Cytogenetic Subgroups Research Articles

Efficacy and Safety of Carfilzomib and Dexamethasone Vs Bortezomib and Dexamethasone in Patients with Relapsed Multiple Myeloma Based on Cytogenetic Risk Status: Subgroup Analysis from the Phase 3 Study Endeavor (NCT01568866)

Efficacy and Safety of Carfilzomib and Dexamethasone Vs Bortezomib and Dexamethasone in Patients with Relapsed Multiple Myeloma Based on Cytogenetic Risk Status: Subgroup Analysis from the Phase 3 Study Endeavor (NCT01568866)

Somatic Genetic and Epigenetic Architecture of Myelodysplastic Syndromes Arising from GATA2 Deficiency

The emergence of GATA2 deficiency as a germline predisposition to myeloid malignancies raises questions about the nature of acquired secondary genetic and epigenetic events facilitating leukemogenesis. Previously, mutations in ASXL1 were implicated as a possible somatic driver in single cases of GATA2-related MDS. However the landscape of secondary changes had not yet been systematically examined in larger MDS cohorts, and accounting for confounding factors. In this study, we used next-generation genomic platforms to investigate targeted mutational landscape and global epigenetic profiles in patients with GATA2 deficiency.In a large cohort of consecutively diagnosed children with MDS we had initially established that GATA2 deficiency accounts for 7% of primary MDS cases. Exploring the known association between GATA2 mutated (GATA2mut) cases and monosomy 7 (-7), the prevalence of GATA2 deficiency was very high in patients with -7 (37%), reaching its peak in adolescence (>70%). We next tested 60 GATA2-deficient patients with MDS for the presence of secondary mutations using targeted NGS for genes involved in myeloid malignancies. Somatic status was confirmed by matched analysis of fibroblasts, hair follicles or T-cells. Single hematopoietic CFU colonies were sequenced to identify subclonal patterns. For comparison, a GATA2 wildtype (GATA2-WT) cohort of 422 children and adolescents with MDS enrolled in the studies of the European Working Group of Childhood MDS were analyzed by targeted NGS. Somatic mutations were detected in 45% (27/60) of GATA2mut as compared to 19% (82/422) GATA2-WT MDS cases (p<0.0001). Recurrently mutated genes in the GATA2mut group included SETBP1, ASXL1, STAG2, RUNX1, CBL, EZH2, NRAS/KRAS, JAK3, and PTPN11. No mutations were found in TP53, BCOR/BCORL and a number of other oncogenes. Because -7 karyotype was significantly overrepresented in GATA2mut cases with somatic mutations (78%), we next focused on this cytogenetic category. Within the -7 subgroup the rate of somatic mutations was the same in GATA2mut (56%) and GATA2-WT (58%) subgroups. However, hotspot SETBP1 mutations were overrepresented in GATA2-deficient patients with -7 (50%) vs. GATA2-WT MDS cohort (22%, p<0.05). Furthermore, STAG2 mutations were found frequently in the GATA2mut group (10%, 6/60) as opposed to only 0.2% (1/422) of the total GATA2-WT cohort (p<0.0001). Next, we aimed to define the clonal hierarchy of concurrent mutations by longitudinal NGS-analysis during disease course in selected patients. Our results indicate that somatic SETBP1 lesions precede the development of ASXL1 mutations. Remarkably, this model of clonal evolution does not depend on preexisting germline GATA2 lesion, as confirmed by sequencing of single CFU colonies cultivated from the bone marrow of 3 GATA2mut and 3 GATA2-WT MDS patients. Finally, to elucidate the epigenetic effects, we compared methylation patterns using methyl-CpG-immunoprecipitation and Illumina-NGS in 25 GATA2mut to 17 GATA2-WT patients and 10 healthy controls. Based on the degree of global methylation, there were no significant alterations allowing for the discrimination of GATA2-deficient patients from the total MDS cohort, when accounted for bias arising from cytogenetic and morphologic subgroups.In summary, somatic SETBP1 and STAG2 mutations are associated with MDS arising from GATA2 deficiency. The remaining targeted clonal landscape is essentially determined by the presence of monosomy 7. Similarly, the global epigenetic changes correlate with morphological and cytogenetic subgroups, rather than with germline GATA2 status. The prospect of potential drug targetability of mutations frequently found in children, particularly in the SETBP1 oncogene, and in histone modifiers ASXL1 and EZH2, warrants further biological studies. DisclosuresNo relevant conflicts of interest to declare.

Clonal Mutational Landscape of Childhood Myelodysplastic Syndromes

Childhood myelodysplastic syndromes (MDS) belong to a rare group of disorders of aberrant clonal hematopoiesis manifesting throughout entire childhood and adolescence. We had previously established that GATA2 germline mutations can be considered the most common "first hit" in pediatric MDS seen in 7% of primary MDS. However the secondary somatic aberrations facilitating leukemogenesis are not elucidated in children. Previous sequencing efforts established that most somatic mutations very frequently encountered in adults, i.e. affecting TET2, DNMT3a, and the spliceosome genes, do not play a role in the pathogenesis of childhood MDS. Here we aim to define the global mutational landscape in childhood MDS using targeted next-generation sequencing (NGS) approaches.We investigated children and adolescents enrolled in the prospective studies of the European Working Group of Childhood MDS. Diverse target enrichment and NGS strategies were established including hybridization capture and Ampliseq PCR, Illumina Miseq/Hiseq and Iontorrent PGM. We first examined a pilot cohort of 68 patients for mutations in 138 myeloid leukemia genes. This allowed for the identification of recurrently mutated genes that were selected to be included in a pediatric MDS panel encompassing 28 genes. Targeted NGS using the Iontorrent PGM identified known recurrent mutations. However, the high indel error rate and coverage gaps in homopolymeric regions i.e. in ASXL1 precluded further studies. Using inhouse-adapted Ampliseq-Miseq approach we then sequenced DNA from bone marrow of 586 MDS patients (469 primary and 117 secondary MDS after radio/chemotherapy or inherited bone marrow failure syndromes) at an average depth exceeding 700 reads per amplicon. Somatic mutations were identified in 22% of primary MDS patients, with 1, 2 and 3 genes affected in 16%, 4.5%, and 1.5% of cases, respectively. In secondary MDS twice as many patients (46%) carried mutations; 1, 2, and 3 genes were concurrently mutated in 32.5%, 9.5%, and 4% of patients, respectively. Longitudinal NGS analyses and single CFU colony sequencing confirmed the presence of multiple somatic clones evolving in a hierarchical manner throughout disease course. Most frequent mutations identified in more than 1% of our study cohort of primary MDS were: SETBP1 (7%), ASXL1 (6%), NRAS/KRAS (5%), RUNX1 (3%), PTPN11 (3%) and BCOR/BCORL (1.5%); and in secondary MDS: RUNX1 (14.5%), TP53 (9%), NRAS/KRAS (8.5%), ASXL1 (8%), SETBP1 (6%), PTPN11 (6%), CBL (5%), BCOR/BCORL1 (3.4%). Other genes mutated at very low frequency of <1% were CTCF, STAG2, RAD21, PTEN, JAK3, FLT3, CSF3R, and EZH2. No somatic mutations were identified in CEBPA, GATA1, GATA2, JAK2, cKIT, VPS45, MPL, CALR, SH2B3. The analysis of individual mutational clusters established that for some mutations strongly associate with monosomy 7; i.e. this cytogenetic subgroup was prevalent in 100% of EZH2, 90% of SETBP1, 79% of RUNX1, and 74% of ASXL1 - mutated children with MDSIn conclusion, we established and cross-platform validated a targeted NGS-panel for pediatric MDS, allowing to identify clonal mutations at a sensitivity of at least 10%. In this, to our knowledge largest systematicaly studied cohort of children with MDS we show that mutational load and clonal complexity differs between primary and secondary MDS, and maintains specific patterns for monosomy 7. Although the biological significance of these genomic changes is currently not understood, the emerging distinctive patterns may already be helpful in establishing therapeutic subgroups. DisclosuresNo relevant conflicts of interest to declare.

