schinke, Grigorios Chrysofakis, Amittha Wickrema, Andrea Pellagatti, Jacqueline Boultwood, Ulrich Steidl, Gang Liu, Alan F. List, Markus Bitzer, Amit Verma. MicroRNA mediated regulation of TGF-β signaling leads to stem cell alterations in myelodysplastic syndromes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5224. doi:10.1158/1538-7445.AM2014-5224

Even though alterations in platelet counts are presumed to be detrimental, their impact on the survival of patients has not been studied in large cohorts. The prevalence of thrombocytopenia and thrombocytosis was examined in a large inner city outpatient population of 36,262 individuals aged ≥65 years old. A significant association with shorter overall survival was found for both thrombocytopenia (HR=1.45; 95% CI: 1.36-1.56) and thrombocytosis (HR=1.75; 95% CI: 1.56-1.97) when compared to the survival of patients with normal platelet counts. This effect persisted across all ethnic groups. However, African-Americans (non-Hispanic Blacks) with either thrombocytopenia or thrombocytosis were at significantly lower risk compared to non-Hispanic Caucasians (HR=0.82; 95% CI: 0.69-0.96 and HR=0.70; 95% CI: 0.53-0.94, respectively). Furthermore, Hispanics with thrombocytosis were found to have a lower mortality risk compared to non-Hispanic Caucasians with thrombocytosis (HR=0.60; 95% CI: 0.44-0.81). A value of <125,000 platelets per microliter was a better prognostic marker for non-Hispanic Blacks and these subjects with this platelet count had similar overall survival to that of Caucasians with a value of <150,000 per microliter. In conclusion, thrombocytosis and thrombocytopenia are independently associated with shorter overall survival in elderly subjects and this effect is modified by ethnicity. Using different thresholds to define the association of thrombocytopenia and thrombocytosis with overall mortality risk among non-Hispanic Blacks may, therefore, be warranted.

Recent studies have shown that an elevated red cell distribution width (RDW) is an important predictor of adverse outcomes. However, the strength of this biomarker has not been tested in a large outpatient elderly population. Also since increased RDW can be due to a variety of etiologies, additional biomarkers are needed to refine the prognostic value of this variable. We assembled a cohort of 36,226 elderly (≥65yo) patients seen at an outpatient facility within the Einstein/Montefiore system from January 1st 1997 to May 1st 2008 who also had a complete blood count performed within 3 months of the initial visit. With a maximum follow‐up of 10 years, we found that an elevated RDW (>16.6) was associated with increased risk of mortality in both non‐anemic (HR = 3.66, p < 0.05) and anemic patients (HR = 1.87, p < 0.05). The effect of RDW on mortality is significantly increased in non‐anemic patients with macrocytosis (HR = 5.22, p < 0.05) compared to those with normocytosis (HR = 3.86, p < 0.05) and microcytosis (HR = 2.46, p < 0.05). When comparing non‐anemic patients with both an elevated RDW and macrocytosis to those with neither, we observed an elevated HR of 7.76 (higher than expected in an additive model). This multiplicative interaction was not observed in anemic patients (HR = 2.23). Lastly, we constructed Kaplan‐Meier curves for each RDW/MCV subgroup and found worsened survival for those with macrocytosis and an elevated RDW in both anemia and non‐anemic patients. Based on our results, the addition of MCV appears to improve the prognostic value of RDW as a predictor of overall survival in elderly patients. Am. J. Heamtol. 88:E245–E249, 2013. © 2013 Wiley Periodicals, Inc.

