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Background:CEBPA-mutated acute myeloid leukemia (AML) is a separate entity in the updated WHO and ICC classifications. Previously identified by double mutations, the clinical profile and favorable outcome were correlated with in-frame mutations in bZIP domain. Beyond mutations recurrent in myeloid neoplasms (involving TET2, FLT3, NRAS), CEBPA-mutated AML is enriched in gene mutations (i.e., GATA2, CSF3R, WT1) rarely observed in CEBPA-wild type AML. No conclusive findings have been drawn for the prognostic impact of additional mutations. Of note, somatic GATA2mutations have been associated with invasive fungal infections in myeloid neoplasms. Although overall correlating with favorable prognosis, an unexpectedly high rate of treatment-related mortality (TRM) in CR has been described in a large multicenter study on intensively treated CEBPA-mutated patients (pts) (Schlenk et alBlood 2013;122(9):1576).

Background:NPM1mutations represent the most common genetic alteration in adult acute myeloid leukemia (AML), often in co-occurrence with FLT3-ITD and mutations in genes influencing DNA methylation ( DNMT3A, IDH1, IDH2and TET2). Whether these genetic alterations correlate with distinct immunophenotypic and clinical features is still a matter of debate. There is growing evidence of distinct immunophenotypic subsets driven by co-occurring genetic mutations in NPM1-mutated AML, potentially accounting for the heterogeneity of clinical presentations and outcome. Mason et alhave described a subtype of NPM1-mutated AML, displaying an immunophenotypic profile resembling that of acute promyelocytic leukemia, specifically, the negativity for both CD34 and HLA-DR, and as such defined APL-like.

Background. New drugs with or without autologous stem cell transplantation (ASCT) can induce deep CR responses. MRD can be now considered in the response evaluation by the IMWG and many studies propose it as surrogate for survivals. Multiparametric flow cytometric assays have now been replaced by advanced assays that permit to assess simultaneously more than 8 markers in a single tube. In particular, Euro-flow consortium has developed NGF, a novel high sensitive and standardized approach for MM MRD evaluation that is based on the use of 2 single 8-color tubes, containing all the markers needed to distinguish normal vs MM PCs. However, it is necessary to work on fresh samples and to acquire 107cell/sample, so to have the possibility to evaluate the Limit Of Quantification (LOQ) and the Limit Of Detection (LOD). The LOQ is calculated as 50 clonal plasmacells among 107nucleated cells; the LOD as 20 clonal plasmacells among 107nucleated cells.

Elena: CELGENE: Other: funding for meeting participation; PFIZER: Membership on an entity's Board of Directors or advisory committees; NOVARTIS: Membership on an entity's Board of Directors or advisory committees; GILEAD: Membership on an entity's Board of Directors or advisory committees. Vannucchi: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

No relevant conflicts of interest to declare.