Clonal Mutations and Clonal Hierarchy in Pediatric Myeloid Neoplasms

Pediatric myeloid neoplasms are a clinically and genetically heterogeneous group of clonal diseases. Unlike in adults inherited predisposition plays a major role in leukemogenesis. A number of germline mutations are known to increase the risk of somatically occurring myeloid transformation. These include the genes associated with the classical inherited bone marrow failure syndromes, but also transcriptopathies resulting from RUNX1, CEBPA or GATA2 deficiency. Herein we review the most significant findings from recent years and our present experience on the clonal landscape of myeloid malignancies in children with a focus on pediatric MDS and JMML. In both disease entities, monosomy 7 is the most frequent chromosomal aberration. It is likely that haploinsufficiency of genes located on chromosome 7 contributes to clonal evolution. Several of the more than 1000 lost genes were postulated as mechanistic drivers. However, in most cases additional somatic mutations not related to chromosome 7 are found at diagnosis, making it difficult to pinpoint the initial oncogenic hit. Driver somatic hits frequently encountered in adults with MDS, affecting TET2, DNMT3A or the spliceosome genes, do not play a major role in the pathogenesis of childhood disease. Also, considering the young age, environmental effects and age-related pre-malignant clonal mutations can be disregarded. In fact, GATA2 germline mutations can be considered the most common "initial hit" in pediatric MDS. In our experience, GATA2 deficiency accounts for 37% of primary pediatric MDS with monosomy 7. The prevalence of GATA2 mutations in MDS with monosomy 7 increases dramatically in adolescence where they are found in up to 72%. In comparison, 16% of pediatric patients with MDS and trisomy 8 have an underlying GATA2deficiency. The somatic mutational landscape in childhood MDS is associated with cytogenetic subgroups, i.e. SETBP1 and ASXL1 mutations are overrepresented in cases with monosomy 7. Interestingly, longitudinal NGS analysis and single colony sequencing indicate that SETBP1 lesions precede the development of ASXL1 mutations. We identified the genes SETBP1, ASXL1, NRAS, KRAS, RUNX1, PTPN11 and BCOR/ BCORL as the most frequent targets of recurrent mutations in a large cohort of pediatric patients with primary MDS. The biological significance of these genomic changes is currently not understood and warrants further studies. In contrast to pediatric MDS, signaling pathways involved in JMML have been better defined during recent years. Somatic aberrations in PTPN11, NRAS, KRAS, CBL, and NF1 genes lead to a constitutively overactive RAS pathway in leukemic cells. Additional activating mutations were identified in some JMML patients, including the RAS pathway gene RRAS, and concomitant mutations in SETBP1 and JAK3. The latter were postulated to develop as subclonal events during disease progression and to confer poor prognosis. However most recently, very rare clones with SETBP1 mutation were reported to be present at diagnosis in as much as 30% of JMML patients. This might indicate that multiple clones with different clonal composition arise during initial stages of disease, and therapy-driven selection pressure results in the expansion of these subclones. It is not clear how the somatic mutations interact with the epigenetic landscape in JMML clones, especially with regard to promoter hypermethylation, and what are the definitive biological consequences. In summary, a number of germline and somatic mutations have been identified which can initiate the development of abnormal hematopoiesis in children. Premalignant stem cells undergo evolutionary selection and produce somatically altered malignant clones resulting in myeloid transformation. DisclosuresNo relevant conflicts of interest to declare.

HFE Gene Mutation Status Predicts Response to Gemtuzumab Ozogamicin in AML

BACKROUND: The protein defective in type 1 hereditary hemochromatosis, called HFE, is similar to MHC class I-type proteins, associates with beta2-microglobulin and is implicated in membranous protein recycling. Gemtuzumab ozogamicin (GO), a monoclonal antibody directed against CD33 linked to a cytotoxic agent, has been used with controversial effects in acute myeloid leukemia (AML). Internalization of GO is required for its anti-leukemic effect. Therefore, we hypothesized that H63D or C282Y HFE gene mutations may impair GO activity by preventing its internalization.METHODS: Wild type, C282Y and H63D HFE leukemic cells and primary cell were used to assess effects of GO alone or in combination with cytarabine on cell proliferation and apoptosis. Flow cytometry, confocal and AMNIS stream analysis were used to evaluate GO internalization. HFE mutations were analyzed by PCR analysis on DNA from patients included in clinical studies. Post-hoc subgroup analysis was performed to assess in vivo the role of HFE status on GO efficacy and toxicity among the 280 patients of the ALFA-0701 study as a study cohort (patients aged 50-70 years; GO 3mg/m² on days 1, 4, and 7 of chemotherapy and on day 1 of the first and second induction ; total dose 15 mg/m² ) and then on the GOELAMS-LAM 2006 IR study (patients aged 18-60 years; GO 6 mg/m² on day 4 of chemotherapy during the induction and the first consolidation; total dose 12 mg/m²) and UK NCRI AML17 study (patients aged 18-81 years; GO 3 vs 6 mg/m² on day 1 of chemotherapy but not during consolidation) as validating cohorts.RESULTS: GO induced cell death by apoptosis in AML cell lines and primary cells in a dose-dependent manner and synergistically in combination with cytarabine. However, the IC5O of GO was significantly higher in HFE mutated cells (125 vs 10 ng/mL p<0.001). To further understand this phenomenon, the CD33 internalization was analyzed upon GO or anti-CD33 antibody exposure. In line with the cytotoxic effect, CD33 was significantly less internalized in HFE mutant cells (17.4% vs 65.1% after 1 hour, p<0.01).HFE mutations were screened in 242 of the 280 ALFA-0701 patients with DNA available. There were 155 non-mutated patients (64%), 68 (28%) heterozygous for H63D, 11 (5%) heterozygous for C282Y, and 8 (3%) homozygous for H63D, which is consistent with the prevalence of the various mutations in the French population. Median age was 62 years (50-71) and the M/F ratio was 0.5, equally distributed among the different groups. No significant difference was observed with respect to the diagnosis of various hematological parameters, including white blood cell count, blasts number, cytogenetic subgroups, molecular mutation incidences (FLT3, NPM1, CEBPA, IDH, DNMT3A).In the ALFA-0701 study cohort, HFE wild-type (WT) patients had a higher overall survival (OS) when treated in the GO arm (median, not reached vs 19.5 months, p=0.0193). In contrast, OS was similar among patients with heterozygotes HFE mutations treated in the GO and the control arm (median, 19.9 vs 21.9, p=0.9675).In confirmatory cohorts, GO treatment led to a trend to increased OS in the GOELAMS-LAM 2006 IR cohort only in HFE WT patients (4-year OS with GO 62% vs 48%, p=0.08) but not in mutated patients (4-year OS with GO 73% vs 64%, p=0.45). Furthermore, in this cohort in the FLT3 WT patients subgroup, GO further improve OS in WT patients (4-year OS with GO 72% vs 50%, p=0.017), but not in patients with heterozygous HFE mutation (4-year OS with GO 80% vs 65%, p=0.23) In the UK NCRI AML17 cohort, which used GO only during induction, 245 patients were randomized between GO at 3mg/m2 and 6mg/m2 and evaluated for HFE status. Overall there was no effect of GO dose on outcomes, and no evidence of either any heterogeneity by HFE, nor any subgroup, which showed a differential effect of GO dose.CONCLUSIONS: Future studies should focus on optimising the fractionated schedule for GO at the 3mg/m2. Fractionated high doses (3x3mg/m2) during induction and single dose during consolidation seems to be the best schedule. Most importantly, the effect of GO treatment differed between HFE WT and heterozygote mutated AML patients. GO only increased OS among HFE WT patients. This is likely related to impaired internalization of the CD33 target. Our data suggest that HFE status should be used as a companion test to predict outcome of AML treated with GO. DisclosuresNo relevant conflicts of interest to declare.

