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DNMT1 protein expression in SKM1 AZA-sensitive (-S) versus AZA-resistant clones (-R). The Figure corresponds to the Figure 1B with signal quantification performed with ImageJ software. The data are expressed as mean {plus minus} SD of three independent experiments, with each performed in triplicate (Mann-Whitney test, p

Pathway enrichment analysis. For ontology analysis, gene lists were analyzed using DAVID software (KEGG pathways). The complete set of genes featured in the microarrays was used as the reference background. The three numbers on the right represent the number of deregulated mRNAs, the overall number of genes within the pathway and the p value, respectively. Data are presented for genes targeted by at least one of the 7 miRNAs and that were found repressed in SKM1 AZA resistant cells.

Purpose: Azacitidine inhibits DNA methyltransferases, including DNMT1, and is currently the standard of care for patients with higher-risk myelodysplastic syndrome (HRMDS) or low blast count acute myeloid leukemia (AML).Experimental Design: The expression of 754 miRNAs was compared in azacitidine-resistant and azacitidine-sensitive myelodysplastic syndrome cells. We investigated the role of differentially expressed miRNAs on DNMT1 expression and azacitidine resistance in vitro. We next evaluated anti-DNMT1 miRNA expression in pretreatment bone marrow samples derived from 75 patients treated with azacitidine for HRMDS or AML.Results: Seven miRNAs, including 5 that in silico targeted the DNMT1 3′ UTR, were repressed in azacitidine-resistant cells in which DNMT1 protein levels were significantly higher. Ectopic anti-DNMT1 miRNA expression decreased DNMT1 expression and increased azacitidine sensitivity, whereas specific inhibition of endogenous anti-DNMT1 miRNAs increased DNMT1 expression and triggered azacitidine resistance. In patients treated with azacitidine, decreased expression of anti-DNMT1 miRNAs was associated with poor outcome. miR-126* had the strongest prognostic impact. Patients with miR-126*low myelodysplastic syndrome had significantly lower response rates (P = 0.04) and higher relapse rates (P = 0.03), as well as shorter progression-free (PFS; P = 0.004) and overall survival (OS; P = 0.004). Multivariate analysis showed that age, miR-126* expression, and revised International Prognostic Scoring System risk independently predicted PFS and OS. In 15 patient samples collected over time, decreased miRNA expression levels were associated with secondary resistance.Conclusions: A decreased expression of anti-DNMT1 miRNAs might account for azacitidine resistance in HRMDS and AML, and measuring miRNA expression before and during treatment might help predict primary or secondary azacitidine resistance. Clin Cancer Res; 23(12); 3025–34. ©2016 AACR.

Obstructive sleep apnea (OSA) is characterized by repeated episodes of intermittent hypoxia (IH) and is recognized as an independent risk factor for vascular diseases that are mediated by a multitude of mechanistic pathophysiological cascades including procoagulant factors. The pro-coagulant state contributes to the development of blood clots and to the increase in the permeability of the blood–brain barrier (BBB). Such alteration of BBB may alter brain function and increase the risk of neurodegenerative diseases. We aim to provide a narrative review of the relationship between the hypercoagulable state, observed in OSA and characterized by increased coagulation factor activity, as well as platelet activation, and the underlying neural dysfunction, as related to disruption of the BBB. We aim to provide a critical overview of the existing evidence about the effect of OSA on the coagulation balance (characterized by increased coagulation factor activity and platelet activation) as on the BBB. Then, we will present the emerging data on the effect of BBB disruption on the risk of underlying neural dysfunction. Finally, we will discuss the potential of OSA therapy on the coagulation balance and the improvement of BBB.

International audience; Molecular tests have become an indispensable tool for the diagnosis and prognosis of hematological malignancies and are subject to accreditation according to the International Standard ISO 15189. National standardization of these techniques is essential to ensure that patients throughout France benefit from the same care. We report here on the experience of the GBMHM (Groupe des Biologistes Moléculaires des Hémopathies Malignes). By organizing External Evaluation of Quality (EEQ) programs and training meetings, the GBMHM has contributed to improvement and standardization of molecular tests in 64 French laboratories. A retrospective analysis of the quality-control results of 11 national campaigns spanning 10 years was performed for the 3 most frequently prescribed tests: BCR-ABL1, JAK2 V617F, and lymphoid clonality. For each test, particular attention was placed on comparing methodologies and their evolution throughout the period. The establishment of the BCR-ABL1, JAK2 V617F, and lymphoid clonality EEQ programs and the associated training meetings have initiated a process of collective standardization concerning the methods of implementation (JAK2 V617F) and the interpretation and formulation of results (lymphoid clonality). In addition, it resulted in objective improvement in technical performance (BCR-ABL1). Our evaluation of the impact of these EEQ programs demonstrates that it is possible to obtain reproducible values across different laboratories in France by applying national recommendations. To our knowledge, this is the first publication that evaluates the impact of a national quality assurance program on improving molecular results in hematology.

