ALBERT

Description: Background: Gene expression profiles have been associated with prognosis in myelodysplastic syndromes. WT1 is a tumor-suppressor gene coding for a transcription factor located on chromosome 11p13, which was originally identified for its involvement in the pathogenesis of the Wilms’ tumor. In normal bone marrow, WT1 expression is low or undetectable, whereas it is aberrantly expressed in hematological malignancies. Evidence indicates that WT1 is important in the lineage-specific differentiation of hematopoietic cells and leukemogenesis. In myelodysplastic syndromes (MDS), WT1 expression has prognostic significance: it is directly correlated with the type of MDS, with IPSS score and with disease progression. Recent data demonstrate that WT1 is a potent activator of the EPO gene under normoxia and it is suggested that WT1 may regulate paracrine EPO synthesis in a tissue-specific manner. Bmi-1 is a transcriptional repressor gene which may be expressed restrictedly in stem cells and progenitors and is required to regulate the adult self-renewing hematopoietic and leukemic stem cells. It appears that it also plays an important role in providing cells the potential for proliferation. A number of reports on Bmi-1 provide perspectives on the close association of its expression with the progression of hematopoietic malignancies. Furthermore, flow cytometry has shown that Bmi-1 positivity in CD34+ cells is positively correlated with IPSS score. Introduction: We have designed a study to evaluate changes in gene expression profiles of bone marrow mononuclear cells of primary low and intermediate-1 IPSS risk MDS patients receiving erythropoetic growth factors (darbepoetin or high-dose rHuEpo alpha). Associations with response, changes in Hb, in percentage of CD34+ and apoptotic cells are evaluated. We present preliminary results in 6 patients. Methods: Bone marrow samples were obtained before and after 12 weeks of treatment. Mononucleated cells were cryopreserved and later thawed for total RNA extraction and cDNA synthesis. Gene expression profiling and the expressions of WT1 and Bmi-1 by RT-PCR were evaluated. Results: Baseline median Hb was 9,5 g/dL (interquartile range 8,7 10,1). Baseline mean Bmi-1 was 14011 (± SD 3927). All patients had a major erythroid response to treatment. Preliminary results demonstrate a significant decrease in WT1 from baseline median 1635 (interquartile range 1251–2150) to 1192 (interquartile range 1005–1365, P=0.046). Fig. 1. Changes in WT1 expression during therapy. Fig. 1. Changes in WT1 expression during therapy. Discussion: Though few patients have yet been studied, it is suggested that WT1 decreases in patients responding to erythropoetic growth factors. Further evaluation with stratification for erythropoetic response in the extended study may furnish novel associations between gene expression, erythropoetic growth factors and prognosis in MDS patients.

Description: Dasatinib was approved for use in the treatment of patients (pts) with chronic myeloid leukaemia (CML) and resistance to Imatinib. We applied a rescue treatment based on Dasatinib therapy to achieve a pharmacological immunomodulation in a setting of CML-relapsed allogenic stem cell transplantated (A-HSCT) pts. Patients were required to have both resistance to Imatinib and unresponsiveness to cellular therapy such as Donor Lymphocyte Induction (DLI). We hypothesized that Dasatinib could potentially improove the disease by immunomodulatory action. Primary aim of this therapeutic design was to address, in single institution trial, therapeutic force of a innovative pharmacologic strategy to induce the cytogenetic response followed by DLI. Therefore, we investigated Dasatinib ability to achieve immuno-effects by targeting key mediators of Th1, Th2, and Treg response. Biological effects were examined on conventional diagnostic parameters such as haematological chimerism, cariotype and Bcr-Abl gene transcript. Herein, we present interim results of a pilot group of 3pts. Patients received dasatinib 70 mg twice daily(140 mg total daily dose). Dose modifications were allowed for the management of toxicity. Treatment was performed until complete cytogenetic, molecular response and haematological full donor chimerism. Materials and Methods: To investigate the immunological changes, we used a TaqMan® Low Density Array, based on comparative CTdd CT method on Applied Biosystems 7900HT, to perform relative quantification of cDNA derived from peripheral venous blood specimens harvested after DLI, before and after starting dasatinib therapy. Assumed that normal control values of all transcripts were = 1, we evaluated over or down regulation of gene expression profile (GEP) of a panel of 48 genes involved in immune response. Results: clinical changes after third month of dasatinib therapy. Case 1: responsive patient, maintained a mixed haematologic chimerism, but showed a complete cytogenetic and molecular remission. Following, patient restarted with DLI therapy. Case 2: responsive patient, showed nearly full-donor haematologic chimerism with complete cytogenetic and molecular remission. Case 3: patient no evalutable because brief treatment (only 1 month). Dasatinib caused early haematological toxicity. Patient maintained a low level of donor T cells with presence of Philadelphia chromosome associated to elevated p210 molecular signal. Gene expression profiles post-dasatinib therapy: According to in vitro experiments (Blood October 25, 2007), in all cases we observed a down regulation of IL-2 and IL-12B (Th1), IL-6 and IL- 18, IL-10 (Th2) cytokines and mediators of apoptosis such as EGR2, EGR1. By contrast, multiple pro-inflammatory factors were up-expressed: IFN-g, IL-17, IL-7. Only in case number 1, TNF-a and IFN-g molecular pathways were not influenced by the drug. In fact their elevated expression was preserved as compared to pre-dasatinib levels. Noterworthy among cases number 2 and 3 (with mixed chimerism), Dasatinib improved a marked inhibition of Th1 effectors in addition to down-regulation of several important molecular transcripts: SERPIN B3–B4, BCL2A1, SELP, PIAS 1, IRF8, IRF1, CCL7, CCL5, CXCL9, CCR4, ICAM. Regards to T regulatory cells, Foxp3 was strongly up-regulated in case number 1 and down-regulated in case2and 3. Conclusion: We think that Dasatinib represent a possibility of cure for for CML pts relapsed after A-HSCT and unresponsive to alternative treatments. For imatinib-resistant CML patients, such as in this study, there are few currently available effective options. The present results strongly emphasize the importance of immune response control to achieve the desired clinical effects.