Analysis of flt3 in Adult AML

FLT3 gene encodes a tyrosine kinase receptor, which is involved in proliferation and differentiation of hematopoietic stem cells. FLT3 mutations are frequently encountered in all AML subgroups specially in AML with t(15;17)- Acute Promyelocytic Leukemia (APL)-, and infers poor prognostic effect. FLT3-ITD mutations represent about 80% of FLT3 mutations. In this study, we aimed to assess frequency and correlation of FLT3-ITD mutation with different AML cytogenetic subgroups, also with MPAL cases and to study the prognostic role of this mutation with focusing on APL.Between 2011 and 2014 in NCI, Cairo University, 212 newly diagnosed acute leukemia patients [198 AML (10 M0, 47 M1, 56 M2, 30 M3, 49 M4, 4 M5, 1 M6 and 1 M7) and 14 MPAL] were included in this study. Thorough Morphological, Immunophenotypic and cytogenetic analysis were done at initial presentation and fixed follow up time points (Day 14, 28, 36) with subsequent monitoring and follow up of patients. FLT3-ITD mutation was detected using RT-PCR.FLT3-ITD mutation was detected in 44/198 AML patients (22.2%) and in 2/14 MPAL patients (14.3%). Correlation with cytogenetic subgroups show significant higher incidence in M3 patients 13/30 (43.3%, P=0.0371) in comparison to other types (20% M0, 20.6% M1, 20.1% M2, 13.4% in Monocytic Leukemia -M4 and M5-). In non-APL patients, favorable cytogenetic changes are more common in FLT3 wild group (19.7%)[27/137 (21 patients with t(8;21) and 6 with inv 16] than in FLT3 mutant group (6.5%) [2 with t(8;21) and none with inv 16]. There were insignificant difference between FLT3 wild and mutant group regarding achieving Compete Remission (CR) by Day 36 (P= 0.261), However Overall Survival (OS) of the wild group was high significantly better than that of mutant group (P=0.002). By the end of the 1st year only 10% of patient with mutant FLT3 were alive versus 30% of those with wild type, with 4 months median survival of mutant group versus 8 months of the wild group.In APL patients, again there was no significance between both groups regarding achieving CR by Day 36 (P=0.06) and still OS of APL patients with wild FLT3 was significantly better than in those with mutant FLT3 (P=0.0182). By the end of 1st year, about 90% of patients with wild group are still alive while only 46% of the mutant group are alive with 2 months median survival.We conclude that FLT3-ITD mutations infers poor prognostic effect even in the presence of favorable cytogenetic changes like t(15;17); which indicate that patient with such mutations should be assigned to aggressive therapy. DisclosuresNo relevant conflicts of interest to declare.

Prognostic Impact of Rare Single Abnormalities in Myelodysplastic Syndromes

Introduction: Since its implementation in 2012, the IPSS-R (Greenberg et al., 2012), defines the latest standard in risk stratification of patients with Myelodysplastic Syndromes (MDS). However, the prognostic impact of rare abnormalities remains unclear as yet because the number of these aberrations was too low to allow a valid statistical analysis. Hence, rare abnormalities were coalesced in one group in the IPSS and IPSS-R and, due to the unknown prognosis of these abnormalities, classified as prognostically intermediate. The main goal of the study presented here was to analyse the type, frequency and prognosis of rare single abnormalities in a large cohort of patients with primary, untreated MDS and integrate them in the existing IPSS-R in order to refine its predictive power.Methods: The data set analyzed was derived from the IPSS-R database and extended by additional data from European centers. In total, 7245 patients with primary, untreated MDS were included. Of these, we identified 410 (6%) pts. with rare single abnormalities. An aberration was defined as rare when it occurred in less than 10 patients in the cytogenetic scoring system that was the basis for the IPSS-R (Schanz et al., 2012). Additionally, further cytogenetic abnormalities not considered in this score were detected. A specific cytogenetic subgroup was defined as having at least n=5 cases with the same abnormality. Survival analyses was performed in cytogenetic subgroups with a minimum number of n=10 exclusively. The participating centers (in numerical order) were: Spain (n=110; 26.8%), MD Anderson Cancer Center (69; 16.8%), Düsseldorf (44; 10.8%), IMRAW (41; 10.0%), France (32; 7.8%), Pavia (21; 5.1%), Vienna (19; 4.6%), Japan (13; 3.2%), Vienna Medical University (12; 2.9%) Italy (11; 2.7%), Cleveland (10; 2.4%), Dundee (9; 2.2%), Brazil (8; 2.0%), Netherlands (5; 1.2%), Czech (2, 0.5%), Freiburg (1; 0.2%), Innsbruck (1; 0.2%), Sweden (1; 0.2%), and Russia (1; 0.2%). The median overall- (OS) and AML-free survival (AFS) was calculated for any specific cytogenetic subgroup.Results: Rare single abnormalities detected were: der(1;7)(n=24; 5.9%), partial or total monosomy 13 (22; 5.4%), partial or total monosomy 9 (22; 5.4%), +21 (20; 4.9%), +mar (14; 3.4%), del(3p) (12; 2.9%), total or partial monosomy 21 (11; 2.7%), total or partial monosomy X (11; 2.7%), total or partial monosomy 18 (10; 2.4%), +1/+1q (10; 2.4%), del(17p) (9; 2.2%), total or partial monosomy 14 (9; 2.2%), total or partial monosomy 16 (9; 2.2%), total or partial monosomy 6 (9; 2.2%), total or partial monosomy 1 (8; 2.0%), t(11q23;varia) (7; 1.7%), total or partial monosomy 19 (6; 1.5%), +11/+11q (6; 1.5%), +13 (6; 1.5%), +14 (6; 1.5%), del(5p) (5; 1.2%), total or partial monosomy 2 (5; 1.2%), +15 (5; 1.2%) and +X (5; 1.2%) The remaining 159 patients (38.7%) showed very rare abnormalities occurring in less than 5 patients each. The median overall survival as well as the AML-free survival in each category will be presented in detail. Furthermore, a multivariate model including all relevant confounders and a proposal to integrate these abnormalities in the cytogenetic module of the IPSS-R will be suggested.Conclusions: In order to overcome the problem of their extremely low frequency, knowledge about rare single abnormalities in MDS can only be gained by large, international cooperative projects. The present study was performed to identify and comprehensively analyze rare abnormalities occurring in MDS, uninfluenced by therapy or additional abnormalities. The results will lead to a further refinement of the cytogenetic prognostic classification in patients with MDS.The study was supported by a grant from the European Leukemia Net (ELN) DisclosuresSchanz:Novartis: Honoraria, Other: Travel Grant; Celgene: Honoraria, Research Funding; Alexion: Other: Travel Grant; Lilly: Other: Travel Grant. Sole:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees. Fenaux:Amgen: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria. Ohyashiki:Kyowa Kirin KK: Honoraria; Novartis Pharma KK: Honoraria, Research Funding, Speakers Bureau; Celegen KK: Consultancy, Honoraria, Research Funding, Speakers Bureau; Jansen Pharma KK: Honoraria, Research Funding, Speakers Bureau; Chugai Pharna KK: Research Funding; Bristol Meyer Squib KK: Research Funding; Taiho Yakuhin KK: Research Funding; Asahikasei: Research Funding; Teijin Pharma KK: Research Funding; Alexion Pharma KK: Research Funding; Asteras: Research Funding; Shinbaio Pharma KK: Honoraria; Toyama Kagaku KK: Speakers Bureau; MSD KK: Honoraria; Nippo Shinyaku KK: Speakers Bureau; Sumitomo Dainippon: Membership on an entity's Board of Directors or advisory committees. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; TetraLogic: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Exploring the Expression Profile of Long Non-Coding RNA (lncRNA) in Different Acute Myeloid Leukemia (AML) Subtypes: t(8;16)(p11;p13)/MYST3-Crebbp AML Harbors a Distinctive Lncrna Signature