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of BCR-ABL1 transcript as a result of reciprocal translocation between chromosome 9 and 22. The most common transcripts subtypes are e13a2 (b2a2) and e14a2 (b3a2). The prognostic impact of the type of BCR-ABL1 transcript has been the subject of controversies over time. In the imatinib era, several studies have suggested a deeper and faster response in patients expressing e14a2. However, the impact on response after first line therapy with a second-generation tyrosine kinase inhibitor, nilotinib, is unknown. We retrospectively evaluated 118 patients newly diagnosed with chronic phase CML and treated frontline with nilotinib inside or outside clinical trial in five French centers. Only patients expressing e14a2 or e13a2 transcripts alone were analyzed. At baseline, 55.3% expressed e14a2, 44.7% expressed e13a2. The median age was 51 years and median follow-up was 49 months. Relative risks of CML at diagnosis were similar according to the ELTS score (p = .87). Complete hematological response and complete cytogenetic response rates were similar among groups. Patients expressing e14a2 transcripts compared to e13a2 transcripts had deeper and faster molecular responses, when considering MMR (100% vs 84.1%, p = .007) with a median time of 6.7 and 17.1 months or MR4.5 (100% vs 59.9%, p = .005) with a median time of 39.7 and 70.9 months, respectively. A sustained treatment free remission was observed in 10/10 patients with e14a2 versus 1/3 with e13a2 transcript (p = .04). In conclusion, even treated with nilotinib first line, patients with chronic phase CML expressing BCR-ABL1 e13a2 transcript have a lower rate of deep molecular responses.

Promyelocytic sarcoma is an uncommon solid tumor made up of myeloblasts. It is characterized, like acute promyelocytic leukemia (APL), by a chromosomal translocation t(15;17) involving the retinoic acid receptor alpha (RARalpha) and the promyelocytic gene (PML). The diagnosis and monitoring of promyelocytic sarcoma is a challenge due to the rarity and severity of the disease. We describe a case with several initial sites and without APL. The patient was monitored with regular 18F-FDG PET/CT from diagnosis to complete response. The evolution of PET/CT imageries was compared to the quantification of PML-RARα fusion gene by RQ-PCR. In promyelocytic sarcoma medical care, 18F-FDG PET/CT appears to be an attractive tool for finding targets for biopsy, for the primary staging, for assessing therapeutic response and for detecting early relapse.

In 2020, accreditation of molecular tests according to ISO 15189 is a requirement for all French medical laboratories. For many years, the GBMHM group (French Group of Molecular Biologists in Hematology) supports this approach through organization of external quality evaluation campaigns, and by publishing recommendations that have allowed the accreditation of the most frequent molecular tests for most laboratories. However, some molecular abnormalities concerns very few patients (and sometimes a single patient), and therefore cannot be evaluated in the same way, because of the lack of external quality controls or inter-laboratory comparisons. In order to allow the accreditation of these rare analyzes, the GBMHM proposes recommendations, based on the fact that analyzes using the same methodology than those already accredited by an extensive validation process, may be accredited without the need for full analytical validation. In particular, assays based on quantitative PCR or endpoint PCR may be accredited after verification of primer specificity, repeatability and/or reproducibility, and the determination of detection or linearity limits. These recommendations, by defining the validation approach for rare molecular abnormalities, make it possible to extend the requirement of accreditation for rare tests, to provide the best patient care.

Myeloid sarcoma (MS) are extramedullary tumors of myeloid precursors, generally occurring in a setting of acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndromes (MD...

Embryonic stem cells typically show properties of long-term self-renewal and lack of differentiation. When appropriately stimulated, they are able to differentiate into all cell lineages, and lose their self-renewal characteristics. These properties are controlled by a series of genes encoding several transcription factors, including OCT4, the product of POU5F1 gene. OCT4 is expressed in germ cell tumors but also aberrantly in cancers developing in differentiated tissues. In a previous study, we observed a high expression of OCT4 in acute myeloid cell lines and primary cells, regardless of the acute myeloid leukemia (AML) subtype. In this study, we investigated the putative oncogenic role of OCT4 in proliferation and differentiation arrest. OCT4 expression was assessed in a panel of myeloid cell lines, together with clonogenic and proliferation properties, before and after differentiation in the presence of retinoic acid (RA). Same experiments were performed under short hairpin RNA (shRNA)-mediated OCT4 inhibition. In the presence of RA, we observed a decrease of OCT4 expression, associated with a loss of clonogenic and proliferation capacities, cell cycle arrest, and upregulation of p21, in HL60, NB4, KASUMI, and Me-1 cell lines. This effect was absent in the KG1a cell line, which did not differentiate. Downregulation of OCT4 by shRNA resulted in the same pattern of differentiation and loss of proliferation. Although KG1a did not differentiate, a decrease in proliferation was observed. Our findings suggest that OCT4 is implicated in the differentiation arrest at least in some types of AML, and that it also plays a role in cell proliferation through different oncogenic mechanisms. OCT4 might be a potential new target for antileukemic treatments.

