ALBERT

Description: We prove that the SH2-containing tyrosine phosphatase 1 (SHP-1) plays a prominent role as resistance determinant of imatinib (IMA) treatment response in chronic myelogenous leukemia cell lines (sensitive/KCL22-S and resistant/KCL22-R). Indeed, SHP-1 expression is significantly lower in resistant than in sensitive cell line, in which coimmunoprecipitation analysis shows the interaction between SHP-1 and a second tyrosine phosphatase SHP-2, a positive regulator of RAS/MAPK pathway. In KCL22-R SHP-1 ectopic expression restores both SHP-1/SHP-2 interaction and IMA responsiveness; it also decreases SHP-2 activity after IMA treatment. Consistently, SHP-2 knocking-down in KCL22-R reduces either STAT3 activation or cell viability after IMA exposure. Therefore, our data suggest that SHP-1 plays an important role in BCR-ABL–independent IMA resistance modulating the activation signals that SHP-2 receives from both BCR/ABL and membrane receptor tyrosine kinases. The role of SHP-1 as a determinant of IMA sensitivity has been further confirmed in 60 consecutive untreated patients with chronic myelogenous leukemia, whose SHP-1 mRNA levels were significantly lower in case of IMA treatment failure (P 〈 .0001). In conclusion, we suggest that SHP-1 could be a new biologic indicator at baseline of IMA sensitivity in patients with chronic myelogenous leukemia.

Description: The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies, but is absent on normal tissues, including hematopoietic progenitor cells, and may therefore be an appropriate candidate for T cell–mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity, PRAME-specific cytotoxic T lymphocytes (CTLs), we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal, PRAME-specific CTL lines and elicited high-avidity CTLs, with a high proportion of cells recognizing a previously uninvestigated HLA-A*02–restricted epitope, P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME+ hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts, but also against leukemic progenitor cells as assessed by colony-forming–inhibition assays, which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors, indicating that this approach may be of value for immunotherapy of PRAME+ hematologic malignancies.

Description: Abstract 2795 In the era of molecular target therapy, whereas Imatinib has shown an overall survival rate of 85% and an estimated event-free survival of 55% in a 8 year result update of IRIS trial, several in vitro data have confirmed that Ph+ stem cells housed within BM niches are resistant to TKI treatments. Second generation TKI Nilotinib has been designed with improved target specificity over Imatinib. Its efficacy in the treatment of patients with CML who are resistant to or intolerant to Imatinib led to the registration of a clinical trial CAMN107EIC01, a phase IIIb, multicentre, open-label study applying Nilotinib in the treatment of newly diagnosed CML. The aim of the current study (CAMN107EIC01 sub-study N10) is to define BM microenvironment markers that nurture and determine leukemic stem cell fate in the BM niches of Nilotinib treated patients. We enrolled 37 patients involving 21 Italian centers, from whom written informed consent has been obtained for sub-study N10. Patients have been monitored by Real Time RT-PCR (RT-qPCR) for the expression of the fusion mRNA BCR-ABL, as specified by the core protocol. Major Molecular Response (MMR) is defined as detectable disease ≤0.1% BCR-ABL according to the international scale (IS). Plasma and mononuclear cells have been collected from BM and PB samples of the enrolled patients at the screening visit (V0) and after 3 months of treatment (V4). We purified total RNA from BM and PB mononuclear cells of the enrolled patients to screen by RT-qPCR the expression of 10 genes (ARF, cKIT, CXCR4, FLT3, LIF, NANOg, PML, PRAME, SET and TIE), involved in the regulation of the stemness and survival signaling of hematopoietic stem cells. RT-qPCR results were normalized by the expression of ABL mRNA (Normalized mRNA copy Number: NCN). Moreover, we evaluated by multiplex ELISA assay BioPlex, the BM plasma concentration level of 20 cytokines (IL1a, IL1b, IL3, IL6, IL7, IL8, IL10, IL12, IL15, G-CSF, M-CSF, SCF, SDF1, TRAIL, HGF, PDGFbb, GM-CSF, MIP-1a, TNFa and VEGF), known to be key factors in the interaction of Ph+ stem cells to BM microenvironment. The interim analysis of MMR until the 12th month is available in 26 out of 37 enrolled patients. We observed that MMR is achieved during the first 12 months of treatments in 17 out of 26 (65%) patients. Molecular analysis showed that the expression of two genes involved in the regulation of stem cell pluripotency (NANOg) and cytokine signaling (SET) were significantly down-regulated at V0 in PB mononuclear cells of patients achieving MMR in comparison to cells from non-responding patients (4vs21 NANOg NCN, p=0.05; 8vs32 SET NCN, p=0.05). These data were also confirmed on BM patient's sample. Moreover, we investigated the concentration level of the above-mentioned soluble factors in BM plasma samples of all 37 enrolled patients at both V0 and V4. We observed that the BM plasma level of several cytokines produced by leukemic cells significantly decreased during the first 3 months of Nilotinib treatment: IL3 (54vs3ng/ml; p=0.02), M-CSF (56vs12 ng/ml; p=0.005), SCF (170vs104 ng/ml; p=0.007), HGF (7565vs705 ng/ml; p