Abstract 1454

Chronic lymphocytic leukemia (CLL) patients bearing 13q14 deletion are known to experience a more favorable clinical course. Recent studies, focusing on patients with loss of 13q as the sole cytogenetic aberration at diagnosis (del13q-only cases), showed that the number of malignant cells carrying this genetic lesion correlates with a more aggressive clinical behavior. However, whether the size of the 13q deletion may also influence the clinical outcome remains to be elucidated.Probes for chromosome 13q (LSI-RB1, LSI-D13S319), 11q (LSI-ATM), 17p (LSI-p53) and chromosome 12 (CEP12) were utilized on nuclei collected at diagnosis from: i) a multi-institutional CLL cohort (342 del13q-only cases) and ii) a consecutive unselected single-institution cohort of 265 cases. RB1 deleted cases (delRB1) were defined as having at least 5% of deleted nuclei. Time to treatment (TTT) intervals, as well as Rai staging, IGHV mutational status, CD38 and ZAP70 expression, B2-microglobulin levels, all evaluated at diagnosis, were also available for all cases that entered the study. Genome wide DNA profile was performed in a pilot series of 90 CLL samples using Affymetrix GeneChip Human SNP6 arrays.According to genome wide DNA analysis, delRB1 occurred in a proportion of del13q-only cases (36/90; 40%), always comprising the deleted region detected with the LSI-D13S319 probe (that covers the miR-15a/16-1 cluster and the DLEU2 gene) and characterized by a larger chromosome loss (median size 2.07 Mb vs. a median size of 0.86 Mb for the canonical del13S319). Maximally selected log-rank statistics identified the 70% of nuclei bearing del13S319 as the most appropriate cut-off value capable of separating del13q-only cases into two subgroups with different TTT distributions. Consistently, del13q-only cases with at least 70% of nuclei bearing del13S319 showed a significantly shorter TTT than del13q-only cases with less than 70% deleted nuclei (p=0.0001). Del13q-only cases were then divided in four subsets according to the percentage of nuclei bearing del13S319 with or without a concomitant delRB1: del13S319 <70% (group 1), 144 cases; del13S319 <70% + delRB1 (group 2), 95 cases; del13S319 >70% (group 3), 64 cases; del13S319 >70% + delRB1 (group 4), 39 cases. The median TTT of group 1 (not reached) was significantly longer than the median TTT of group 2 (92 months, p=0.012), group 3 (68 months, p<0.0001), and group 4 (82 months, p=0.0025; see Fig. 1A). Multivariate Cox proportional hazard analyses selected the presence of delRB1 (p=0.029), along with the IGHV mutational status (p<0.0001), as an independent negative prognosticator in the context of del13q-only cases with low/intermediate Rai risk (Rai stage of 0/I at diagnosis) and <70% of del13S319. Cases belonging to the consecutive unselected single-institution CLL cohort were divided into subsets according to the classification proposed by Döhner et al (NEJM, 2000). Notably, the presence of del13S319 in <70% of cells in the absence of delRB1 identified a patient subset with particularly stable and benign clinical course (group A in Fig. 1B, 48 cases; median TTT not reached). Conversely, patients characterized by del13S319 in <70% of cells but with a larger deletion, as determined by concomitant delRB1 (group B, 24 cases), or del13S319 in >70% of cells (with or without delRB1, group C, 25 cases) or a normal karyotype (group D, 75 cases) had shorter median TTT intervals (ranging from 105 to 129 months, p<0.01 in all the comparisons). Finally, patients affected by CLL bearing trisomy 12 (group E, 48 cases) and del11q or del17p (group F, 45 cases) experienced the worst clinical courses (p<0.0001).In the context of del13q-only cases, different clinical outcomes were associated to the percentage of 13q14 deleted cells, as well as to the size of the 13q14 deletion, as detected by the LSI-RB1 probe. Moreover, the presence of delRB1 emerged as a feature capable of refining the prognostic assessment in the context of CLL cases with <70% del13S319. The underlying genetic mechanisms correlated with the different clinical outcomes and associated with the size of the 13q deletion are presently under investigation.No relevant conflicts of interest to declare.