Background Approximately 70%-80% of kidney cancers are clear cell renal cell carcinomas (CCRCCs). Patient management is based on imaging (abdominal ultrasound and computerized tomography), surgical excision of the tumor, and pathological analysis. A tissue biopsy is therefore necessary to confirm the diagnosis and avoid unnecessary nephrectomy. For metastatic cancers, a tissue biopsy is essential for establishing the targeted therapy. This biopsy of tumor material is invasive and painful. Other techniques such as liquid biopsy would help reduce the need for tissue biopsy. The development of a simple biological test for diagnosis is essential. CA9 is a powerful marker for the diagnosis of CCRCC. Exosomes have become a major source of liquid biopsy because they carry tumor proteins, RNA, and lipids. Urine is the most convenient biological liquid for exosome sampling. Objective The aim of this study (PEP-C study) is mainly to determine whether it is possible to detect urinary exosomal CA9 for the molecular diagnosis of CCRCC. Methods This study will include 60 patients with CCRCC and 40 noncancer patients. Exosomes will be isolated from urine samples and exosomal CA9 will be detected by transmission electron microscopy, flow cytometry, and reverse transcription-quantitative polymerase chain reaction. Results This study is currently underway with funding support from the CHU Saint-Etienne of France. Conclusions We expect to demonstrate that urinary tumor exosomes could be a novel liquid biopsy to diagnose CCRCC and to guide clinicians in treatment decision-making. Trial Registration ClinicalTrials.gov NCT04053855; https://clinicaltrials.gov/ct2/show/NCT04053855 International Registered Report Identifier (IRRID) DERR1-10.2196/24423

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.

In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), and favorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2Shigh patients were found to be younger than TET2low patients without a difference in the rate of complete remission. However, TET2E2Shigh patients exhibited a significantly lower CIR (p

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of BCR-ABL1 transcript as a result of reciprocal translocation between chromosome 9 and 22. The most common transcripts subtypes are e13a2 (b2a2) and e14a2 (b3a2). The prognostic impact of the type of BCR-ABL1 transcript has been the subject of controversies over time. In the imatinib era, several studies have suggested a deeper and faster response in patients expressing e14a2. However, the impact on response after first line therapy with a second-generation tyrosine kinase inhibitor, nilotinib, is unknown. We retrospectively evaluated 118 patients newly diagnosed with chronic phase CML and treated frontline with nilotinib inside or outside clinical trial in five French centers. Only patients expressing e14a2 or e13a2 transcripts alone were analyzed. At baseline, 55.3% expressed e14a2, 44.7% expressed e13a2. The median age was 51 years and median follow-up was 49 months. Relative risks of CML at diagnosis were similar according to the ELTS score (p = .87). Complete hematological response and complete cytogenetic response rates were similar among groups. Patients expressing e14a2 transcripts compared to e13a2 transcripts had deeper and faster molecular responses, when considering MMR (100% vs 84.1%, p = .007) with a median time of 6.7 and 17.1 months or MR4.5 (100% vs 59.9%, p = .005) with a median time of 39.7 and 70.9 months, respectively. A sustained treatment free remission was observed in 10/10 patients with e14a2 versus 1/3 with e13a2 transcript (p = .04). In conclusion, even treated with nilotinib first line, patients with chronic phase CML expressing BCR-ABL1 e13a2 transcript have a lower rate of deep molecular responses.

Chronic myelomonocytic leukemia (CMML) is a myelodysplastic/myeloproliferative neoplasm, characterized by persistent monocytosis and dysplasia in at least one myeloid cell lineage. This persistent monocytosis should be distinguished from the reactive monocytosis which is sometimes observed in a context of infections or solid tumors. In 2015, Selimoglu-Buet et al. observed an increased percentage of classical monocytes (CD14+/CD16− >94%) in the peripheral blood (PB) of CMML patients. In this study, using multiparametric flow cytometry (MFC), we assessed the monocytic distribution in PB samples and in bone marrow aspirates from 63 patients with monocytosis or CMML suspicion, and in seven follow-up blood samples from CMML patients treated with hypomethylating agents (HMA). A control group of 12 healthy age-matched donors was evaluated in parallel in order to validate the analysis template. The CMML diagnosis was established in 15 cases in correlation with other clinical manifestations and biological tests. The MFC test for the evaluation of the repartition of monocyte subsets, as previously described by Selimoglu-Buet et al. showed a specificity of 97% in blood and 100% in marrow samples. Additional information regarding the expression of intermediate MO2 monocytes percentage improved the specificity to 100% in blood samples allowing the screening of abnormal monocytosis. The indicative thresholds of CMML monocytosis were different in PB compared to BM samples (classical monocytes >95% for PB and >93% for BM). A decrease of monocyte levels in PB and BM, along with a normalization of monocytes distribution, was observed after treatment in 4/7 CMML patients with favorable evolution. No significant changes were observed in 3/7 patients who did not respond to HMA therapy and also presented unfavorable molecular prognostic factors at diagnosis (ASXL1, TET2, and IDH2 mutations). Considering its simplicity and robustness, the monocyte subsets evaluation by MFC provides relevant information for CMML diagnosis.

