Cytogenetic Groups Research Articles

The expression and regulation of HOX genes and membrane proteins among different cytogenetic groups of acute myeloid leukemia.

BackgroundThe cytogenetic aberrations were considered as markers for diagnosis and prognosis in acute myeloid leukemia (AML), while the expression and regulation under different cytogenetic groups remain to be fully elucidated.MethodsIn this paper, for favorable, poor, and cytogenetically normal groups of AML patients, we performed comprehensive bioinformatics analyses including identifying differentially expressed genes (DEGs) and microRNAs (miRNAs) among them, functional enrichment and regulatory networks.ResultsWe found that DEGs were enriched in membrane‐related processes. Eleven genes and two miRNAs were significantly differentially expressed among these three AML groups. In survival analysis, membrane‐related genes and several miRNAs were significant on prognostic outcome. Notably, six HOXA and three HOXB genes were significantly in low expression and high methylation in AML with favorable cytogenetics. Meanwhile, the miRNA‐HOX gene co‐regulatory networks revealed that HOXA5 was a hub node and regulated an AML oncogene SPARC.ConclusionOur work may provide novel insights to the molecular characteristics and classification between AML with different cytogenetics.

Insights into the heterochromatin evolution in the genus Melipona (Apidae: Meliponini)

The species of the four Melipona subgenera can be subdivided into two cytogenetic groups: species belonging to Group I are characterized by having low heterochromatin content in their karyotypes ( 50%). To study the patterns of heterochromatin accumulation in this genus, we isolated moderate to highly repetitive sequences in M. (Michmelia) mondury (Mmon probe) and M. (Melikerria) fasciculata (Mfas probe) obtained based on the renaturation kinetics of C0t − 1 DNA and used these fragments as probes to hybridize in thirteen species belonging to the four Melipona subgenera. The Mmon probe marked all chromosomes in all Michmelia species and additionally the secondary constriction of M. (Eomelipona) puncticollis. The Mfas probe showed positive markings only within Melikerria. These results indicate the independence of the heterochromatin growth between Michmelia and Melikerria. Absence of markings in the regular set of chromosomes in M. (Melikerria) quinquefasciata and positive markings in the Bs, with the Mfas probe, suggests an interspecific origin of these chromosomes. We also hypothesize about the possible scenario that led to the heterochromatin growth in the genus Melipona.

Cytogenetic abnormalities in MM: Association with disease characteristics and treatment response.

e20520 Background: Cytogenetic abnormalities detected by FISH are found in the majority of multiple myeloma (MM) patients. Although their prognostic value has been studied extensively, less is known about their association with disease characteristics and treatment response. Methods: To address these questions, we designed a retrospective study including 2031 Mayo Clinic patients diagnosed with MM from 2004 to 2018. We compared baseline characteristics and treatment outcomes between primary cytogenetic groups: t(11;14), t(4;14), t(14;16), (14;20), t(6;14), unknown IgH translocation/del and trisomy (without IgH translocation). These included 373, 177, 78, 20, 18, 228 and 791 patients respectively. Kruskal-Wallis and Fisher’s exact tests were used for categorical and continuous variables respectively. Time to next treatment (TTNT) was estimated using Kaplan-Meier method and compared using Log-Rank test. Results: t(4;14), t(14;16), t(6;14) and t(14;20) groups were associated with hemoglobin < 10 g/dL, beta2microglobulin > 5.5 µg/ml, ISS stage 3 and ≥50% bone marrow plasma cells. The latter 3 groups were also associated with renal dysfunction (Cr ≥2 mg/dL) and higher urinary monoclonal protein. t(4;14) was associated with IgA isotype, serum monoclonal protein ≥1g/dL and plasma cell labeling index ≥1%. Light chain myeloma was more prevalent in patients with t(11;14). Overall response rate (ORR) to proteasome inhibitor (PI) induction was higher for those with IgH translocations (any) compared to trisomies (85% vs 77% P = 0.02), while ORR was higher for those with trisomies with immunomodulatory drug (IMiD) induction (90% vs 78% P < 0.01). The rate of ≥ very good partial response was higher for patients with high risk IgH translocations [t(4;14), t(14;16) or t(14;20)] compared to standard risk with PI-IMiD combination treatment (88% vs 65% P < 0.01). Otherwise, response rates did not differ between these 2 groups. TTNT was longer in patients with trisomies compared to those with IgH translocation with IMiD or PI-IMiD treatments (32.2 vs 19 and 44 vs 27.4 months, respectively P < 0.01). For all cytogenetic groups, better treatment responses and longer TTNT were seen with PI-IMiD combinations compared to other treatments. Conclusions: t(4;14), t(14;16), t(6;14), and t(14;20) are associated with high risk disease characteristics. Patients with IgH translocations may have better response to PI induction compared to those with trisomies, while those with trisomies may have better response to IMiD treatment, with best outcomes for both seen with PI-IMiD combinations.

NEAT1 Long Isoform Is Highly Expressed in Chronic Lymphocytic Leukemia Irrespectively of Cytogenetic Groups or Clinical Outcome.

The biological role and therapeutic potential of long non-coding RNAs (lncRNAs) in chronic lymphocytic leukemia (CLL) are still open questions. Herein, we investigated the significance of the lncRNA NEAT1 in CLL. We examined NEAT1 expression in 310 newly diagnosed Binet A patients, in normal CD19+ B-cells, and other types of B-cell malignancies. Although global NEAT1 expression level was not statistically different in CLL cells compared to normal B cells, the median ratio of NEAT1_2 long isoform and global NEAT1 expression in CLL samples was significantly higher than in other groups. NEAT1_2 was more expressed in patients carrying mutated IGHV genes. Concerning cytogenetic aberrations, NEAT1_2 expression in CLL with trisomy 12 was lower with respect to patients without alterations. Although global NEAT1 expression appeared not to be associated with clinical outcome, patients with the lowest NEAT1_2 expression displayed the shortest time to first treatment; however, a multivariate regression analysis showed that the NEAT1_2 risk model was not independent from other known prognostic factors, particularly the IGHV mutational status. Overall, our data prompt future studies to investigate whether the increased amount of the long NEAT1_2 isoform detected in CLL cells may have a specific role in the pathology of the disease.