Description: Abstract 2622 Poster Board II-598 Introduction: Genetic alterations reported in myelodysplastic syndromes (MDS) are not disease-specific and the underlying molecular causes of the disease remain poorly understood. It has been suggested that one or more of the genes mapping within the commonly deleted region of the 5q syndrome, together with other distant genes, may be critical to the development of the 5q syndrome. Potential candidate genes have been identified including the tumor suppressor gene SPARC, and the ribosomial protein gene, RPS14. Haploinsufficiency of RPS14 has been demonstrated and recent evidence indicates RPS14 as a causal gene for the 5q syndrome. Lenalidomide has proven efficacy in MDS patients with del(5q). Rapid and durable responses include transfusion-independence, with a rise in Hb, suppression of the 5q-deletion clone and improvement in bone marrow morphologic features. Methods: In a multicenter Italian phase II trial to evaluate safety, changes in quality of life and efficacy of lenalidomide in primary MDS patients with del(5q) and low or Int-1 risk IPSS, we investigate changes in bone marrow cytogenetics and gene expression patterns during treatment. The starting dose of lenalidomide is 10 mg p.o once daily on a continuous daily schedule for a maximum of 12 months. Dosing is based upon clinical and laboratory findings. Bone marrow cytogenetics and gene expression profiling are performed on study entry and every 12 weeks up to end of study (week 52). Gene expression assays of 51 candidate genes from the published literature and genomic databases have been selected and are carried out with TaqMan® Low Density Array Fluidic card (TaqMan® Human Array, Applied Biosystems, Foster City, CA, USA) based on Applied Biosystems PRISM® 7900HT comparative dd CT method, according to manufacturer's instructions. Using an 18S mRNA gene pre-designed assay from Applied Biosystems to detect the expression of the housekeeping gene 18S in each sample, target gene expression is normalized with 18S gene expression derived from a bone marrow pool of normal healthy subjects and for each sample the ratio between the target and 18S are expressed. Results: Baseline values for 23 patients (mean age 73 ± 10 years) are available and 16 have been re-evaluated after 12 weeks. Mean Hb was 8.6 ± 0.9 g/dL and 20 patients were transfusion-dependent. Seven patients had additional cytogenetic abnormalities. At baseline, RPS14 was under-expressed in 19 out of 21 patients evaluated. After 12 weeks RPS14 was re-evaluated in 13 patients: all had erythroid responses and RPS14 increased significantly from 0.07 (IQ Range 0.03–0.13) to 76.1 (0.73– 304.0, p=0.002). SPARC expression was under-expressed in 15/23 patients and variations during treatment were not significant. Baseline FAS gene was under-expressed in all patients and increased above reference values (p=0,006) after 12 weeks in 7/14 cases. IL7R was over-expressed in all patients at baseline (median 3263.3, IQ range 1998.3–5027.1) and was significantly reduced after 12 weeks (median 0.17, IQ range 0.05–2.20, p