Search

Supplementary Methods from Persistent Activation of the Fyn/ERK Kinase Signaling Axis Mediates Imatinib Resistance in Chronic Myelogenous Leukemia Cells through Upregulation of Intracellular SPARC

Supplementary Figure 4 from Resveratrol Promotes Autophagic Cell Death in Chronic Myelogenous Leukemia Cells via JNK-Mediated p62/SQSTM1 Expression and AMPK Activation

Supplementary Figure Legends 1-4 from Resveratrol Promotes Autophagic Cell Death in Chronic Myelogenous Leukemia Cells via JNK-Mediated p62/SQSTM1 Expression and AMPK Activation

Supplementary Materials and Methods from Resveratrol Promotes Autophagic Cell Death in Chronic Myelogenous Leukemia Cells via JNK-Mediated p62/SQSTM1 Expression and AMPK Activation

Supplementary Figure 1 from Resveratrol Promotes Autophagic Cell Death in Chronic Myelogenous Leukemia Cells via JNK-Mediated p62/SQSTM1 Expression and AMPK Activation

Supplementary Figure 2 from Resveratrol Promotes Autophagic Cell Death in Chronic Myelogenous Leukemia Cells via JNK-Mediated p62/SQSTM1 Expression and AMPK Activation

Supplementary Figure Legends 1-6 from Persistent Activation of the Fyn/ERK Kinase Signaling Axis Mediates Imatinib Resistance in Chronic Myelogenous Leukemia Cells through Upregulation of Intracellular SPARC

Supplementary Figure 3 from Resveratrol Promotes Autophagic Cell Death in Chronic Myelogenous Leukemia Cells via JNK-Mediated p62/SQSTM1 Expression and AMPK Activation

Personalized medicine is a challenge for patients with acute myeloid leukemia (AML). The identification of several genetic mutations in several AML trials led to the creation of a personalized prognostic scoring algorithm known as the Knowledge Bank (KB). In this study, we assessed the prognostic value of this algorithm on a cohort of 167 real life AML patients. We compared KB predicted outcomes to real-life outcomes. For patients younger than 60-year-old, OS was similar in favorable and intermediate ELN risk category. However, KB algorithm failed to predict OS for younger patients in the adverse ELN risk category and for patients older than 60 years old in the favorable ELN risk category. These discrepancies may be explained by the emergence of several new therapeutic options as well as the improvement of allogeneic stem cell transplantation (aHSCT) outcomes and supportive cares. Personalized medicine is a major challenge and predictions models are powerful tools to predict patient's outcome. However, the addition of new therapeutic options in the field of AML requires a prospective validation of these scoring systems to include recent therapeutic innovations.

Acute myeloid leukemia (AML) is a hematological malignancy with an undefined heritable risk. Here we perform a meta-analysis of three genome-wide association studies, with replication in a fourth study, incorporating a total of 4018 AML cases and 10488 controls. We identify a genome-wide significant risk locus for AML at 11q13.2 (rs4930561; P = 2.15 × 10−8; KMT5B). We also identify a genome-wide significant risk locus for the cytogenetically normal AML sub-group (N = 1287) at 6p21.32 (rs3916765; P = 1.51 × 10−10; HLA). Our results inform on AML etiology and identify putative functional genes operating in histone methylation (KMT5B) and immune function (HLA)., Genome wide association studies in cancer are used to understand the heritable genetic contribution to disease risk. Here, the authors perform a genome wide association study in European patients with acute myeloid leukemia and identify loci associated with risk of developing the disease.

Background Core-binding factor acute myeloid leukemia (CBF AML) with recurrent genetic abnormalities inv(16)(p13.1q22) or t(16;16)(p13.1;q22)/ CBFB-MYH11 are usually prognostically favorable but heterogeneous group and additional abnormalities change their prognosis. Materials and Methods To evaluate the impact of a complex karyotype on CBF AML prognosis, we included 24 patients with a median age of 56.4 years (23.2-83.3) and a median number of abnormalities of 5 (4-13). Results Median follow-up was 110.4 months. Among patients with a primary clone, complete remission (CR) was reached in 66.7% of patients. 31.3% of patients experienced AML relapse with a median of 8.5 months. Median OS for transplanted patients was 80.7 versus 40.5 months for non-transplanted patients, excluding the 4 patients with early death. Among patients harboring AML with clonal evolution, CR was reached in 62.5% of patients. 50% of patients underwent allogeneic stem cell transplantation (ASCT). In these, median RFS was 19.3 versus 0 months in non-transplanted patients. Median OS seemed also longer in transplanted patients with 23.5 versus 2.95 months in non-transplanted. Conclusion Use of new treatment and tailored strategy based on measurable residual disease monitoring may now improve these results. However, these data allow us to reconsider the good prognosis historically associated with CBF patients despite of karyotype and the place of ASCT in the strategy.