FBXW4 Is Highly Expressed and Associated With Poor Survival in Acute Myeloid Leukemia.

The F-box and WD repeat domain-containing (FBXW) proteins play an important role in ubiquitin proteasome by inducing protein degradation. Ten FBXW proteins have been identified in humans. The functions of FBXW proteins, like FBXW7, have been well-established in many human cancers. However, little is known about their transcriptional expression profiles and relationship with prognosis in acute myeloid leukemia (AML). Here we investigated the roles of FBXW proteins in AML by analyzing their mRNA expression profiles and association with clinical features using data from EMBL-EBI, the Cancer Cell Line Encyclopedia, Gene Expression Profiling Interactive Analysis, and cBioPortal databases. Our results showed that the mRNA level of FBXW proteins were highly detected by microarray in 14 AML cell lines, although there were no obvious differences. The expression of FBXW4 was significantly higher in AML patients compared with that in normal controls (P < 0.01). Patients whose age was ≥60 years old had a higher FBXW4 expression when compared with those who were <60 years old (P < 0.05). Cytogenetic favorable-risk group patients had a much lower FBXW4 expression than the intermediate- and poor-risk group patients (P < 0.0001). Moreover, patients with high FBXW4 expression exhibited significantly shorter event-free survival (EFS) and overall survival (OS) than those with low FBXW4 expression (median EFS: 5.3 vs. 10.0 months, P = 0.025; median OS: 8.1 vs. 19.0 months, P= 0.015). A multivariate analysis indicated that high FBXW4 expression was an independent risk factor for poor EFS in AML patients who received intensive chemotherapy followed by allo-SCT. In summary, our data suggested that FBXW4 is aberrantly expressed in AML and high FBXW4 expression might be a poor prognostic biomarker; future functional and mechanistic studies will further illuminate the roles of FBXW4 in AML.

Lenalidomide (len) Maintenance (maint) after Autologous Stem Cell Transplant (ASCT) for Multiple Mmyeloma (MM) Improves Outcomes of Patients (pts) with Both Standard- and High-Risk Cytogenetics: A Single Institutional Experience of over 1000 ASCT Pts.

<h3>Introduction</h3> A meta-analysis of three large randomized trials has demonstrated that len maint prolongs progression free survival (PFS) and overall survival (OS) in pts with MM who have undergone ASCT. While len maint benefits pts with both high-risk and standard-risk cytogenetics, it does not appear to provide equal magnitude of benefit to patients with high-risk cytogenetics. We conducted a retrospective analysis comparing the outcomes of pts with MM who underwent ASCT at our institution and len maint compared to no maint. We further evaluated the impact of len maint on the two cytogenetic groups. <h3>Methods</h3> We performed a retrospective analysis of 1002 consecutive pts with newly diagnosed MM who underwent ASCT at The Ohio State University between 1992 through 2016. Data were collected regarding patient demographics, disease characteristics, therapy received including maint, and clinical outcomes. Pts were excluded from the analysis if they received len combination or other drug maint therapies. High-risk cytogenetics was defined as having t(4;14), t(14;16), gain 1q, or del17p. PFS and OS were estimated using Kaplan-Meier method. Log-rank test was used to compare PFS and OS between the groups and Cox proportional hazard models were used to estimate the hazard ratios (HR). <h3>Results</h3> Pt characteristics are listed in Table 1. 484 pts (53.3%) received no maint while 423 pts (46.7%) received len maint. 245 pts (24.4%) had high-risk cytogenetics while 561 pts (56%) had standard-risk cytogenetics with the remaining unknown. Both PFS and OS were significantly better in the len maint group compared to the no maint group (Figure 1). Adjusting for pts age at transplant, cytogenetic risk, pre-transplant and post-transplant disease status, and ISS staging, the superiority in PFS and OS remained statistically significant, HR=0.50 (95% CI: 0.41-0.62), p<0.001 for PFS, HR=0.48 (95% CI: 0.36-0.63), p<0.001 for OS, respectively. Furthermore, both high-risk and standard-risk groups derived benefit from len maint (p<0.001, Figure 2). <h3>Conclusion</h3> Consistent with previously published data, our results demonstrate an improvement in outcomes in MM pts treated with len maint following ASCT compared to no maint regardless of cytogenetic risk group. Larger studies are needed to assess the individual benefit obtained with len maint with each high-risk cytogenetic abnormality. Several trials incorporating newer drugs with novel mechanisms of action are currently being done that could further optimize maint therapy and lead to better outcomes in high-risk group.

Balancing Retrospection and Visions: The Cytogenetics Group of the Society of Plant Breeding (GPZ) Came Together in Dresden

Balancing Retrospection and Visions: The Cytogenetics Group of the Society of Plant Breeding (GPZ) Came Together in Dresden

Prognostic impact of Annexin A1 expression in acute myeloid leukemia

Objective The aim was to assess expression of Annexin A1 (ANXA1) on malignant myeloid blast cells and its correlation with clinical outcome, overall survival (OS), and other prognostic factors among adult Egyptian patients with acute myeloid leukemia (AML). Patients and methods A total of 60 patients with de novo AML were treated and followed up in Ain Shams University Hospitals, Hematology Unit and compared with 20 age-matched and sex-matched normal healthy controls. Expression of ANXA1 was detected by flow cytometry in bone marrow samples. Results Patients with AML had significant higher mean ANXA1 expression than mean ANXA1 expression in control at D0. The mean ANXA1 level in the favorable cytogenetics group was significantly higher than the mean ANXA1 level in the unfavorable cytogenetics group. ANXA1 was positive in 76.7% patients with AML and negative in 23.3% patients with AML. There was a significant difference between ANXA1-positive group and ANXA1-negative group of patients with AML regarding different outcomes. In ANXA1-positive group, 52.2% of patients with AML achieved complete remission (CR), whereas in ANXA1-negative group, 14.3% of patients with AML achieved CR. Highest mean ANXA1 expression was in the group of patients who had CR. High ANXA1 expression was associated with longer OS. Conclusion ANXA1 is significantly expressed in Egyptian patients with de novo AML, and its expression associated with favorable prognostic effect on clinical outcome and longer OS.

