Search

Introduction : Hematological complications (HC) as Aplastic anemia (AA) and myelodysplasia and acute leukemia (MDS/AL) are frequent and life threatening in patients with Shwachman Diamond Syndrome (SDS) with SBDS mutations. The therapy of such events is based on Hematopoetic stem cell transplantation (HSCT), which results remain quite poor, especially in case of malignancy. So far, it is difficult to anticipate to such HC and a lot is expected from the study of clonal evolution prior HC. Methods: A Targeted panel of 43 genes involved in MDS/AL (sensibility 1%) has been evaluated in 80 patients with SBDS mutation, representative of a nation based cohort of 154 patients. This cross sectional study has been completed by a prospective study for 40 patients evaluated at several time points. Results: The evaluation was performed in various situations: steady state i.e. no haematological complication, in MDS/AL and AA and lastly after HSCT. At the first evaluation, somatic mutation was found in 21 patients (30%) among the 70 in steady state and in 7 of the 8 cases with HC (6/6 cases with MDS/AL, in 1 among the 2 cases with AA) while the 1 of the 2 patients long term survivors after HSCT have no mutation and the other one kept a TP53 clone with a normal blood count and a low (1.5%) variant allele frequency (VAF). Among the 40 patients with several time points, 17 have a mutation at the first time points, but 10 others had additional mutation later. Globally, the most frequent gene involved was TP53 (82%) while mutations in other genes have been observed rarely. VAF in patients with vs without HC is lower (median VAF 0% vs 22.8% respectively p < 0.001) . Complex caryotype, monosomy 7, Iso7q were associated with P53 clone while in Del20q, 8 patients out 14 have a P53 mutations. The comparison between blood and bone marrow results allow the possibility to monitor such mutations in blood. Clonal evolution in one patient who presents a MDS in the course of the follow up had shown a competition between clones. Conclusion: Acquired TP53 is extremely frequent in patients with SBDS mutations, even in the absence of HC, but the prevalence as well as the VAF increased in case of HC. When sequential evaluation could be performed, competition between clones is frequent and a clinical decision remains therefore difficult, just on the evaluation of a time point. Acknowledgments: The French SCN registry is supported by grants from Amgen, Chugai, Prolong Pharma, X4 Pharma, Inserm, the Association 111 les Arts, the Association RMHE, the Association Sportive de Saint Quentin Fallavier. The authors thank the association IRIS and Mrs Grosjean and Mr Gonnot(ASSQF), the association Barth France for their support. Disclosures Sicre de Fontbrune: Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Renard:Jazz Pharmaceuticals: Research Funding. Tournilhac:ABBVIE: Consultancy, Honoraria, Other: Travle grant; INNATE Pharma: Consultancy, Honoraria; GILEAD: Consultancy, Honoraria, Other: Travel Grant; Takeda: Consultancy, Honoraria, Other: Travel grant; Janssen: Consultancy, Honoraria, Other: Travel grant.

Context: Shwachman Diamond disease (SDS) is caused by an SBDS mutation, is typically associated with neutropenia and exocrine pancreas deficiency. Pancytopenia, myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML), are life-threatening complications of SDS. To date, the sole risk factors identified for SDS are early symptoms (before age of 3 months) and mild chronic anemia or thrombocytopenia(1). Methods: To determine which mutations underlie clonal development and leukemic changes in SDS, we screened a series of patients with congenital neutropenias at various time points of follow-up. We used a consensus NGS panel of 41 genes involved in the development of myeloid malignancies (Haloplex® Agilent) (2, 3). Patients with SBDS mutations included in the French Severe Congenital Neutropenia registry or followed in the Leuwen University were screened when bone marrow samples were available as well as other subtypes of congenital neutropenia. Results: Among the 139 SDS patients, bone marrow samples of 23 patients were available for screening at various time points. We found isolated somatic TP53 mutations in 10 cases. In addition one patient had concomitant FLT3 TKD and TP53 mutations, and another patient had the recurrent IDH1 p.Arg132Cys variant. Strikingly, no TP53 mutations were observed when the screening was extended to 70 non-SDS neutropenia patients. None of the 11 SDS patients without any detectable mutations (with a threshold of detection of 0.5%) had any severe hematological expression nor presented any major hematological complications at time of sampling. By contrast, among the 12 SDS patients with somatic mutations, AML or MDS were observed in 3 cases, 1 with the IDH1 mutation (Variant allelic frequency (VAF): 42%), 1 with the recurrent TP53 p.Gly245Ser mutation (VAF:53%), 1 with two TP53 mutations (VAFs : 19% and 37%). Severe cytopenias without MDS or AML were found in 2 other cases, one with an isolated TP53 mutation at 24%, and one with both TP53 and FLT3 mutations around 45%. In the 7 remaining patients, allele frequencies of TP53 variants were found below 2% in four cases, and at 3%, 14%, and 37% in the 3 other patients. By sequential analysis in one patient we found a p.Val272Met variant (1.1% at 9 years) which was no more observed but was replaced by the recurrent p.Arg175His mutation (4% at 12 years and 14% at 15 years). Figure 1 depicts the allele frequencies of the variants with time among the 23 patients. The three patients with MDS / AML died despite hematopoietic stem cell transplantation (HSCT) in two of them. In contrast, the two patients with severe cytopenias and TP53 mutations who were transplanted are doing well three years after HSCT, and no TP53 mutation was detected one year after transplant. Conclusion: TP53 mutations are associated with hematological complications and specifically acquired in SDS when compared to other congenital neutropenias. This is in line with the frequency of complex karyotype MDS/AML in SDS(1). Routine evaluation of TP53 load in SDS patients may offer a powerful tool to screen SDS who may be susceptible to have severe hematological complications in a preemptive transplantation strategy setting. References: 1. J. Donadieu et al., Haematologica 97, 1312 (2012). 