Acute myeloid leukemia (AML) is a hematological malignancy with an undefined heritable risk. Here we performed a meta-analysis of three genome-wide association studies, with replication in a fourth study, incorporating a total of 4018 AML cases and 10488 controls. We identified a genome-wide significant risk locus for AML at 11q13.2 (rs4930561; P = 2.15 × 10-8; KMT5B). We also identified a genome-wide significant risk locus for the cytogenetically normal AML sub-group (N=1287) at 6p21.32 (rs3916765; P = 1.51 × 10-10; HLA). Our results inform on AML etiology by identifying putative functional genes operating in histone methylation (KMT5B) and immune function (HLA).

To determine the prevalence of the V617F Janus Kinase 2 (JAK2) mutation in patients with thrombosis without other biological signs of underlying myeloproliferative neoplasm (MPN) and identify associated risk factors for thrombosis. Over a 10-year period, data were collected from patients with thrombotic events and who had also been screened for the V617F JAK2 mutation. Patients with signs of underlying MPN, such as haematocrit levels ≥ 50% and/or platelet counts ≥ 450 × 109/L and/or splanchnic thrombosis were excluded from the study. Of 340 patients fulfilling inclusion criteria, JAK2 mutation was found in 9 (2.65%), the allele burden being at least 2% in 4 (1.1%). Upon follow-up, MPN was diagnosed in the latter 4. Univariate analysis of the whole cohort showed that age (54 ± 15 vs. 64 ± 13, p = 0.027), platelet count (317 ± 111 vs. 255 ± 75, p = 0.017), C-reactive protein level > 5 mg/L (OR 7.29, p = 0.014), and splenomegaly (OR 54.5, p = 0.0002) were significantly associated with JAK2 mutation. There was also a trend for an increased risk of cerebral venous thrombosis (OR 6.54, p = 0.064). Logistic regression confirmed a significant association between splenomegaly and JAK2 mutation (OR 43.15 [95%CI, 3.05–610.95], p = 0.0054). The V617F JAK2 mutation is rarely found in patients with thrombotic events without overt MPN. Splenomegaly, however, is a statistically and clinically relevant indicator of a potential JAK2 mutation in patients with non-splanchnic thrombotic events. Such patients should require further assessment and a close follow-up.

Isolated trisomy 8 (+8) is a frequent cytogenetic abnormality in the myelodysplastic syndromes (MDS), but its characteristics are poorly reported. We performed a retrospective study of 138 MDS patients with isolated +8, classified or reclassified as MDS (excluding MDS/myeloproliferative neoplasm). Myeloproliferative (MP) features were defined by the repeated presence of one of the following: white blood cell count >10 × 109 /l, myelemia (presence of circulating immature granulocytes with a predominance of more mature forms) >2%, palpable splenomegaly. Fifty-four patients (39·1%) had MP features: 28 at diagnosis, 26 were acquired during evolution. MP forms had more EZH2 (33·3% vs. 12·0% in non-MP, P = 0·047), ASXL1 (66·7% vs. 42·3%, P = 0·048) and STAG2 mutations (77·8% vs. 21·7%, P = 0·006). Median event-free survival (EFS) and overall survival (OS) were 25 and 27 months for patients with MP features at diagnosis, versus 28 (P = 0·15) and 39 months (P = 0·085) for those without MP features, respectively. Among the 57 patients who received hypomethylating agent (HMA), OS was lower in MP cases (13 months vs. 23 months in non-MP cases, P = 0.02). In conclusion, MP features are frequent in MDS with isolated +8. MP forms had more EZH2, ASXL1 and STAG2 mutations, responded poorly to HMA, and tended to have poorer survival than non-MP forms.

