Myelodysplasic Syndrome Research Articles

Neurological Facets of Hematological Malignancies: A Case Series

Background: Central nerve involvement is a serious complication in hemopathies. Clinical presentations are heterogeneous; it can affect the central nervous system as well as the peripheral. The pathophysiological mechanisms are multiple. Aims: To illustrate the diversity of these neurological manifestations affecting both the central and peripheral nervous systems. We study the pathophysiological mechanisms and diagnostic strategies. We discuss the therapeutic alternatives and the prognostic value of neurological damage. Methods: To illustrate the clinical and etiological polymorphism, we report a series of clinical cases of seven patients followed for malignant hemopathies and who consulted for different neurological manifestations. Results: We have seven patients followed for malignant hemopathies: Acute lymphoblastic Leukemia (ALL), Acute myeloblastic Leukemia (AML), Myelodysplasic syndrome (MDS), Chronic myeloid Leukemia (CML). Patients consult for different neurological manifestations (optic neuritis, neuropathic pain, sensory and motor deficit, myopathic syndrome. The etiological assessment identified different causes and mechanisms: leukemic medullary infiltration (myelitis) and optic nerve (optic neuritis). Acute post infectious polyradiculoneuritis in a patient undergoing chemotherapy. The neurological manifestations related to drugs were painful peripheral neuropathy and myopathic syndrome secondary to cytarabine syndrome. The fatal outcome was associated with extensive infiltration and fulminant neurovascularitis induced by MDS. Conclusion: We discuss here through our series the different epidemiological, physiopathological, clinical, evolutionary and prognostic aspects of neurological complications of hematological malignancies.

PB1845: ACUTE MYELOID LEUKEMIA IN THE ELDERLY: A MULTICENTER EXPERIENCE IN COLOMBIA, ON BEHALF OF ACHO’S RENEHOC INVESTIGATORS

Background: The overall survival (OS) of acute myeloid leukemia (AML) decreases as age increases among patients. There are clinical and biological differences in AML in older patients. Even with actual treatments, 70% of patients older than 65 years will die of their disease within 1 year of diagnosis. Aims: Our aim was to characterize the elderly patients (>65 years old) with acute myeloid leukaemia (AML), who were attended in 13 health institutions in Colombia, from 2009 to 2021, including RENEHOC (online platform) and PETHEMA date base (Spanish Program for Hematology Treatments). Methods: An ambispective registry of elderly AML patients, treated in approved centers over the last 12 years. Descriptive statistics were used for patient’s demographic and clinical characteristics. The Kaplan-Meier method was used to assess RFS and OS. Results: A total of 175 patients were included. 54,86% were male and the median age at diagnosis was 73 years old. Most of the patients were classified according to the Eastern Cooperative Oncology Group (ECOG) as ECOG 0-1 (52,7%), ECOG 2 (25 %) and ECOG > 2 (22.3%). 24,26% presented as secondary AML, from these 34% had previous history of myelodysplasic syndrome. At presentation, fever was the main symptom (39,16), followed by bleeding (18,56%). The most frequent comorbidities were hypertension in 20% and diabetes in 9,4%. 24.34% of the patients had one comorbidity while 21.6% of the population had 3 or more. For treatment: 32,9% of the patients received hypomethylating agents, 23,2% received intensive chemotherapy with cytarabine and idarrubicine, 14,1 % had FLUGA (fludarabine low dose and cytarabine) and supportive therapy in 16,7% of the patients. After induction, complete remission (CR) was achieved in 16,9% of patients; CR with incomplete hematologic recovery (Cri) in 7% and 20,9% had primary refractory disease. Mortality during in induction therapy was 12,9%. The median age of patients in the intensive chemotherapy group was 66 years old (66-70), with ECOG 0-2 in 63,8%. The OS was 36.1% (95%CI: 27.0%–45%) at 1 year and 0 % at 5 years of follow-up, the relapse-free survival (RFS) was 38.3 % (95%CI: 27.4%- 49.1%) at 1 year and 0% at 5 years. According to the ECOG. the OS at 1 year was 50,9 % (95%CI: 36.1%–63.9%) for patients with ECOG <2, while the OS at 1 year was 22.7% (95%CI: 12.7%–34.4%) for ECOG >2 with p<0.001. Image:Summary/Conclusion: Management of AML in older patients remains a medical challenge, not only because of the age, but also because older adults are more likely to have impaired functional status and comorbidities that limit treatment options. More refined assessments tools and scales are needed to differentiate between fit and fragile older adults. This multicenter report analyzing real world data from AML patients in Colombia confirmed the impact of clinical factors: age, ECOG on OS and RFS. Challenges includes access to newer treatments with novel, targeted and less intensive therapies. Keywords: acute myeloid leukemia, survival, elderly

Preliminary Results of the Filo Phase 2 Trial for Untreated Fit Patients with Intermediate Risk Chronic Lymphocytic Leukemia Comparing Ibrutinib Plus Venetoclax (IV) Versus FCR

Preliminary Results of the Filo Phase 2 Trial for Untreated Fit Patients with Intermediate Risk Chronic Lymphocytic Leukemia Comparing Ibrutinib Plus Venetoclax (IV) Versus FCR

Hypoplastic Myelodysplastic Syndrome: Clinical, Histopathological and Molecular Characterization

