Myeloid Neoplasms In Patients Research Articles

Hematologic Malignancies Arising in Patients with Germ Cell Tumors: Secondary Somatic Differentiation of Hematopoietic Malignancies from Germ Cell Precursors

Genomic analyses have recently illuminated our understanding of therapy-associated myeloid neoplasms in patients receiving therapy for other cancers. One of the most intriguing relationships between solid tumors and myeloid neoplasms involves a unique clinical entity of patients with germ cell tumors (GCT) and myeloid neoplasms. One in 17 patients with primary mediastinal germ cell tumor (PMGCT) develops a hematologic malignancy (most commonly AML, MDS, or histiocytosis) and the median survival in such patients is poor at only 5 months. Intriguingly, the presence of isochromosome 12p [i(12p)], a clonal marker common to GCTs but absent in de novo myeloid neoplasms, has been demonstrated as shared across GCTs and myeloid neoplasms in such individuals. While these data suggest a clonal relationship between the two, the exact nature of the shared origin is unknown. There are two competing hypotheses to explain this: (1) an embryonic progenitor with the capacity to differentiate into germ cell and hematopoietic lineages harbors the initiating genetic alterations and drives development of both malignancies or (2) the leukemia is derived directly from GCTs with the capacity to differentiate into hematopoietic lineages. To trace the clonal evolution of these seemingly unrelated cancer types and identify recurrent genomic lesions in leukemias present in GCT patients, we applied whole exome sequencing (WES), targeted genomic analyses, and/or RNA-seq to leukemias, GCTs, and germline DNA in a series of patients with myeloid neoplasms and concurrent GCTs.We collected 12 patients with GCT and synchronously or metachronously occurring myeloid neoplasms (8 AML, 5 MDS/CMML, 2 histiocytic sarcoma (some had >1 hematologic malignancy)) with an average of 4 months between the two diagnoses. Consistent with prior reports, all were young men (median age 26) with PMGCT and nonseminomatous histology and a 3-month median survival from leukemia diagnosis (Fig. A). In each case, at least one copy number alteration or coding mutation was shared across the GCT and hematopoietic neoplasm, demonstrating the shared origin of both lesions. For example, half of the patients (6/12) carried i(12p) in both the GCT and hematopoietic neoplasm. In the i(12p) negative cases, somatic genetic alterations identified in the GCT were also found in the leukemia. The most common genomic alterations in leukemias beyond i(12p) included mutations activating RAS-PI3K-AKT signaling (including PTEN, RAS and PI3K isoform mutations) or inactivating TP53 (Fig. B). The only exception was a testicular-only GCT patient who developed clonally distinct acute promyelocytic leukemia; however, further analysis identified this as a chemotherapy-induced neoplasm with the PML-RARa breakpoint mapped to an etoposide sensitive area and this patient was not counted amongst the 12 cases.We next traced the evolutionary history of clonally related GCTs and leukemias based on cancer cell fraction of all coding mutations and copy number alterations using WES of DNA from each tumor type and finger nails. In each instance, we identified clonal evolution of the hematopoietic malignancies from antecedent precursors within the GCT. To illustrate this, a 19-year-old male developed successive diagnoses of histiocytic sarcoma, CMML, and AML within 18 months of GCT diagnosis. Lineage tracing by WES of each of these four individual cancers revealed that all four were clonally related, and the histiocytic sarcoma, CMML, and AML were all derived from the GCT with a common precursor giving rise to the three hematopoietic malignancies (Fig. C-D). Moreover, the histiocytic sarcoma evolved separately from CMML/AML in this patient, where the AML represented leukemic transformation from the CMML.These data conclusively demonstrate that myeloid neoplasms developing in patients with PMGCT represent secondary somatic differentiation of a hematologic progenitor from totipotent aberrant cells that are present in the GCT. Thus, GCT-associated leukemias have a unique ontogeny from de novo and/or secondary myeloid neoplasms, which originate from progenitors within the bone marrow. As such, GCT-associated leukemias have characteristic genomic alterations hallmarked by frequent i(12p) in combination with mutations activating RAS-PI3K-AKT signaling and inactivating TP53, and these patients do poorly even when treated with aggressive contemporary chemotherapy. DisclosuresRampal:Jazz: Consultancy, Honoraria; Celgene: Honoraria; Constellation: Research Funding; Incyte: Honoraria, Research Funding; Stemline: Research Funding. Tallman:ADC Therapeutics: Research Funding; Orsenix: Other: Advisory board; AROG: Research Funding; Cellerant: Research Funding; AbbVie: Research Funding; Daiichi-Sankyo: Other: Advisory board; BioSight: Other: Advisory board.