2. P. Hirsch et al., Nat. Commun. 7, 12475 (2016). 3. E. Papaemmanuil et al., N. Engl. J Med. 374, 2209 (2016). Acknowledgments: The French SCN registry is supported by grants from Amgen, Chugai, Prolong Pharma, Inserm, the Association 111 les Arts, the Association RMHE, the Association Sportive de Saint Quentin Fallavier and « Le Fond de dotation Contre la Leucémie". The authors thank the association IRIS and Mrs Grosjean and Mr Gonnot (ASSQF) for their support. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: The genetic landscape of adult acute myeloid leukemias (AML) has been recently unravelled. This makes achievable the determination of a comprehensive profile of driver lesions for virtually all patients at diagnosis. Recent studies using multi-target minimal residual disease (MRD) strategies with around 1% sensitivity indicate that the clearance of all molecular events after chemotherapy is associated with better survival. To improve the clono-specificity and the sensitivity of this approach, after a precise determination of AML clonal composition, we combined cytogenetic, FISH, and high sensitivity deep sequencing technologies to monitor the MRD in a series of 69 patients. Methods: Forty-five consecutive patients reflecting the genetic diversity of AML were prospectively included and 24 patients were retrospectively studied. All patients received an anthracycline + cytarabine based regimen. The clonal architecture was established at diagnosis based on NGS-targeted resequencing (122 gene panel) and cytogenetic data. Lesions were next investigated in complete remission (CR). Based on the initial clonal composition, targeted resequencing panels were designed to improve the sensitivity by the use of unique molecular barcodes (Haloplex High Sensitivity, or HS-NGS assay). Cytogenetic events were evaluated by FISH. Results: In the 69 patients, a median of 4 genetic or chromosomal events were identified per patient (range 0-10). One patient had no evaluable target allowing MRD evaluation in 68/69 patients. To determine the threshold of detection of the HS NGS assay, we analyzed the frequency of mutant reads in multiple samples expected to be wild type for 31 given SNVs and 2 indels. A consensus threshold of detection was set at a variant allele frequency (VAF) of 0.2% for all lesions. In CR samples, early initiating events frequently persisted after treatment, especially mutations in DNMT3A, TET2, ASXL1, EZH2, IDH1, TP53, SRSF2, and U2AF1. Mutations in FLT3, NRAS, KIT, NPM1, CEBPA, WT1, IDH2 and BCOR were the most frequently cleared events. Seven patients did not reach CR after one course, and two had no available material after one course. In the 59 remaining patients, we tested whether the global response level of all targets was associated with prognosis. We used the median VAF of the first events of all clonal architectures to separate good responder from poor responders (i.e. VAF = 1.66%). At 2 years, there was a trend to lower leukemia free survival (LFS) probability in poor responders (31.7+/-9.9% vs 51.7+/-9.8%, p=0.08) with no translation in overall survival (OS). We next investigated if the persistence of two or more detectable markers was associated with prognosis. The 58 patients with more than one evaluable event were consequently separated in two groups: patients with 0 or 1 marker above the detection threshold after treatment (n=31), and patients with 2 or more detectable lesions (n=27). At 2 years, DFS was 64.9+/-9.3 % in patients with 0 or 1 detectable marker vs 19.8+/-8.7% in patients with 2 or more detectable markers (p=0.001). OS probability was higher in patients with 0 or 1 detectable marker 84+/-6.6% vs 57.1+/-10.5% (p=0.023). When focusing on the 40 patients with intermediate cytogenetics, persistence of 2 or more markers was associated with lower LFS (57+/-11.8% vs 19.4+/-10.5 p=0.0048) and with a trend to lower OS (85+/-8% vs 61+/-11.9% p=0.07). Similar results were observed when restricting the analyses to the 42 prospectively included patients (At 2 years: LFS 73+/-10% vs 24+/-10%, p=0.0026 and OS 90.2+/-6.6% vs 62.8+/-11.5%, p=0.036). In 50 patients with 3 or more identified events, the persistence of 3 or more markers after one course was associated with a very high risk of relapse (DFS 23.5+/-10.3 % vs 75.8 +/-7.5% at one year, p Conclusion Our study shows the high prognostic value of a personalized multi-target clono-specific MRD evaluation that can be used in nearly all AML patients. Detection of two or more events in more than 0.4% cells after one course is associated with lower survival, in particular in intermediate cytogenetic patients. Larger studies are needed to confirm the results and to evaluate if this strategy could be useful to guide treatment decisions. Disclosures No relevant conflicts of interest to declare.