Introduction and AimsHypoplastic Myelodysplasic Syndrome (h-MDS) has distinct features compared to normo- or hypercellular MDS, with a higher response rate to immunosuppressive therapy and more favorable prognosis. However, to date, reproducible diagnostic criteria for h-MDS have not been clearly defined, making differential diagnosis from other bone marrow failure (BMF) syndromes challenging. Based on a large and well-annotated consecutive cohort of patients with BM hypocellularity from two tertiary centres, we aimed to delineate the clinical, histopathological and molecular features of h-MDS.MethodsOf 1262 consecutive adult patients analyzed at King's College Hospital, London, UK and IRCCS Policlinico San Matteo & University of Pavia, Italy: 533 patients had a hypocellular BM, including 205 MDS patients with BM cellularity ≤25%, 77 MDS with reduced age-adjusted BM cellularity, 139 with Aplastic Anemia (AA), 97 with a diagnosis of Idiopathic Cytopenia of Undetermined Significance (ICUS) and 15 with congenital BMF (c-BMF) and 729 with normo- or hypercellular MDS (n-MDS).ResultsComparison of clinical features and outcome of patients with h-MDS as defined by a BM cellularity ≤25% (n=205) and those with a reduced age-adjusted cellularity (n=77) did not detect any significant differences. Therefore, a reduced age-adjusted BM cellularity was adopted as a criterion to define h-MDS (n=282). Compared with patients with n-MDS, those with h-MDS were significantly younger, displayed higher levels of hemoglobin (p=.01), lower neutrophil and platelet counts and lower percentage of BM blasts, ring sideroblasts (RS) and CD34+cells as determined by immunohistochemistry (all p<.001). No significant difference was observed in the distribution of chromosomal abnormalities. A significantly higher prevalence of PNH clones was observed in h-MDS (24% vs 2%, p<.001). Those patients with h-MDS showed a significantly lower risk of leukemic evolution compared with n-MDS (4-year cumulative incidence 14% vs 26%, p=.003). Comparing h-MDS with AA, h-MDS showed a significantly higher percentage of RS (p=.004), BM blasts, dysmegakaryopoiesis and dysgranulopoiesis (p<.001), BM fibrosis (p=.002), clusters of CD34+ cells and chromosomal abnormalities (p<.001). H-MDS patients showed a significantly higher risk of leukemic evolution compared with AA (4-year cumulative incidence 14% vs 0%, p<.001).Targeted sequencing of 24 commonly mutated myeloid genes was performed on 358 patients (73 h-MDS, 45 AA and 240 n-MDS). Twenty-five patients with h-MDS (34%) showed one or more somatic mutation (median 1; range 1-4). Comparing h-MDS to n-MDS revealed a significantly lower number of mutations per subject and variant allele frequency (VAF) (p<.001). Moreover, a significantly lower number of mutations per patient and VAF were observed in AA compared to h-MDS (p=.031 and p=.003, respectively). Thirty-seven patients with a hypocellular BM had a peripheral blood telomere length (TL) ≤1st percentile and/or a germline mutation consistent with a diagnosis of c-BMF, leading to an estimated prevalence of unidentified c-BMF of 13% in cases without robust morphological evidence of MDS.Based on these findings, we defined a diagnostic score (h-score), including variables with the highest specificity for MDS: dysmegakaryopoiesis, dysgranulopoiesis, BM fibrosis, clusters of CD34+ cells, RS 2-14%, BM blasts 2-4% (score 1); RS≥15% and BM blasts ≥5% (score 2). By applying ROC analysis, a cut off value of 2 was associated with the highest specificity for MDS (0.98). Notably, 71% of patients with a hypoplastic BM and low h-score (0-1) had no evidence of clonal disease by cytogenetic or mutation analyses or had a mutation pattern consistent with age-related clonal hematopoiesis. These patients showed a significantly better OS and lower risk of leukemic evolution (P<.001).ConclusionsIntegration of cyto-histological and genetic features in patients with a hypocellular BM identifies two distinct groups, one with clinical and genetic features highly consistent with a clonal disease with high risk of leukemic evolution, and one with features more consistent with a non-malignant bone marrow failure. Furthermore, we identified relevant enrichment of patients with previously undiagnosed c-BMF. DisclosuresKulasekararaj:Akari Therapeutics Plc: Consultancy, Honoraria; Alexion Pharmaceuticals: Consultancy, Honoraria; Ra Pharmaceuticals: Consultancy, Honoraria; Achillion pharmaceuticals: Consultancy, Honoraria.

Clinical, histopathological and molecular characterization of hypoplastic myelodysplastic syndrome

Diagnostic criteria for hypoplastic myelodysplasic syndrome (h-MDS) have not been clearly established, making the differential diagnosis from other bone marrow failure syndromes (BMF) challenging. In this study, we aimed to delineate clinical, histopathological, and molecular features of h-MDS, based on a large and well-annotated cohort of patients with bone marrow (BM) hypocellularity. The study included 534 consecutive adult patients with hypocellular BM (278 h-MDS and 136 aplastic anemia), and 727 with normo- or hypercellular MDS (n-MDS). Comparison of clinical features of patients with h-MDS as defined by BM cellularity ≤25% (n = 204) or reduced age-adjusted cellularity (n = 74) did not reveal significant differences. We developed a diagnostic score to discriminate h-MDS from non-malignant BMF based on histological and cytological variables with the highest specificity for MDS (h-score). The information from chromosomal abnormalities and somatic mutation patterns was then integrated into a cyto-histological/genetic score (hg-score). This score was able to segregate two groups of h-MDS with a significantly different risk of blast progression (P < 0.001). The integration of cyto-histological and genetic features in adult patients with hypocellular BM facilitated segregation into two distinct groups, one with clinical and genetic features highly consistent with myeloid neoplasm, and one with features more consistent with non-malignant BMF.

Treosulfan, Fludarabine and Cytarabine As Conditioning before Allogeneic Hematopoietic Stem Cell Transplantation