TP53 aberrations, including somatic mutations of TP53 gene or 17p deletion leading to the loss of the TP53 locus, are a major predictive factor of resistance to fludarabin based chemotherapy in chronic lymphocytic leukemia (CLL) and remain an adverse prognostic factor in the chemofree era. Therefore, detection of TP53 alteration before each new line of treatment is required for theranostic stratification. In order to better characterize the distribution and combination of the TP53 variants in CLL, we collected the TP53 sequencing data of 343 patients harboring TP53 mutations from centers of the French Innovative Leukemia Organization-CLL (FILO) and established a large data base of 573 TP53 mutations. Mutations were identified through NGS sequencing (covering exon 2 to 11) allowing the detection of low frequency variants down to 1% VAF. Several distinct low VAF mutations were orthogonally confirmed by digital PCR. TP53 variants were analyzed through UMD_TP53 data gathering 90 000 TP53 mutations from all type of cancers. IGHV mutational status and FISH analysis were available for 224 and 176 patients respectively. Using ACMG criteria from the UMD_TP53 database, we confirmed that 523 could be classified as pathogenic, 42 were likely pathogenic and 8 were VUS (Variants of Unknown Significance). As expected, the mutation distribution along the p53 protein exhibited a clustering of variants in the DNA binding domain of the protein. We also confirmed the presence of a specific hotspot at codon 234 (6%) which is noticeable in other CLL cohorts but absent in solid tumors. 431 TP53 variants led to the expression of a mutant protein whereas the remaining 142 led a TP53 null phenotype. For 8 patients without 17p deletion and a mutation VAF larger than 50%, SNP analysis indicate that these tumors had a copy number neutral loss of heterozygosis at 17p with a duplication of the mutant allele leading to homozygous mutations of TP53. When focusing on the allele burden of TP53 mutations, 264/573 (46%) variants had an allele frequency Among the 343 patients, 113 (33%) were poly-mutated and harbored more than one pathogenic TP53 variants (2 to 11 variants per patient): 57 (16,7 %) had 2 variants, 32 (9,3%) had 3, 10 had 4 (3%) and 14 patients (4%) had 5 to 11 variants. Using both long range sequencing and in silico analysis, we could show that all these variants were distributed in different alleles supporting an important intratumoral heterogeneity and a strong selection for TP53 loss of function during tumor progression in these patients. Null variants were rarely found as single alteration: only 46 patients (13,4%) patients harbored a single null mutation. Null mutations were predominantly found in patients with multiclonal mutations (87% with 4 or more). Median size of variants significantly decreased with the number of mutations and most of low VAF (less than 10%) variants were found in multiclonal combinations. Multiclonal mutations were predominantly found in previously treated patients (41% treated versus 10 % untreated) but whether all these variants preceded treatment and were further selected is currently unknown. We observed that 71,5 % of patients were IGHV unmutated and multiclonal mutations were surprisingly more frequent in mutated IGHV cases than in unmutated ones. Only 50% of cases carried a 17p deletion, highlighting again the importance of testing for TP53 mutations in addition to FISH analysis. Presence or absence of 17p deletion was unrelated to the number of TP53 mutations. Taken together these observations suggest that the TP53 mutational landscape in CLL is very complex and can involve multiple mechanisms, converging to a total loss of TP53 function and tumor progression. NGS provides a powerful tool for detecting all these alterations including variants with low VAF and should become a standard for CLL screening prior to each line of treatment. Disclosures Leblond: Amgen: Honoraria, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Astra Zeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Letestu:Abbvie: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Roche: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Alexion: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts. Cymbalista:Abbvie: Honoraria; Roche: Research Funding; Sunesis: Research Funding; Gilead: Honoraria; Janssen: Honoraria; AstraZeneca: Honoraria.

Myelodysplastic syndromes (MDS) are hematopoietic stem cell malignancies. Known predisposing factors to adult MDS include rare germline mutations, cytotoxic therapy, age-related clonal hematopoiesis, and autoimmune or chronic inflammatory disorders. To date, no published studies characterizing MDS-associated germline susceptibility polymorphisms exist. We performed a genome-wide association study of 2 sample sets (555 MDS cases vs 2964 control subjects; 352 MDS cases vs 2640 control subjects) in non-del(5q) MDS cases of European genomic ancestry. Meta-analysis identified 8 MDS-associated loci at 1q31.1 (PLA2G4A), 3p14.1 (FAM19A4), 5q21.3 (EFNA5), 6p21.33, 10q23.1 (GRID1), 12q24.32, 15q26.1, and 20q13.12 (EYA2) that approached genome-wide significance. Gene expression for 5 loci that mapped within or near genes was significantly upregulated in MDS bone marrow cells compared with those of control subjects (P < .01). Higher PLA2G4A expression and lower EYA2 expression were associated with poorer overall survival (P = .039 and P = .037, respectively). Higher PLA2G4A expression is associated with mutations in NRAS (P < .001), RUNX1 (P = .012), ASXL1 (P = .007), and EZH2 (P = .038), all of which are known to contribute to MDS development. EYA2 expression was an independently favorable risk factor irrespective of age, sex, and Revised International Scoring System score (relative risk, 0.67; P = .048). Notably, these genes have regulatory roles in innate immunity, a critical driver of MDS pathogenesis. EYA2 overexpression induced innate immune activation, whereas EYA2 inhibition restored colony-forming potential in primary MDS cells indicative of hematopoietic restoration and possible clinical relevance. In conclusion, among 8 suggestive MDS-associated loci, 5 map to genes upregulated in MDS with functional roles in innate immunity and potential biological relevance to MDS.