Background: AZA is the standard of care for patients with either HRMDS or AML with 20-30% blasts (Oligoblastic AML). The response rate is ~50% with a median response duration of 12-15 months. The mechanisms underlying primary and secondary resistance are poorly understood and it remains difficult to accurately predict which patients will respond and in responders, to predict secondary resistance. The main factors demonstrated or suggested to interfere with response to AZA and/or survival include marrow blast percentage, performance status, IPSS cytogenetic risk, presence of peripheral blasts, transfusion dependency, IPSS-R risk, mutations of TET2, P53, IDH1/2, and DNMT3A, elevated expression of BCL2L10, Fas, or PI-PLCbeta1. Deregulation of microRNAs is a hallmark of MDS, yet its consequences on AZA efficacy have not been specifically addressed in HRMDS. Methods: We measured the expression of 754 miRNAs in SKM1 MDS cells resistant or sensitive to AZA. miRNAs of interest were ectopically expressed or specifically inhibited in HEK293T cells which were assayed for AZA sensitivity (MTT). Seven miRNAs were quantified in bone marrow mononuclear cells deriving from 75 patients with HRMDS (n= 50) or oligoblastic AML treated with AZA [median age 74, 24 females, median cycle number 6, (1-39)]. Results: Seven miRNAs (miR-125a-5p, miR-99b-5p, miR-126, miR-126*, hsa-let-7c, miR-34b-3p, and miR-10b*) that include 6 tumor-suppressor miRNAs, were found differentially expressed between AZA-sensitive versus -resistant cells; all being transcriptionally repressed in resistant cells. In silico, 5 of these miRNAs were found to target the 3' UTR of DNA methyltransferase 1 (DNMT1) while Zhao et al. [Arthritis & rheumatism, 2011; 63(5)] have shown that miR-126 directly inhibits DNMT1 translation. DNMT1 is one of the main AZA molecular target, the higher the level of DNMT1 expression, the lower the AZA sensitivity [Li et al., Cancer biology & therapy, 2010; 9(4)]. Microarray and western blotting showed that levels of DNMT1 protein, but not mRNA, were significantly higher in AZA-resistant cells. In HEK293T cells, specific endogenous miR-126/126* inhibition significantly augmented DNMT1 protein expression and triggered AZA-resistance, as measured through MTT proliferation assay. In the same cells, ectopic expression of miR-126/126* decreased DNMT1 protein expression in a concentration-dependent manner. In the 75 patients treated with AZA, a decreased expression level of all but miR-10* was associated with decreased response rate (IWG 2006 criteria) and poor outcome; miR-126/126* having the strongest prognostic impact. When compared with miR-126*High MDS, miR-126*Low MDS had significantly lower overall response rate (37% versus 60%, p=0.04), higher relapse rate (100% versus 71%, p=0.034), shorter progression-free (PFS) (8±8% versus 49±12% at 3 years, p=0.004, log rank test), and overall survival (OS) (16±6% versus 46±9%, p=0.004). Baseline patient characteristics of the 2 groups were similar. Multivariate analyses were performed using the Cox proportional hazards model. Table 1 shows that age, miR-126* expression, and IPSS-R risk independently predicted PFS and OS. miRNAs expression was analyzed over time in 15 patients. The mean expression level of 5/7 miRNAs increased over time in the 10 responders without statistically significant difference (DNS) between the first and latest values. In contrast, the expression of 7/7 miRNAs decreased over time in the 5 patients with treatment failure, the difference being statistically significant for 2 miRNAs (p²0.043, Wilcoxon test). Secondary resistance occurred in 7/10 responders and was found associated with a decreased expression of 6/7 miRNAs (p²0.044 for 4 miRNAs) versus 2/7 (DNS) in the 3 long-term responders. Conclusion: Our results suggest that in HRMDS and oligoblastic AML, i. primary or secondary resistance to AZA is associated with the decreased expression of anti-DNMT1 tumor-suppressor microRNAs that trigger AZA resistance in vitro; ii. measuring miRNAs expression before and under treatment might help to predict primary or secondary AZA resistance and thereby to rapidly offer alternative treatment; iii. increasing AZA exposure might help to circumvent AZA resistance in case of either low or decreased anti-DNMT1 miRNA concentration while using anti-DNMT1 microRNAs as a therapeutic tool might increase or restore AZA sensitivity. Disclosures Fenaux: Janssen: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Wattel:AMGEN: Consultancy, Research Funding; PIERRE FABRE MEDICAMENTS: Research Funding; CELGENE: Research Funding, Speakers Bureau; NOVARTIS: Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding.