Background: The combination of treosulfan witth fludarabine was successfully introduced into toxicity-reduced conditioning regimens for hematopoietic stem cell transplantation (HCT). However, the risk of post-HCT relapse remains of concern. Here we report for the first time on the results of an individual treatment approach with treosulfan, fludarabin and cytarabine as conditioning for allogeneic HCT in patients with AML, MPN or MDS.Methods: 22 patients were treated with fludarabine 30 mg/m² given on day -6 to day -2, treosulfan 14 g/m² administered on days -4 to day -2 and cytarabin 2g/m² given on days -6 and -5. GvHD-prophylaxis consisted of cyclosporine A and methotrexate or MMF. In addition, antithymocyte globulin was applied in case of an unrelated donor. One patient received bone marrow and the remaining patients received peripheral blood stem cells from matched related donors (9%), matched unrelated donors (73%) or mismatched unrelated donors (18%). All patients were considered to have high risk of relapse because of unfavourable cytogenetic features and/or insufficient or missing response to previous treatment. Three patients (14 %) with CML after blast crisis received the combination because of the reported high relapse rate (46%) after three-day scheduled conditioning with treosulfan [1]. In addition, patients were considered to be ineligible for myeloablative standard conditioning because of multi-morbidity (n = 4; 18% with HCT-CI >2) and/or age >55 years (n = 14; 64%).Results: The median age of patients was 59 (35-68) years. Patients suffered from acute myeloid leukemia (n = 14, 64%), myeloproliferative neoplasia (n = 6, 27%) or myelodysplasic syndrome (n = 2, 9%). The conditioning regime was well tolerated and nearly all patients engrafted and achieved complete donor-type chimerism, except for one who died very early from sepsis. Another patient with underlying myelofibrosis suffered from secondary graft failure on day 100. Two patients developed aGVHD °III/ IV. None of the patients suffered from veno-occlusive disease or severe chronic GVHD. Overall survival and event-free survival at one year reached 60.2% and 59.6%, respectively. Six patients died from infectious disease, two from relapse and one patient from acute GVHD °IV.Conclusion: The combination of cytarabine with the established conditioning of treosulfan and fludarabine is feasible in patients with high risk of relapse and ineligible for myeloablative standard conditioning.Holowiecki J, Giebel S, Wojnar J et al. Treosulfan and fludarabine low-toxicity conditioning for allogeneic 334 haematopoietic stem cell transplantation in chronic myeloid leukaemia. Br J Haematol 335 2008; 142(2): 284-92. DisclosuresHilgendorf:Novartis: Other: Travel support, Research Funding; Medac: Other: Travel support, Research Funding. Frietsch:Deutsche Krebshilfe: Research Funding. Scholl:Abbivie: Other: Travel support; MDS: Other: Travel support; Novartis: Other: Travel support; Carreras Foundation: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Deutsche Krebshilfe: Research Funding; Alexion: Other: Travel support. Hochhaus:Novartis: Research Funding; Bristol-Myers Squibb: Research Funding; Incyte: Research Funding; Takeda: Research Funding; Pfizer: Research Funding. Casper:Medac: Membership on an entity's Board of Directors or advisory committees, Other: travel grant, Research Funding.

Causes de polyarthrite - polyarthralgies pouvant simuler une polyarthrite rhumatoïde

Rheumatoid arthritis (RA) is a frequent disease. However, various diseases could mimicking RA, and have to be known, even if the disease is rare. It is the case with several infectious manifestations, like Whipple or Poncet's diseases. Paraneoplastic polyarthritis could happen before a tumor, frequently a lung cancer, sometime before myelodysplasic syndrome or a lymphoma. Some arthritis could reveal amyloid deposit in the joints, en relation with a myeloma, or a proliferation of hystiocytes cells. Without the exhaustively, the evocations of the very rare diagnostic could help the physicians to recognize these uncommon pathologies.

Ribosome Biogenesis Controls Erythroid Differentiation Via a p53-Dependent Transcriptional Checkpoint and Its Limitation By RPS14 Haploinsufficiency Results in Selective Defects in Translation

Haploinsufficiency in genes encoding ribosomal proteins (RP) or ribosome-associated proteins either by mutation or deletion leads to a predominant erythroid phenotype. In acquired 5q- myelodysplasic syndrome (MDS), the macrocytic anemia has been linked to the monoallelic deletion of RPS14 gene which results in altered ribosome biogenesis. Because of the defective maturation of the small 40S ribosome subunit, the RPL5/RPL11/5SrARN complex, normally involved in the assembly of the large 60S subunit assembly, accumulates and inhibits E3-ligase-HDM2 leading to the stabilization and activation of p53 resultig in cell cycle arrest, increased apoptosis and defective differentiation of maturing erythroblasts. In the present work, we hypothesized that p53 could play a key role in the control of normal erythroid differentiation by ribosome biogenesis and we further investigated the involvement of the decreased pool of ribosome on erythroid defects in the 5q- syndrome.To investigate the first hypothesis, we studied the kinetics of ribosome biogenesis in human primary erythroblasts by mass spectrometry after pulse-SILAC. We noted that ribosome renewal collapses begining at the polychromatophilic erythroblast stage. We subsequently used the pharmacological agent CX-5461 to inhibit RNA polymerase I. When ribosome biogenesis in proerythroblasts is blocked by CX-5461, p53 is activated and proerythroblasts enter the terminal differentiation by expressing GATA1-erythroid target genes without apoptosis. By ChIP-seq in primary erythroblasts, we demonstrated that p53 binds to 1289 genes including 263 genes specifically activated by CX-5461, 6 of them being upregulated during CX-5461-induced erythroid differentiation and 3 of them being known GATA1 targets. We further used an shRNA strategy to demonstrate that one of these genes is required to permit entry into terminal erythroid differentiation when ribosome biogenesis is abrogated. We thus showed that normal erythroid differentiation is controlled by ribosome biogenesis through a p53-dependent checkpoint.In 5q- syndrome, ribosome biogenesis is continuously decreased along all stages of erythropoiesis and in contrast to the normal conditions, erythroid differentiation is defective with an excess of apoptosis affecting mature erythroblasts. We previously reported that GATA1 is targeted by a caspase-dependent cleavage since GATA1 is not protected by its chaperone HSP70 in the nucleus of MDS erythroblasts. We confirmed that GATA1 protein is decreased in 5q- primary erythroblasts and in RPS14 shRNA-expressing normal erythroblasts. To obtain further insights into the defective erythroid maturation of RPS14-deleted erythroblasts, we developed an inducible shRNA to RPS14 in the UT7/Epo cell line. Polysome profiling confirmed the decrease of 40S subunit and absolute quantification of RP by deep proteomics demonstrated a 50% decrease of RPS in conjunction with 50% reduction of ribosome content in these cells. GATA1 expression was decreased and was only partially rescued by treatment with either caspase inhibitor qVD or proteasome inhibitor, bortezomib. We then tested the hypothesis of a decrease in GATA1 translation, as previously shown in a shRNA RPS19 Diamond-Blackfan model, by analyzing and comparing the global transcriptome and the translatome corresponding to transcripts present in high molecular weight polysomes using Affymetrix HTA 2.0 microarrays. We observed a decoupling between transcriptome and translatome suggesting a selectivity of translational defects. Thermodynamic characteristics i.e. the fold energy, energy per base and length of the 3'UTR and the energy per base of the 5'UTR (Vienna RNA Package, UCSC genome browser) were the determinants of transcript selection on the polysome. The shortest transcripts with a highly structured 3'UTR including GATA1 were the transcripts which were less effectively translated. Consistently, the diminution of GATA1 protein was associated with a decrease of its target genes. Our results suggest that GATA1 is a potentially interesting therapeutic target.In summary, our results show that ribosome biogenesis controls erythroid differentiation via a p53-dependent transcriptional regulation and that a reduction of the ribosome pool leads to a selective translation at the expense of erythroid master gene GATA1. DisclosuresFontenay:Celgene: Research Funding.