Introduction: Long non-coding RNAs (lncRNAs) have recently emerged as important actors in the regulation of multiple cellular processes including cancer. Acute myeloid leukemia (AML) is a heterogeneous disease; most of the main cytogenetic AML subgroups harbor a specific gene expression profile. AML with translocation t(8;16)(p11;p13) (t(8;16) AML) is a subtype with specific clinical and biological characteristics including a distinctive gene (Camós et al, Cancer Research 2006) and microRNA (Díaz-Beyá et al, Leukemia 2013) expression profile. In this translocation, MYST3 on chromosome 8p11 fuses with CREBBP on chromosome 16p13.3. The MYST3-CREBBP fusion protein is able to interact with multiple transcription factors (TF) producing a disturbed transcriptional program. However, the lncRNA expression pattern of different cytogenetic AML subtypes, including t(8;16) AML, have not been described yet.Aims: To examine the expression profile of lncRNAs within different AML subtypes, and to characterize the expression pattern of lncRNAs in t(8;16) AML in comparison to other AML subtypes.Patients and Methods: 46 AML patients, 4 normal bone marrow (NBM) and 3 CD34+ NBM samples were included in the study. Samples included different AML subtypes: intermediate-risk cytogenetic AML (IR-AML, n=18), t(15;17) (APL, n=4), t(8;21) AML (n=4), inv(16) AML(n=2), t(6;9) AML (n=7), AML with monosomal karyotype (n=4), t(3;3) AML (n=1), t(9;11) AML (n=1) and t(8;16) AML (n=5). Within IR-AML patients with a different mutational profile: FLT3-ITD (n=7), NPM1 (n=5), CEBPA (n=7) and DNMT3A (n=6) were included. The lncRNA expression was studied using Affymetrix® Human Gene 2.1 ST platform which includes 9698 lncRNAs transcripts. The filtering and normalization of the array data was performed using oligo package from Bioconductor. Statistical analyses were performed with TiGR MultiExperiment Viewer, BRB tools and R. The Transcription factor Affinity Prediction Web Tool was used to determine the putative transcription factors binding to the differentially expressed lncRNAs promoters.Results: The hierarchical cluster analysis showed that all 4 NBM as well as all 3 CD34+ NBM clustered together according to their lncRNA expression. Interestingly, all 5 t(8;16) AML samples clustered together, as well as the 3 APL, the 7 t(6;9) AML and 5 out of 7 cases with CEBPA mutations. The specific lncRNA signature of APL was composed of 79 differentially expressed lncRNA and t(6;9) AML lncRNA signature comprised of 15 differentially expressed lncRNAs. When we focused on t(8;16) AML lncRNA profile, we identified an specific 113-lncRNA signature in the supervised analysis (Figure).Interestingly, when we analyzed which (TF) had motifs overrepresented in the promoters regions of the t(8;16) AML lncRNA signature, we identified GATA2 as the TF with significantly overrepresented motifs for GATA2 (p<0.001). Interestingly, levels of GATA2 were differentially expressed in t(8;16) AML samples in comparison with other AMLs samples (p<0.001). GATA2 has been described to interact with CREBBP, one of the partners involved in t(8;16) AML. Of note, 4 overexpressed lncRNAs of the signature (linc-HOXA11, HOXA11-AS, HOTTIP and NR_038120) were located in the HOXA genomic region, previously found upregulated in t(8:16) AML. Since several studies suggest an active crosstalk between microRNAs and lncRNAs, we also correlated the expression of these lncRNAs with the microRNA t(8;16) AML profile. We found significant correlation between linc-HOXA11 and miR-222* (R2 =0.996, p=0.003), HOXA11-AS and miR-let-7c (R2=0.994, p=0.006), HOTTIP and miR-196b*(R2=0.958, p=0.041), and NR_038120 with miR-486-3p (R2=0.999, p=0.0004) and miR-19a (R2=0.953, p=0.04).Conclusions: LncRNAs expression profile seems specific of several AML subtypes, including t(8;16) AML. Some of the lncRNAs of this distinctive signature in t(8;16) AML are located in the HOXA genomic region, and correlate with several of the characteristic microRNAs previously described in this entity. Interestingly, we have predicted in silico GATA2, which interacts with CREBBP, as the most significant TF that could potentially regulate this lncRNAs signature. Nonetheless, further investigation is warranted to determine the mechanisms leading to this lncRNA signature and to identify the specific targets of these lncRNAs.Río Hortega CM13/00205, FIS PI13/00999 DisclosuresNo relevant conflicts of interest to declare.