Deciphering the mutational patterns and/or the biomarkers that might predict clinical response in patients with myelofibrosis is primordial to make treatment decisions. In this report, we discuss the clinical history, pathological evaluation, and genomics findings in a patient with JAK2-positive myelofibrosis who developed a secondary myelodysplasia after hematopoietic stem cell transplantation and JAK1/2 inhibitor treatment. Using next-generation sequencing, a paired comparison of relapse-specific versus primary tumour mutations highlighted the dynamic clonal evolution at relapse, showing concurrently the complete eradication of the JAK2-positive clone and the expansion of a second JAK2-negative clone with additional mutations. Importantly, another unexpected finding was that myelodysplasia was not secondary to allogeneic transplantation as relapse was driven by the overgrowth of a preexisting mutated clone, probably fostered by initial treatment options. This case underlines the fact that determining the genetic changes associated with the primary disease and its evolution is crucial to accurately correlate variants frequency to treatment decision and/or treatment response.

Over recent years, the development of molecular biology techniques has improved the hematological diseases diagnostic and follow-up. Consequently, these techniques are largely used in the biological screening of these diseases; therefore the Hemato-oncology molecular diagnostics laboratories must be actively involved in the accreditation process according the ISO 15189 standard. The French group of molecular biologists (GBMHM) provides requirements for the implementation of quality assurance for the medical molecular laboratories. This guideline states the recommendations for the pre-analytical, analytical (methods validation procedures, quality controls, reagents), and post-analytical conditions. In addition, herein we state a strategy for the internal quality control management. These recommendations will be regularly updated.

// Aminetou Mint Mohamed 1 , Marie Balsat 1 , Morgan Thenoz 1 , Catherine Koering 1 , Lea Payen-Gay 2 , Meyling Cheok 3 , Hussein Mortada 4 , Didier Auboeuf 4 , Christiane Pinatel 5 , Mohamed El-Hamri 6 , Charles Dumontet 7 , Emeline Cros 7 , Pascale Flandrin-Gresta 1, 8 , Olivier Nibourel 4 , Claude Preudhomme 4 , Mauricette Michallet 1, 6 , Xavier Thomas 6 , Franck Nicolini 6 , Francoise Solly 1, 8 , Denis Guyotat 1, 9 , Lydia Campos 1, 8 , Eric Wattel 1, 6, * , Franck Mortreux 1, * 1 Universite Lyon 1, CNRS UMR5239, Oncovirologie et Biotherapies, Faculte de Medecine Lyon Sud, ENS – HCL, Pierre Benite, France 2 INSERM, UMR-S1052, Centre de Recherche en Cancerologie de Lyon, Lyon, France 3 Jean-Pierre Aubert Center, INSERM U837, Facteurs de persistance des cellules leucemiques, Institute for Cancer Research in Lille, Lille cedex, France 4 Centre de Recherche sur le Cancer de Lyon, Inserm, Epissage alternatif et progression tumorale, Lyon, France 5 Centre de Recherche sur le Cancer de Lyon, Inserm, Echappement aux systemes de sauvegarde et plasticite cellulaire, Lyon, France 6 Universite Lyon I, Service d’Hematologie, Pavillon Marcel Berard, Centre Hospitalier Lyon-Sud, Pierre Benite, France 7 Centre de Recherche sur le Cancer de Lyon, Inserm, Anticorps anticancer, Lyon, France 8 Universite de Saint Etienne, Laboratoire d’Hematologie, CHU de Saint-Etienne, Saint-Etienne, France 9 Institut de Cancerologie de la Loire, CHU de Saint-Etienne, Saint Priest en Jarez, France * These authors have contributed equally to this work Correspondence to: Eric Wattel, e-mail: eric.wattel@chu-lyon.fr Franck Mortreux, e-mail: franck.mortreux@ens-lyon.fr Keywords: acute myeloid leukemia, alternative splicing, WT1, DEK, multidrug resistance Received: March 16, 2015 Accepted: April 28, 2015 Published: May 12, 2015 ABSTRACT In addition to spliceosome gene mutations, oncogene expression and drug resistance in AML might influence exon expression. We performed exon-array analysis and exon-specific PCR (ESPCR) to identify specific landscapes of exon expression that are associated with DEK and WT1 oncogene expression and the resistance of AML cells to AraC, doxorubicin or azacitidine. Data were obtained for these five conditions through exon-array analysis of 17 cell lines and 24 patient samples and were extended through qESPCR of samples from 152 additional AML cases. More than 70% of AEUs identified by exon-array were technically validated through ESPCR. In vitro , 1,130 to 5,868 exon events distinguished the 5 conditions from their respective controls while in vivo 6,560 and 9,378 events distinguished chemosensitive and chemoresistant AML, respectively, from normal bone marrow. Whatever the cause of this effect, 30 to 80% of mis-spliced mRNAs involved genes unmodified at the whole transcriptional level. These AEUs unmasked new functional pathways that are distinct from those generated by transcriptional deregulation. These results also identified new putative pathways that could help increase the understanding of the effects mediated by DEK or WT1, which may allow the targeting of these pathways to prevent resistance of AML cells to chemotherapeutic agents.

