Abstract 2789 Background:The bone marrow microenvironment provides a crucial hematopoietic niche for survival and differentiation of hematopoietic progenitor/stem cells (HPSCs). The dysfunctional microenvironment in myelodysplastic syndrome (MDS) may provide a new source of prognostic and diagnostic information and even provide insight into the pathophysiology of the disease. We used tissue microarray (TMA) techniques combined with semi-automated image acquisition using double immunofluorescence and confocal microscopy to methodically and quantitatively analyze the interrelationships among key components of the bone marrow microenvironment and their relationships to CD34+ hematopoietic progenitor/stem cells (HPSCs) in intact human bone marrow derived from archival diagnostic paraffin-embedded core biopsy specimens representing benign (NL), MDS, and acute myeloid leukemia (AML) diagnoses. Methods:Whole bone marrow core biopsies (13 NL, 11 MDS, 7 AML) and 1 mmTMA cores (5 NL, 6 MDS, 4 AML) were evaluated. The area occupied by stromal cell populations was assessed using freely available ImageJ software. Adjacency of CD34+ HPSCs to specific cell populations was manually quantitated on double immunofluorescent-stained whole TMA core images. Results:CD271 (low-affinity nerve growth factor receptor/pNGFR) highlights an extensive arborizing MSC population, whereas nestin and CD146 (melanoma cell adhesion molecule, MCAM) localize to specific vascular compartments. In MDS, CD271+ MSCs are markedly increased as compared to benign marrow (p=0.012), whereas there is no significant change in CD146, nestin, or CD163+ macrophage populations (Table 1). We document an intimate and conserved relationship of the CD271+ MSC population to CD34+ HPSCs and quantitate the preservation of this relationship in MDS (Fig 1, Table 2). Remarkably, the CD271+ MSC population is in intimate contact with the majority of CD34+ HPSCs (86% on average) across NL, MDS and AML marrows, whereas no significant contact is seen with nestin+ or CD146+ vascular elements. In benign bone marrow expression of the pro-survival chemokine CXCL12 is largely restricted to the vasculature, whereas in MDS and AML CD271+ MSCs abnormally express CXCL12. [Display omitted] Conclusions:We propose that CD271+ MSCs are candidates for a pathophysiologic role in MDS, and are candidate prognostic/diagnostic markers. We hypothesize that the relationship of MDS and AML CD34+ blasts with abnormal CD271+ MSC likely exposes neoplastic CD34+ HPSCs to aberrant contact-dependent signaling via the chemokine CXCL12 or other signals. We propose that the increased, dysfunctional, and spatially disarranged CD271+ arborizing MSC infrastructure may contribute to the ineffective hematopoiesis that is a defining feature of MDS by promoting inappropriate proliferation/survival of CD34+ HPSCs and by failing to support trafficking of maturing hematopoietic cells to microenvironmental compartments where they receive specific contact-dependent signals.Table 1:Quantification of % bone marrow area encompassed by CD271, CD146, nestin, and CD163+ cellsCD271CD146nestinCD163Nmean+/−std devnmean+/−std devnmean+/−std devnmean+/−std devNL143.1 +/− 3.2%160.6 +/− 0.9%140.0 +/− 0.0%612.6 +/− 4.9%MDS139.1 +/− 8.7%100.6 +/− 0.6%100.1 +/− 0.1%523.5 +/− 17.8%AML107.1 +/− 5.3%100.6 +/− 1.0%90.7 +/− 0.2%411.5 +/− 2.2%Total376.3 +/− 6.6%360.6 +/− 0.8%330.1 +/− 0.4%1515.9 +/− 11.4%Mann-Whitney U test p valuesNormal vs MDS0.0120.280.290.40Normal vs AML0.0640.790.160.40CD34+ HPSCs are in intimate and conserved contact with CD271+ MSCs at baseline and in myeloid neoplasia. CD34+ HPSCs (red) are in intimate contact with NGFR+ arborizing stromal cells (green). Representative photomicrographs of benign (NL), low-grade myelodysplastic syndrome/5Q minus (RA5Q), and high grade myelodysplastic syndrome/refractory anemia with excess blasts 2 (RAEB2).Table 2:Quantification of % CD34+ HPSCs in contact with CD271, CD146, nestin, and CD163+ cell typesCD271CD146nestinCD163nmean+/−std devnmean+/−std devnmean+/−std devnmean+/−std devNL682 +/− 10%50.0 +/− 0.0%41.0 +/− 2.1%627 +/− 23%MDS588 +/− 9%50.2 +/− 0.5%41.3 +/− 1.6%516 +/− 16%AML488 +/− 5%40.2 +/− 0.3%30.5 +/− 0.8%415 +/− 6%Total1586 +/− 7%140.1 +/− 0.3%121.0 +/− 0.2%1520 +/− 17%Kruskall Wallis test p values, adjusted for multiple comparisonsversus CD271—<0.001<0.0010.003 Disclosures:Greenberg:amgen: Consultancy, Research Funding; onconova: Research Funding; glaxosmithkline: Research Funding; novartis: Consultancy, Research Funding.