Molecular Study of FLT3 gene mutations in Acute Myeloid Leukemia from Pakistan : Correlation with clinicopathological parameters.

Introduction: FLT3 mutations are common genetic changes reported to have prognostic significance in acute myeloid leukemia (AML). Bone marrow/peripheral blood samples of 63 AML Pakistani patients were collected and DNA was isolated.Materials and Methods: The FLT3 internal tandem duplication (ITD) and the D835 activating mutation in the tyrosine kinase domain (TKD) were analyzed by polymerase chain reaction (PCR).Results: Among 63 AML patients, 42 were males and 21 were females with male to female ratio 2.1:1. The age ranged between 15 to 75 years with a median age of 32 years. AML-M2 was the predominant French-American-British (FAB) subtype (32%) followed by M3 (27%), M4 (19%), M5 (6.3%) and M1 (6.3%). The incidence of FLT3/ITD and TKD was 22% and 6.3% respectively. Majority of the FLT3/ITD mutation were detected in AML-M4 (38%) patients while D835 mutation was common in both FAB M1, M2. Presence of mutation was significantly associated with age but significance was not achieved for hyperleukocytosis.Conclusion: This study constitutes the first report from Pakistan reporting significant presence of FLT3/ITD mutations in our adult AML patients with different FAB subtypes Molecular mutation analysis in different cytogenetic groups with follow-up is required to understand the pathogenesis of leukemias and their role as a valuable prognostic marker in our patients.

A review on tumor heterogeneity and evolution in multiple myeloma: pathological, radiological, molecular genetics, and clinical integration.

Recent research has dramatically advanced our understanding of the genetic basis of multiple myeloma (MM). MM displays enormous inter- and intratumoral heterogeneity, and underlies a clonal evolutionary process driven and shaped by diverse factors such as clonal competition, tumor microenvironment, host immunity, and therapy. Two main cytogenetic groups are distinguished: MM with recurrent translocations involving the immunoglobulin heavy chain locus and MM with hyperdiploidy involving the odd chromosomes. The disease virtually always starts with a preneoplastic prodromal phase-monoclonal gammopathy of undetermined significance-that variably progresses to symptomatic MM within a few months or many years. Tumor heterogeneity and its evolution in space and time have important consequences for the clinical management and outcome of MM patients. At diagnosis, spatial intratumoral heterogeneity poses a challenge for classification and risk stratification. During maintenance therapy, clonal evolution may complicate disease monitoring and promote drug resistance. Upon progression or transformation, identifying the dominant disease-driving neoplastic clones and elucidating their properties are key to tailor personalized therapy. In this review, we discuss tumor heterogeneity and clonal evolution in MM, integrating pathological, radiological, molecular genetics, and clinical data. Current and prospective classification schemes and prognostic parameters, incorporating new genetic and proteomic discoveries and advances in imaging, are highlighted. In addition, the roles of the tumor microenvironment, host immunity, and resistance mutations, and their effects on therapy, are discussed. An improved understanding of high-risk disease, tumor heterogeneity, and clonal evolution will guide future therapies and may ultimately lead towards a cure for MM.

Differential Induction Among Younger Patients Diagnosed with Secondary Acute Myelogenous Leukemia Is Feasible By Cytogenetic/Molecular Risk Stratification

Background: Secondary acute myeloid leukemia (s-AML) [therapy-related (t-AML) and evolving from antecedent hematologic disorder, i.e. myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN)] are highly aggressive disorders with 20% overall survival [OS] at 1 year. Challenges for initial therapy selection include 1. Difficulty for precise discrimination of pre-existing disorders [MDS vs MPN vs t-AML] and 2. Lack of reliable karyotypic/molecular risk stratification [i.e. European-leukemia-network-2017 [ELN 2017], originally designed for de novo AML]. In s-AML, enrichment for high-risk karyotypic and molecular abnormalities [i.e. complex karyotype (CK), -17p, -7/7p, p53, RUNX1, ASXL1 mutations] suggests that systematic stratification is feasible to adopt tailored therapy for complete remission [CR] success and OS estimation. In this study, our primary aim was to investigate clinical, cytogenetic and molecular predictors for CR and OS outcome among s-AML patient (pt). Methods: After IRB approval, 178 AML pt diagnosed in Baylor College of Medicine institutions [Michael E. DeBakey VA Houston and Baylor St. Luke's Medical Center] were screened. 68/178 [38%] s-AML pt were selected, however, 55/68(81%) had data available for analysis. Variables with known predictive value for AML prognosis were investigated including age, hemoglobin, white blood cell count [WBC], marrow blast percentage, pre-existing disease [MDS v MPN], karyotypic abnormalities and mutations obtained by next generation sequencing [NGS]. To identify cytogenetic subgroups with differential effect on survival, individual karyotypic abnormalities were "aggregated" into groups exhibiting similar OS [inferior v intermediate v superior survival, see Figure 1, group 0 v 1 v 2]. Kaplan Meier method was used to evaluate differential significant OS among cytogenetic groups. Linear regression model examined independent prognostic effect of "proposed cytogenetic stratification" when adjusting for potential confounders selected from our univariate analysis. Statistical analysis was performed with SAS software, NC, USA and Prism 5. Result: Median age of pt was 67 years (range 42-91). S-AML pt was Male 52/55 (96%), and white 36/55 (65%). Baseline characteristic for the 3 cytogenetic groups are depicted in Table 1. Median OS for all s-AML pt was 90.5 days [d] (range: 3-644). Pre-existing disease was available in 51/55 (93%) pt [10/51 (20%) MPN, 36/51 (70%) MDS, and 5/51(10%) t-AML (p=0.69)]. Intense [7+3] v less intense [hypomethylating agents (HMA)] therapy was delivered in 24/43 (56%), and 16/43 (37%), respectively. Karyotypic abnormalities were 5/55 (9%) monosomy 7, 23/55 (42%) CK [typical 14/23 (61%), and atypical 9/23 (39%)], and 27/55 (49%) abnormalities not classifiable as favorable or adverse (NC or &lt;3 abnormalities not including -5q, -7q, and/or -17p). Median OS for group 0 [all -7] was 70 d (5-78). In intermediate cytogenetic, median OS was 122 d (3-405) [typical + atypical CK were aggregated in group 1 given equal OS (140 d vs 102 d, respectively, p= 0.3)]. Group 2 [NC +others] median OS was 160 d (3-644) [ANOVA, 0 v 1 v 2, p= 0.03] (Figure 1). Complete remission/complete remission incomplete [CR/CRi] rate was 0%, 30%, and 39% for 0 v 1 v 2 [0 v 1 p=0.1 and 0 v 2, p=0.08]. Importantly, in group 2, CR/CRi was 66% v 36%, in pt &lt; and &gt;60 y, respectively, p=0.05. Superior CR/CRi in pt &lt;60 y was associated with absence of high-risk mutations [p53, ASLX1, RUNX1], p=0.008. Proposed cytogenetic stratification was the only independent predictor for survival among pt diagnosed with s-AML when controlled for all potential confounders (p= 0.015; 95% CI, 0.05-0.48). Conclusion: In s-AML with heterogeneous pre-existing disease and cytogenetic background, our "proposed cytogenetic stratification" retains ability to discriminate differential OS and odds for CR/CRi success. Our data adds "body of evidence" for selection of "differential" induction in s-AML cytogenetic subgroups predicted to have inferior CR/CRi and OS [i.e. HMA+ Venetoclax vs CPX 351 formulation, instead of standard 7+3]. It is conceivable that in pts with s-AML younger than 60 y who are assigned into cytogenetic subgroup 2 without high-risk mutations, 7+3 is reasonable induction while bridging toward allogeneic stem cell transplant. Disclosures Yellapragada: Novartis: Employment, Other: Spouse Employment ; Celgene: Research Funding; BMS: Research Funding; Takeda: Research Funding.