Introduction Therapy-related myelodysplastic syndrome and acute myeloid leukemia (t-MDS/AML) are defined as complications of previous cytotoxic therapy. Azacitidine (AZA), a hypomethylating agent, has showed activity in t-MDS/AML. Objectives We evaluated the clonal dynamics of AZA-treated t-MDS/AML. Methods We collected bone marrow samples, at diagnosis and during treatment, from AZA-treated t-MDS/AML patients. NGS on 19 myeloid genes was performed, and candidate mutations with a variant allele frequency >5% were selected. Results Seven t-AML and 12 t-MDS were included with median age of 71 (56-82) years old, median number of AZA cycles of 6 (1-15), and median overall survival (OS) of 14 (3-29) months. We observed correlation between AZA response and clonal selection. Decrease of TP53-mutated clone was correlated with response to AZA, confirming AZA efficacy in this subgroup. In some patients, emergence of mutations was correlated with progression or relapse without impact on OS. Clones with mutations in genes for DNA methylation regulation frequently occurred with other mutations and remained stable during AZA treatment, independent of AZA response. Conclusion We confirmed that the molecular complexity of t-MNs and that the follow-up of clonal selection during AZA treatment could be useful to define treatment combination.

Introduction The standard first-line treatment for acute myeloid leukemia (AML) is a combination of cytarabine and anthracyclines. To date, there is no commonly agreed-on regimen for patients who are ineligible for this therapy because of cardiac comorbidities or prior exposure to anthracyclines. We compared 3 anthracycline-free regimens currently used in France. Patients and Methods Two patients with newly diagnosed or relapsed/refractory AML were treated intensively in 3 French centers. All patients had at least one contraindication to the receipt of anthracyclines. Three regimen types were used: fludarabine, cytarabine, and granulocyte-colony stimulating factor (FLAG); clofarabine and cytarabine (CLARA); and topotecan plus cytarabine (TA). Results Thirty patients (58%) had de novo AML. The European LeukemiaNet 2013 risk categories were favorable, intermediate, and adverse in 4 (8%), 27 (52%), and 20 (39%) patients, respectively. Twenty-four patients received TA and 28 FLAG/CLARA regimens. Fifty percent of patients had cardiac dysfunction, and 50% had prior anthracycline exposure above the maximum tolerated dose. The rate of cardiac events was similar after TA (17%) and FLAG/CLARA (25%) (P = .78). The 5-year nonrelapse mortality was 17.9% and 12.5% in the TA and FLAG/CLARA groups, respectively (P = .59). In patients with previously untreated AML, complete response occurred in 18 (72%) of 25, but median overall survival was only 9.7 months. Conclusion TA, FLAG, and CLARA regimens are efficient and are associated with acceptable toxicity in AML patients ineligible for the 3 + 7 regimen as a result of cardiac comorbidities. However, long-term outcome remains disappointing, thereby highlighting the need for the development of less toxic regimens.

The karyotype is a strong independent prognostic factor in myelodysplastic syndromes (MDS). Since the implementation of the new comprehensive cytogenetic scoring system for MDS, chromosome 7 anomalies are no longer generally assigned to poor risk features but are thoroughly separated. However, der(1;7)(q10;p10), hereinafter der(1;7), is merged into the group labeled "any other single" and belongs to the intermediate risk group, just by definition due to lack of adequate clinical data. The aim of our international collaborative was to clarify the "real" prognostic impact of der(1;7) on a homogenous and well-documented data base. We performed detailed analysis of 63 MDS patients with isolated der(1;7) constituting the largest cohort hitherto reported. Furthermore, clinical data are compared with those of patients with isolated del(7q) and isolated monosomy 7. Median overall survival (OS) of patients with der(1;7) is 26 months (hazard ratio (HR) 0.91 for del(7q) vs der(1;7) and 2.53 for monosomy 7 vs der(1;7)). The der(1;7) is associated with profound thrombocytopenia most probably causing the reduced OS which is in striking contrast to the low risk for AML transformation (HR 3.89 for del(7q) vs der(1;7) and 5.88 for monosomy 7 vs der(1;7)). Molecular karyotyping indicates that der(1;7) is generated in a single step during mitosis and that a chromosomal imbalance rather than a single disrupted gene accounts for malignancy. Thus, the current cytogenetic scoring system assigning isolated der(1;7) to the intermediate risk group is now confirmed by a sufficient data set.