Acute myeloid leukemia is driven by leukemic stem cells which can be identified by cross lineage expression or arrest of differentiation compared to normal hematopoietic stem cells. Self-renewal and lack of differentiation are also features of stem cells and have been associated with the expression of embryonic genes. The aim of our study was to evaluate the expression of embryonic antigens (OCT4, NANOG, SOX2, SSEA1, SSEA3) in hematopoietic stem cell subsets (CD34+CD38− and CD34+CD38+) from normal bone marrows and in samples from acute myeloid leukemia patients. We observed an upregulation of the transcription factors OCT4 and SOX2 in leukemic cells as compared to normal cells. Conversely, SSEA1 protein was downregulated in leukemic cells. The expression of OCT4, SOX2, and SSEA3 was higher in CD34+CD38− than in CD34+CD38+ subsets in leukemic cells. There was no correlation with biological characteristics of the leukemia. We evaluated the prognostic value of marker expression in 69 patients who received an intensive treatment. The rate of complete remission was not influenced by the level of expression of markers. Overall survival was significantly better for patients with high SOX2 levels, which was unexpected because of the inverse correlation with favorable genetic subtypes. These results prompt us to evaluate the potential role of these markers in leukemogenesis and to test their relevance for better leukemic stem cell identification.

CXCR4, receptor of the chemokine SDF-1 (stromal cell-derived factor 1) plays a major role in the normal hematopoiesis but also in the biology of the leukaemic cell. This receptor is expressed on the surface of blasts and is a key molecule in "the anchoring" of the leukaemic stem cell (LSC) within the bone marrow niche. The interactions of the LSC with the bone marrow microenvironment promote survival signals and drug resistance. Recent flow cytometry analyses reported that CXCR4 expression levels have a major prognostic impact in acute myeloid leukaemia (AML). CXCR4 expression is associated with poor prognosis and can be useful to stratify patients, according to their phenotype, in order to establish risk-adapted strategies. Newly diagnosed AML are now routinely stratified according to molecular markers which guide prognosis and treatment. Many leukaemia are composed of multiples subclones with differential susceptibility to treatment and specific targeted therapies are missing. Despite therapeutic improvements for the treatment of AML, long term survival remains poor. Targeting CXCR4 is a novel promising approach of therapy. CXCR4 antagonists are used in combination with chemotherapy in preclinical and clinical studies. This review summarises our current knowledge regarding the key role of CXCR4 in AML and discusses how targeting this pathway could provide an interesting approach to eradicate the LSC.

Purpose: Azacitidine inhibits DNA methyltransferases, including DNMT1, and is currently the standard of care for patients with higher-risk myelodysplastic syndrome (HRMDS) or low blast count acute myeloid leukemia (AML). Experimental Design: The expression of 754 miRNAs was compared in azacitidine-resistant and azacitidine-sensitive myelodysplastic syndrome cells. We investigated the role of differentially expressed miRNAs on DNMT1 expression and azacitidine resistance in vitro. We next evaluated anti-DNMT1 miRNA expression in pretreatment bone marrow samples derived from 75 patients treated with azacitidine for HRMDS or AML. Results: Seven miRNAs, including 5 that in silico targeted the DNMT1 3′ UTR, were repressed in azacitidine-resistant cells in which DNMT1 protein levels were significantly higher. Ectopic anti-DNMT1 miRNA expression decreased DNMT1 expression and increased azacitidine sensitivity, whereas specific inhibition of endogenous anti-DNMT1 miRNAs increased DNMT1 expression and triggered azacitidine resistance. In patients treated with azacitidine, decreased expression of anti-DNMT1 miRNAs was associated with poor outcome. miR-126* had the strongest prognostic impact. Patients with miR-126*low myelodysplastic syndrome had significantly lower response rates (P = 0.04) and higher relapse rates (P = 0.03), as well as shorter progression-free (PFS; P = 0.004) and overall survival (OS; P = 0.004). Multivariate analysis showed that age, miR-126* expression, and revised International Prognostic Scoring System risk independently predicted PFS and OS. In 15 patient samples collected over time, decreased miRNA expression levels were associated with secondary resistance. Conclusions: A decreased expression of anti-DNMT1 miRNAs might account for azacitidine resistance in HRMDS and AML, and measuring miRNA expression before and during treatment might help predict primary or secondary azacitidine resistance. Clin Cancer Res; 23(12); 3025–34. ©2016 AACR.