Imatinib mesylate is highly effective in inducing rapid hematologic and cytogenetic responses in the vast majority of chronic myeloid leukemia (CML) patients. Yet, discontinuation of treatment is associated with disease relapse probably due to the persistence of resistant leukemic stem cells representing a reservoir of the disease. On this regard it has been reported that BCR-ABL positive progenitor cells can still be detected in patients in complete cytogenetic response (CCyR) after short term of imatinib treatment (Bhatia R, et al. Blood. 2003;101:4701-4707) but also after a stable long lasting CCyR (Bocchia M, et al. Leukemia. 2008;22:426-428). Compared to imatinib, the second generation Tyrosin Kinase Inhibitor (TKI) nilotinib appears to eradicate more rapidly the bulk of CML cells, inducing high rate of CCyR and major molecular response (MMolR) after a very short period of treatment (78% CCyR and 52% MMolR at 3 months) (Rosti G, et al. Blood. 2009;114(24):4933-8). Despite nilotinib is a more potent TKI, it didn't appear to be more effective in eliminating in vitro CML progenitors than imatinib (Konig H, et al Leukemia. 2008;22:748-755). Up to date no data evaluating the persistence of Ph+ stem cells in early chronic phase CML patients during first line treatment with nilotinib have been reported.We investigated the presence of residual CD34+/Ph+ cells in 24 CML patients in CCyR during first line nilotinib treatment. Patients were enrolled in 2 clinical trials (GIMEMA CML0307 and CAMN107A2303) and evaluation of residual leukemic stem cells was performed during a routine bone marrow aspirate after receiving specific patients informed consent. Bone marrow purified CD34+ cells were evaluated for BCR-ABL fusion gene by fluorescent in situ hybridization (FISH) analysis. A minimum of 100 interphase nuclei of purified CD34+ cells was considered optimal for FISH analysis.All 24 patients have been treated exclusively with nilotinib since diagnosis (17/24 at 400mg bid; 5/24 at 300mg bid; 2/24 at 400mg/day). At the time of analysis all 24 patients were in CCyR for a median time of 27 months (range 6–29) after being treated for a median time of 30 months (range 9–30) with this second generation TKI. Regarding molecular response 20/24 (83%) were in MMolR while only 1/24 (4%) was in CMolR. Harvest, purification and subsequent FISH analysis of bone marrow CD34+ cells was optimal in 15/24 (63%) patients, suboptimal in 5/24 (21%) patients (less than 100 interphase nuclei analyzed) and not adequate in 4/24 (16%) patients (less than 50 interphase nuclei). With respect to leukemic stem cells, residual CD34+/Ph+ cells were found only in 1/20 (5%) evaluable patients. Of note, in this patient 140 CD34+ interphase nuclei were analyzed and only 1 was found bcr-abl positive (0.7%).Our study shows for the first time that in patients in CCyR during front line Nilotinib treatment residual CD34+/Ph+ stem cells are very rarely detected. These results quite differ from what was previously found in imatinib treated patients (Bocchia M, et al. Leukemia. 2008;22:426-428). In fact in the present series, only 1/20 (5%) patients treated with nilotinib in CCyR for a median time of 27 months showed residual CD34+/Ph+ cells, while in our prior study residual leukemic CD34+ cells were still detectable in 14/31 (45%) imatinib treated patients in stable CCyR (median of 39 months). Despite the limited number of patients studied, this novel evidence may support the better short term clinical results observed with nilotinib as first line treatment in CML.Rosti: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Honoraria, Speakers Bureau; Roche: Speakers Bureau.

Abstract 3578

Abstract 3413

Standard induction chemotherapy for Acute Myeloid Leukaemia (AML) is represented by a combination of cytarabine and antracyclin with or without the addition of a third drug (e.g. etoposide), with a Complete Remission (CR) rate of about 60-65%.The main goal of adding further drugs to induction therapy is to increase the CR rate in order to extend the opportunity of intensification therapies (e.g. autologous or allogeneic stem cell transplantation).Eighty-four consecutive AML patients younger than 65 yrs were treated with a four-drug induction regimen Fludarabine (25 mg/sqm), Ara-C (2 g/sqm), Etoposide 100 mg/sqm on days 1-5, Idarubicin (6 mg/sqm) on days 1, 3, and 5, between 2002 and 2008 in two Italian Hematological Centres. The median age of this series of patients was 45 years (range 18-65), 39 were males and 44 were females. According to karyotype at diagnosis, white blood count (WBC > 30.000/mmc) and secondary disease, 58/84 patients (69%) were considered at high risk Patients were evaluated for response rate and treatment-related adverse events. Stem cell transplantation was performed according to the risk stratification and donor availability.After induction with FLAIE, CR occurred in 62 of 84 patients (74%). There were two death during induction (DDI 2 %). Six out of 22 resistant patients (7%) achieved CR after the second course of therapy, that was HD-Ara-c (2 g/sqm) on days 1-5 + idarubicine (12 mg/sqm) on days 1-3. Three patients were lost to follow up after induction, while in CR. The toxicity of FLAIE was acceptable, with grade III/IV hematological and extra-hematological adverse events comparable with standard induction therapy. Fifty-five patients of the 81 evaluable (68%) underwent transplant procedures while in first CR (22/81, 27 %, auto-SCT and 33/81, 41 %, allo-SCT). Five patients received allo-SCT in second CR, after early relapse. Three patients received allo-SCT with active disease and died because of disease progression. Sixteen patients (20%) received only chemotherapy-based consolidation because of either resistant disease or lack of donors or unsuccessful peripheral blood harvestt. After a median follow-up of 46 months (range 1-81), 40 of 81 evaluable patients (49%) are alive (40/40 in CR). Relapse rate is 32% at 4 yrs. These results suggest that a four drugs induction chemotherapy is feasible in young AML patients and can effectively permit intensification chemotherapy in at least 2/3 of the patients. A larger number of patients, in the context of a randomized trial may better define its efficacy with respect to standard 2 – 3 drugs induction therapy.Baccarani: Novartis Pharma: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Mayer Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Abstract 4147