Prognostic Relevance of Recurrent Genetic Aberrations in Pediatric Acute Megakaryoblastic Leukemia

Genetic abnormalities and early treatment response are the main prognostic factors in pediatric acute myeloid leukemia (AML). Non-Down Syndrome (non-DS) acute megakaryoblastic leukemia (AMKL) is a rare subtype of AML, with a poor prognosis. These cases present with diverse cytogenetic aberrations, which might be important for risk-group stratification. A well-known genetic aberration in non-DS-AMKL is t(1;22)(p13;q13), resulting in expression of the RBM15/MKL1(OTT/MAL) fusion transcript. In recent years, targeted and genome-wide sequencing has identified novel, recurrent molecular abnormalities in pediatric AMKL. New fusions identified are inv(16)(p13.3q24.3) resulting in CBFA2T3/GLIS2 (Gruber et al, Cell, 2012; Masetti et al, Blood, 2013) and t(11;12)(p15;p13) resulting in NUP98/KDM5A (de Rooij et al, Leukemia, 2013) as specific aberrations in pediatric non-DS AMKL. Also KMT2A(=MLL)-rearrangements are recurrent in non-DS AMKL. The prognostic relevance of these novel abnormalities is not determined in a large cohort, since this disease is rare.To assess frequencies, clinical characteristics and outcome parameters of recurrent cytogenetic aberrations of pediatric non-DS AMKL, databases of the BFM-SG, DCOG, AIEOP, and COG were combined. In this study, we analyzed 151 newly diagnosed pediatric non-DS AMKL cases diagnosed between 1998 and 2014 of whom a sample was available. All patients included were screened for NUP98/KDM5A, CBFA2T3/GLIS2 and RBM15/MKL1 with reverse transcriptase(RT-) PCR,and for KMT2A-rearrangements using split signal FISH and RT-PCR. To assess outcome, probability of event-free survival (pEFS) and probability of overall survival (pOS) were estimated by the Kaplan-Meier method, and groups were compared with the log-rank test; the cumulative incidence of non-response or relapse (pCIR) was analysed by the Kalbfleisch and Prentice method, and groups were compared with the Gray's test. A Cox regression analysis was done for EFS and relapse incidence with as co-variables cytogenetic subgroup, age, sex, WBC and stem cell transplantation (time dependent variable).Patients were treated with different protocols; however, all protocols consisted of intensive chemotherapy using an anthracycline and cytarabine backbone for both induction and consolidation; 26% of patients received stem cell transplantation (SCT) in first remission. Median age was 1.6 years (range 0.1-17.1 years), with 62% of the patients younger than 2 years at diagnosis; 45% of patients were males, and median white blood cell count was 13.7x109/L (range 1.1-378.5x109/L). Translocation NUP98/KDM5A was identified in 9%; the CBFA2T3/GLIS2 translocation in 16%; RBM15/MKL1 in 12% and KMT2A-rearrangements in 9%, and hence in 54% none of these abnormalities were detected. All these aberrations were mutually exclusive. Comparing patients with NUP98/KDM5A, CBFA2T3/GLIS, RBM15/MKL1, or KMT2A-rearrangements with other pediatric AMKL patients, no significant differences in sex, age, and white blood cell count were found. Outcome between the included collaborative groups did not differ significantly. The 5-year pOS of the entire pediatric non-DS AMKL cohort was 56±4%, 5-year pEFS was 51±4%. NUP98/KDM5A, CBFA2T3/GLIS2 and KMT2A-rearranged positive patients showed a poor outcome (pOS 36±13%, 38±10%, and 38±13% respectively), compared to RBM15/MKL1 positive cases and other pediatric non-DS AMKL (70±11% and 65±5% respectively, p=0.04, Figure 1). Harboring NUP98/KDM5A, CBFA2T3/GLIS2 or a KMT2A-rearrangement resulted in an increased risk of experiencing an event (EFS; HR1.65, 95%CI;1.03-2.66, p=0.039) or relapse (RFS; HR2.14, 95%CI;1.12-4.11, p=0.022). SCT was not an independent factor for event or relapse free survival (HR1.20, 95%CI;0.65-2.20, p=0.565 and HR 1.13, 95%CI;0.57-2.24, p=0.717, respectively), nor did sex, age at diagnosis or WBC.Our results indicate that non-DS AMKL is a heterogeneous group within pediatric AML. Although the overall survival is poor for non-DS AMKL in general with a 5-yr pOS of ~55%, the poor outcome is specifically determined by cases with NUP98/KDM5A, CBFA2T3/GLIS2 and KMT2A-rearrangements. Other variants, including those with RBM15/MKL1, are associated with a better outcome. These data show that international collaboration allows the identification of prognostic subgroups, which may lead to new and more refined risk-group stratification. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Additional Analyses of a Randomized Phase II Study of Azacitidine Combined with Lenalidomide or with Vorinostat Vs. Azacitidine Monotherapy in Higher-Risk Myelodysplastic Syndromes (MDS) and Chronic Myelomonocytic Leukemia (CMML): North American Intergroup Study SWOG S1117

Additional Analyses of a Randomized Phase II Study of Azacitidine Combined with Lenalidomide or with Vorinostat Vs. Azacitidine Monotherapy in Higher-Risk Myelodysplastic Syndromes (MDS) and Chronic Myelomonocytic Leukemia (CMML): North American Intergroup Study SWOG S1117

The Complexity of Interpreting Genomic Data in Patients with Primary and Secondary Acute Myeloid Leukemia (AML)