The nucleoside analogue cytarabine (AraC) has served as the backbone of acute myeloid leukemia (AML) treatment for nearly forty years. About one-third of expressed genes are abnormally spliced in AML yet alternative exon usage (AEU) plays a role in the plasticity of tumor cells and may influence the response to treatment. Here the exon expression profiles of the erythroleukemia K562 cell line were compared to that of its AraC-resistant variant K562/AraC through Affymetrix HTA2 exon arrays. 5140 exon events harbored by 2583 genes distinguished the 2 cell lines. Among these, the skipping of TET2 exon 2 was identified in K562 cells sensitive to AraC whereas TET2 gene expression remained unchanged at the whole transcript level. The results were confirmed by exon-specific RTPCR (ESPCR). Microarray analysis did not evidenced any significant change in mRNA splicing for the 10 remaining exons of the TET2 gene. TET2 is a dioxygenase that catalyzes the conversion of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and promote DNA demethylation. TET2 somatic mutations occur in about 25% AML, distributing across the whole coding sequence without obvious hot spots. These mutations decrease TET2 enzymatic activity by truncating the protein or affecting its catalytic activity. TET2 exon 2 is spliced in a mutually exclusive manner with exon 1 yet it is used as an alternative promoter (https://fasterdb.lyon.unicancer.fr/). However, TET2 exon 2 is not translated into protein and its role in TET2 regulation is still unknown. Having found that AraC sensitive cells harbor the spliced TET2 isoform, we investigated whether or not skipping of TET2 exon 2 correlate with disease outcome in AML patients treated with AraC-based intensive chemotherapy (AraC-IC). The discovery cohort included 106 consecutive AML patients treated with AraC-IC (median age 57.91, 64 males). RNA was extracted from bone marrow MNCs and assayed for TET2 exon 2 skipping through real-time quantitative ESRTPCR (qESRTPCR) amplification of E1E3 (spliced) and E2E3 (unspliced) TET2 isoforms. TET2 exon 2 skipping was quantified by calculating the ratio E1E3/E2E3. For statistical analysis, the ratio E1E3/E2E3 was dichotomized using median value as the cutoff value. Skipping of TET2 exon 2 was associated with a significantly lower response rate: 65% vs 92%, p = 0.001 but with a significantly lower relapse rate: 39% vs 85%: p In conclusion TET2 exon 2 skipping is associated with a low relapse rate and possesses a strong favorable prognostic impact in AML treated with AraC-CTI. The mechanism linking this alternative exon usage with resistances to AraC are currently investigated while our results suggest that the determination of TET2 exon 2 splicing status might assist risk stratification. Disclosures Nicolini: Novartis: Consultancy.