Cytogenetic Characteristics and Outcomes of Patients Receiving CTL019 CAR T Cell Therapy

Background: CTL019 is a therapy derived from autologous T cells expressing a CD19-specific chimeric antigen receptor (CAR) that was approved by the FDA in August 2017 (tisagenlecleucel). Complete and durable remissions have been seen in the setting of pediatric and young adult patients with relapsed and refractory B cell acute lymphoblastic leukemia (ALL) (Maude NEJM 2018). Initial case reports suggested that there may be differential outcomes mediated by cytogenetic characteristics of the leukemia at CAR T cell infusion. Here, we report results from a single institution experience of 112 patients. Methods: Patients with relapsed/refractory ALL were identified as having received CTL019 either in the context of a clinical trial (NCT02906371) or commercial product (tisagenlecleucel) at Children's Hospital of Philadelphia from October 2016 to April 2019. Patients who received prior CAR T therapy were excluded. Demographic, cytogenetic, and outcome data were manually abstracted from the medical record or clinical trial datasets. High risk lesions were defined as MLL(KMT2A) rearrangements, Philadelphia-chromosome (Ph+), Ph-like, hypodiploidy, and TCF3/HLF fusion. Favorable cytogenetics were defined as the presence of hyperdiploidy or ETV6/RUNX1fusion and intermediate were defined as iAMP21, IKZF1deletion, or TCF3/PBX1. Patients were classified according to their highest risk cytogenetic characteristic and stratified by cytogenetic risk category present at CAR T cell infusion. Relapse-free survival (RFS) and overall survival (OS) was described for cohorts with more than 10 patients. Results: One hundred and twelve patients were included in the analysis, with a median age of 11 years (range 1-29) at infusion, of which 32% had had a previous allogeneic hematopoietic stem cell transplant (alloHSCT). Disease burden at the time of CTL019 infusion was heterogenous, with 61% having detectable disease in the bone marrow and 21% having more than 25% blasts by flow cytometry. Thirty-six patients (32%) had leukemias with high-risk genetic lesions at infusion, including 12 with MLL rearrangements and 18 with Ph+ or Ph-like lesions (Table 2). Thirty-one patients (28%) had hyperdiploidy or ETV6/RUNX1; 3 additional were in conjunction with high-risk cytogenetics (t(17;19) and 2 with Ph+), and 3 in the setting of intermediate-risk cytogenetics (iAmp21, TCF3/PBX1, IKZF1deletion). Figure 1 demonstrates RFS for those patients in remission at day 28 following infusion, stratified by cytogenetic risk category. Complete remission (CR) rate in the high-risk cytogenetics group was 94%. RFS at 12 months was 69% (0.50-0.82), 69% (0.40-0.86), and 67% (0.48-0.80) for non-informative, favorable, and high-risk cytogenetic groups, respectively. Figure 2 shows OS of patients infused with CTL019, again stratified by cytogenetic categories of interest, with a maximum follow-up time of 30 months. OS at 12 months was 84% (0.68-0.93) and 76% (0.56-0.88) for the non-informative and high-risk cytogenetic groups, respectively. There were no deaths in that time period for the favorable risk category. There was no statistically significant difference in RFS or OS for patients with high-risk cytogenetics. The intermediate-risk cytogenetics group (n&amp;lt;10) was excluded from these analyses. Conclusion: Durable remissions can be achieved with CTL019 across several high-risk cytogenetic subtypes of B-ALL. Stratifying outcomes by cytogenetic risk category in this unadjusted analysis does not show a statistically significant difference in either RFS nor OS. Further investigation is needed to parse out the contribution of individual cytogenetic lesions as well as the effects of other relapse and survival risk factors at play. Figure Disclosures Rheingold: Novartis: Consultancy; Pfizer: Research Funding. Callahan:Novartis: Consultancy. Hunger:Bristol Myers Squibb: Consultancy; Amgen: Consultancy, Equity Ownership; Jazz: Honoraria; Novartis: Consultancy. Grupp:Novartis: Consultancy, Research Funding; Roche: Consultancy; GSK: Consultancy; Cure Genetics: Consultancy; Humanigen: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards. Maude:Kite: Consultancy; Novartis: Consultancy.