First case of B ALL with KMT2A-MAML2 rearrangement: a case report

BackgroundA large number of chromosomal translocations of the human KMT2A gene, better known as the MLL gene, have so far been characterized. Genetic rearrangements involving KMT2A gene are frequently involved in lymphoid, myeloid and mixed lineage leukemia. One of its rare fusion partners, the mastermind like 2 (MAML2) gene has been reported in four cases of myeloid neoplasms after chemotherapy so far: two acute myeloid leukemias (AML) and two myelodysplasic syndrome (MDS), and two cases of secondary T-cell acute lymphoblastic leukemia (T-ALL).Case presentationHere we report the case of a KMT2A - MAML2 fusion discovered by Next-Generation Sequencing (NGS) analysis in front of an inv11 (q21q23) present in a 47-year-old female previously treated for a sarcoma in 2014, who had a B acute lymphoid leukemia (B ALL).ConclusionIt is, to our knowledge, the first case of B acute lymphoblastic leukemia with this fusion gene. At the molecular level, two rearrangements were detected using RNA sequencing juxtaposing exon 7 to exon 2 and exon 9 to intron 1–2 of the KMT2A and MAML2 genes respectively, and one rearrangement using Sanger sequencing juxtaposing exon 8 and exon 2.

Novel Molecular Findings in Protein Tyrosine Phosphatase Receptor Gamma (PTPRG) Among Chronic Myelocytic Leukemia (CML) Patients Studied By Next Generation Sequencing (NGS): A Pilot Study in Patients from the State of Qatar and Italy

Background: Chronic Myelocytic Leukemia (CML) is a clonal myeloproliferative disorder characterized by constitutive phosphorylation of Protein Tyrosine kinases (PTKS) that continuously activates multiple proliferative and antiapoptotic signaling pathways.Protein Tyrosine Phosphatases (PTPs) on the other hand is potential natural inhibitory mechanism for regulating the tyrosine kinase activities in which phosphorylation is reciprocally controlled and maintained in equilibrium state by PTKs and PTPs.As a member of PTPs family, Protein Tyrosine Phosphatase Receptor Gamma (PTPRG) was found to act as a tumor suppressor gene. This negative regulatory mechanism of PTPRG was observed to be down-regulated and disabled in CML and one of the possible mechanisms that alter the negative regulatory effect of PTPs is mutations.Several mutations have been identified in PTPs in many different leukemias such as Acute Myeloid Leukemia (AML), Juvenile MyeloMonocytic Leukemia (JMML), Myelodysplasic Syndrome (MDS), B-cell Acute Lymphoblastic Leukemia (B-ALL) and these mutations are associated with hyper-cellular proliferation, disease progression and poor outcome.However, relatively little is known about PTPRG mutations and no studies on CML are available in the literature while mutations inBCR-ABL1tyrosine kinase have been extensively characterized.Thus, understanding the role of PTPRG in antagonizing the PTK phosphorylation of BCR-ABL1 will be important to determine its role in CML development and progression.Aim:1) To identify potential genetic alterations causing inactivation of PTPRG and 2) correlate the PTPRG findings with patients' response to the Tyrosine kinase Inhibitors.Methods:16 CML patients, 9 from Qatar and 7 from Italy respectively, were studied for PTPRG mutations by exome sequencing. Custom primers were designed for Human PTPRG gene (5 Kb of exonic region of interest) using Ion AmpliSeq Designer. Target regions were enriched and amplified for the 16 DNA samples using Ion AmpliSeq Library kit 2.0.The amplicons were partially digested with FuPa reagent and phosphorylated prior to ligation of Ion Xpress Barcode Adapters followed by cleanup using HighPrep reagent. The adapter ligated molecules were enriched with adapter specific primers using a limited cycle PCR followed by a cleanup using HighPrep reagent. The final libraries were quantified on Qubit Flurometer using Qubit dsDNA HS Assay Kit and Agilent Bioanalyzer using Agilent High Sensitivity DNA Kit. All samples were pooled according to the concentrations on the Bioanalyzer and loaded on Ion 318TM Chip kit V2 to be sequenced on Ion Personal Genome Machine (PGM) system.European Leukemia Net (ELN) 2013 criteria were employed to assess the response/resistance of patients to treatment. Responses are defined at the hematological, cytogenetic and molecular levels. Patients response was classified into optimal and failureResults:Four mutations/variants were identified in PTPRG genes, three were missense Y92H, G574S, S561Y and 1 was frameshift Y285fs in the 16 CML patients.PTPRG Y92H was identified in 5 (1 Homozygous and 4 heterozygous alleles) patients and the 5 patient failed the Imatinib Mesylate (IM) treatment.On the other hand, The PTPRG G574S was identified in 6 (2 homozygous and 4 heterozygous alleles) patients. Out of the 6 patients, 4 were classified as failure to the treatment and 2 responded optimally.In addition, the PTPRG S561Y and Y285fs were identified on 1 and 3 patients respectively and these patients responded optimally to IM treatment.Discussion and Conclusions:This is the first prospective pilot study to investigate PTPRG gene mutations as underlying mechanism to explain treatment failure.Our preliminary data showed that the identified variant PTPRG Y92H might be associated with IM failure although it has been reported as Single Nucleotide Polymorphisms (SNPs) (rs62620047) and this could be attributed that some polymorphisms might behave like a mutation.On the other hand, PTPRG G574S variant (rs2292245) showed various clinical outcomes regardless to its allele zygosity as 67% (4/6) of patients failed the TKIs treatment.From the results of our pilot study we recommend carrying out PTPRG sequencing in a significantly larger cohort of patients to further explore and pinpoint the crucial mutations that can be correlated with CML resistance/response to treatment. DisclosuresNo relevant conflicts of interest to declare.