To the editor: A family history of chronic lymphocytic leukemia (CLL) is one of the best characterized risk factors for the development of CLL. First-degree relatives of individuals with CLL have a 3- to 8-fold increased risk for CLL.[1][1],[2][2] CLL often has an indolent behavior, but some

Abstract 1721 Introduction: CD133 is a transmembrane protein expressed by hematopoietic stem cells and other cell types, whose physiological functions remain unknown. To determine its potential prognostic value in AML, we analyzed its expression on blasts from 76 patients at diagnosis. Moreover, since leukemic stem cells play a critical role in the resistance to chemotherapeutic drugs and may be responsible for relapse, we compared the growth potential and activated cellular pathways of CD133+ and CD133- cells from 20 AML patients. Methods: First, bone marrow samples were collected at diagnosis from 76 AML patients (median age: 65, range: 1–84). CD133 expression was evaluated on blasts cells and CD34+ cells using 6-color flow cytometry (FC) and the monoclonal antibody AC133. The mean fluorescence intensity ratio (MFIR) was calculated by dividing the MFI of CD133 by the MFI of its isotypic control. Second, purified CD34+, CD34+CD133+ and CD34+CD133- cells were isolated from 20 AML bone marrows using the magnetic separation system Minimacs (Miltenyi Biotec). These fractions were submitted to an in vitro colony forming assay during 14 days. Finally, the level of expression of survival proteins involved in the PI3K/AKT, Mitogen-Activated Protein Kinase (MAPK) and Bcl-2 pathways, as well as CXCR4 and Focal Adhesion Kinase (FAK) migration proteins, was analyzed by FC in these fractions. Results: The level of CD133 expression on blasts cells was significantly different between AML patients with favorable (n=12), intermediate (n=38) and unfavorable karyotypes (n=23) (p=0.007). In particular, it was significantly higher in patients with unfavorable karyotype than in patients with intermediate karyotype (p=0.032) or favorable karyotype (p=0.005). Moreover, when considering NPM1 and FLT3 mutation status in patients with intermediate karyotype, the level of CD133 expression was significantly higher in patients with an intermediate molecular profile than in patients with a favorable molecular profile (p=0.004). Most interestingly, we showed that the level of CD133 expression has an impact on overall survival in multivariate analysis, independently of leukocytosis, karyotype and achievement of complete remission after induction therapy (p=0.04). Second, in 20 patients, we found that AML progenitor cells (AML CFU) were present in all sorted fractions, but the CD34+CD133+ fraction gave rise to larger and more numerous colonies (74.9±39.6) than the CD34+CD133- fraction (11,25±6). Moreover, the CD34+CD133+ fraction was the only one able to form secondary colonies (52.1±31.8). Finally, we found that the expression of PI3K (mean percentage of positive cells: 78±20.1), pAKT (80±19.8), pERK (80.1±19), CXCR4 (71.6±22.7), pFAK (81.2±19.8), Bcl-2 (72.9±26.1), Bcl-xL (45.7±24.8), and pBad (63.9±36.1) was significantly upregulated in CD34+CD133+ cells compared to CD34+CD133- cells, whereas Bax was downregulated. Discussion: CD133 is known to be expressed in hematopoietic progenitors, but its role in the resistance to chemotherapy is not well studied. In our work, the level of expression of CD133 on AML blasts appeared as an independent immunophenotypic prognostic factor, encouraging its further evaluation in a larger number of cases. We also showed that CD133+ cells have greater replicative properties than CD133- cells, and exhibit activation of various signaling pathways. Since CD133+ cells might escape from apoptosis through activation of PI3K/AKT and ERK, these proteins could represent potential therapeutic targets. In conclusion, our results revealed that CD133+ cells exhibit more aggressive behavior than CD133- ones, and that CD133+ AML progenitors might contribute to resistance to chemotherapy through preferential activation of PI3K/AKT, MAPK and Bcl-2 survival response. Disclosures: No relevant conflicts of interest to declare.

Abstract 3226 Heat Shock Protein 90 (HSP 90), a 90 Kda molecular weight protein, is one of the most abundant cytosolic chaperone, acting in protein folding, refolding and degradation and of particular importance in cell survival. HSP 90 is overexpressed in acute leukemia cells and this high expression is related with a poor prognosis. This ATP-dependent chaperone could be an interesting potential drug target. 