Abstract Abstract 5150 Recent studies have shown that an elevated red cell distribution width (RDW) is an important predictor of adverse outcomes in a variety of clinical settings. The etiologies of an elevated RDW are different depending on mean corpuscular volume (MCV). If the effect of RDW on mortality differs by MCV (i. e., presence of interaction between RDW and MCV), studies of RDW using MCV as a simple covariate may result in spurious findings. In this study, we set out to confirm the importance of RDW in predicting mortality in a large outpatient cohort of elderly patients and to assess potential interaction between RDW and MCV by anemia status. Our cohort consisted of 36, 292 elderly (>=65yo) patients seen at an outpatient facility within the Einstein/Montefiore system from January 1st 1997 to May 1st 2008 who also had a complete blood count performed within 3 months of the initial visit. Using multivariate regression analyses, we examined the effect of RDW and MCV by anemia status in predicting overall mortality. With a maximum follow-up of 10 years, we found that an elevated RDW (>16. 6) increased risk of mortality in both non-anemic (adjusted HR=3. 55, p<0. 05) and anemic patients (adjusted HR=2. 07, p<0. 05). The effect of RDW on mortality is significantly increased in non-anemic patients with macrocytosis (MCV>96 fL; HR=5. 22, p<0. 05) compared to those with normocytosis (MCV 80–96 fL; HR=3. 86, p<0. 05) and microcytosis (MCV<80 fL; HR=2. 46, p<0. 05). To confirm multiplicative interaction, we calculated individual adjusted HRs for an elevated RDW, an elevated MCV and for elevations in both. In non-anemic patients, an elevated RDW by itself resulted in a HR of 3. 82 (p<0. 05). Similarly, an elevated MCV alone resulted in a HR of 1. 36 (p<0. 05). When comparing patients with both an elevated RDW and macrocytosis to those with neither, we observed an elevated HR of 7. 76 (higher than expected in an additive model). We repeated this analysis for anemic patients and this multiplicative effect was not observed (HR=1. 27 for elevated RDW, 2. 26 for elevated MCV, 2. 23 for both). To assess the predictive value of using both RDW and MCV for risk stratification, we constructed Kaplan-Meier survival curves for each RDW and MCV category by anemia status. In both non-anemic (Fig 1a) and anemic patients (Fig 1b), survival was worse for those with macrocytosis and an elevated RDW. The median survival for this group of patients was 2. 7 years in non-anemics and 2. 2 years in anemics (vs. >10 years and 7. 1 years, respectively, in patients with normal RDW and MCV). For both non-anemic and anemic patients, stratifying by RDW and MCV allowed us to create subgroups with clear survival differences. Patients with an elevated RDW had worse survival than those without. Among both groups, survival was worst in patients with macrocytosis, followed by normocytosis then microcytosis. This pattern remained consistent regardless of anemia status. In conclusion, our results suggest a strong interaction between RDW and MCV in predicting overall mortality in non-anemic patients. Stratification by MCV or the addition of an interaction term to account for multiplicative effect between RDW and MCV is needed in future studies of RDW to avoid spurious results. Anemia status must also be taken into account when assessing RDW as a predictor of adverse outcomes as it affects the strength of the interaction. For both non-anemic and anemic patients, the addition of MCV appears to improve the prognostic value of RDW as a predictor of overall survival in elderly patients. Further studies are needed to better understand the mechanisms in which anisocytosis, especially in the setting of macrocytosis, predicts increased mortality. Figure 1a Figure 1a. Figure 1b Figure 1b. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4645 Introduction: Platelets play a critical role in hemostasis and are also important in the development of pathologic processes including atherosclerosis and arterial thrombosis. Both thrombocytopenia and thrombophilia are common findings in several illnesses including liver diseases, infections, autoimmune disorders and malignancies. However, to date no study has directly investigated the effect of thrombocytopenia and thrombocytosis on the overall survival of the general population. The present study was thus undertaken to elucidate the independent association of abnormal platelet counts with subsequent mortality among a large inner city outpatient elderly cohort comprised of different ethnicities and comorbid conditions. Methods: All 36,262 non-acutely ill individuals >65 years of age who were seen at an outpatient clinic within the Montefiore medical system from January 1st 1997 to May 1st 2008 and who underwent a complete blood count within 3 months of the visit were included in our cohort. Thrombocytopenia and thrombocytosis were defined as a platelet count of <150,000 per microliter or >450,000/microliter respectively. Data on 19 co-morbidities that could affect