The Wilms tumor gene WT1 is overexpressed in hematopoietic malignancies such as Myelodysplastic syndromes and leukemias and the WT1 protein was demonstrated to be an attractive target antigen for an immunotherapeutic approach to these diseases. Most of the efforts have been focused to the search for immunogenic peptides suitable for inducing cytotoxic T lymphocytes (CTLs) and to less extent for CD4+ T lymphocytes with potential cytotoxic activity. On this matter, in our previous experience with a p210-derived peptide vaccine developed for chronic myeloid leukemia patients with minimal residual disease, the main immune and therapeutic effect observed after vaccinations appeared to be mediated by peptide-specific CD4+ T cells induced by the longest peptide (25 mer) included in our vaccine. CML-peptide specific T cells were found to be either CD4+/perforin+ or CD4+/CD25+/Foxp3+ and we recently showed their direct cytotoxicity against a CML cell line. Thus to pursue a vaccine strategy mainly devoted to a similar CD4+ T cell immune response, we screened WT1 protein through Syfpeithi database to identify original peptides with a suitable length (23–25 amino acids) to be processed by several HLA class II molecules and to induce a strong CD4+ T cell stimulation. Additionally, in order to maximize the immunogenic potential of the novel peptides, we focused our attention on areas of the protein with known CTLs/CD4 T cells immunogenic epitopes. We identified two peptides that fulfilled these requirements: SEPQQMGSDVRDLNALLPAVPSLGG (WT1-iso5 64–88) which includes 5 amino acid from the alternative splicing derived isoform 5 of WT1 and the first 20 aa of “canonical” WT1 sequence and RPFMCAYPGCNKRYFKLSHLQMHSR (WT1321–345). Both 25mer peptides showed strong HLA binding properties for HLA-DRB1*0101, HLADRB1* 0401, HLA-DRB1*0701, HLA-DRB1*1101, HLA-DRB1*1501 and HLADRB1* 0301( DR17). We first tested them in vitro for their capability to induce peptide-specific CD4+ T cells. Briefly, CD4+ T cells freshly isolated from PBMC were cultured for 21 days in 5% AB human serum media while undergoing to 3 rounds of stimulation with autologous CD14+ cells and both WT1-iso5 64–88 and WT1 321–345 peptides at 20μg/ml in the presence of IL-15. This in vitro stimulation was performed in 3 normal subjects and in 3 MDS patients with high levels of bone marrow WT1 transcript (2 patients presenting a low-International Prognostic Scoring System (IPSS) refractory anemia (RA) and 1 with intermediate IPSS RA). In all 3 healthy donors tested, both peptides were able to induce peptide specific CD4+ T cell proliferation as measured by standard 3HThymidine assay, with a stimulation index (SI) ranging from 2.0 to 2.5 regardless of their HLA-DR phenotype ( SI= cpm CD4+ T cells plus test peptides/CD4+ T cell alone or CD4+ T cells plus control peptides; peptide-specific T cell proliferation was considered positive for SI≥2). Similar results were obtained in all 3 MDS patients in which WT1-iso5 64–88 and WT1 321–345 induced peptide-specific CD4+ T cell proliferation with a SI value of 2.5, 2.9 and 3.0 respectively. In conclusion the present study identified 2 novel WT1-derived 25 mer peptides which were able to easily induce in vitro a peptide-specific CD4+ T cell response in MDS patients. WT1-specific CD4+ T cells proliferated with similar SI values in normal donors and in WT1 positive MDS patients, the latter being highly exposed to this antigen and thus potentially tolerant to it. A possible cytotoxic activity of these WT1-specific CD4+ T cells is under evaluation and in vivo vaccinations of low-intermediate IPSS MDS patients with these peptides are planned.

