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Significantly higher basal levels of Stat3 in patients with poor-risk cytogenetics.

Multivariate analysis of prognostic factors for overall survival in the patient cohort (n=74)

Molecular characterization of the G-CSF-inducible Stat3/5 double positive (DP) and double negative (DN) subpopulations.

Flow cytometric analyses of cellular hierarchy in Lin-CD34+ selected cells.

Application of the GFM prognostic score on the patient cohort of the study (n=72).

Purpose: Azacitidine is the mainstay of high-risk myelodysplastic syndromes (MDS) therapy, but molecular predictors of response and the mechanisms of resistance to azacitidine remain largely unidentified. Deregulation of signaling via Stat3 and Stat5 in acute myeloid leukemia (AML) is associated with aggressive disease. Numerous genes involved in cell signaling are aberrantly methylated in MDS, yet the alterations and the effect of azacitidine treatment on Stat3/5 signaling in high-risk MDS have not been explored.Experimental Design: We assessed longitudinally constitutive and ligand-induced phospho-Stat3/5 signaling responses by multiparametric flow cytometry in 74 patients with MDS and low blast count AML undergoing azacitidine therapy. Pretreatment Stat3/5 signaling profiles in CD34+ cells were grouped by unsupervised clustering. The differentiation stage and the molecular properties of the CD34+ G-CSF–inducible Stat3/5 double-positive subpopulation were performed by flow cytometry and quantitative real-time PCR in isolated MDS progenitors.Results: The pretreatment Stat3/5 signaling profiles in CD34+ cells correlated strongly with response and cytogenetics and independently predicted event-free survival. We further identified a CD34+ G-CSF–inducible Stat3/5 double-positive subpopulation (DP subset) whose pretreatment levels were inversely associated with treatment response and cytogenetics. The kinetics of the DP subset followed the response to azacitidine and the disease course, whereas its molecular characteristics and cellular hierarchy were consistent with a leukemia propagating cell phenotype.Conclusions: Our findings provide a novel link among Stat3/5 signaling and MDS pathobiology and suggest that the Stat3/5 signaling biosignature may serve as both a response biomarker and treatment target. Clin Cancer Res; 22(8); 1958–68. ©2015 AACR.

Mutations analysis of the TET2 and TP53 coding regions using Next-Generation Sequencing (NGS)

Multivariate analysis of prognostic factors for event-free survival in the patient cohort (n=74)

Purpose: Azacitidine is the mainstay of high-risk myelodysplastic syndromes (MDS) therapy, but molecular predictors of response and the mechanisms of resistance to azacitidine remain largely unidentified. Deregulation of signaling via Stat3 and Stat5 in acute myeloid leukemia (AML) is associated with aggressive disease. Numerous genes involved in cell signaling are aberrantly methylated in MDS, yet the alterations and the effect of azacitidine treatment on Stat3/5 signaling in high-risk MDS have not been explored. Experimental Design: We assessed longitudinally constitutive and ligand-induced phospho-Stat3/5 signaling responses by multiparametric flow cytometry in 74 patients with MDS and low blast count AML undergoing azacitidine therapy. Pretreatment Stat3/5 signaling profiles in CD34+ cells were grouped by unsupervised clustering. The differentiation stage and the molecular properties of the CD34+ G-CSF–inducible Stat3/5 double-positive subpopulation were performed by flow cytometry and quantitative real-time PCR in isolated MDS progenitors. Results: The pretreatment Stat3/5 signaling profiles in CD34+ cells correlated strongly with response and cytogenetics and independently predicted event-free survival. We further identified a CD34+ G-CSF–inducible Stat3/5 double-positive subpopulation (DP subset) whose pretreatment levels were inversely associated with treatment response and cytogenetics. The kinetics of the DP subset followed the response to azacitidine and the disease course, whereas its molecular characteristics and cellular hierarchy were consistent with a leukemia propagating cell phenotype. Conclusions: Our findings provide a novel link among Stat3/5 signaling and MDS pathobiology and suggest that the Stat3/5 signaling biosignature may serve as both a response biomarker and treatment target. Clin Cancer Res; 22(8); 1958–68. ©2015 AACR.