In acute myeloid leukaemia (AML) initiating pre-leukaemic lesions can be identified through three major hallmarks: their early occurrence in the clone, their persistence at relapse and their ability to initiate multilineage haematopoietic repopulation and leukaemia in vivo. Here we analyse the clonal composition of a series of AML through these characteristics. We find that not only DNMT3A mutations, but also TET2, ASXL1 mutations, core-binding factor and MLL translocations, as well as del(20q) mostly fulfil these criteria. When not eradicated by AML treatments, pre-leukaemic cells with these lesions can re-initiate the leukaemic process at various stages until relapse, with a time-dependent increase in clonal variegation. Based on the nature, order and association of lesions, we delineate recurrent genetic hierarchies of AML. Our data indicate that first lesions, variegation and treatment selection pressure govern the expansion and adaptive behaviour of the malignant clone, shaping AML in a time-dependent manner., Pre-leukaemic clones, together with the propensity to cause disease in mice, are characterized by appearing early in myeloid leukaemia and being found at relapse. Here, the authors identify clones in human samples and find that they are characterized by hierarchically organized genetic lesions, which can be used to track evolution of the disease.

In AML, approximately one-third of expressed genes are abnormally spliced, including aberrant TET2 exon 2 expression. In a discovery cohort (n=99), TET2 exon 2 skipping (TET2E2S) was found positively associated with a significant reduction in the cumulative incidence of relapse (CIR). Age, cytogenetics, and TET2E2S were independent prognostic factors for disease-free survival (DFS), and favorable effects on outcomes predominated in cytogenetic normal (CN)-AML and younger patients. Using the same cutoff in a validation cohort of 86 CN-AML patients, TET2E2S

// Pascale Flandrin-Gresta 1,2 , Francoise Solly 1,2 , Carmen Mariana Aanei 1 , Jerome Cornillon 3 , Emmanuelle Tavernier 2,3 , Nathalie Nadal 1 , Franck Morteux 2 , Denis Guyotat 2,3 , Eric Wattel 2 , Lydia Campos 1,2 , 1 Laboratoire d’Hematologie, University Hospital of Saint-Etienne, University Hospital of Saint-Etienne, 42055 Saint-Etienne Cedex 2, France. 2 UMR5239 CNRS, Universite Claude Bernard Lyon 1, Faculte deMedecine J Lisfranc Saint-Etienne, 42023 Saint-Etienne Cedex 2, France. 3 Institut de Cancerologie de la Loire, University Hospital of Saint-Etienne, 42271 Saint Priesten Jarez cedex, France. Correspondence: Pascale Flandrin-Gresta, email: // Keywords : HSP90, MDS, FAK, 17-AAG Received : July 18, 2012, Accepted : September 25, 2012, Published : September 28, 2012 Abstract Myelodysplastic syndromes are characterized by a high risk of evolution into acute myeloid leukaemia which can involve activation of signalling pathways. As the chaperone heat shock protein 90 (HSP90) has a key role in signal transduction, we investigated its role in the pathogenesis and evolution of myelodysplastic syndromes. Expressions of HSP90 and signalling proteins clients (phosphorylated-AKT (pAKT), Focal Adhesion Kinase (FAK) and phosphorylated-FAK (pFAK)), were assessed in bone marrow mononuclear and CD34-positive (CD34 + ) cells from 177 patients with myelodysplasia. Effects of HSP90 inhibition were also evaluated in 39 samples. The levels of all proteins studied were significantly higher in patients with high grade disease, than those with low grade myelodysplastic syndrome or chronic myelomonocytic leukaemia. High levels of HSP90, FAK, pFAK and pAKT were associated with shorter survival and increased risk of progression into acute leukaemia. A down regulation of pFAK and pAKT and increased apoptosis was observed in mononuclear and CD34 + cells after 12 hours of incubation with 17-AAG. In conclusion, our data suggest the implication of HSP90 and FAK and AKT activation in the pathogenesis of myelodysplastic syndromes with excess of blasts and evolution to leukaemia. Moreover this signalling network could be a therapeutic target through HSP90 inhibition.