17-AAG, a Geldanamycin derivates, is a HSP 90 inhibitor with a promising antitumor activity. Very few reports have evaluated the effects of 17-AAG in acute lymphoblastic leukemia (ALL). The aim of this work is to study the cytotoxicity of 17-AAG and to compare Philadelphia positive (Ph+) ALL to common B-cell ALL. Two human leukemia cell lines, Reh (common B-cell ALL) and SUPB15 (Ph+ ALL) were used in this study. We identified 63 consecutive patients treated in our institution for ALL (44 common B-cell ALL and 19 Ph+ ALL) and leukemic samples were collected at diagnosis from bone marrow aspiration after patient's consent. The expression of HSP 90, pro-apoptotic BAX protein, anti-apoptotic bcl-2 and bcl-xl proteins was studied by flow cytometry. Cell lines and ALL patient's cells were cultured in RPMI 1640 and exposed to various concentrations of 17-AAG. Apoptosis was evaluated by an Annexin V and an activated caspase-3 staining by flow cytometry. Results were expressed as percentage of positive cells. Pro-apoptotic effect of 17-AAG in Ph+ ALL cells was superior when compared to common B-cells with a 100% mortality rate after exposure to [10μM] 17-AAG for 24 hours for SUPB15 cell line whereas 24% of cell surviving was noted for Reh cell line. Similar results were observed with patient's leukemic samples (as shown in figure). The susceptibility of Ph+ ALL cells to 17-AAG was confirmed by the assessment of the IC50, estimated at 1.1 μM for SUPB15 cells versus 2.9 μM for ReH cells. As assessed by Annexin V binding and activated caspase-3 staining, 17-AAG induced apoptosis in ALL cells. As regard Ph+ ALL, the median percentage of Annexin-V positive cells and caspase-3 positive cells after exposure to [5μM] 17 AAG for 24 hours was 54% and 57% respectively and increased up to 99% and 99,5% after exposure for 48 hours. Spontaneous apoptosis in control cell culture was measured in 1% at 24 hours and 3% at 48 hours. The pattern of expression of HSP90 and apoptotic proteins was different between common B-cell ALL and Ph+ ALL cells. The percentage of HSP90 positive cells was 62% for Reh cell line whereas it reached 100% for SUP B15 cell line. As shown in the Table, the percentage of HSP90-positive cells and anti-apoptotic bcl-2 and bcl-xl proteins expression were higher for Ph+ ALL samples when compared to common B-cell ALL samples. HSP90 Bcl-2 Bcl-xl Bax Common B-cell ALL samples n=44 32.5% (±19.8) 33% (± 21.4) 28.5% (±19.5) 33% (±17) Ph+ ALL samples n = 19 79% (±7.5) 82% (±8.4) 84% (±7.8) 12% (±4.3) When ALL cells were exposed in culture to various concentrations of 17-AAG, there was no change in HSP90 expression but we observed a down-regulation in bcl-2 and bcl-xl expression and an up-regulation in bax expression. In summary, we showed that HSP90 and anti-apoptotic proteins were expressed at a higher level on Ph+ ALL cells when compared to common B-cell ALL. High percentage of HSP90-positive cells was associated with high sensitivity to 17-AAG. 17-AAG is a new targeted therapy that induces the apoptotic death of leukemic cells via a caspase-3 dependant way. It would be interesting to test its antileukemic activity in combination with chemotherapeutic agents to study additional or synergistic effects. Despite therapeutic improvement with the development of tyrosine kinase inhibitors (TKI) in the treatment of Ph+ ALL, relapse remains a major problem. Considering that Bcr-Abl constitutes HSP 90 substrates and depends on this chaperone for its maturation and conformational maintenance, 17-AAG could be of particular interest for Ph+ ALL disease, in combination with TKI. We can hypothesise that this drug could restore the sensitivity to TKI treatment for patients with Bcr-Abl mutation. Disclosures: No relevant conflicts of interest to declare.

Abstract 289 MDS are characterized by a high risk of evolution into acute myeloid leukemia (AML). The pathogenesis of this evolution is still unclear. Some studies indicate that aberrant activation of survival signaling pathways is involved. The 90-kDa heat shock protein (HSP90) is implicated in the conformational maturation and stabilization of protein kinases and has key roles in signal transduction, protein folding, and protein degradation. HSP90 levels are increased in AML cells, and associated with resistance to chemotherapy induced apoptosis. Moreover, HSP90 is involved in the formation of focal adhesions. Focal Adhesion Kinase (FAK), a non-receptor tyrosine kinase, and a client of HSP90, is a member of the integrin-mediated signal transduction pathway. FAK was found over-expressed and constitutively activated in solid tumors. In AML, FAK expression is associated with enhanced blast migration and poor prognosis. FAK also exerts a potent antiapoptotic effect through adhesion to extracellular matrix and stromal cells. Finally 27-kDa heat shock protein (HSP27), a small HSP, prevents apoptosis by interfering with the mitochondrial pathway of apoptosis. In a cancer cell line, HSP27 has been shown to regulate cell adhesion via modulation of FAK. The aim of our study was to investigate the role of HSP90 in high-risk MDS and its potential role on focal adhesion. The expression of HSP90, HSP27, phosphorylated FAK (pFAK), and phosphorylated Akt (pAkt) was assessed by multicolor flow cytometry in bone marrow (BM) mononuclear (MNC) and CD34+ cells from 177 MDS samples at diagnosis: 96 refractory anemias with excess of blasts (RAEB), 58 refractory anemias (RA) and 23 chronic myelomonocytic leukemias (CMML). The levels of HSP90, HSP27, FAK, pFAK, and pAkt in MNC from RAEB patients, were significantly higher than in those measured in RA or CMML patients (p Disclosures: No relevant conflicts of interest to declare.