Oncogenic MAF in Co-Operation with IRF4 Confers Extensive Chromatin Re-Arrangement in Plasma Cells and Generates ‘Neo-Enhancers’ That Regulate Genes Critical for Myeloma Biology

Overexpression of the transcription factor MAF, as a result of its juxtaposition to the IgH enhancer [MAF-translocated t(14;16)], is a myeloma-initiating event in 3-5% of patients with multiple myeloma (MM) and confers a poor prognosis. MAF is also overexpressed in another 40% of cases, often in co-operation with the oncogene MMSET. The mechanisms by which MAF overexpression impacts on the regulatory genome to generate the MAF-driven oncogenic transcriptome and its direct targets are not known. To address this, we employed a multi-layer -omics approach using primary myeloma plasma cells (PC) as well as myeloma cell lines (MMCL). First, we determined the chromatin accessibility and transcriptome profiles of MAF-translocated myeloma by performing ATAC-seq and RNA-seq, respectively, in purified bone marrow CD138+ PC from two patients with t(14;16) and three healthy donors. We identified 6,640 differentially accessible regions, 87% of which displayed enhanced chromatin accessibility in MAF samples compared to normal PC. Secondary analysis comparing this with ATAC-seq data from a set of 28 other MM samples, including hyperdiploid, MMSET and CCND1-translocated MM, revealed 33% of those regions to be MAF subgroup specific (1,949 regions), with the rest shared between MAF and other cytogenetic groups. Gene annotation and pathway enrichment analysis using GREAT confirmed overrepresentation of the MF myeloma patient signature, as previously identified in microarray datasets. RNA-seq detected significant upregulation of approximately 900 genes in MAF samples compared to normal counterparts, including MAF itself (top 4th hit) as well as its presumed targets (CCND2, ITGB7 and NUAK1). Next, we obtained the MAF cistrome using ChIP-seq in the MAF-translocated MMCL MM1.S and integrated it with the primary PC ATAC-seq data. This revealed that 31% (618/1,949) of the differentially accessible regions in MAF-translocated MM PC are also MAF-bound. Additional overlay with ENCODE ChromHMM epigenome map showed that 47% of MAF binding sites are on active enhancers and 42% on active promoters signifying potential direct regulation of the corresponding genes. Next, we superimposed the accessible and MAF-bound loci on the epigenomic landscapes of normal PC and other B-cell types using their corresponding ChromHMM maps (Blueprint consortium data). Interestingly, 56% (345/618) of the MAF-specific regions were not active in any stage of B cell development. This suggests that aberrant MAF overexpression and chromatin binding in PC is associated with de novo activation of these chromatin regions, over half of which (200/345; 58%) are enhancers; we termed these 'neo-enhancers'. Upon de novo motif analysis of MAF ChIP-seq in MAF-translocated JJN3 and MM1.S MMCL, we confirmed MAF as the first and, interestingly, IRF4 as the second top hit, suggesting a possible MAF-IRF4 functional interaction in myelomagenesis. Indeed, overlay of the accessible MAF-bound loci with IRF4 ChIP-seq data in MM1.S revealed 63% co-occupancy (including 62% of "neo-enhancers"), proposing a novel and extensive co-operative chromatin-based network between the two transcription factors. Final integration of the accessible MAF-bound regions with the paired transcriptomes of primary myeloma PC revealed a set 206 candidate enhancer-gene pairs. Strikingly, we identified two IRF4-cobound "neo-enhancers" linked to overexpression of TLR4 and CCR1, two genes known for their roles in myeloma cell proliferation and migration. We confirmed significant downregulation of both genes upon shRNA-mediated knockdown of MAF in the two MAF-translocated MMCL, MM1.S and JJN3, as well as the lethality of MAF depletion. Further, MAF overexpression in MAF-negative myeloma backgrounds led to transcriptional upregulation of these genes, further validating them as MAF targets. While CRISPR/Cas9i experiments targeting TLR4 are ongoing, preliminary results validated the functional role of the "neo-enhancer" in CCR1 gene expression. In conclusion, we demonstrate for the first time an extensive re-organisation of the PC chromatin conferred by oncogenic MAF in MM; we reveal its extensive co-operation with IRF4 in this process; we validate the directly MAF-regulated genes and functionally characterise neo-enhancers of key MAF-dependent genes that in addition to MAF itself are also critical for myeloma biology. Disclosures Hatjiharissi: Janssen: Honoraria. Caputo:GSK: Research Funding. Karadimitris:GSK: Research Funding.

TP73 As Novel Determinant of Resistance to BCL-2 Inhibition in Acute Myeloid Leukemia

TP73 As Novel Determinant of Resistance to BCL-2 Inhibition in Acute Myeloid Leukemia

Influence of Cytogenetics in Patients with Relapsed Refractory Multiple Myeloma Treated with Oral Selinexor and Dexamethasone: A Post-Hoc Analysis of the STORM Study

Influence of Cytogenetics in Patients with Relapsed Refractory Multiple Myeloma Treated with Oral Selinexor and Dexamethasone: A Post-Hoc Analysis of the STORM Study

Efficacy of Upfront Daratumumab Combination Regimen in Patients with Newly Diagnosed Multiple Myeloma