The Wilms tumor gene WT1 is overexpressed in hematopoietic malignancies such as Myelodysplastic syndromes and leukemias and the WT1 protein was demonstrated to be an attractive target antigen for an immunotherapeutic approach to these diseases. Most of the efforts have been focused to the search for immunogenic peptides suitable for inducing cytotoxic T lymphocytes (CTLs) and to less extent for CD4+ T lymphocytes with potential cytotoxic activity. On this matter, in our previous experience with a p210-derived peptide vaccine developed for chronic myeloid leukemia patients with minimal residual disease, the main immune and therapeutic effect observed after vaccinations appeared to be mediated by peptide-specific CD4+ T cells induced by the longest peptide (25 mer) included in our vaccine. CML-peptide specific T cells were found to be either CD4+/perforin+ or CD4+/CD25+/Foxp3+ and we recently showed their direct cytotoxicity against a CML cell line. Thus to pursue a vaccine strategy mainly devoted to a similar CD4+ T cell immune response, we screened WT1 protein through Syfpeithi databaseto identify original peptides with a suitable length (23–25 amino acids) to be processed by several HLA class II molecules and to induce a strong CD4+ T cell stimulation. Additionally, in order to maximize the immunogenic potential of the novel peptides, we focused our attention on areas of the protein with known CTLs/CD4 T cells immunogenic epitopes. We identified two peptides that fulfilled these requirements: SEPQQMGSDVRDLNALLPAVPSLGG (WT1-iso5 64–88) which includes 5 amino acid from the alternative splicing derived isoform 5 of WT1 and the first 20 aa of “canonical” WT1 sequence and RPFMCAYPGCNKRYFKLSHLQMHSR (WT1321–345). Both 25mer peptides showed strong HLA binding properties for HLA-DRB1*0101, HLADRB1* 0401, HLA-DRB1*0701, HLA-DRB1*1101, HLA-DRB1*1501 and HLADRB1* 0301( DR17). We first tested them in vitro for their capability to induce peptide-specific CD4+ T cells. Briefly, CD4+ T cells freshly isolated from PBMC were cultured for 21 days in 5% AB human serum media while undergoing to 3 rounds of stimulation with autologous CD14+ cells and both WT1-iso5 64–88 and WT1 321–345 peptides at 20μg/ml in the presence of IL-15. This in vitro stimulation was performed in 3 normal subjects and in 3 MDS patients with high levels of bone marrow WT1 transcript (2 patients presenting a low-International Prognostic Scoring System (IPSS) refractory anemia (RA) and 1 with intermediate IPSS RA). In all 3 healthy donors tested, both peptides were able to induce peptide specific CD4+ T cell proliferation as measured by standard 3HThymidine assay, with a stimulation index (SI) ranging from 2.0 to 2.5 regardless of their HLA-DR phenotype ( SI= cpm CD4+ T cells plus test peptides/CD4+ T cell alone or CD4+ T cells plus control peptides; peptide-specific T cell proliferation was considered positive for SI≥2). Similar results were obtained in all 3 MDS patients in which WT1-iso5 64–88 and WT1 321–345 induced peptide-specific CD4+ T cell proliferation with a SI value of 2.5, 2.9 and 3.0 respectively. In conclusion the present study identified 2 novel WT1-derived 25 mer peptides which were able to easily induce in vitro a peptide-specific CD4+ T cell response in MDS patients. WT1-specific CD4+ T cells proliferated with similar SI values in normal donors and in WT1 positive MDS patients, the latter being highly exposed to this antigen and thus potentially tolerant to it. A possible cytotoxic activity of these WT1-specific CD4+ T cells is under evaluation and in vivo vaccinations of low-intermediate IPSS MDS patients with these peptides are planned.