CXC chemokine receptor 4 (CXCR4) is the receptor for stromal derived factor (SDF-1 or CXCL12) and plays a crucial role in the homing of leukemia cells within the marrow microenvironment. Adhesion to marrow stromal cells is essential for the survival and proliferation of acute myelogenous leukemia (AML) cells, and protect AML cells from chemotherapy-induced apoptosis. In the same way, heat shock proteins (HSP) act as molecular chaperones and are involved in signalling pathways for cell proliferation and survival. HSP have a role in the modulation of apoptosis and are implicated in the resistance of leukemia cells to therapeutic drugs. The aim of this work was to assess the prognostic impact of CXCR4, adhesion molecules such as Very Late Antigen-4 (VLA-4) and Focal Adhesion Kinase (FAK), and HSP expression (HSP 27, HSP 70, HSP 90). In this study, we retrospectively analysed, by flow cytometry, the “adhesive” phenotype and the HSP expression in AML cells from 36 patients treated between 04/1998 and 03/2002. These patients presented de novo AML, and favourable, intermediate and adverse cytogenetics were observed in 7, 19 and 10 patients respectively. Study of the adhesive phenotype was repeated for 10 patients at time of relapse and compared to adhesive phenotype at diagnosis. The flow cytometry analysis was performed with a FACS Canto: conjugated antibodies were used in combination with anti-CD34-FITC and anti-CD45-APC. Twenty-eight of the 36 patients achieved complete remission (CR) whereas seven patients were refractory to the chemotherapy and one patient died from toxicity during aplasia. Median overall survival (OS) was 19.4 months (95% CI: 0.25–102 months). In univariate analysis, the two main prognostic factors in terms of CR achievement were lower CXCR4 and VLA-4 expression on leukemia cell surface (p = 0.03 and p = 0.05 respectively). Overall survival (OS) was negatively influenced by higher CXCR4 expression (p = 0.01), higher VLA-4 expression (p = 0.01), higher FAK expression (p = 0.04) and higher HSP90 expression (p = 0.04) by leukemia cells at diagnosis. Patients were secondarily distributed in two prognostic groups: - Group A including 26 patients presenting overexpression of 0, 1 or 2 factors within CXCR4, VLA-4, FAK, HSP 90 - Group B including 10 patients with overexpression of 3 or 4 factors. Group B is associated with significantly shorter OS. In multivariate analysis, FAK overexpression and HSP90 overexpression remained of prognostic value for OS (p = 0.01 and p = 0.03 respectively). Moreover, when comparing the adhesive phenotype for 10 patients between diagnosis and relapse, we noted that CXCR4 and VLA-4 are overexpressed at time of relapse (91% positive cells versus 26% at diagnosis for CXCR4 (p = 0.008), and 88% versus 21% for VLA-4 (p = 0.008)). In conclusion, CXCR4, adhesion molecules and HSP90 are new phenotypic prognostic markers. These data indicate that it should be interesting to determine CXCR4, VLA-4, FAK and HSP 90 in the routine flow cytometry diagnostic of AML in order to establish risk-stratified therapeutic strategies. A prospective study with a larger series of patients may lead to confirm the prognostic value of these markers. Figure Figure

Deregulation of signal transduction pathways (STPs) including JAK/STAT, RAS/Raf/MEK/ERK and PI3K/AKT may promote leukemogenesis by conferring cells proliferation and survival advantages in acute myelogenous leukemia (AML). The activation of these pathways had an adverse prognosis in AML and development of targeted therapies seems to be promising. Heat-shock proteins (HSP) are involved in the conformational maturation of a number of signaling proteins, and HSPs expression in AML is associated with other adverse prognostic factors (Bcl2, MRP). The aim of this work was to study STPs expression in AML, and there correlation to other adverse prognostic factors and complete remission rate. Sixty five patients with primary AML were analyzed by flow cytometry for constitutive ERK, PI3K and AKT activation, HSP90, Bcl2 and P170 expression. FAB subtypes were M0=3, M1=19, M2=16, M4=10, M5=16, M6=1. All patients received an induction treatment, 44 patients reached complete remission. Cytogenetics was available in 63 cases (Intermediate = 36, favorable = 3, unfavorable= 24). We performed a 3 color flow cytometry protocol using CD45 and CD34 to identify blast cells, and used a third antibody to the following transduction proteins (ERK, pERK, AKT, pAKT, PI3K) or intracytoplasmic proteins (Bcl2, HSP90, p170). Spontaneous growth of leukemic progenitor cells (CFU-L) was investigated in 44 samples. In AML, activated proteins were found in CD34+ cells. ERK and PI3K/AKT were frequently co-activated. Flow cytometry results showed that levels of STPs were significantly higher (p