Introduction: Management of newly diagnosed multiple myeloma (NDMM), which accounts for 1% of all cancers, is an area in dire need of therapeutic innovation. In recent years, the introduction of novel agents is one of the major advances in the management of patients with NDMM, in both transplant- eligible and transplant- ineligible candidates. Studies have combined daratumumab, a human IgGκ monoclonal antibody that targets CD38 which is highly expressed on myeloma cells, with proteasome inhibitors and immunomodulatory agents-based regimens in the first-line treatment of NDMM. The purpose of our study is to explore and consolidate the efficacy of upfront daratumumab combination regimen in patients with NDMM. Methods: We systematically conducted a comprehensive literature search using MEDLINE, EMBASE databases and meeting abstracts from inception through June 2019. Phase III RCTs utilizing daratumumab in patients with newly diagnosed/ untreated multiple myeloma were incorporated in the analysis. A generic inverse variance method was used to calculate the estimated pooled hazard ratio (HR) for progression-free survival (PFS) with 95% confidence interval (CI). Mantel-Haenszel (MH) method was used to calculate the estimated pooled risk ratio (RR) for overall response rate (ORR), including stringent complete response (sCR), CR and very good partial response (VGPR). Heterogeneity was assessed with Cochran's Q -statistic. Random effects model was applied. Results: Three phase III RCTs with a total of 2,528 patients with NDMMwere included.Studies compared daratumumab (D) + bortezomib (V) + melphan (M) + prednisone (P) vs VMP, D + lenalidomide (R) + dexamethasone (d) vs Rd, and DVd + thalidomide (T) vs VTd. The randomization ratio was 1:1 in all studies. The I2statistic for heterogeneity was 0, suggesting homogeneity among RCT. The pooled HR for PFS was statistically significant at 0.52 (95% CI: 0.44-0.61; P &lt; 0.0001). The PFS benefit was observed in all ISS categories, types of immunoglobulin (Ig) and standard risk cytogenetic; ISS I cohort (HR, 0.55; 95% CI: 0.37- 0.82; P = 0.003), ISS II cohort (HR, 0.43; 95% CI: 0.33- 0.55; P &lt; 0.0001), ISS III cohort (HR, 0.63; 95% CI: 0.48- 0.82; P = 0.0006), IgG cohort (HR, 0.56; 95% CI: 0.40- 0.77; P = 0.0003), non-IgG cohort (HR, 0.52; 95% CI: 0.28- 0.97; P = 0.04), and standard risk cytogenetic cohort (HR, 0.43; 95% CI: 0.35- 0.53; P &lt; 0.0001). The pooled HR for PFS in high risk cytogenetic cohort was not statistically significant at 0.76 (95% CI: 0.53- 1.10; P = 0.15). The pooled RR for ORR was 1.13 (95% CI: 1.01-1.26; P = 0.03), sCR was 2.02 (95% CI: 1.33-3.08; P = 0.001), CR was 1.46 (95% CI: 1.20-1.79; P = 0.0002),and VGPR was 1.01 (95% CI: 0.82-1.25; P = 0.93). The pooled RR for negative minimal residual disease (MRD) was 2.54 (95% CI: 1.24-5.20; P = 0.01). Conclusions: Upfront combination regimen with daratumumab significantly improved PFS, ORR, sCR and CR along with negative MRD, compared to control arm in patients with NDMM. The improvement in PFS was noted across all subgroups except in high-risk cytogenetic group. More randomized studies are required to explore further novel agents and to formulate optimal combination regimen to improve survival in this high-risk cytogenetic subset. Disclosures No relevant conflicts of interest to declare.

A Comprehensive Study of Human Integrins in Pediatric Lymphoblastic Leukemia Supports a Role of CD49f (Integrin α6) in the Localization to Bone Marrow but Not Spinal Fluid

Introduction: The overall survival of children with ALL (Acute Lymphoblastic Leukemia) has improved markedly over the last decade, yet relapse occurs in about 20% of patients. The spread of leukemic blasts to the Central Nervous System (CNS) and increased resistance to therapy due to cell adherence within the Bone Marrow (BM) or CNS constitutes major challenges to treatment. For these reasons, adhesion molecules governing the homing and adhesion of leukemic cells are perceived to be of extraordinary importance, both as potential biomarkers and therapeutic targets. Integrins constitute a large family of heterodimeric receptors composed of alpha and beta subunits, which play important roles during homing and migration of normal leucocytes by facilitating adhesion to both stromal cells and components of the extracellular matrix. Increased expression of CD49d (integrin subunit α4) is a marker for adverse prognosis in ALL and recently, CD49f (integrin subunit α6) was shown to facilitate metastasis of ALL xenografts to the central nervous system in mice (Yao H et al., Nature 2018). Methods Previous studies of integrins in BCP-ALL have focused on individual alpha integrins in xenograft models and were based on limited numbers of clinical samples. The present study was based on a large number of Danish pediatric BCP-ALL patients stratified between 2012-2018 using Minimal Residual Disease (MRD) according to the Nordic NOPHO-2008 protocol (Toft N et al., Eur J Haematol 2013). Diagnostic BM samples were subjected to flowcytometric analysis (FCM) of CD49f (n=246) and CD49d (n=135), using a backbone of lineage-specific B-cell markers (CD45, CD10, CD19, CD20). Leukemic blasts were detected in Cerebrospinal Fluid (CSF) using high-sensitivity FCM and the following markers CD45, CD10, CD19, CD20, CD34 and CD38 (n = 246, with matching BM and CSF samples). Results Our data provided us with a unique possibility to identify the role of CD49d and CD49f with respect to minimal residual disease (MRD) at the end of induction therapy (day 29), which is considered the most important prognostic factor in paediatric lymphoblastic leukemia. We found that CD49f was more highly expressed in patients with MRD ≥ 0,1% at day 29 than patients with MRD &lt; 0,1% (p = 0,01), whereas no difference was seen with respect to CD49d. We also investigated the correlation between white blood cell (WBC) and surface expression of CD49d and CD49f in diagnostic BM blasts with respect to different cytogenetic subtypes. A Kruskall-Wallis test showed that the expression varies according to genetic subtypes (p‹0.0001). We found that the expression of CD49d was highest among the high hyperdiploid and iAMP21 (intrachromosomal amplification of chromosome 21) patients, whereas the expression of CD49f was highest among the t12,21 and iAMP21 patients. Notably, the expression of CD49f was inversely correlated to WBC (r=0,17, p=0.01), which was most pronounced among the patients in the B-other cytogenetic subgroup defined as leukaemia that could not be classified into the existing cytogenetic groups (r=0,42, P‹0.0001). In case of strong adherence to BM, lower levels of leukemic blasts might be expected in circulation resulting in high MRD but low WBC. Therefore, both MRD and WBC data are consistent with a prominent adhesive role of CD49f within BM. In contrast, we found significantly lower CD49f surface expression in diagnostic BM samples in patients with leukemic blasts within CSF (p=0.0297). Conclusions: Recently, Yao et al. (2018) showed that ALL cells in circulation are unable to breach the blood-brain barrier in mice and instead employ CD49f (integrin α6) to migrate into the CNS along vessels that connect vertebral or calvarial bone marrow and the subarachnoid space. Potentially, this mechanism could account for cases of ALL relapse within CNS. Our work shows a strong association between high MRD and the expression of CD49f which is a function that would have been anticipated for CD49d due to previous works with ALL and CLL. Furthermore, we found significantly lower CD49f surface expression in leukemic blasts within the BM in patients with CSF involvement and therefore no support for the recently proposed role of CD49f in facilitating the spread of leukemic cells to the CNS. Disclosures No relevant conflicts of interest to declare.