Cytogenetics of Patients with Myelodysplasic Syndrome in Venezuela

The myelodysplastic syndromes are clonal hematological disorders of the stem Cell, and clinically characterized by ineffective hematopoiesis and morphological dispoyesis. The affected patients have a high risk of leukemic transformation. In a total of (n=416) patients studied, (n=247) patients (59.3%) corresponded with a female gender, with an average age of = 59 years (limits 15-99 yrs), and (n=169) male patients (40.6%), with average age of =62 years (limits 09-89 yrs). In (n = 295) studied patients, 71% had normal karyotype 46, XX or 46, XY, where in 16% (n = 66) of the samples cell growth was not attained. In (n = 54) of patient samples, 13% showed karyotype changes, including those frequently reported for MDS; and 11.1% (n = 6) with -Y, 11q 1.8% (n = 1), del20q 3.7% (n = 2), +8 3.7% (n = 2), - 21 1.8% (n = 1), del 7q 1.8% (n = 1), inv3 1.8% (n = 1), complex karyotypes 12.9 % (n = 7) cases. Other alterations include, simple anomalies: +2 1.8% (n = 1), +13 5.5% (n = 3), del17q 1.8% (n = 1), del4p 1.8 %(n=1), del9q 3.7% (n = 2), der21q 3.7 %(n = 2), del17p3 3.7% (n = 2), t (1; 7) 1.8% (n = 1), t (4; 12) 1.8% (n = 1), rob13 1.8% (n = 1); 14, r2 1.8%(n = 1); double anomalies, delXq and del10q (n = 1), del18p and del13q (n = 1). Among other findings, polymorphisms in (n=15) patients (27.7%), such as 1qh + (n = 2), 9qh + (n = 4), 16qh + (n = 3), 14PS + (n = 1), 15ps + (n = 2) , 22ps + (n = 1), inv9 (n = 2). According with IPSS-R karyotypes, samples were classified as very favorable 12.9% (n = 7), pro 3.7% (n = 4), Intermediate 40.7% (n = 22), 1.8% unfavorable ( n = 1) and very favorable 18.5% (n = 12.9). DisclosuresNo relevant conflicts of interest to declare.

French consensus on myelodysplasic syndrome and chronic myelomonocytic leukemia: diagnostic, classification and treatment 2015 update by the Myelodysplasia French Group

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 [...]

Natural Killer Cells Enhance Transplantation Outcomes

Natural killer (NK) cells, the first lymphocyte subset to reconstitute after hematopoietic cell transplantation (HCT), may enhance transplant outcomes by killing virally-infected or malignant cells to reduce relapse and treatment-related mortality by promoting engraftment and by reducing graft vs. host disease. The function of NK cells is regulated by the net balance of signaling via several families of activating or inhibitory receptors. The killer-cell immunoglobulin-like receptor (KIR) family is of particular importance in HCT because of its interactions with class I human leukocyte antigen (HLA) molecules. HLA-C, HLA-Bw4 and some HLA-A allotypes function as ligands for the inhibitory KIR receptors, thus mediating tolerance to self. HLA-mismatched HCT may generate alloreactive NK cells in the recipient when there is a KIR-ligand mismatch at HLA-C, B or A. The proof of concept for this principle was established by the Perugia group in haploidentical transplants, where KIR-ligand mismatch was associated with reduced relapse in patients with acute myeloid leukemia. Subsequent groups have studied this and other algorithms, including KIR ligand absence or KIR-KIR ligand genotyping to evaluate the role of NK cells in mediating protection after allogeneic HCT. Human KIR haplotypes are defined by gene content, where the A haplotype contains mainly inhibitory KIR, and B haplotypes contain more activating KIR. Unrelated donors with more KIR B haplotype genes have been associated with protection from relapse and improved survival in AML, an effect which is enhanced in recipients expressing HLA-C1. Although NK cells mediate beneficial effects after HCT, compared to adult donor NK cells, engrafting NK cells are immature and hyporesponsive when exposed to tumor targets. Recently human cytomegalovirus (CMV) has been shown to drive the expansion of a population of long-lived CD57+NKG2C+ NK cells with attributes of immunologic memory. These cells have heightened capacity for cytokine production or cytolytic response to tumor targets. Several groups have shown that CMV reactivation correlates with reduced relapse after allogeneic HCT, suggesting that these CMV “adaptive” NK cells may contribute to the graft vs. leukemia effect. Strategies to isolate and expand “adaptive” NK cells without clinical CMV reactivation are being explored. Lastly, adult donor NK cells are an appealing population to exploit for adoptive cellular therapy. Donors can be chosen based on predicted NK cell alloreactivity or based on KIR gene content. Adoptively transferred NK cells which expand and proliferate in vivo in response to cytokine stimulation (IL-2 or IL-15) are potent anti-tumor effectors which do not mediate graft vs. host disease. Several groups are using haploidentical adult NK cell products to treat acute myeloid leukemia, multiple myeloma, lymphoma, myelodysplasic syndrome and a variety of solid tumors. Various approaches using adoptive transfer of NK cells, together with HCT, targeting antibodies which mediate antibody dependent cellular cytotoxicity, or cytokine stimulation are being tested clinically. In summary, we are just beginning to define the complexity of NK cell interactions with HLA and other ligands and to describe different properties of various NK cell subsets to develop more sophisticated strategies to exploit NK cells to treat cancer. Disclosures No relevant conflicts of interest to declare.

Anti-D auto-immunization in a patient with weak D type 4.0

We report the case of a 56-year-old patient with blood group O+C−c+E−e+K−, followed for a myelodysplasic syndrome and treated by regular pheno-identical and compatible (RBCs) transfusion since December 2007. In June 2009, a positive crossmatch was found with 2 RBCs O+C−c+E−e+K−. A positive anti-body screening with a positive autocontrol was detected and anti-D was unidentified in the patient's serum. The DAT was positive (IgG) and elution identified an anti-D. The following assumptions were then made: it could be a partial D phenotype with anti-D alloantibodies or RH: 1 phenotype with an anti-D auto-antibodies. Molecular analysis by multiplex PCR and sequencing have depisted a weak D type 4.0 phenotype. In October 2009, over three months of RH:−1 RBC transfusion, the antibody screening and DAT (IgG) remained positive, and an eluate made from the patient's erythrocytes contained an anti-D. All these funding confirmed the autoimmune nature of the anti-D. This case report illustrates the importance of a well-conducted and immunohematological laboratories test in order to distinguish between auto- or allo-immune of anti-D in a RH: 1 poly-transfused patients. This distinction is of great importance for transfusion support.