Direct cell-cell contact between haematopoietic progenitor cells (HPCs) and their cellular microenvironment is essential to maintain 'stemness'. In cancer biology, focal adhesion (FA) proteins are involved in survival signal transduction in a wide variety of human tumours. To define the role of FA proteins in the haematopoietic microenvironment of myelodysplastic syndromes (MDS), CD73-positive mesenchymal stromal cells (MSCs) were immunostained for paxillin, pFAK [Y(397)], and HSP90α/β and p130CAS, and analysed for reactivity, intensity and cellular localisation. Immunofluorescence microscopy allowed us to identify qualitative and quantitative differences, and subcellular localisation analysis revealed that in pathological MSCs, paxillin, pFAK [Y(397)], and HSP90α/β formed nuclear molecular complexes. Increased expression of paxillin, pFAK [Y(397)], and HSP90α/β and enhanced nuclear co-localisation of these proteins correlated with a consistent proliferative advantage in MSCs from patients with refractory anaemia with excess blasts (RAEB) and negatively impacted clonogenicity of HPCs. These results suggest that signalling via FA proteins could be implicated in HPC-MSC interactions. Further, because FAK is an HSP90α/β client protein, these results suggest the utility of HSP90α/β inhibition as a target for adjuvant therapy for myelodysplasia.

Background: Congenital acute leukemia is a rare event, presenting usually as an aggressive disease with a poor prognosis. A differential diagnosis is the transient myeloproliferative disorder observed in Down syndrome. We describe the case of an apparently healthy newborn male child presenting with normal peripheral blood (PB) counts but with a blast population on differentials. The child's condition and the blast population remained unchanged during the first year of life. Methods: Bone marrow and PB were morphologically analyzed. Multiparametric flow cytometry was performed at the time of diagnosis and repeated at 1 year. These studies were completed by cytogenetic and molecular analyses. Results: Bone marrow contained 40% of undifferentiated blasts. Multiparametric flow cytometry showed low expression of CD38, HLA-DR, and CD33 markers. All other markers were negative. Constitutional and blast cell karyotypes were normal. Fluorescence in situ hybridization analysis showed no rearrangement. Molecular studies were negative. The blast percentage remained stable during several months. After 1 year, the PB counts showed thrombocytopenia, with an increase of blast cells exhibiting the same phenotype. Clinically, an enlarged spleen was found. The child did not respond to chemotherapy and only partially to gemtuzumab ozogamicin. A cord blood cell transplantation was finally performed. With a follow-up of 12 months, the child is doing well. Conclusions: To our knowledge, this is the first case of congenital acute leukemia with initially indolent course in a newborn without Down syndrome. This observation emphasizes the importance of a careful follow-up. © 2010 International Clinical Cytometry Society

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.

We analysed, by flow cytometry, the “adhesive” phenotype of acute myelogenous leukemia (AML) cells from 36 patients treated in our institution. In univariate analysis, the main prognostic factor for CR achievement was lower CXCR4 expression (p = 0.03). Overall survival (OS) was negatively influenced by higher CXC chemokine receptor 4 (CXCR4) (p = 0.01), very late antigen-4 (VLA-4) (p = 0.01), and focal adhesion kinase (FAK) expression (p = 0.04). Combination of these markers allowed to distinguish two prognostic groups: patients overexpressing 2 or 3 factors had a significantly shorter OS (p = 0.015). CXCR4, VLA-4 and FAK are new phenotypic markers which could be helpful to establish risk-stratified therapeutic strategies.

CXCR4, receptor of the chemokine SDF-1 (stromal cell-derived factor 1) plays a major role in the normal hematopoiesis but also in the biology of the leukaemic cell. This receptor is expressed on the surface of blasts and is a key molecule in "the anchoring" of the leukaemic stem cell (LSC) within the bone marrow niche. The interactions of the LSC with the bone marrow microenvironment promote survival signals and drug resistance. Recent flow cytometry analyses reported that CXCR4 expression levels have a major prognostic impact in acute myeloid leukaemia (AML). CXCR4 expression is associated with poor prognosis and can be useful to stratify patients, according to their phenotype, in order to establish risk-adapted strategies. Newly diagnosed AML are now routinely stratified according to molecular markers which guide prognosis and treatment. Many leukaemia are composed of multiples subclones with differential susceptibility to treatment and specific targeted therapies are missing. Despite therapeutic improvements for the treatment of AML, long term survival remains poor. Targeting CXCR4 is a novel promising approach of therapy. CXCR4 antagonists are used in combination with chemotherapy in preclinical and clinical studies. This review summarises our current knowledge regarding the key role of CXCR4 in AML and discusses how targeting this pathway could provide an interesting approach to eradicate the LSC.

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.