PF264 TREATMENT INTENSIFICATION WITH FLAG‐IDA MAY IMPROVE DISEASE CONTROL IN YOUNGER PATIENTS WITH SECONDARY AML WHICH MANIFESTS ITSELF WITH LONGER FOLLOW‐UP: LONG‐TERM ANALYSIS OF THE MRC AML15 TRIAL

Background:Typically patients with Secondary AML do badly, even with intensive chemotherapy (typically Ara‐C + anthracycline). A recent phase 3 trial of CPX‐351, a liposomal daunorubicin/ara‐C combination, found significant benefit compared to standard DA 3+7 chemotherapy in a secondary AML population age 60–75 with prior MDS or CMML, therapy related AML, or an MDS‐related karyotype (mOS 9.6 vs 6.0 m p = 0.005). It is unclear whether DA is an appropriate comparator for younger patients or if they do better with more intensive chemotherapy such as FLAG‐Ida.Aims:To evaluate the outcomes of secondary AML patients aged 16–59 enrolled in the UK MRC AML15 trial which randomized 1736 patients between DA/DAE (daunorubicin, araC ± etoposide) and FLAG‐Ida.Methods:Patients aged 16–59 with secondary AML were included, with performance status of ≤2. In AML15 patients were randomised between 2 courses of either DAE/DA (3+10/3+8) or FLAG‐Ida (Burnett et al JCO, 2013). Survival was evaluated using the Kaplan‐Meier method and compared using stratified logrank tests and Cox regression for multivariate analyses. Median follow‐up was 115.6 months (range 8.4–145.2). Outcomes are reported at 5 years. An odds ratio or hazard ratio &lt;1 indicates better outcomes for FLAG‐Ida.Results:A total of 115 patients were identified (73 DA/DAE, 42 FLAG‐Ida). Median age was 52 (range 16–59) y; 52% were male; median WBC was 6.4x109/L (range 0.2–260.0); 10%, 65% and 25% had favourable, intermediate and adverse karyotype. There was some evidence that patients given FLAG‐Ida had worse risk karyotype (p = 0.10): analyses are presented both raw and stratified by cytogenetics. CR/CRi rate was 80% (DAE/DA 79%, FLAG‐Ida 81%, unadj. OR 0.91 (0.35–2.34) p = 0.8; strat. OR 0.54 (0.19–1.55) p = 0.3). 5‐year survival was better for patients treated with FLAG‐Ida (median OS 20.2 m/5‐y OS 27% vs 20.6 m/37%, unadj. HR 0.81 (0.52–1.26) p = 0.4) which was significant in stratified analyses (strat HR 0.54 (0.33–0.90) p = 0.02). RFS was improved with FLAG‐Ida (22% vs 41%; unadj. HR 0.70 (0.43–1.15) p = 0.16; strat HR 0.54 (0.31–0.96) p = 0.04). In a multivariable analysis of RFS, only cytogenetic group (HR 2.06 (1.21–3.51) p = 0.03) and FLAG‐Ida (HR 0.52 (0.29–0.95) p = 0.03) emerged as significant prognostic factors. Allografts in first CR occurred in 24% of cases (21% vs 31% p = 0.2). In a landmark analysis at 120 days post remission, survival in the transplanted group was better than the non‐transplanted group after adjustment for cytogenetics (43% vs 32%, unadj. HR 0.80 (0.45–1.44) p = 0.3, strat HR 0.48 (0.23–0.98) p = 0.04).Summary/Conclusion:Outcomes in this group of patients are poor with DA/DAE chemotherapy, with median OS of 20.2 m and 5‐y/10‐y survival of 27%/21%. Despite similar median OS (20.6 m) FLAG‐Ida showed a significant improvement in survival in analyses adjusted for the imbalance in cytogenetic risk group with 5‐y/10‐y survival of 37%/31%. While numbers are small, and a greater number of patients received an allograft in first remission in the FLAG‐Ida arm than in the DA/DAE group, there is a suggestion that recipients of an allograft had better outcomes, after adjustment for cytogenetic risk. The emergence of a survival difference after 2 years shows the need for long‐term follow‐up when considering questions of treatment intensification. The improved results with FLAG‐Ida show that DA may not be an appropriate standard of care in younger patients with secondary AML, and that thought should be given to FLAG‐Ida as comparator in this younger population. The NCRI AML19 is currently randomising CPX‐351 versus FLAG‐Ida.image

PF216 ROLE OF MEASURABLE RESIDUAL DISEASE (MRD) IN REDEFINING COMPLETE RESPONSE (CR) IN ELDERLY PATIENTS WITH ACUTE MYELOID LEUKEMIA (AML): RESULTS FROM THE PETHEMA‐FLUGAZA PHASE III CLINICAL TRIAL