Translocations of 14q32/IgH have a low incidence in CLL. Partner chromosomes and the prognostic significance are poorly defined. Four hundred thirty cases of CLL seen at the Hospitals of Ferrara, Salamanca and Siena between 1992 and 2006 were studied. Inclusion criteria were: diagnosis of CD5/CD19+ CLL with k/λ restriction, cytogenetic/FISH data, immunophenotypic data, complete hematological and clinical data. Lymphomas in leukemic phase were excluded. FISH was performed for 17p13/TP53, 11q22.3/ATM, 6q21, chr 12 centromere, 13q14 and 14q32/IGH. Patients with no detectable aberration or isolated 13q− were included into a favourable cytogenetic group (group 1), those with +12, 6q− or 1–2 aberrations into an intermediate risk group (group 2) and those with 17p−, 11q−, ≥ 3 aberrations into an unfavourable group (group 3). Cases with 14q32/IGH translocation as primary chromosome change represented a specific category (group 4) and were studied with FISH probes for the detection of partners (BCL1, BCL2, BCL3, BCL6, c-MYC, BCL11A, PAX5, CCND3, CDK6). One hundred eighty-six cases were allocated into group 1; 158 into group 2; 64 into group 3, and 22 into group 4. Additional aberrations were found in a minority of cells in 8 patients in group 4. Being the aim of the study to assess whether the 14q32/IGH translocation represented an unfavourable parameter as compared with cases in the favourable or intermediate cytogenetic risk groups, cases within group 3 were excluded from the analysis and the data presented here will refer to 366 patients belonging to groups 1,2 and 4. Translocation partners of 14q32/IGH were identified in 9/22 cases: 2p13/BCL11A, (n 1); 6p21/CCND3 (n 1); 7q21/CDK6 (n 1); 18q21/BCL2 (n 6). Thirteen cases did not show involvement of the loci studied. Cases with 14q32/IGH translocations were characterized by typical morphology and classical immunophenotype (score 4–5 in 92% of the cases). Unmutated IGVH genes were found in 11/18 cases tested (61%); ZAP-70 was positive in 3/5 cases tested. Median age was 63.5 years (range 18–97), male:female ratio 240/126; 345 patients were in Rai stage 0-II, 21 were in stage 3–4; CD38 was positive in 137/366 cases. There was no difference between groups 1,2 and 4 for age, stage, male:female ratio, hematological parameters at diagnosis, IGVH mutations and ZAP70. CD38 was more positive in group 4 than in group 1 (p=0.028). There was no difference in survival and treatment free interval (TFI) when comparing cases in group 4 with and without additional aberrations. Cases in group 4 had a shorter TFI and a shorter survival when compared with group 1 (p=0.02) and group 2 (p=0.02). The difference maintained its statistical significance at multivariate analysis for TFI (p=0.02) along with stage (p<0.0001) and CD38 positivity (p<0.0001) and for survival (p=0.02) along with sex (p=0.006) and stage (p=0.0001). In conclusion, the 14q32/IGH translocation in CLL shows heterogeneity of partner chromosomes and it represents a cytogenetic marker predicting for an evolutive form of CLL.