BackgroundAcute myeloid leukemia (AML) is a complex, heterogeneous neoplasm characterized by the accumulation of complex genetic alterations that are responsible for the initiation and progression of the disease. Secondary AML (sAML) represents a progression from antecedent hematologic disorders such as myelodysplastic syndromes (MDS) or myeloprolifrative neoplasms (MPN). Certain acquired mutations have been reported to be specific for sAML when compared to primary AML (pAML), but many limitations exist when cytogenetic grouping or other parameters are taken into account. In addition, some mutations have been shown to impact survival in some studies, but not others.MethodsWe performed targeted deep sequencing on samples from bone marrow and peripheral blood of pts diagnosed with sAML and pAML and treated at our institution between 1/2003-1/2013. Additional data on pAML was added from The Cancer Genome Atlas (TCGA). A panel of 62 gene mutations described as frequently recurrent mutations in myeloid malignancies were assessed. Cytogenetic grouping was defined by CALGB/Alliance criteria. Differences were compared using Fisher's exact test and the Mann-Whitney U test for categorical and continuous variables, respectively. Overall survival (OS) was calculated from the time of diagnosis to last follow up or death.Results:A total of 496 pts included: 273 with pAML and 223 with sAML. Comparing pAML to sAML, pts were younger (median age 59 vs. 68 years, p<.001) and had a higher WBC at diagnosis (13.5 vs. 3.9 X 109/L, p<.001), respectively. Cytogenetic analysis showed significant differences: 58% of pAML pts had normal karyotype (NK) compared to 37% of sAML (p=.002), whereas 24% and 26% of sAML had intermediate risk (other than NK) and complex karyotype (> 3 abnormalities) compared to 11% and 16% for pAML (p< .001, .009), respectively. Mutations in ASXL1 (p<.001), JAK2 (P =.014), CBL (P =.05), BCOR (P =.02), STAG2 (p =.003), SF3B1 (P =.04), SRSF2 (P =.001 ), and U2AF1 (P =.03) were highly specific for the sAML phenotype, whereas mutations in NPM1 (p<.001 ), FLT3 (p< .001), DNMT3A (p<.001), and IDH2 (P =.02) were more specific for pAML. When the analysis was restricted to pts with NK cytogenetics, only ASXL1 (p<.001) remained specific for sAML and DNMT3A (p<.001) for pAML.Further, when the analysis was restricted to pts with unfavorable risk cytogenetics, only ASXL1 (p=.01) remained specific for sAML. No other mutations were specific for pAML.We then evaluated whether the mutations that were specific to each AML phenotype had an impact on OS. We observed different mutations that impacted OS in each phenotype: DNMT3A (HR 1.81, 95% CI 1.28-2.57, p<.001), TP53 (HR 3.1, 95% 1.74-5.53, p< .001), and SUZ12 (HR 3.18, 95% CI 1.01-10, p=.05) led to worse OS in pAML, whereas mutations in EZH2 (HR 2.12, 95% CI 1.07-4.21, p =.03), PRPF8 (HR 2.32, 95% CI 1.20-4.46, p=.01), and TP53 ( HR 2.92, 95% CI 1.69-5.04, p<.001) lead to worse OS in sAML. Different mutations had a different impact on OS when cytogenetic analysis was taken into account. Mutations in FLT3 (HR 2.15, 95% CI 1.37- 3.35, p<.001) and DNMT3A (HR 2.41, 95% CI 1.57-3.70, p<.001) led to worse OS in NK pAML, whereas none of the mutations impacted OS in NK sAML. Further, in pAML with unfavorable cytogenetics, BCOR (HR 2.41, 95% CI 1.57-3.70, p<.001) and TP53 (HR 2.41, 95% CI 1.57-3.70, p<.001) had led to worse OS, whereas BOCR (HR 2.95, 95% CI 1.03-8.50, p<.001), SF3B1 (HR .19, 95% CI .05-.82, p<.001), SUZ12 (HR .12, 95% CI .01-.99, p<.001),and TP53 (HR 1.9, 95% CI 1.09-3.46, p<.001) only impacted OS in sAML.ConclusionClear genomic variations exist between sAML and pAML. Although some of these genomic changes are more specific to each phenotype in general, this specificity and the impact on OS differed for each cytogenetic subgroup, highlighting the complexity of interpreting genomic information in pts with AML and the need to incorporate both cytogenetic and molecular data in prognosis-driven treatment decisions. DisclosuresSekeres:TetraLogic: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Characterization of Del(14)(q24q32) in Mature B-Cell Neoplasms By Array-CGH, Cytogenetics and Molecular Mutations

Background: Deletions of 14q occur recurrently in mature B-cell neoplasms at a low frequency of 1.5% (Reindl et al., BJH 2010). In about one-third of these cases breakpoints show a clustering at 14q24.1 (centromeric) and at 14q32.3 (telomeric). Limited genetic data is available on this rare subgroup.Aim: To characterize del(14)(q24q32) using array based comparative genomic hybridization (aCGH) and to analyze cytogenetical and molecular characteristics.Patients and Methods: 34 patients with mature B-cell neoplasms and del(14)(q24q32) by chromosome banding analysis were analyzed. Median age was 72 years (range: 45-94 years). Male:female ratio was 1.4:1. All cases were analyzed by aCGH (Agilent, Waldbronn, Germany) and for mutations in MYD88, NOTCH1, TP53, and SF3B1 as well as for IGHV mutational status by direct Sanger sequencing. IGHV mutated (mut) cases without stereotypic VH3-21 were classified as IGHV favourable (IGHV fav). For statistical analysis of CLL patients data were compared with a cohort of 1,136 untreated CLL patients without del(14q).Results: Patients with del(14)(q24q32) were immunophenotypically classified as follows: 26 (59%) had CLL (n=20) or CLL/PL (n=6), 6 (18%) had splenic marginal zone lymphoma (SMZL), one patient had two B-cell neoplasms (SMZL and CLL/PL: 66% and 10% infiltration, respectively) and one patient had monoclonal B-cell lymphocytosis that progressed to CLL within two years.Analysis with aCGH showed that centromeric breakpoints were detected within a region of 970 kb (69,135,775 - 70,106,558) and telomeric breakpoints localized within a region of 712 kb (105,618,017 - 106,329,974). The median length of the deletions was 36.9 Mb (range: 35.5-37.1). Interestingly, the same breakpoints were present in 4 patients, each: 69,248,772 - 106,329,974 (cluster 1) and 69,271,436 - 106,329,974 (cluster 2). Genes located at the centromeric breakpoint that may be activated by juxtaposition to the IGH locus include FUT8. Its expression contributes to cancer malignancy. Interesting candidate genes located within the deleted region are PPP1R13B (p53 interaction) and NUMB (NOTCH1 interaction). In addition to del(14)(q24q32) 39 cytogenetic aberrations were detected in 23 patients. Two changes were recurrent: trisomy 12 (+12; n = 14) and del(13q) (n = 3). All patients of cluster 1 had +12 (cluster 1 vs. non-cluster 1: 100% vs. 33%, p=0.022).Mutations were detected in TP53 (n = 5, 15%) and NOTCH1 (n = 12, 35%). In SF3B1 a variant and in MYD88 no mutation were found. All mutated cases were CLL or CLL/PL cases only (n.s.). No molecular or cytogenetic differences were detected between CLL or CLL/PL and SMZL. Additionally, the IGHV mutational status was determined in CLL and CLL/PL cases (unmutated in 18 (69%), mutated in 8 (31%)).Further statistical analyses were performed in cases with CLL or CLL/PL with vs. without del(14)(q24q32). NOTCH1 mut (42% vs. 12%, p<0,001) and +12 (46% vs. 14%, p<0.001) were more frequent in patients with del(14q) vs. without. Of note, NOTCH1 mut were not associated with +12 in patients with del(14q) (with vs. without +12: 21% vs. 45%, n.s.), whereas patients without del(14q) showed a significant association (31% vs. 9%, p<0.001). Furthermore, del(13q) was rare in patients with del(14q) (4% vs. 61%, p<0.001) and IGHV fav was detected in less cases (35% vs. 60%, p=0.014). In 978 cases (events = 373; del(14q): n=13, events = 11) data on time to treatment (TTT) was available. TTT was shorter in patients with vs. without del(14q) (4 vs. 95 months, p<0.001). This was also the case when TP53 disrupted (del(17p) and TP53 mut) and +12 cases were excluded from the del(14q) cohort (17 vs. 95, p<0.001). For Cox regression analyses cytogenetic aberrations were hierarchically classified as follows: del(14q), del(17p), del(11q), +12, del(13q) sole. Univariate analysis of cytogenetic subgroups and molecular mutations identified that del(14q), TP53 disrupted, del(11q), +12, NOTCH1 mut, and SF3B1 mut have significant negative impact on TTT and del(13q) sole and IGHV fav are positive prognosticators. Multivariate analysis showed independent impact of del(14q) (HR: 5.2, p<0.001), del(11q) (HR:1.5, p=0.037), SF3B1 mut (HR: 1.5, p=0.008), and IGHV fav (HR: 0.3, p<0.001).Conclusions: del(14)(q24q32) are deletions with a very low variability in breakpoints in mature B-cell neoplasms. In CLL patients they are associated with +12, NOTCH1 mut and independently with a very short TTT. DisclosuresJeromin:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Integration of Minimal Residual Disease with Other Patient Risk Factors Identifies a Population with Very Poor Overall Survival in Pediatric ALL: Results from the UKALL 2003 Trial