Targeted Next Generation Sequencing for Myeloid Neoplasm in Pakistani Patients

Introduction:Myeloid malignancies are heterogenous diseases caused by excessive accumulation of apparently myeloid clone of cells. Genomic studies on myeloid malignancies in recent years have identified new genetic alterations with biological and clinical significance. In addition to cytogenetics and morphological examination these genetic mutation play an important role in diagnosis, prognosis and treatment of the patient. We assessed the frequency and clinicopathologic significance of 54 genes in myeloid neoplasm patients by using targeted next-generation sequencing.Methods:About 50 samples were collected from OPD at National Institute of Blood Diseases (NIBD), that consisting of 17 MDS, 18 AML and other myeloid neoplasms. They comprises of 33 males and 17 females with median age of 33 years (range: 5-69 years). The myeloid sequencing panel of 54 genes (complete coding exons of 15 genes and exonic hotspots of 39 genes) was sequenced. The panel total coverage was 141 kb in genomic sequence. TruSight myeloid sequencing (Illumina, CA) libraries were prepared and runs were performed on a MiSeq (Illumina) genome sequencer. The generated data were analyzed by on-instrument software or TruSeq Amplicon® and BaseSpace Apps®.Results:Overall 3092 variants were identified, after excluding intronic and synonymous variants, 380 missense variants were found in 50 patients. Around 38 mutations in 22 genes were identified in 23 out of 50 samples (46 %). The recurrent mutations found in RUNX1, ASXL1, GATA2 and CEPBA genes in our cohort.Conclusion:Most of the myeloid neoplasms are not easily manageable with limited treatment options. Therefore, targeted gene panel by next generation sequencing was an appropriate method for precise identification of mutations in myeloid neoplasms at our institution. Based on the obtained findings we will be able to design patient management plan with respect to individualize genetic mutations in the clinical setting. DisclosuresNo relevant conflicts of interest to declare.

TP53 Mutations in Myeloid Neoplasm Patients with and without Significant Personal and Family History of Cancer

TP53 Mutations in Myeloid Neoplasm Patients with and without Significant Personal and Family History of Cancer

Next-Generation Sequencing of DDX41 in Myeloid Neoplasms Leads to Increased Detection of Germline Alterations

Introduction: Germline predispositions to hematologic malignancies were historically thought to be rare; however growing awareness has raised clinical challenges regarding how to identify, test, and manage these patients. Germline mutations in the gene DDX41 predispose to moderately increased lifetime risks of MDS and AML with a later age of onset. Optimal clinical care of these patients relies on identifying germline mutations and innovative strategies are needed to improve clinical detection.Methods: 1,262 individuals with myeloid malignancies underwent next-generation sequencing (NGS)-based molecular sequencing of DDX41. Individuals identified to have ≥1 DDX41 alterations present at >40% variant allele frequency (VAF) in the bone marrow were flagged for potential referral to genetic counseling (GC). All individuals referred for GC underwent standard genetic counseling evaluation and were offered DDX41 germline analysis on cultured skin fibroblasts.Results: Of 1,262 individuals, 32 (2.5%) were identified to have ≥1 somatic DDX41 mutation(s). Fourteen (44%) were referred for GC and germline confirmation testing. Eleven patients were male (78.5%) and 13/14 (93%) were Caucasian. Average age at diagnosis of myeloid neoplasm was 65 years (range 53-77 years). Fifty-seven percent (8/14) individuals were diagnosed with AML, 6/14 presented with MDS, including therapy-related MDS. 12/14 patients had diploid cytogenetics at presentation. A second somatic DDX41 mutation (biallelic) was identified in 10/14 (71%). There were no other significantly recurrent concomitant somatic mutations. Thirteen patients underwent germline evaluation and 12/13 (92%) were confirmed to have a germline DDX41 mutation. Six individuals underwent hematopoietic stem cell transplantation (SCT); five from a matched related donor, and in four cases, the related donor was negative for the familial DDX41 mutation. Six patients (43%) reported antecedent cytopenias: five with leukopenia and one with anemia. Five patients had a prior history of malignancy: three with prostate cancer, one with Non-Hodgkin's lymphoma and melanoma, and one with MGUS. 13/14 (93%) patients reported a family history of cancer, six (43%) of which included hematologic malignancies and/or cytopenias. From the 12 DDX41 germline-positive patients, 11 unaffected relatives underwent genetic testing. Four (36%) tested positive for the familial DDX41 mutation and seven (64%) tested negative.Conclusions: The detection of somatic DDX41 mutations at near-heterozygous frequencies on NGS panel testing is highly suggestive of a germline mutation and germline testing is strongly recommended. Our data validates existing reports in DDX41 germline patients including primarily high grade myeloid neoplasms, diploid cytogenetics, and later age at diagnosis. Interestingly nearly half of our patients had antecedent cytopenias, most often leukopenia. NGS screening for DDX41 mutations through multi-disciplinary collaboration is a useful and feasible tool to screen unselected myeloid neoplasm patients for high likelihood of germline DDX41 mutations enabling timely and appropriate care of these patients. DisclosuresDaver:Novartis: Consultancy; Incyte: Research Funding; Pfizer: Consultancy; ImmunoGen: Consultancy; Pfizer: Research Funding; Sunesis: Consultancy; Alexion: Consultancy; Novartis: Research Funding; Sunesis: Research Funding; Kiromic: Research Funding; Karyopharm: Research Funding; ARIAD: Research Funding; Daiichi-Sankyo: Research Funding; BMS: Research Funding; Incyte: Consultancy; Otsuka: Consultancy; Karyopharm: Consultancy. Oran:AROG pharmaceuticals: Research Funding; Celgene: Consultancy, Research Funding; ASTEX: Research Funding. Kadia:Abbvie: Consultancy; Abbvie: Consultancy; Jazz: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy; Celgene: Research Funding; Celgene: Research Funding; BMS: Research Funding; BMS: Research Funding; Jazz: Consultancy, Research Funding; Novartis: Consultancy; Takeda: Consultancy; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Takeda: Consultancy. DiNardo:Karyopharm: Honoraria; Celgene: Honoraria; Bayer: Honoraria; Abbvie: Honoraria; Medimmune: Honoraria; Agios: Consultancy.