Low CD34 Cell Dose Is Associated With Higher Non-Relapse and Overall Mortality After Reduced Intensity Conditioning Hematopoietic Cell Transplantation For Acute Myeloid Leukemia and Myelodysplastic Syndrome

In allogeneic hematopoetic cell transplantation (HCT) using myeloablative conditioning, the nucleated and CD34 cell doses were found to be of importance for several outcome parameters including survival, relapse and graft- versus- host disease (GVHD). Reduced intensity conditioning (RIC) HCT is increasingly used for older patients and for younger patients with co-morbidities. Peripheral blood progenitor cells (PBPC) is the predominant graft for RIC HCT. There is no large study of the effect of CD34 cell dose in the setting of RIC transplantation. Therefore, we studied the effect of CD34 dose on 1057 patients aged 45 – 75 years with acute myeloid leukemia (AML) or myelodysplasic syndrome (MDS) who received RIC regimen HCT between 2000 and 2011. Patients received grafts from HLA-matched siblings (n = 370) or from volunteer adult unrelated donors matched at the allele-level at HLA-A, -B, -C and –DRB1 (8/8; n = 522) or mismatched at 1 HLA-locus (7/8; n = 178). All patients received peripheral blood progenitor cells (PBPC), low dose TBI regimens (200 cGy) or alkylating agent plus fludarabine containing regimens. Separate analyses were conducted for HLA-matched and unrelated donor transplants. For HLA-matched sibling transplants (AML n=301; MDS n=69), exploratory analysis identified differences in survival for CD34 dose less than 4 x 106/kg. Multivariate modeling, adjusting for performance score, disease status, cytogenetic risk and transplant period, showed transplantation of PBPC with CD34 dose < 4 x 106/kg was associated with higher risks of overall mortality (HR 1.48, p=0.008) and non-relapse mortality (HR 2.03, p=0.004) compared to transplantation of PBPC with CD34 dose ≥ 4 x 106/kg. The effect of CD34 dose was independent of the other factors associated with mortality. The likelihood of neutrophil (HR 0.76, p=0.03) and platelet recovery (HR 0.76, p=0.03) was also lower with CD34 dose < 4 x 106/kg containing PBPC grafts. CD34 dose was not associated with acute or chronic GVHD or disease recurrence. In contrast, after unrelated donor transplantation (AML n=557; MDS n=130), the effect of CD34 cell dose on mortality was marginal. Exploratory analysis identified differences in survival for CD34 dose less than 6 x 106/kg. After adjusting for age, performance score, disease status, interval from diagnosis to HCT, and HLA-match, transplantation of PBPC with CD34 dose < 6 x 106/kg was associated with marginally higher risks for overall mortality (HR 1.20, p=0.05) and non-relapse mortality (HR 1.38, p=0.02) compared to transplantation of PBPC with CD34 dose ≥ 6 x 106/kg. There were no significant differences in hematopoietic recovery, acute or chronic GVHD and disease recurrence by CD34 dose. Further, we did not identify a CD34 dose for either donor source above which acute or chronic GVHD risks were higher. In conclusion, in the setting of RIC transplants for AML and MDS, the data support optimizing PBPC collections such that the CD34 dose delivered is in excess of 4 x 106/kg for HLA-matched sibling and CD34 dose in excess of 6 x 106/kg for unrelated donor transplants. Disclosures:No relevant conflicts of interest to declare.