The myelodysplastic syndromes (MDS) are preleukemic diseases of elderly patients characterized by defective maturation of clonal hematopoietic progenitor cells resulting in peripheral blood cytopenias. Clonal chromosomal abnormalities are heterogeneous and can be detected in less than 50% of patients with de novo MDS and more frequently in secondary MDS (up to 80%). The karyotype plays an important role in the pathogenesis, diagnosis, and prognosis to evaluate the risk of leukemic transformation and, more recently, in treatment allocation. The gold standard for cytogenetic diagnosis in MDS is conventional chromosome banding analyses of bone marrow metaphases. The most frequent abnormalities are deletions and losses of chromosomes 5 (-5/5q-) and 7 (-7/7q-) and various isolated or combined abnormalities. Fluorescent in situ hybridization and array comparative genomic hybridization can reveal cryptic genetic abnormalities but are not recommended in routine diagnosis. New techniques including next generation sequencing revealed somatic driver mutations especially those affecting genes involved in RNA splicing or those harboring important prognostic value (TP53, ASXL1…) with potential applications in clinical practice in the future.

We report five chronic myeloid leukaemia (CML) patients in whom we identified and characterized undescribed BCR-ABL1 fusion transcripts. We investigated the precise features of the molecular rearrangements and the minimal residual disease follow-up for these five patients. Three resulted from new rearrangements between the BCR and ABL1 sequences (the breakpoints being located within BCR exon 13 in two cases and within BCR exon 18 in one case). The other two cases revealed a complex e8-[ins]-a2 fusion transcript involving a third partner gene, PRDM12 and SPECC1L, respectively. Moreover, single nucleotide polymorphism-array analysis performed in the latter two cases showed copy number alterations shared by the two patients, thus identifying genes that were deleted during rearrangement and suggesting their potential role in CML pathogenesis. Interestingly, we highlight that the prognosis of alterations, such as the presence of an e8a2 transcript or the deletion of various genes, which have been controversial, may be definitively erased by the introduction of tyrosine kinase inhibitors (TKIs).

Les syndromes myelodysplasiques (SMD) sont des affections clonales des cellules souches pluripotentes, caracterisees par une hematopoiese inefficace, responsable de cytopenies sanguines qui contrastent avec une moelle generalement riche. De plus, les SMD evoluent frequemment en leucemie aigue myeloide (LAM) et constituent les plus frequents des etats preleucemiques de l’adulte.Les SMD predominent chez le sujet âge, avec une mediane d’âge au diagnostic de l’ordre de 70 [...]

International audience; Myeloproliferative neoplasms are clonal hematopoietic stem cell disorders characterized by aberrant proliferation and an increased tendency toward leukemic transformation. The genes JAK2, MPL, and CALR are frequently altered in these syndromes, and their mutations are often a strong argument for diagnosis. We analyzed the mutational profiles of these three genes in a cohort of 164 suspected myeloproliferative neoplasms. JAK2 V617F mutation was detected by real-time PCR, whereas high-resolution melting analysis followed by Sanger sequencing were used for searching for mutations in JAK2 exon 12, CALR, and MPL. JAK2 V617F mutation was associated with CALR (n Z 4) and MPL (n Z 4) mutations in 8 of 103 essential thrombocytosis patients. These cases were harboring a JAK2 V617F allelic burden of

// Thomas Cluzeau 1,2,3,4,6,* , Alix Dubois 1,2,3,4,* , Arnaud Jacquel 1,2,3,4 , Frederic Luciano 1,2,3,4 , Aline Renneville 5 , Claude Preudhomme 5 , Jean Michel Karsenti 6 , Nicolas Mounier 6 , Pierre Rohrlich 6 , Sophie Raynaud 7 , Bernard Mari 8 , Guillaume Robert 1,2,3,4 and Patrick Auberger 1,2,3,4,6 1 INSERM U1065, Mediterranean Center for Molecular Medicine (C3M), Nice, France 2 INSERM U1065, Team 2: Cell Death, Differentiation, Inflammation and Cancer 3 Equipe labellisee par la Ligue Nationale Contre le Cancer (2011-2013), Paris, France 4 University of Nice, Nice, France 5 Laboratory of Hematology, Biology and Pathology Center, CHRU of Lille, Lille, France 6 Department of Clinical hematology and Transplantation, CHU of Nice, Nice, France 7 Oncohematology laboratory, CHU of Nice, Nice, France 8 Molecular and Cellular Pharmacology Institute, CNRS, Sophia-Antipolis, France * These authors participated equally to this work Correspondence: Patrick Auberger, email: // Keywords : MDS, AML, Azacitidine, Polyploidy, TET2, ASLX1 Received : May 5, 2014 Accepted : May 26, 2014 Published : May 27, 2014 Abstract In the present study, we provide a comparative phenotypic and genotypic analysis of azacitidine-sensitive and resistant SKM-1 cell lines. Morphologically, SKM1-R exhibited increase in cell size that accounts for by enhanced ploidy in a majority of cells as shown by cell cycle and karyotype analysis. No specific Single Nucleotide Polymorphism (SNP) alteration was found in SKM1-R cells compared to their SKM1-S counterpart. Comparative pangenomic profiling revealed the up-regulation of a panel of genes involved in cellular movement, cell death and survival and down-regulation of genes required for cell to cell signaling and free radical scavenging in SKM1-R cells. We also searched for mutations frequently associated with myelodysplastic syndromes (MDS) and found that both cell lines harbored mutations in TET2, ASLX1 and TP53. Collectively, our data show that despite their different morphological and phenotypic features, SKM1-S and SKM1-R cells exhibited similar genotypic characteristics. Finally, pangenomic profiling identifies new potential pathways to be targeted to circumvent AZA-resistance. In conclusion, SKM1-R cells represent a valuable tool for the validation of new therapeutic intervention in MDS.