Background:Overall survival (OS) for children with ALL treated on contemporary protocols is now > 90%. To further improve outcomes, stratification must direct intensive treatment to those patients with the highest risk of relapse. Whilst minimal residual disease (MRD) has emerged as the most powerful predictor of outcome, its use in isolation is insufficient to identify those patients with the greatest risk. To address this, we performed a detailed analysis of the entire patient cohort from the recently reported large multi-center randomized controlled trial, UKALL 2003. Integration of end of induction (EOI) MRD with other predictive factors - cytogenetics, age and white cell count (WCC) - allowed the identification of very high risk groups that are likely to benefit from early treatment intensification or novel therapies.Methods:The UKALL 2003 trial recruited 3126 patients aged 1-25 years with Philadelphia-negative ALL between Oct 1, 2003, and June 30, 2011. Treatment was initially stratified based on NCI risk and cytogenetic results. Subsequent treatment was directed by EOI MRD measured using EuroMRD approved real-time quantitative PCR for immunoglobulin and T-cell receptor gene rearrangements. Analysis was performed on 2666 patients with available MRD results and focused on the systematic integration of MRD results with other patient characteristics to identify subgroups with a very poor OS (<75% at 5 years). MRD was considered positive at a level ≥0.005% and low risk if <0.005% unless otherwise stated.Results:Both MRD and cytogenetic risk group were independently predictive of survival. In particular, patients with high risk (HR) genetic abnormalities - low hypodiploidy, near haploidy, KMT2A (MLL) rearranged, iAMP21 and t(17;19)(q23;p13) (n=100) had a poor outcome with 5 yr OS of 76.0%. Combining these factors showed that MRD could stratify the HR cytogenetic subgroup with a significant difference in survival between those with low risk MRD and positive MRD (5yr OS 93.0 vs. 63.1%, p=0.002, n=43 vs. 57). Crucially, this demonstrates that HR cytogenetics are only truly poor risk in the context of poor treatment response. Importantly, there were almost no survivors following relapse in the HR cytogenetic/MRD positive group, whilst 50% of those with low risk MRD could be salvaged after relapse. In addition, whilst t(1;19)(q23;p13)was not considered a HR cytogenetic factor in this trial, combination with positive EOI MRD identified an additional subgroup of 20 patients with a 5yr OS of only 74.7% (vs. 96.7% in MRD low risk patients, p=0.003).Whilst WCC at presentation was also predictive of outcome, this difference was significant only in those with B-ALL and not T-ALL. In B-ALL, WCC>100 x109/L was associated with significantly poorer survival (5yr OS 95.1 vs. 84.0%, p<0.0001) whilst high WCC in T-ALL had little effect on survival, even at WCC >200 x109/L (5yr OS 90.2 vs. 86.7%, p=0.283). Integration of MRD results allowed the identification of a very high risk group with B-ALL, WCC>100 and positive EOI MRD (n=80) which had a 5yr OS of only 71.3%. Further breakdown by cytogenetic risk group showed those patients with good risk cytogenetics (ETV6-RUNX1, high hyperdiploidy) (n=19) had a remarkably good survival despite WCC>100 (5yr OS 94%). In contrast, those with intermediate (n=34) or HR cytogenetics (n=15) had a very poor 5yr OS (65.7% and 60.0% respectively).Older age (≥10yr) was also associated with poorer outcomes compared to those <10yr (5yr OS 95.8 vs. 87.4%, p<0.0001). Integration with MRD results further separated these groups but did not identify a very high risk subgroup. However, combining older age with a higher EOI MRD cut-off of 1% successfully identified a subgroup (n=96) with a very poor 5yr OS of 51.3% (vs. 91.4% in those with MRD<1%, p<0.0001). Interestingly, this was independent of cytogenetic risk group.Conclusions:Through the integration of EOI MRD with other risk factors in a large pediatric ALL cohort we have identified a population of patients with a very poor OS. When combined, these criteria select only 6% of all patients, but capture 35% of all deaths suggesting current treatment is suboptimal in this group. This information is essential for the planning of future trials to ensure that treatment intensification and novel therapies are targeted at those patients at greatest risk, whilst avoiding unnecessary treatment in those patients that already experience good outcomes. DisclosuresNo relevant conflicts of interest to declare.

Establishment of Primary Adult MDS Nested Case-Control Study Cohort and Study of Risk Factors Associated with MDS Evolution to Leukemia

To establish a nested case-control study cohort in myelodysplastic syndrome (MDS) patients and investigate the clinical characteristics, WHO subtype and risk factors associated with MDS evolution to leukemia of this cohort. All patients, ≥18 years of age, provided by 24 Shanghai hospitals with initial clinical findings consistent with a hematopoietic abnormality between June 2003 and April 2007, were the candidates for inclusion in this study. The blood and bone marrow samples of every patient should be provided at baseline. Diagnosis was made by incorporating morphologic, immunophenotypic, cytogenetic and molecular features according to WHO classification criteria. Cytogenetic analysis was performed using conventional G-banding karyotyping and fluorescence in situ hybridization (FISH) techniques. Cumulative risk of evolution was estimated by Kaplan-Meier method. Prognostic factors were evaluated by univariate Log-rank method and multivariate Cox proportional hazard models. A total of 435 patients were diagnosed as MDS. The median age of MDS onset was 58(18-90) years, with 248 male patients and 187 female patients (male: female 1.33: 1). The percentage of cases with refractory cytopenia with multilineage dysplasia (RCMD) was the highest (65.5%), while that of refraetory anemia (RA) (2.3%), refractory anenia with ring sideroblast (RARS) (1.1%) and 5q-syndrome (0.5%) was lower. Trisomy 8 (+8) was the most common chromosome abnormalities (71 cases, 12.7%). The mean follow-up time was 20.3 (4.2-57.1) months. Cases were patients with evolution by the end of follow-up, while controls were patients without evolution by that time. Case group included 41 patients and control group included 342 patients. Univariate analysis showed that the age, sex, WHO subtype, WBC count, absolute neutrophil count (ANC), IPSS cytogenetic subgroup, IPSS group and bone marrow blast percentage were significant risk factors for leukemia-free survival (LFS). Multivariate analysis of COX model showed that the age, sex, WHO subtype, IPSS cytogenetic subgroup and bone marrow blast were independent risk factors for LFS. A nested case-control study cohort of MDS patients is established. The clinical characteristics and WHO subtype of MDS patients in Chinese Shanghai are different from that in Western countries. The independent risk factors for MDS evolution are age, sex, WHO subtype, IPSS cytogenetic subgroup and bone marrow blast percentage.