Description: Introduction Interferon-alpha 2 (IFN) is able to induce hematological response in about 70-80% of ET patients but some of them could be defined as bad responders. IFN binding its receptor results in tyrosine cross-phosphorylation and auto-phosphorylation of the JAKs proteins (Tyk2 and Jak1). These phosthyrosines recruit and activate STAT family member such as STAT1 and STAT3. These proteins induce the transcription of SOCSs, whose role is to extinguish cytokine signaling by inhibition of JAK kinase-activity directly through the KIR-domain, and indirectly promoting the proteasomal degradation of Jak2, by SOCS-box-motif. In summary, IFN induces the expression of SOCSs, which inhibit TPO mediated signaling through Jak2 double inhibition. This allows IFN-α and TPO pathway to cross-talks by means of the JAK-STAT-SOCS cascade. Aims To identify molecular markers that identify those patients who respond to IFN, we analyzed bone marrow cells transcript levels of specific genes involved in the IFN receptor pathway, whose signal cross-talks with the TPO dependent JAK-STAT pathway. In particular we investigated the mRNA expression of JAK1, TYK2, STAT1, STAT3, SOCS1 and SOCS3. Methods We analyzed 60 ET patients treated with 3 million units of IFN-α-2b 5 times a week as induction (3 months), and 3 times a week as maintenance. Responses were classified as follow: Good-Responders(R) (n=44), those who achieved complete response according to European Leukemia Net criteria, and Bad-Responders(NR) (n=17) who didn’t reach the criteria. The mRNA expression of genes of interest was measured in bone marrow samples from ET patients by RTq-PCR and tested for their predictive value using receiver operating characteristics (ROC) curves. Data were normalized as following: [mRNA normalized copy number (NCN)=mRNA target gene/mRNA GUSB]. An IFN score was calculated as an average in log2 of mRNA levels of genes differently expressed between Good-R and Bad-R. Results Main clinical characteristics were similar between the two groups of response. JAK2 V617F mutation was detected in 56,8% of Good-R and 58,8% of Bad-R (p=0,81) and no difference was found in JAK2V617F allele burden (p=0,17) and mRNA expression (p=0,2). Patients showed a median spleen volume of 500 ml in Good-R and 250 ml in Bad-R group (p=0.01). Bad-R compared with Good-R showed higher mRNA expression of JAK1 (13.4 vs 4.7; p

Description: To date no single molecular biomarker, with proven clinical utility, that is predictive and identifies patients with more aggressive chronic myeloid leukaemia (CML) at diagnosis, has been reported. Such a molecular biomarker would enable stratification of patients according to aggressiveness of the malignancy and therefore at greater risk of progressing to blast crisis (BC). If these patients could be identified a more potent TKI could be prescribed at presentation or be considered for stem cell transplant up front. Recently a common polymorphic variant of growth factor independent 1 (GFI1), a zinc finger DNA binding transcriptional repressor, whereby serine is substituted by asparagine at residue 36 (S36N), was reported (Khandanpour et al. Blood. 2010; 115:22462) to be associated with a 1.6 fold increased risk of developing acute myeloid leukemia (AML). In addition, investigators reported a frequency of 11% of the S36N GFI1 allele among AML patients, compared with 3-7% among the European populations. Reports suggest S36N GFI1 variant fails to repress the target genes, including HOXA9, and as a consequence their expression levels are elevated. These observations are consistent with a critical role for GFI1 in myeloid differentiation. In view of these findings we hypothesized that the GFI1 S36N allele might influence CML progression, via up-regulation of HOXA9, from a relatively indolent chronic phase (CP) to invariably fatal BC, characterized by presence of leukaemic stem cells, resistant to tyrosine kinase inhibitors in peripheral blood. Therefore, we assessed if S36N allele frequency was increased in CML and quantified GFI1expression. We retrospectively studied 106 cDNA samples, synthesized using 1 µg RNA extracted from nucleated cellular pellets isolated from peripheral blood or bone marrow aspirate. Total RNA was reverse transcribed using reverse transcriptase primed with random hexamers oligonucleotides. Of the 196 sample 89 were from highly heterogeneous CML patients and the remaining 17 comprised of randomly selected normal control samples from healthy adult volunteer blood donors. The 89 CML patients included 24 (accelerated phase: 2 ; BC:22) with 51 years median age (range 20-75) who had progressed to advanced disease (AD) of these 13 were males. The remaining 65 CML patients (M:43 ; F: 22) were in chronic phase (CP) with median age 74 years (range 7-90). Polymerase chain reaction products, containing GFI1 exons 2, 3 and 4 were subjected to restriction fragment length polymorphism using BfaI restriction enzyme to test for the S36N GFI1 allele. Samples with the S36N allele were confirmed by Sanger sequencing. Total GFI1 (i.e. serine and asparagine alleles), HOXA9 and GUSß (endogenous control gene) mRNA transcripts were quantified by quantitative real time using Taqman assay. Each Taqman assay included a standard curve constructed using 2 plasmids containing HOXA9 or GUSß insert. Serially diluted cDNA derived from MEG01 hematopoietic cell line was used to prepare the standard curve for GFI1quantification. S36N GFI1 allele frequency was significantly increased (p

Description: Introduction. Interferon alpha (IFN-α) is an attractive agent for the treatment of Essential Thrombocythemia (ET) due to its ability to induce clonal complete remission, sometimes lasting beyond treatment discontinuation, and to its recognized non-leukemogenicity. However, despite decades of clinical experience with IFN-α in patients with MPNs, optimal dose schedules, treatment duration and the ultimate molecular basis of the heterogeneous response still remain undefined. Hence, the early identification of IFN-sensitive patients may help limit IFN-α exposure to those who really benefit from treatment. Aim. Here we report the results of a trial involving 61 ET patients treated with IFN-α, aimed to identify the baseline molecular and clinical parameters able to predict response to treatment. Methods. IFN treatment schedule implied an initial induction phase with 3MU/five times a week; in patients who reached a platelet count 600x109/L or platelet reduction was