hma.2012.0744 Auteur(s) : Therese Aurrant, Evelyne Callet-Bauchu, Florence Cymbalista, Alain Delmer, Brigitte Dreyfus, Florence Nguyen Khac, Veronique Leblond, Stephane Lepretre, Vincent Levy, Sophie Raynaud, Xavier Troussard1 troussard-x@chu-caen.fr Pour l’intergroupe GCFLLC/MW/GOELAMS 1 Laboratoire d’hematologie, CHU de Caen, France Tires a part : X. Troussard Incidence La leucemie lymphoide chronique (LLC) est la plus frequente des leucemies de l’adulte. Avec 3 800 nouveaux [...]

Myelodysplastic syndromes (MDSs) are hematopoietic stem cell disorders in which recurrent mutations define clonal hematopoiesis. The origin of the phenotypic diversity of non-del(5q) MDS remains unclear. Here, we investigated the clonal architecture of the CD34+CD38- hematopoietic stem/progenitor cell (HSPC) compartment and interrogated dominant clones for MDS-initiating cells. We found that clones mainly accumulate mutations in a linear succession with retention of a dominant subclone. The clone detected in the long-term culture-initiating cell compartment that reconstitutes short-term human hematopoiesis in xenotransplantation models is usually the dominant clone, which gives rise to the myeloid and to a lesser extent to the lymphoid lineage. The pattern of mutations may differ between common myeloid progenitors (CMPs), granulomonocytic progenitors (GMPs), and megakaryocytic-erythroid progenitors (MEPs). Rare STAG2 mutations can amplify at the level of GMPs, from which it may drive the transformation to acute myeloid leukemia. We report that major truncating BCOR gene mutation affecting HSPC and CMP was beneath the threshold of detection in GMP or MEP. Consistently, BCOR knock-down (KD) in normal CD34+ progenitors modifies their granulocytic and erythroid differentiation. Clonal architecture of the HSPC compartment and mutations selected during differentiation contribute to the phenotypic heterogeneity of MDS. Defining the hierarchy of driver mutations provides insights into the process of transformation and may guide the search for novel therapeutic strategies.

AXL is a receptor tyrosine kinase of the TAM family, the function of which is poorly understood. We previously identified AXL overexpression in Imatinib (IM)-resistant CML cell lines and patients. The present study was conducted to investigate the role of AXL and the mechanisms underlying AXL overexpression in Tyrosine Kinase Inhibitor (TKI)-resistant CML cells. We present evidence that high AXL expression level is a feature of TKI-resistant CML cells and knockdown of AXL sensitized TKI-resistant cells to IM. In addition, expression of wild-type AXL but not a dominant negative form of AXL confers IM-sensitive CML cells the capacity to resist IM effect. AXL overexpression required PKCα and β and constitutive activation of ERK1/2. Accordingly, GF109203X a PKC inhibitor, U0126 a MEK1 inhibitor and PKCα/β knockdown restore sensitivity to IM while PKCα or PKCβ overexpression in CML cells promotes protection against IM-induced cell death. Finally, using luciferase promoter activity assays we established that AXL is regulated transcriptionally through the AP1 transcription factor. Our findings reveal an unexpected role of AXL in resistance to TKI in CML cells, identify the molecular mechanisms involved in its overexpression and support the notion that AXL is a new marker of resistance to TKI in CML.

Philadelphia (Ph)-negative myeloproliferative neoplasms (MPNs) are known to harbor alterations of the tyrosine kinase JAK2 (9p24), resulting in the constitutive autoactivation of the encoded protein. Here, we report an unclassifiable MPN case, BCR/ABL1-negative, showing a three-way t(9;18;22)(p23;p11.3;q11.2) translocation, which generates a 5'BCR/3'JAK2 gene by fusing BCR at intron 1 to JAK2 at intron 14 on the derivative chromosome 22. The fusion gene produced two alternatively spliced 5'BCR/3'JAK2 transcripts, fusing in-frame BCR exon 1 to JAK2 exon 15 and exon 17. This is the first report of the simultaneous occurrence of two BCR/JAK2 fusion transcripts in the same sample and of the longer transcript isoform (BCR exon 1 fused to JAK2 exon 15). Notably, both BCR/JAK2 encoded fusion proteins are predicted to juxtapose the coiled-coil dimerization domain of BCR to the catalytically inactive pseudokinase domain (JH2), entirely or partially deprived of the inhibitory region 1 (IR1). Interestingly, IR1 is involved in the auto-inhibitory interaction with the JAK2 kinase domain (JH1), which may result in deregulation of JAK2 activity.