Heterogeneous cytogenetic subgroups and outcomes in childhood acute megakaryoblastic leukemia: a retrospective international study

AbstractComprehensive clinical studies of patients with acute megakaryoblastic leukemia (AMKL) are lacking. We performed an international retrospective study on 490 patients (age ≤18 years) with non–Down syndrome de novo AMKL diagnosed from 1989 to 2009. Patients with AMKL (median age 1.53 years) comprised 7.8% of pediatric AML. Five-year event-free (EFS) and overall survival (OS) were 43.7% ± 2.7% and 49.0% ± 2.7%, respectively. Patients diagnosed in 2000 to 2009 were treated with higher cytarabine doses and had better EFS (P = .037) and OS (P = .003) than those diagnosed in 1989 to 1999. Transplantation in first remission did not improve survival. Cytogenetic data were available for 372 (75.9%) patients: hypodiploid (n = 18, 4.8%), normal karyotype (n = 49, 13.2%), pseudodiploid (n = 119, 32.0%), 47 to 50 chromosomes (n = 142, 38.2%), and >50 chromosomes (n = 44, 11.8%). Chromosome gain occurred in 195 of 372 (52.4%) patients: +21 (n = 106, 28.5%), +19 (n = 93, 25.0%), +8 (n = 77, 20.7%). Losses occurred in 65 patients (17.5%): –7 (n = 13, 3.5%). Common structural chromosomal aberrations were t(1;22)(p13;q13) (n = 51, 13.7%) and 11q23 rearrangements (n = 38, 10.2%); t(9;11)(p22;q23) occurred in 21 patients. On the basis of frequency and prognosis, AMKL can be classified to 3 risk groups: good risk—7p abnormalities; poor risk—normal karyotypes, –7, 9p abnormalities including t(9;11)(p22;q23)/MLL-MLLT3, –13/13q-, and –15; and intermediate risk—others including t(1;22)(p13;q13)/OTT-MAL (RBM15-MKL1) and 11q23/MLL except t(9;11). Risk-based innovative therapy is needed to improve patient outcomes.

Immunophenotypic Characterization of Cytogenetic subgroups in Egyptian Pediatric B-Acute Lymphoblastic Leukemia Patients

Immunophenotypic Characterization of Cytogenetic subgroups in Egyptian Pediatric B-Acute Lymphoblastic Leukemia Patients

Azacitidine treatment for patients with myelodysplastic syndrome and acute myeloid leukemia with chromosome 3q abnormalities

Acute Myeloid Leukemia (AML) and myelodysplasia (MDS) with chromosome 3q abnormalities have a dismal outcome either untreated or with conventional treatments. Azacitidine (AZA) is now considered as the standard of care in high-risk MDS and oligoblastic AML patients. The objective of this study was to evaluate the impact of azacitine treatment in this cytogenetic subgroup. We report here a multicentre retrospective study of 157 patients treated with AZA for AML/MDS with chromosome 3q abnormalities and 27 patients with isolated EVI-1 overexpression. Median age was 65 years, 40 patients (25%) had inv(3)(q21q26.2) or t(3;3)(q21;q26.2), 36 patients (23%) had other balanced 3q26 rearrangements, 8 patients (5%) had balanced 3q21 rearrangements and 73 patients (46%) had other 3q abnormalities. The overall response rate was 50% (29% CR). Median overall survival was 10.6 months. By multivariate analysis, patients with lower bone marrow blast counts, higher platelet counts, non-complex cytogenetics, and absence of prior treatment with intensive chemotherapy had a better outcome. 27 patients were allo-transplanted and achieved a 21-month median OS. Balanced 3q21 translocations were associated with a better response rate and overall survival. Outcome of patients with isolated EVI-1 overexpression was comparable to that of patients with chromosome 3q lesions. Thus, AML/MDS patients with 3q abnormalities appear to be a heterogeneous group in their response to AZA, and AZA may represent a suitable option in particular as a bridge to allogeneic transplantation.

Frequency of copy number abnormalities in common genes associated with B-cell precursor acute lymphoblastic leukemia cytogenetic subtypes in Brazilian children

Copy number alterations (CNAs) in genes committed to B-cell precursors have been associated with poor survival in subgroups of patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We investigated submicroscopic alterations in a series of 274 Brazilian children with BCP-ALL by multiplex ligation-dependent probe amplification and evaluated their correlation with clinical and laboratory features. The relevance of overlapping CNA abnormalities was also explored. Deletions/amplifications in at least one gene were identified in 83% of the total series. In children older than 2 years, there was a predominance of CNAs involving deletions in IKZF1, CDKN2A, and CDKN2B, whereas the pseudoautosomal region 1 (PAR1) had deletions that were found more frequently in infants (P <0.05). Based on the cytogenetic subgroups, favorable cytogenetic subgroups showed more deletions than other subgroups that occurred simultaneously, specifically ETV6 deletions (P <0.05). TCF3-PBX1 was frequently deleted in RB1, and an absence of deletions was observed in IKZF1 and genes localized to the PAR1 region. The results corroborate with previous genome-wide studies and aggregate new markers for risk stratification of BCP-ALL in Brazil.

Regulation of the C/EBPα signaling pathway in acute myeloid leukemia (Review).

The transcription factor CCAAT/enhancer binding protein α (C/EBPα), as a critical regulator of myeloid development, directs granulocyte and monocyte differentiation. Various mechanisms have been identified to explain how C/EBPα functions in patients with acute myeloid leukemia (AML). C/EBPα expression is suppressed as a result of common leukemia-associated genetic and epigenetic alterations such as AML1-ETO, RARα-PLZF or gene promoter methylation. Recent data have shown that ubiquitination modification also contributes to its downregulation. In addition, 10-15% of patients with AML in an intermediate cytogenetic risk subgroup were characterized by mutations of the C/EBPα gene. As a transcription factor, C/EBPα can translocate into the nucleus and further regulate a variety of genes directly or indirectly, which are all key factors for cell differentiation. This review summarizes recent reports concerning the dysregulation of C/EBPα expression at various levels in human AML. The currently available data are persuasive evidence suggesting that impaired abnormal C/EBPα expression contributes to the development of AML, and restoration of C/EBPα expression as well as its function represents a promising target for novel therapeutic strategies in AML.

The protein expression profile of meningioma cells is associated with distinct cytogenetic tumour subgroups

Limited information exists about the impact of cytogenetic alterations on the protein expression profiles of individual meningioma cells and their association with the clinicohistopathological characteristics of the disease. The aim of this study is to investigate the potential association between the immunophenotypic profile of single meningioma cells and the most relevant features of the tumour. Multiparameter flow cytometry (MFC) was used to evaluate the immunophenotypic profile of tumour cells (n = 51 patients) and the Affymetrix U133A chip was applied for the analysis of the gene expression profile (n = 40) of meningioma samples, cytogenetically characterized by interphase fluorescence in situ hybridization. Overall, a close association between the pattern of protein expression and the cytogenetic profile of tumour cells was found. Thus, diploid tumours displayed higher levels of expression of the CD55 complement regulatory protein, tumours carrying isolated monosomy 22/del(22q) showed greater levels of bcl2 and PDGFRβ and meningiomas carrying complex karyotypes displayed a greater proliferation index and decreased expression of the CD13 ectoenzyme, the CD9 and CD81 tetraspanins, and the Her2/neu growth factor receptor. From the clinical point of view, higher expression of CD53 and CD44 was associated with a poorer outcome. Here we show that the protein expression profile of individual meningioma cells is closely associated with tumour cytogenetics, which may reflect the involvement of different signalling pathways in the distinct cytogenetic subgroups of meningiomas, with specific immunophenotypic profiles also translating into a different tumour clinical behaviour.