Purpose: We hypothesized that targeting both Akt and heat shock protein (HSP) 90 would induce cytotoxic activity against multiple myeloma (MM) cells and target the bone marrow (BM) microenvironment to inhibit angiogenesis, osteoclast formation, as well as migration and adhesion of MM cells. Experimental Design: MM cell lines were incubated with perifosine (5 and 10 μmol/L) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG; 50 and 100 nmol/L) alone and in combination. Results: The combination of Akt inhibitor perifosine and HSP90 inhibitor 17-DMAG was synergistic in inducing MM cell cytotoxicity, evidenced by inhibition of DNA synthesis and induction of apoptosis. In addition, perifosine and 17-DMAG almost completely inhibited osteoclast formation: perifosine interfered with both early and late stages of osteoclast progenitor development, whereas 17-DMAG targeted only early stages. We next showed that combined therapy overcomes tumor growth and resistance induced by BM stromal cells and endothelial cells as well as the proliferative effect of exogenous interleukin-6, insulin-like growth factor-I, and vascular endothelial growth factor. Moreover, the combination also induced apoptosis and growth inhibition in endothelial cells and inhibited angiogenesis. Finally, we showed that the two agents prevented migration of MM cells toward stromal-derived factor-1 and vascular endothelial growth factor, which are present in the BM milieu, and also prevented adhesion of MM cells to fibronectin. Conclusions: This study provides the preclinical framework for treatment protocols targeting both the Akt and HSP pathways in MM.

INTRODUCTION: The procurement of NHC, for use as controls in hematologic research, is often problematic. The once-conventional source of a voluntary bone marrow [BM] aspirate is now widely prohibited, forcing researchers to purchase these specimens from only a few retailers worldwide. Having to both compensate a donor and generate a profit, the current cost of healthy, human BM is, on average, $40/mL or $4000 for a typical experiment [3 Donors X 50mL]. These specimens are also plagued by several other shortcomings, including: long and erratic lead times, legal restrictions which prohibit age-matching, and variability in their preanalytical handling due to shipping. More than 750,000 joint replacements are now performed annually in the United States alone, and as part of the preparation for these procedures, femoral BM is commonly aspirated and discarded; however, this phenomenon is largely overlooked as a source of controls due to the excess adipocytes found in adult long bones. As such, we sought to develop a method for the acquisition of NHC from the intramedullary aspirates routinely discarded during TJA. METHODS: In collaboration with the Department of Orthopedics and IRB at the Massachusetts General Hospital, Boston, we obtained the discarded intramedullary aspirates of 50 patients undergoing primary, elective TJA of either the hip or knee [Ratio-1:1]. Eligible patients were free of active leukemia or lymphoma and a history of pertinent illness. Aspirates were collected via an in-line sputum trap and standard medical suction just prior to the placement of the femoral prosthesis, heparinized [50 U/mL] and stored at 4oC for < 3 hours before being processed. A novel, hydrophobicity-based methodology was developed to remove the bone, reticulocytes, and excess adipose which dominate these specimens, while sparing the mononuclear cell compartment [MNC]. Specific lymphocytes were immunomagnetically selected, analyzed for purity by flow cytometry [FACS] and compared to commercial BM. RESULTS: Hips produced a median 593×106 MNC (SD=54×106), knees yielded 387×106 MNC (SD=54×106); viability was > 93% by Trypan Blue. The median for commercial marrow was 354×106 MNC (SD=24×106) [n=12,25mL]. Selection of CD34+, CD19+, CD138+ and CD3+ cells from processed TJA BM resulted in substantial populations of enriched cells; the purity of each subset was determined to be > 90% by FACS. Age-matching to hematologic malignancies [median age at diagnosis: 67 - NCI's SEER database] was possible with TJA controls, the median age was 66.6, whereas for commercial donors, it was 27.2 [n=37]. CONCLUSIONS: Using our novel methodology, the intramedullary aspirates routinely discarded during primary, elective TJA represent a viable, and underutilized, reservoir of NHC, and are superior to purchased marrow specimens in terms of cost, yield and appropriateness. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3991.