mortality and confound the analysis were extracted from the electronic medical records of each patient. These included the presence of anemia, neutropenia, congestive of heart failure, baseline anemia and/or neutropenia, prior myocardial infarction, peripheral vascular disorders, cerebrovascular disease, dementia, chronic pulmonary disease, rheumatologic disease, peptic ulcer disease, liver disease, diabetes, renal disease, malignancy, neurologic deficits and HIV infection. A Cox proportional hazard model was constructed to assess the independent association of abnormal platelet counts with subsequent mortality after controlling for all extracted co-morbidities, age, gender and race. Results: As shown in table 1, caucasians had higher thrombocytopenia rates compared to blacks, Hispanics and other ethnicities (p<0.001 for all comparisons). Ethnicities other than Caucasians, Blacks or Hispanics had significantly lower rates of thrombocytosis compared to Caucasians or Blacks (p values <0.01). The median platelet count was 237,000/microliter. The median follow-up period was 3.3 years with a total of 134,132 person-years of observation. Fully adjusted hazard ratios (HR) using normal platelet counts (≥150,000 and ≤450,000) as the reference group showed a significant association of thrombocytopenia (HR=1.43, 95% CI 1.34–1.53; p<0.001) and thrombocytosis (HR=1.73, 95% CI 1.54–1.94; p<0.001) with shorter overall survival. A statistically significant interaction term was also identified between race and thrombocytopenia (Table 1). More specifically, we found that Hispanics with thrombocytopenia are at a higher risk compared to Caucasians with thrombocytopenia (HR=1.34, 95% CI 1.13–1.59; p=0.001). Conclusions: Thrombocytopenia and thrombocytosis are independently associated with increased mortality in our study population. The effect of thrombocytopenia on overall survival is more prominent on Hispanics. These findings suggest that abnormal platelet count may be a mediator of increased mortality risk in older patients and this effect may differ by ethnicity. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 3813 Myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis that leads to peripheral cytopenias. TGF-b is a hematopoietic inhibitory cytokine that has been indirectly linked to the pathogenesis of some subsets of MDS. We have shown that smad2, a component of the TGF- signaling pathway, is constitutively activated and upregulated in MDS progenitors (Blood, 112(8):3434; 2008). Since there is conflicting data about upregulation of TGF- b levels in MDS, we next sought to determine the molecular basis of TGF- b pathway activation in this disease. We observed that smad-7, a negative regulator of TGF- b receptor-I kinase, is markedly down regulated in MDS and leads overactivation of the receptor and subsequent smad2 phosphorylation / activation in this disease (Cancer Res, 71(3):955–63). In the present study we wanted to determine the cause of smad7 reduction in MDS. Since microRNA dysregulation has been reported in many malignancies, we explored the 3'UTR of the smad7 gene for putative microRNA binding sites and observed predicted mir-21 and mir-15/16 binding sites that were conserved across species. mir-21 was found to be elevated in a microarray screen in MDS (British J. Haem. 153(1):24–32) and was subsequently found by us to be significantly elevated by qPCR in MDS marrow samples when compared with age matched controls (TTest, N=11 in each group, P Value= 0.02). Luciferase reporters containing wild type and mutant 3' UTR of the smad7 gene were then used to determine whether mir21 was able to directly bind to the predicted sequence in the gene. Enforced expression of mir-21 was able to inhibit the wild type smad7 reporter expression and a mutation of 4 complementary residues in the 3'UTR led to abrogation of this effect, thus demonstrating direct effects of mir-21 on smad7 gene. To test the role of mir-21 in regulating TGF-b signaling in vivo, we used chemically modified, locked nucleic acid (LNA) inhibitors of mir-21. These were used in a TGF-overexpressing transgenic mouse model that develops progressive anemia and dysplasia and thus serves as a model of human bone marrow failure. Treatment with the mir-21 inhibitor led to significant increases in RBC counts when compared to placebo (P value<0.01, T test) and led to increase in smad7 expression and decrease in smad2 phosphorylation in bone marrow progenitors of the treated mice. Finally, mir-21 inhibitor treatment led to increases in erythroid and myeloid colony formation from human MDS bone marrow stem cells, demonstrating its ability in stimulating hematopoiesis in vitro. Taken together, these studies demonstrate that mir-21 mediated reduction in smad-7 contributes to ineffective hematopoiesis in MDS by activating TGF-beta signaling in bone marrow progenitors. Most importantly, these studies illustrate the therapeutic potential of mir-21 inhibitors in this disease. Disclosures: List: Celgene: Consultancy.