HSP90 targeting is a promising therapeutic approach in cancer. 17-AAG is an HSP90 inhibitor with completed Phase I trials in patients with advanced cancer and recently published Phase II trials. The aim of this work was to study the expression of HSP 90 and apoptotic proteins, the effects in culture of 17-AAG on cell survival and apoptosis and to compare Philadelphia-positive (Ph+) ALL to common B cell ALL, in ALL cell lines and in patients’ cells collected at ALL diagnosis. We analysed 2 ALL cell lines and 63 leukaemic samples from patients treated in our institution (44 common B cell ALL and 19 Ph+ ALL). We performed flow cytometry analysis of bone marrow aspiration and cell lines with a combination of anti-HSP90, Bax, Bcl-2 and Bcl-xl antibodies. Apoptosis after cell culture (in presence or not of 17-AAG) was assessed using Annexin V and activated caspase-3 staining. Ph+ ALL cells appeared to be more sensitive to 17-AAG cytotoxicity with a 100 % mortality rate after exposure to 10 μM for 24 h (vs. 62 % for B-common ALL). A high percentage of HSP90-positive cells (in Ph+ ALL samples) was associated with high sensitivity to 17-AAG. 17-AAG induced apoptosis in a dose-dependent manner and was associated with down-regulation of Bcl-2 and Bcl-Xl expression and up-regulation of Bax expression. Considering that Bcr-Abl constitutes HSP 90 substrates, HSP 90 inhibition could be of particular interest for Ph+ ALL disease, even in patients harbouring resistance to tyrosine kinase inhibitor therapy.

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.

Myelodysplastic syndromes (MDSs) are clonal disorders of hematopoietic stem cells (HSCs) characterized by ineffective hematopoiesis. MDSs are responsible for 1 or several peripheral cytopenias. The evidence accumulated in recent years demonstrates that in addition to HSC defects, a particular role is also played by stromal microenvironment dysfunctions, which mediate the direct contact with hematopoietic precursor cells (HPCs). These interactions help regulate different adhesion-related processes, such as progenitor cell proliferation, apoptosis, clonogenic growth, and maintenance in in vitro cultures. As previously reported, these interactions are responsible for altering the microenvironment in MDS. Herein, we present a novel selection protocol for obtaining a standards-compliant mesenchymal stromal cell (MSC) preparation. This method allowed us to comparatively analyze 2 subpopulations of bone marrow MSCs (BM-MSCs) in terms of their adhesion profiles and growth abilities: BM-MSCs selected from MDS settings and their normal counterparts. Functional assays revealed that the MSCs from MDS are intrinsically pathological, thus showing a continuous decline of proliferation and a reduced clonogenic capacity during 14 days of culture and in the absence of signals from hematopoietic cells. The MSC growth defects were significantly correlated with decreases in CD44 adhesion molecules and CD49e (α5-integrin).

Background: The knowledge of normal marrow is mandatory to assess the malignant counterpart of normal cells and define leukemia-associated immunophenotypes (LAIPs). In this study, the expression of a variety of antigens expressed in normal and postchemotherapy bone marrow (BM) was analyzed to provide a frame of reference for the identification of myeloid LAIPs. Methods: Multiparameter four- and six-color flow cytometry was used to define antigen combinations totally absent or present at very minimal levels in marrow cells of normal individuals (n = 20) and patients receiving chemotherapy for acute lymphoblastic leukemia (n = 20). Immature (blast) cells were gated according to CD45/SSC properties. Fifty-three acute myeloid leukemia (AML) samples were studied in six-color combinations. Results: In six-color flow cytometry, 47 phenotypes were totally absent from blast gate in all normal samples. Forty-one other phenotypes were identified in less than 0.05% of blast cells. There was no difference between normal and postchemotherapy BMs. The four-color panel allowed to identify only 30 phenotypes present at a frequency

The 90-kDa heat shock protein (HSP90) is implicated in the conformational maturation and stabilization of a variety of client proteins with receptor and signal transduction functions. The objective of this study was to assess its expression in primary acute myeloid leukemia (AML) cells and to evaluate its biological and clinical significance. The in vitro effects of 17-AAG, a selective inhibitor of HSP90, was also evaluated. Cells from 65 patients with newly diagnosed AML were studied. The expression of HSP90 correlated with that of CD34, p170, and bcl-2 proteins but not with white cell counts, FAB or WHO subtype, or cytogenetics. HSP90 levels were also higher in samples exhibiting an autonomous growth in liquid culture or forming spontaneous colonies. A concomitant constitutive activation of the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/AKT pathways was observed in a majority of samples and was significantly correlated with HSP90 expression. All patients received induction chemotherapy. The percentages of HSP90-, CD34-, bcl-2-, and p170-positive cells were higher in patients who did not attain complete remission. Survival was also shorter in patients with high levels of HSP90. In vitro exposure of leukemic cells to 17-allylamino-demethoxy geldanamycin (17-AAG) resulted in inhibition of growth in liquid and clonogeneic cultures and in apoptosis, at concentrations which in most cases were not toxic for normal CD34-positive or progenitor cells. The concentration inhibiting 50% growth at 72 h in liquid culture correlated with HSP90 expression. Our study suggests that HSP90 is overexpressed in poor-prognosis AML cells and plays a role in cell survival and resistance to chemotherapy. Targeted therapy with 17-AAG represents a promising antileukemic strategy in adult AML.