Leukemogenesis in Seven Donor Cell Derived Myeloid Neoplasms Patients. Whole Exome Sequencing Reveals Clonal Dynamics

IntroductionDonor cell derived myeloid neoplasm (DCMN), defined as the development of de novohematological malignancies from cells of donor origin,is a late complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We report on seven cases of DCMN in which whole-exome sequencing (WES) at different time-points after allo-HSCT, as well as in a sample from each donor, was performed. The ultimate objective was to accurately described the clonal architecture, spatial heterogeneity and identify somatic mutations that are induced in the process of leukemogenesis and clonal evolution of myeloid neoplasm. Donors were also analyzed to detect underlying condition predisposing to the development of DCMN.Patient and MethodsSeven patients with a confirmed diagnosis of DCMN and their donors were recruited from different Spanish institutions. This cohort included a total of 32 BM samples at different time points after allo-HSCTand one PB sample from each donor (one case received dual allo-HSCT, CB and PB from both donors were obtained), which provided a total of 40 samples.Genomic DNA samples were prepared according to Agilent SureSelect-XT Human exon 50Mb enrichment kit (Agilent Technlogies, Santa Clara, CA) preparation guide and libraries were sequenced on Illumina HiSeq platform (Illumina, San Diego, CA). DNA sequencing data from recipient post-transplant BM samples, were matched against their donor PB sample and previous post-transplant BM samples to identify the acquisition of mutations along the post allo-HSCT period.Germline variants in donors were studied in order to detect mutations that predisposed to the development of a myeloid neoplasm.The research protocol was approved by the Ethic Committee of Gregorio Marañón General University Hospital. Patients´ and donors´ information was collected from their medical records.ResultsClinical and biological characteristics of the 7 patients with DCMN and their donors are shown in Table 1.Mutational profiles obtained from the follow-up samples at different time-points post-HSCT demonstrated high intra-tumor genetic heterogeneity and clonal dynamic for all cases. The number of variants are increased over time and at the moment of DCMN diagnosis, the median number of variants was 28, ranging from 18 to 92 variants (Figure 1).WES identified in DCMN patients gene mutations commonly seen in adult AML or MDS, such as in SETBP1, DNMT3A, TET2, RUNX1, CSF3R, EP300and IDH2.In addition, others non-silent variants were acquired in all cases. Among the additional novel alterated genes, we found 23 strong candidateswith oncogenic potential. LUC7L2, NOP14, LAMA5, SKOR2, EML1, SNX13, RHPN2, IRS1, MTG2, TENM2, MEFV, GSE1, NOTCH4, DTX1, CNOT4, PNKP, GRB7,SENP7,TAF1L, ZKSCAN2, ZBTB20, ZNF461 and MEGF10.Analysis of CNVs revealed numerical alterations across the post allo-HSCT samples in patients 1, 2, 3, 4, 5 and 7. The most common chromosomal alterations in DCMN were monosomy 7 and other chromosome 7 abnormalities, which detected in the 86% (6/7) of the patients.Although none of the donors developed a myeloid neoplasm at the moment of diagnosis of DCMN in recipient, donor 1 revealed an abnormal karyotype (45,XY,-7) at the moment of the allo-HSCT. All other donors harbored at least one pathogenic or probably-pathogenic variants, most probably of germline origin, in genes involved in hematological or solid tumor predisposition.ConclusionsThe development of DCMN involves dynamic genomic processes that begin months before the clinical onset. In this study, our integrated multi-step analysis revealed the intra-tumor heterogeneity and evolutionary history of seven DCMN.The present study reveals a process of sequential clonal expansions promoted by the acquisition of somatic mutations in donor hematopoietic cells.Detection of heritable or acquired gene mutations in donors associated with predisposition to haematological malignancies could have clinical implications for the patients undergoing to allo-HSCT.Leukemic transformation ofdonor hematopoietic stem cells provides a useful in vivomodel to study the mechanisms involved in leukemogenesis.Novel approaches based on high-depth next generation sequencing to study consecutive samples from post-transplant period in these patients, appear promising to discover new genes involved in myeloid neoplasm and to decipher the mechanisms of leukemogenesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Treatment of 30 elderly patients with advanced myeloid neoplasm by allogeneic hematopoietic stem cell transplantation