Flow cytometry analysis of minimal residual disease (MRD) in acute myeloid leukemia (AML) is based on the detection of aberrant phenotypes responsible for the relapse. Until now, all studies were performed by 3 or 4 color immunostaining, allowing the identification of LAIP in 80% of cases. Moreover, no data is available regarding the existence of such phenotypes in regenerating bone marrow. The new generation of cytometers allows the study of 8 parameters that permit a better distinction of malignant from normal phenotypes. In our study we analyzed 20 bone marrow samples from allogeneic donors, 20 ALL regenerating bone marrows after chemotherapy and 53 AML samples at diagnosis. Multiparameter 4 colour and 6 colour flow cytometry was used in order to define antigen combinations which are totally absent or present at very minimal levels in normal and regenerating hematopoiesis. “Blast cells” were gated according to CD45/SSC properties.For the first time we describe by 6 color flow cytometry 47 phenotypes totally absent from “blasts” gate in all normal bone marrow (ex: CD34+DR−117+33−15+, CD34+38+33−56+19−, CD14−DR+4+11B+64+). Another 41 phenotypes were identified as presents at a frequency < 0,05% of total cells (ex: CD34+DR+117−33+15+, CD14−DR+4+11B+64−, CD34+65−56+4−16−). There was no significant difference between normal and regenerating marrows. The 4 color panel of moAbs allowed us to identify only 30 phenotypes presents at a frequency < 0,05% of total cells (ex: CD34+33−13+, CD34+117+11b+, CD34+DR−13+). 53 AML at diagnosis were studied using 6 color immunophenotyping and 58 % of phenotypes described as aberrant or infrequent in normal myeloid hematopoiesis were found in at least one AML at diagnosis in more than 1% of total cells. All AML cases show at least one LAIP but frequently we observed more than one LAIP blast subpopulation in the same sample. Some examples of LAIP observed are CD34+ 38+ 33+ 56+ 19−, CD34+ 38+ 33+ 56− 19+, CD34− DR− 117+ 33+ 15−. In conclusion our results shows that (1) the ability to clearly distinguish leukemic from the healthy cells is considerably increased by 6 color approach (8 parameters analyzed) than 4 color. (2) Furthermore that these aberrant or infrequent phenotypes in normal or regenerating bone marrow samples are identified in AML cases and can be utilized in AML minimal residual disease study. (3) Knowledge of the expression of different markers in normal hematopoietic development provides a frame of reference for identification of abnormal differentiation patterns.

The 90-Kda heat shock protein (Hsp90), together with a number of other chaperones are involved in normal cellular differentiation and cancerogenesis. Hsp90 is implicated in the conformational maturation of a large variety of protein kinases. We have shown its overexpression in a series of 116 acute myeloid leukemias (AML). Geldanamycin and its analogue 17-AAG selectively block the activities of Hsp90, but do not interact with other members of the Hsp family. The objective of the study was to test the effect of 17-AAG on cell lines (Jurkat, U-937, HL60, HL60R : resistant to daunorubicin (DNR), and KG1a) and on 24 AML samples tested positive for Hsp90 (14 strongly positive and 10 moderately positive). CD34+ cells from bone marrow donors were used as controls. Cells were maintained in liquid culture in the presence or absence of 17-AAG. Moreover hematopoietic and leukemic progenitor assays were performed in semi-solid media containing or not 17-AAG. CD34+ cells were non affected by 17-AAG. Jurkat, U937, HL60 were partially sensitive to 17-AAG. Addition of low dose DNR to 17-AAG induced an apoptotic death in 48 hours in all samples, including HL60R. KG1a (exhibiting high HSP90 levels) was more sensitive to 17-AAG alone with a death rate of 100% at higher doses. A similar effect was observed in AML samples : 17-AAG induced cell death in all 14 cases highly positive for HSP90, but a partial death in 5 cases, and no death in 5 cases mildly positive for HSP90. DNR increased apoptosis in only 5/10 of these cases. The other 5 patients were also positive for bcl-2. When they were first incubated with 17-AAG and then treated with bcl-2 antisens oligodeoxynucleotides (bcl-2AS), a complete apoptosis was obtained. So, in the 10 samples with moderate HSP90 expression a synergystic effect was found when a combination of 17-AAG with either chemotherapy or bcl -2AS was used. While normal progenitors were not affected by 17-AAG, leukemic progenitors were completed inhibited in the 14 patients who responded to 17-AAG in liquid culture. This apoptosis was caspase -3 dependant. Finally, we investigated the implication of the ERK signal pathway in Jurkat line and in 14/24 17-AAG responder samples using an ERK inhibitor (PD98059). When this inhibitor was added prior to 17-AAD and DNR, the apoptotic effect was completely inhibited. So, ERK signal pathway may interfere with the Hsp90 function. In conclusion, these results raise the possibility that Hsp90 antagonists represent a novel antileukemic strategy in sequential addition to conventional chemotherapy