Background:The general outcome of elderly patients with AML is poor, even for those achieving morphological CR. However, the role of MRD in redefining CR remains poorly investigated in elderly AML due to the reluctance of treating with intensive chemotherapy and the renewed interest in low‐intensity therapy such as hypomethylating agents (HMA).Aims:To help defining the role of MRD assessment by multidimensional flow cytometry (MFC) and therapeutic decision making in older AML patients treated with semi‐intensive chemotherapy vs HMA.Methods:285 AML patients (median age, 75) were included in the phase 3 PETHEMA‐FLUGAZA trial and were randomized to receive 3 induction cycles with fludarabine and cytarabine (FLUGA) followed by 6 consolidation cycles with reduced intensity FLUGA, vs 3 induction cycles with 5‐azacitidine (AZA) followed by 6 consolidation cycles with AZA. After consolidation, patients continued with the same treatment if MRD ≥0.01% or stopped if MRD &lt;0.01%. MRD was prospectively assessed after induction and consolidation among patients in CR with or without incomplete blood count recovery. At diagnosis, the EuroFlow AML panel was used to identify leukemia‐associated immunophenotypes (LAIP). At CR, bone marrow samples were stained with 8‐color combinations based on previously identified maturation arrest, lineage commitment and LAIPs. MRD was assessed with an estimated sensitivity of 0.01%.Results:On intention‐to‐treat, 38/141 (27%) patients achieved CR after 3 cycles of FLUGA and 31/144 (21.5%) after 3 cycles of AZA (p = .33). Among patients in CR with previously identified LAIP, 14/69 (20%) achieved a negative MRD status whereas the remaining 55 (80%) had persisting MRD: 56% with ≥0.1% MRD and 24% with &lt;0.1% MRD. Regarding the effect of semi‐intensive chemotherapy vs HMA on depth of response, we observed a non‐significant trend toward higher MRD‐negative rates among patients in CR after FLUGA vs AZA (25% vs 15%, respectively; p = .38).The 2‐year rates of cumulative incidence of relapse (CIR) for MRD‐positive ≥0.1%, &lt;0.1% and MRD‐negative patients were 95%, 74% and 45%, respectively (HR, 0.45; p &lt; .001). Of note, the CIR of patients in CR but persistent MRD were similarly poor as compared to those in partial remission (HR, 1.2; p = .54). Furthermore, MRD‐positive patients with adverse cytogenetics displayed the poorest outcome with significantly higher 2‐year CIR rates than MRD‐positive cases with intermediate/favorable cytogenetics (HR, 2.0; p = .01). Among patients in CR and persistent MRD, 2‐year CIR rates showed a non‐significant trend towards slightly more frequent relapses in those treated with FLUGA vs AZA (91% vs 77%, respectively; HR, 1.6; p = .12). On multivariable analysis for CIR including MFC‐MRD and cytogenetics, both retained significant prognostic value. The median overall survival (OS) for MRD‐positive ≥0.1%, &lt;0.1% and MRD‐negative patients was 13, 12 and 21 months, respectively. On multivariable analyses for OS including age, WBC count, cytogenetic grouping and secondary disease, persistent MRD showed a trend for independent prognostic value (HR, 2.4; p = .08).Summary/Conclusion:This study reveals that sensitive MFC‐MRD assessment supersedes CR and is an independent prognostic factor in older patients with AML, treated with semi‐intensive chemotherapy or HMA. Nevertheless, the risk of relapse among the few patients with no MRD (5%) remains high after stopping treatment, and warrants innovative approaches aimed at maintaining an MRD‐negative CR status. Updated follow‐up and molecular data will be presented at the meeting.

PB2094 A SINGLE CENTRE EXPERIENCE WITH AZACITIDINE FOR MDS, AML AND CMML IN RELATION TO CYTOGENETIC RISK AND PERFORMANCE STATUS

Background:Azacitidine is a hypomethylating agent which is used in the treatment of myelodysplastic syndromes (MDS), acute myeloid leukaemia (AML) and chronic myelomonocytic leukaemia (CMML). In the AZA‐001 trial, azacitidine altered the natural history of patients with higher‐risk MDS (Fenaux et al., Lancet 2009) by significantly improving overall survival (OS) compared to conventional care regimens (24.5 months vs 15.0 months) and in particular improved OS in the poor risk cytogenetics group (17.4 months vs 6 months) which led to its NICE approval in 2011.Aims:We report a single centre experience over 4 years of using azacitidine in the licensed NICE indications and compared results to the published data.MethodsWe retrospectively reviewed patients with MDS, AML and CMML who received azacitidine at Ipswich Hospital NHS Trust between November 2014 and November 2018. Data was collected on demographics, disease characteristics, treatment, blood and platelet transfusion response, disease progression and cause of deathResults:Thirty‐nine patients met the NICE criteria for treatment and were included in the analysis, irrespective of the number of cycles received. Median age was 72.2 years. Male: Female ratio was 2:1. Median number of cycles was 9 (range from 1‐43). 51% of patients had a diagnosis of MDS, 31% AML and 18% CMML. 25.6% had poor risk cytogenetics as per the IPSS criteria. Median percentage of blasts on bone marrow aspirate was 15.7%. Causes of death were either disease progression or sepsis with only one death unrelated to the underlying condition (lung cancer). Median Kaplan‐Meier OS for all groups from the start of treatment with azacitidine was 18 months, with MDS patients OS of 22 months compared to 18 months for AML and 13 months for CMML (p = 0.22). There was a significant difference noted in the group who had poor risk (IPSS) cytogenetics versus other cytogenetic risks, with a median OS of 8 months and 20 months respectively (p = 0.002). A significant difference was also seen in the median OS based on performance status (PS), with an OS of 7 months, 20 months and not yet reached for PS 2, 1 and 0 respectively (p = 0.005).Summary/Conclusion:In the AZA‐001 trial median OS on azacitidine was 24 months vs. 18 months seen in our cohort. The AZA‐001 trial also showed improved OS for poor risk cytogenetics at 17.4 months vs. only 8 months seen in our experience. Although our data does support an overall improvement in median OS for all groups, our data showed less impressive results, with a 6 months less survival advantage in all groups and 9 months less OS in the poor cytogenetic group as compared to the trial data. As previously shown, we observed that the most significant factors in predicting OS were cytogenetic risks and PS. Interestingly, a large real world registry collection in Canada of 1101 patients on azacitidine over 6 years only showed a median OS of 12 months for all groups which further highlights the difference we often see between real world patients and trial data.