Ten-eleven translocation 2 (TET2) mutations have been involved in myeloid malignancies. This retrospective study aims at evaluating the frequency and impact of TET2 mutations in 247 secondary acute myeloid leukemia cases referred to as myelodysplasia-related changes (n=201) or therapy-related (n=46) leukemias. Mutation of at least one copy of the TET2 gene was detected in 49 of 247 (19.8%) patients who presented with older age, higher hemoglobin level, higher neutrophil and monocyte counts, and lower platelet count. TET2 mutations were significantly less frequent in therapy-related (8.7%) than myelodysplasia-related changes (22.3%; P=0.035) leukemias and strongly associated with normal karyotype (P

The impact of ten-eleven-translocation 2 (TET2) mutations on response to azacitidine (AZA) in MDS has not been reported. We sequenced the TET2 gene in 86 MDS and acute myeloid leukemia (AML) with 20-30% blasts treated by AZA, that is disease categories wherein this drug is approved by Food and Drug Administration (FDA). Thirteen patients (15%) carried TET2 mutations. Patients with mutated and wild-type (WT) TET2 had mostly comparable pretreatment characteristics, except for lower hemoglobin, better cytogenetic risk and longer MDS duration before AZA in TET2 mutated patients (P=0.03, P=0.047 and P=0.048, respectively). The response rate (including hematological improvement) was 82% in MUT versus 45% in WT patients (P=0.007). Mutated TET2 (P=0.04) and favorable cytogenetic risk (intermediate risk: P=0.04, poor risk: P=0.048 compared with good risk) independently predicted a higher response rate. Response duration and overall survival were, however, comparable in the MUT and WT groups. In higher risk MDS and AML with low blast count, TET2 status may be a genetic predictor of response to AZA, independently of karyotype.

Background: Myelodysplastic Syndromes (MDS) are genetically and hematologically diverse stem cell malignancies pathogenetically linked to constitutive innate immune activation. Other than rare germline mutations and age, the only known predispositions to adult MDS include prior cytotoxic therapy, clonal hematopoiesis, and autoimmune or chronic inflammatory disorders. Few studies investigating the genetic susceptibility to MDS have been performed owing to the limitations of SNP-array sample size. Here, we report the results from the first unbiased genome wide analysis of germline polymorphisms associated with non-del(5q) MDS using a multinational curated data set. Methods: Association analyses were performed on 2 sample sets (set 1: 555 cases, 2,964 controls; set 2: 352 cases, 2,640 controls) and combined by meta-analysis. Standard SNP- and sample-level QC was applied. Haplotype reference consortium (HRC) imputation was done by the Michigan imputation server (Rsq>0.4) providing 23,278,269 markers for analysis. Gene expression sequencing was performed on an independent sample set from the National Taiwan University Hospital (213 MDS cases, 20 healthy donors; HumanHT-12 v4 Expression BeadChip; Chuang et al. Leukemia 2015). Functional analyses were performed as described. Results: Eight MDS associated loci were identified with lead variants, rs6683416, rs34539210, rs341274, rs1634783, rs7099032, rs2947170, rs4404050, and rs1206818, at 1q31.1 (PLA2G4A), 3p14.1 (FAM19A4), 5q21.3 (EFNA5), 6p21.33, 10q23.1 (GRID1), 12q24.32, 15q26.1, and 20q13.12 (EYA2), respctively. Odds ratio (OR) and p-values of each are listed in Table 1. Using gene expression profiling in an independent MDS sample set, we found expression of these five candidate genes was significantly increased in MDS vs controls (p Conclusion: We describe here the first MDS susceptibility loci ever identified the majority of which have a direct relationship to innate immune activation, a driver of MDS pathogenesis. Expression of genes housing these loci is increased in MDS with demonstrable prognostic and biological relevance. Further, functional studies implicate EYA2 as a novel, biologically rational target for MDS treatment. The direct functions of each polymorphism as well as the potential relationship between these predisposition loci to age-related clonal hematopoiesis merits further investigation. Disclosures Cluzeau: Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Speakers Bureau; AbbVie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Menarini: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Speakers Bureau. Sallman:Celgene: Research Funding, Speakers Bureau. Sokol:Spectrum Pharmaceuticals: Consultancy; Seattle Genetics: Consultancy; Mallinckrodt Pharmaceuticals: Consultancy. Maciejewski:Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. List:Celgene: Research Funding.