Although the success of imatinib mesylate therapy represents an exciting advance in targeted cancer therapy, it has still to be determined whether responses to this p210 inhibitor in chronic myeloid leukemia (CML) patients will be durable. In fact most of clinical studies agree on the evidence of a persistent molecular disease in the majority of treated patients and altough the absolute level of bcr-abl transcript may vary over the treatment, yet a molecular complete response is of rare observation. In addition, discontinuation of imatinib exerts always in rapid loss of response. In accordance to this the persistence of malignant progenitors in patients in complete cytogenetic response (CCR) after short term imatinib treatment, has been recently demonstrated. In particular, Bathia et al. showed in 12/15 patients studied after a median time of 10 months of imatinib treatment a median of 11% of residual CML CD34+ progenitors in the bone marrow (by FISH Dual Fusion bcr/abl analysis)while only 3/15 patients had no detectable residual CD34+ cells. Less is known about residual Ph+/CD34+ cells surviving after a prolonged therapy with this targeting drug. Thus, we evaluated the amount of bone marrow residual CD34+ cells in 17 CML patients in stable CCR after a long lasting treatment with imatinib. At the time of evaluation, the patients were on conventional dose (400mg) Imatinib for a median time of 48 months (range 36–58 months) having achieved a CCR status (conventionally defined as the complete absence of t(9;22) on caryotypic analysis) within 3 to 6 months of treatment. However all of them still showed molecular disease as detected by nested RT-PCR. Bone marrow CD34+ cell-enriched populations were selected from mononuclear cells using immunomagnetic column separation and were evaluated after cytospin by FISH using a bcr-abl Dual Color Extra Signal Probe(LSI bcr-abl ES, Vysis), that is able to detect bcr-abl fusion in interphase nuclei with a false positive signal rate close to 0. A minimum of 100 CD34+ nuclei per each sample were evaluated. Interestingly, in 8/17 patients no Ph+/CD34+ cells were detected, while in the remaining 9/17 patients a median of 2% (range 0.5–11%) of bcr-abl positive progenitors were still observed. In this small selected serie of patients prolonged treatment with imatinib appears to be correlated with a lower, yet detectable, amount of residual bone marrow Ph+/CD34+ cells when compared to previously published data. This result could be partly explained with the different specificity and sensitivity of the probe used (bcr/abl ES<1% false positive; bcr-abl Dual Fusion 8–10% false positive) The clinical significance of these data as well as the role of this cell target to monitor minimal residual disease in CML needs to be evaluated on a larger serie of patients.

Although the success of imatinib mesylate therapy represents an exciting advance in targeted cancer therapy, it has still to be determined whether responses to this p210 inhibitor in chronic myeloid leukemia (CML) patients will be durable. In fact most of clinical studies agree on the evidence of a persistent molecular disease in the majority of treated patients and altough the absolute level of bcr-abl transcript may vary over the treatment, yet a molecular complete response is of rare observation. In addition, discontinuation of imatinib exerts always in rapid loss of response. In accordance to this the persistence of malignant progenitors in patients in complete cytogenetic response (CCR) after short term imatinib treatment, has been recently demonstrated. In particular, Bathia et al. showed in 12/15 patients studied after a median time of 10 months of imatinib treatment a median of 11% of residual CML CD34+ progenitors in the bone marrow (by FISH Dual Fusion bcr/abl analysis)while only 3/15 patients had no detectable residual CD34+ cells. Less is known about residual Ph+/CD34+ cells surviving after a prolonged therapy with this targeting drug. Thus, we evaluated the amount of bone marrow residual CD34+ cells in 17 CML patients in stable CCR after a long lasting treatment with imatinib. At the time of evaluation, the patients were on conventional dose (400mg) Imatinib for a median time of 48 months (range 36–58 months) having achieved a CCR status (conventionally defined as the complete absence of t(9;22) on caryotypic analysis) within 3 to 6 months of treatment. However all of them still showed molecular disease as detected by nested RT-PCR. Bone marrow CD34+ cell-enriched populations were selected from mononuclear cells using immunomagnetic column separation and were evaluated after cytospin by FISH using a bcr-abl Dual Color Extra Signal Probe(LSI bcr-abl ES, Vysis), that is able to detect bcr-abl fusion in interphase nuclei with a false positive signal rate close to 0. A minimum of 100 CD34+ nuclei per each sample were evaluated. Interestingly, in 8/17 patients no Ph+/CD34+ cells were detected, while in the remaining 9/17 patients a median of 2% (range 0.5–11%) of bcr-abl positive progenitors were still observed. In this small selected serie of patients prolonged treatment with imatinib appears to be correlated with a lower, yet detectable, amount of residual bone marrow Ph+/CD34+ cells when compared to previously published data. This result could be partly explained with the different specificity and sensitivity of the probe used (bcr/abl ES<1% false positive; bcr-abl Dual Fusion 8–10% false positive) The clinical significance of these data as well as the role of this cell target to monitor minimal residual disease in CML needs to be evaluated on a larger serie of patients.