An IHC Screen For EphA3 Positive FFPE Tumors

Eph receptors are the largest subgroup of the receptor tyrosine kinases (RTK). Unlike other RTK, these function principally during development. Quiescent in postembryonic tissues, Eph expression by adult tissues is abnormal and implicated in tumor initiation, invasion and metastasis.Aberrant EphA3 expression is seen in solid and hematologic tumors, particularly advanced stage lymphoproliferative and myeloproliferative diseases. In this regard, targeting EphA3 may constitute a novel treatment for hematological malignancy.With clinical utility in mind, i.e., patient selection for anti-EphA3 therapy, a panel of commercial and proprietary (KaloBios) antibodies was screened by Western Blot for reactivity to recombinant Eph receptors (EphA3, EphA4, EphA5, EphA7, EphB2, EphB3).Those with EphA3 binding selectivity were further screened (QualTek) by immunohistochemistry (IHC) using formalin-fixed paraffin-embedded (FFPE) normal and diseased human bone marrow (NLBM, AML) as well the LK63 pre-B-ALL cell line from which EphA3 was originally isolated.Different tissue pretreatments were used for antigen/epitope retrieval of EphA3, including steam-heating in citrate-based or Tris & chelator-based buffers, subsequent/or protease digestion, or neither.Following each antigen/epitope retrieval procedure, antibody reactivity for EphA3 was assessed by light microscopy using enzymatic biotin, tyramide and polymer-based detection techniques. In each instance, the location of the EphA3/antibody complex was visualized with 3,3-diaminobenzidine that precipitates a discrete insoluble reaction product in presence of enzyme (HRP). Nuclei were counterstained with hematoxylin to assess cell/tissue morphology.From this screen, one antibody (with an epitope in the cytoplasmic CT-domain of EphA3) was chosen for further assay optimization based on its selective reactivity towards LK63 and AML and low selective reactivity towards NLBM.Assay optimization included, amongst other aspects, evaluation of EphA3 expression in a panel of NLBM and hematopoietic tumors (200+ specimens inclusive of acute & chronic leukemia, peripheral T-cell & B-cell neoplasm, Hodgkin & non-Hodgkin lymphoma, multiple myeloma, myelodysplasic syndrome, myeloproliferative neoplasm) by a board-certified hematopathologist.In NLBM, the frequency of EphA3+ immature-blast cells (CD34+ or CD117+) was insignificant. Less than 10% CD34+/CD117+ cells were EphA3+.Elevated EphA3 expression (percentage EphA3+ nucleated cells) was observed in most hematopoietic tumors.For example, in multiple myeloma, tumor cells were typically EphA3+/CD138+ plasma cells. For AML, leukemic CD34+ or CD117+ blasts/initiating cells were typically EphA3+. Some CD138- plasma cells or leukemic CD34-/CD117- cells were also EphA3+.Correlation was made between EphA3 expression and specific tumor maturation stages, differentiation status and/or tumor aggressiveness.Tumors in blast crisis presented elevated EphA3 expression, e.g., CML in accelerated or blastic crisis but not chronic phase.Elevated EphA3 expression was noted in pre-B-ALL & pro-B-ALL (early pre-B-ALL) rather than mature B-cell ALL.EphA3 expression for some peripheral B-cell neoplasms correlated well with tumor grade: high grade, poorly differentiated (typically aggressive) B-cell lymphomas or follicular lymphomas were EphA3+. A similar relationship was noted for non-Hodgkin lymphoma (Hodgkin lymphoma was EphA3-).Preclinical screening also provided evidence for EphA3 expression by stroma/fibroblast (mostly lymphoma) and vasculature/endothelium, further rationale for development of reliable tools for profiling EphA3 in hematologic tumors and other malignancies.Using well-characterized normal and diseased FFPE bone marrow biopsies, this IHC assay for EphA3 has subsequently been validated (QualTek) to provide data that is not directly available from routine histopathology review and that supports use of the assay for profiling EphA3 in specific hematologic tumors and for patient selection in early Phase clinical trial/s of an anti-EphA3 monoclonal antibody (KB004, KaloBios).Beyond this, EphA3 targeted therapy with KB004 is anticipated for treatment of solid tumors. Recent genome-wide surveys identify EphA3 amongst the most frequently overexpressed genes in pancreatic, colon and lung carcinoma, melanoma and glioblastoma. Disclosures:Locke:QualTek Molecular Labs: Employment. Bernstein:QualTek Molecular Laboratories: Employment, Equity Ownership. Lynch:QualTek Molecular Laboratories: Employment, Equity Ownership. Siami-Namini:QualTek Molecular Laboratories: Consultancy. Walling:KaloBios: Consultancy; Corcept Therapeutics: Consultancy; Prothena: Consultancy; New Gen Therapeutics: Consultancy; Valent Technologies: Consultancy; LBC Pharmaceuticals: Consultancy; Amgen: Equity Ownership; BioMarin: Equity Ownership; Crown BioScience: Membership on an entity's Board of Directors or advisory committees. Yonker:KaloBios: Employment, Equity Ownership. Yarranton:KaloBios: Employment, Equity Ownership; Glaxo: Equity Ownership; EnGen: Equity Ownership, Science Advisor, Science Advisor Other; StemLine Therapeutics: Equity Ownership.

Long-Term Follow-Up Of Invasive Aspergillosis In Allogeneic Stem Cell Transplantation Recipients and Leukemia Patients: Differences In Risk Factors and Outcoumes

Invasive aspergillosis (IA) remains a major concern among patients with hematological malignancies who receive cytotoxic chemotherapy or allogeneic stem cell transplantation (allo-SCT). With a view toward better understanding the differences in terms of clinical significance and outcomes of IA between allo-SCT recipients and patients treated for leukemia, we report a single-center study of 739 unselected consecutive patients treated in our unit between August 2000 and February 2004. IA episodes were classified according to the 2008 EORTC/MSG criteria. Patients were hospitalized either for allo-SCT (n=135), or for the treatment of acute leukemia (n=378), chronic lymphocytic leukemia (n=116), or myelodysplasic syndrome (n=105). Probable or proven IA were observed in 29 patients, among whom 7 were undergoing allo-SCT (5.2%), 20 were treated for acute leukemia (5.3%), 1 for chronic lymphocytic leukemia (0.8%), and 1 for myelodysplastic syndrome (0.95%). Most of the clinical signs were mild and nonspecific, especially in allo-SCT recipients. Chronologically, thoracic computerized tomography (CT) showed the first signs of IA in 38% of cases, and was positive the same day as at least one biological test (bronchoalveolar lavage [BAL] and serum galactomannan antigen) in 41% of cases. BAL was contributive to the diagnosis in 66% of the 29 cases. Comparison of patients undergoing allo-SCT to the others revealed significant differences. In allo-SCT recipients, the onset of IA occurred later than in the other patients, after the neutropenic period. The median time between the last hematologic treatment and the diagnosis of IA were 231 days (range, 68-341) in allo-SCT recipients and 17 days (6-57) in the other patients (p&lt;0.001). At the time of AI onset, the median absolute neutrophil count were 1.8 x 109 cells/L (0.7-5) and 0 x 109cells/L (0-2.1) in allo-SCT patients and the others, respectively (p&lt;0.0001). In addition, the median C-reactive protein level was lower in allo-SCT recipients (53 mg/L, 13-146) compared to the other patients (143.5 mg/L, 22.5-285), p=0.023. It is noteworthy that the 7 cases of IA in allo-SCT recipients occurred only in patients treated with corticosteroids for graft-versus-host disease (GVHD) (n=42). First line therapy was liposomal amphotericin B (n=22), caspofungin (n=8), voriconazole (n=6), or itraconazole (n=1), either alone (n=21) or in combination (n=8). Complete remission of IA on CT was observed in 12 cases, stable lesions in 8 cases and progression in 9 cases. Mortality directly related to IA was 24% in our study and occurred early after the onset of infection. The 100-day, 1-year, 2-year and 10-year overall survival (OS) were 42.9%, 28.6%, 0%, 0% for allo-SCT recipient, respectively, compared to 68.1%, 40.9%, 18.2%, 13.6% for the other patients, respectively (P-values ≥ 0.05). These poor outcomes were mainly attributable to the non-relapse mortality (NRM), especially in allo-SCT recipients, who had a 2-year NRM of 100% compared to 54.6% in the other patients, p=0.034 (2-year cumulative incidence of relapse were 0% and 70.2%, respectively). In conclusion, our data seem to distinguish 2 types of IA. One has an early onset in neutropenic leukemia patients. The second, observed in allo-SCT recipients, occurs later, after GVHD development and is associated with a very high NRM incidence, suggesting that those patients remain fragile. Thus, systematic broad-spectrum anti-fungal prophylaxis is highly recommended in patients undergoing allo-SCT who develop GVHD. Disclosures: Berthon: CELGENE: Research Funding.