SPARC is an extracellular matrix protein that exerts pleiotropic effects on extracellular matrix organization, growth factor availability, cell adhesion, differentiation, and immunity in cancer. Chronic myelogenous leukemia (CML) cells resistant to the BCR-ABL inhibitor imatinib (IM-R cells) were found to overexpress SPARC mRNA. In this study, we show that imatinib triggers SPARC accumulation in a variety of tyrosine kinase inhibitor (TKI)–resistant CML cell lines. SPARC silencing in IM-R cells restored imatinib sensitivity, whereas enforced SPARC expression in imatinib-sensitive cells promoted viability as well as protection against imatinib-mediated apoptosis. Notably, we found that the protective effect of SPARC required intracellular retention inside cells. Accordingly, SPARC was not secreted into the culture medium of IM-R cells. Increased SPARC expression was intimately linked to persistent activation of the Fyn/ERK kinase signaling axis. Pharmacologic inhibition of this pathway or siRNA-mediated knockdown of Fyn kinase resensitized IM-R cells to imatinib. In support of our findings, increased levels of SPARC mRNA were documented in blood cells from CML patients after 1 year of imatinib therapy compared with initial diagnosis. Taken together, our results highlight an important role for the Fyn/ERK signaling pathway in imatinib-resistant cells that is driven by accumulation of intracellular SPARC. Cancer Res; 70(23); 9659–70. ©2010 AACR.

Philadelphia chromosome–negative myeloproliferative neoplasms (MPNs) including polycythemia vera, essential thrombocythemia, and primary myelofibrosis show an inherent tendency for transformation into leukemia (MPN-blast phase), which is hypothesized to be accompanied by acquisition of additional genomic lesions. We, therefore, examined chromosomal abnormalities by high-resolution single nucleotide polymorphism (SNP) array in 88 MPN patients, as well as 71 cases with MPN-blast phase, and correlated these findings with their clinical parameters. Frequent genomic alterations were found in MPN after leukemic transformation with up to 3-fold more genomic changes per sample compared with samples in chronic phase (P < .001). We identified commonly altered regions involved in disease progression including not only established targets (ETV6, TP53, and RUNX1) but also new candidate genes on 7q, 16q, 19p, and 21q. Moreover, trisomy 8 or amplification of 8q24 (MYC) was almost exclusively detected in JAK2V617F− cases with MPN-blast phase. Remarkably, copy number–neutral loss of heterozygosity (CNN-LOH) on either 7q or 9p including homozygous JAK2V617F was related to decreased survival after leukemic transformation (P = .01 and P = .016, respectively). Our high-density SNP-array analysis of MPN genomes in the chronic compared with leukemic stage identified novel target genes and provided prognostic insights associated with the evolution to leukemia.

Using array-based CGH, we identified 2p gain in 22/78 (28%) untreated Binet stages B/C CLL, which was the second most frequent copy number change after 13q deletion. It never occurred as a sole abnormality and was associated with other changes (6q deletion; 1p gain). The region of 2p gain frequently included two oncogenes, REL and MYCN. All patients with gain of REL were unmutated for IGHV (p=0.03). Gain of MYCN was associated with increased mRNA expression (p=0.005), suggesting a pathogenic role for MYCN. Gain of 2p appears to be a marker of progression and may contribute to the poor prognosis.

Imatinib is the leading compound to treat patients with chronic myelogenous leukemia (CML) but the exact mechanism of its anti-leukemic effect is incompletely elucidated. Through inhibition of BCR-ABL, Imatinib blocks several downstream pathways and induces apoptosis of BCR-ABL positive cells. In this study, we analyzed further the mode of action of Imatinib in different appropriate cellular models of CML either sensitive or resistant to Imatinib and in CD34+ cells from CML patients. Pharmacological or short hairpin RNA-mediated inhibition of BCR-ABL triggers lysosomal membrane permeabilization (LMP) that culminates in activation and redistribution of Cathepsin B (CB) into the cytoplasm of CML cells, in which it triggers directly BCR-ABL degradation. Pharmacological inhibition of CB by CA-074Me or small interfering RNA-mediated knock-down of CB partly protects K562 cells from Imatinib-induced cell death and CB overexpression sensitizes these cells to Imatinib killing. Strikingly, Imatinib-triggered LMP, CB activation and BCR-ABL cleavage in CD34+ cells from CML patients and inhibition of CB confers protection against cell death in clonogenic assays of CD34+ primary cells from CML patients. Hence, we describe an original pathway by which Imatinib participates to the elimination of CML cells through LMP and CB-mediated specific degradation of BCR-ABL.

The most frequent t(14;19)(q32;q13)-positive B-cell malignancy corresponds to an aggressive subgroup of atypical chronic lymphocytic leukemia