Association between multiple myeloma and acute myeloid leukemia secondary to myelodysplastic syndrome

We report a rare case of association of two distinct hematologic malignancies: refractory cytopenia with multilineage dysplasia associated with del(5q) and symptomatic multiple myeloma associated with del(17p) and del(13q). After 16 months, the patient presented an acute leukemic transformation of the myelodysplasic syndrome.

Post-Transplant Outcome in Patients with Acute Myeloid Leukemia and Myelodysplasic Syndrome Who Received Conditioning Regimen Based on Fludarabin, Busulfan and Anti-Thymoglobulin Prior to Allogeneic Hematopoietic Stem Cell Transplantation

Post-Transplant Outcome in Patients with Acute Myeloid Leukemia and Myelodysplasic Syndrome Who Received Conditioning Regimen Based on Fludarabin, Busulfan and Anti-Thymoglobulin Prior to Allogeneic Hematopoietic Stem Cell Transplantation

Post-Transplant Outcome in Patients with Acute Myeloid Leukemia and Myelodysplasic Syndrome Who Received Conditioning Regimen Based On Fludarabin, Busulfan and Anti-Thymoglobulin Prior to Allogeneic Hematopoietic Stem Cell Transplantation

AbstractAbstract 2013 Purpose:The introduction of reduced intensity conditioning (RIC) regimens has allowed performing allogeneic hematopoietic cell transplantation (HSCT) in previously discarded old or unfit patients. In this fragile population, non-relapse mortality (NRM) has been dramatically reduced, making the post-transplant disease control the remaining challenge. Over the last years, we have tuned the chemotherapy dose intensity of a busulfan based RIC regimen with the aim to better control disease while retaining a low toxicity profile. In this perspective, we retrospectively analyzed a cohort of patients with myeloid malignancies treated identically in two French centers. Patients and methods:From 2005 to 2010 in Marseille and Nantes we transplanted 165 patients (median age: 56.8 years (range:18–71); males: 83, females: 82) presenting AML (N=124) or MDS (N=41) following a similar conditioning regimen but for the dose of busulfan (Bu): 1/Fludarabine (30 mg/m2/day over 5 days) 2/, rabbit anti-thymocyte-globulins (r-ATG) (2.5mg/kg/day over 2 days) and 3/ i.v. or oral Busulfan (Bu) (130 mg/m2/day or 3.2 mg/kg respectively over 2 to 4 days) (table 1). Ciclosporin A was given as post graft immunosuppression. Initially higher Bu doses were proposed to patients under the age of 55, without comorbidities and with higher risk diseases. These indications were progressively extended over time when it became evident that toxicity was not dramatically increased. Results:With a median follow up of 20.7 months for surviving patients, 2 year overall survival (OS) and progression free survival (PFS) were 59.6% (95% CI: 51.8–68.6) and 54.9% (95% CI: 47–64) respectively. Cumulative incidence of grade 2–4 and 3–4 acute graft-versus-host diseases (aGvhD) at day 100 was 19.4% and 7.9% respectively. Cumulative incidence of chronic GvhD (all grades) and extensive chronic GvhD was 25% and 14.4% at 1 year post transplantation respectively. NRM was 11.7 and 15.4% at 12 and 24 months. Cumulative incidence of relapse (CIR) was 24% and 29.7% at 1 and 2 years.Overall neither the patient age, nor the donor type or the dose of Bu influenced significantly OS, PFS or CIR in the whole cohort. Surprisingly, NRM was lower after 3 or 4 days Bu conditioning regimen (3.6% vs 21.3%, p=0.009).AML patients transplanted in 1st complete remission (n=93): those patients had 2-year OS and PFS of 63.4% (95% CI: 53.3–75.3) and 60.9% (95%CI: 50.8–72.9) respectively. Two-year OS reached 66.4% (95%CI: 56.4–81.8) for patients with favorable/intermediate karyotype and 50.5% (95%CI: 33–77.3) for unfavorable, p=0.08. Patients with favorable or intermediate karyotype tend to have a better 2-year PFS (63.8%, 95%CI: 52.2–78.1) than unfavorable karyotype (52%, 95%CI: 34.5–78.3), p=0.2.Patients under the age of 55 (n=76): this population presents overall a better outcome than older (OS: 63.9 (CI: 52.7–74.4) vs 56% (CI: 45.6–68.7), p=0.2, PFS: 60.5 (49.7–73.8) vs 50.4% (CI: 40–63.5), p=0.2, NRM: 9.4 vs 20.3%, p=0.07). In addition higher doses of Bu improved OS (77.5% vs 47.9%, p=0.058), PFS (71.6 vs 46.4%, p=0.1), without increase of NRM (2.2% vs 18.7%, p=0.03). Conclusion:This conditioning regimen using Fludarabin, Bu and r-ATG can result in good OS and PFS probabilities in myeloid malignancies. Given the patient population characteristics', the NRM can be considered as low. Interestingly enough even if patients who received 3 or 4 days of Busulfan were younger and had less comorbid conditions, which is a bias, they had a lower NRM than those who received only 2 days of bu. In younger patients higher doses of Bu seem to be associated with better prognosis. These encouraging results led us setting up a prospective study comparing different doses of Bu in poor risk myeloid malignancies undergoing HLA identical allo HSCT and ineligible for standard double alkylating agent or high dose TBI based conditioning.Table 1Patients and transplant's characteristics.Patients characteristicsN%centerMarseille10865,5Nantes5734,5gendermale8350,3female8249,7age at transplantation< 55 years7646,1≥55 years8953,9diagnosisAML12475,2MDS4124,8karyotype (AML)Favorable118,9Intermediate8165,3Unfavorable3225,8NR1disease statusAML CR19356,4AML CR> =22313,9AML refractory84,8MDS CR106,1MDS PR/SD3118,8transplants' characteristicsdonor typeMRD9255,8MUD3420,6MMUD148,5cell sourcePBSC15795,2BM84,8Day nb of Bx/BU210664,233521,242414,5 Disclosures:No relevant conflicts of interest to declare.