目的评估异基因造血干细胞移植（allo-HSCT）治疗高龄髓系肿瘤的疗效。方法回顾性分析2014年9月至2017年9月接受allo-HSCT的30例髓系肿瘤患者的临床资料，男16例、女14例，中位年龄58（50~67）岁。急性髓系白血病（AML）18例，慢性粒-单核细胞白血病（CMML）1例，骨髓增生异常综合征（MDS）11例。移植时6例患者完全缓解，其余均为未缓解。亲缘全相合（MRD）12例，非血缘全相合（MUD）6例，单倍型12例。18例患者采用减低剂量预处理方案，12例采用了清髓性预处理方案。结果5例患者在预处理期死亡，24例患者获得造血重建，中性粒细胞、血小板中位植活时间分别为14（10~18）、15（10~19）d。6例患者发生急性GVHD（均为Ⅱ度），8例（32%）患者发生慢性GVHD（中、重度慢性GVHD各1例）。发生巨细胞病毒血症7例，肺部感染7例，皮肤带状疱疹5例，治疗后好转，未出现因感染而死亡的患者。中位随访7（0.5~38）个月，存活21例，预计2年总生存（OS）率为62.5%（95%CI 39.2%~85.8%），无白血病生存率为59.2%（95%CI 26.9%~91.5%）。单因素分析显示，移植时的合并症指数是影响OS的唯一因素。结论allo-HSCT是高龄髓系肿瘤患者比较理想的治疗选择。

A novel mutation in PTPN11 in an extremely preterm infant with suspected juvenile myelomonocytic leukemia

Juvenile myelomonocytic leukemia is a myelodysplastic/myeloproliferative neoplasm with a poor prognosis if left untreated. We report a missense mutation (c.794G>A [p.Arg265Gln]) discovered in the protein tyrosine phosphatase, nonreceptor type 11 (PTPN11) gene in an extremely preterm male infant with a mild Noonan syndrome (NS) phenotype and suspected juvenile myelomonocytic leukemia (JMML) whose clinical course was notable for severe, persistent thrombocytopenia, progressive leukocytosis, and profound respiratory failure resulting in death. This is the first such mutation described in an extremely low-birth-weight infant and the first reported case of this variant in association with both NS and JMML. Neonatologists and other clinicians, who work with preterm neonates, should maintain a high index of suspicion for myeloid neoplasms in patients with unusual, unremitting hematologic abnormalities.

TP53 gene and myeloid neoplasms

TP53 gene, an important tumor suppressor gene, has been a hot topic in tumor molecular biology. The assessment of TP53 mutations/deletions is becoming a routine clinical practice for chronic lymphocytic leukemia patients. In recent years, a number of studies have shown that TP53 mutations/deletions in myeloid neoplasms also suggests poor prognosis. Myeloid neoplasms patients with TP53 mutations/deletions show a good response to hypomethylation agents such as decitabine and azacitidine, and some novel experimental drugs targeting TP53 mutations/deletions, such as galectin inhibitor GCS-100 and BH3 mimetics are also being studied. To further understand prognostic significance of TP53 mutations/deletions in myeloid neoplasms and elaborate its potential as novel therapy target, this article reviews literatures on the relationship between TP53 mutations/deletion and prognosis of myeloid neoplasms, related molecular mechanisms and treatment progress. Key words: Genes, p53; Prognosis; Myeloid neoplasms

Down syndrome, RASopathies, and other rare syndromes

In this article we discuss the occurrence of myeloid neoplasms in patients with a range of syndromes that are due to germline defects of the RAS signaling pathway and in patients with trisomy 21. Both RAS mutations and trisomy 21 are common somatic events contributing to leukemogenis. Thus, the increased leukemia risk observed in children affected by these conditions is biologically highly plausible. Children with myeloid neoplasms in the context of these syndromes require different treatments than children with sporadic myeloid neoplasms and provide an opportunity to study the role of trisomy 21 and RAS signaling during leukemogenesis and development.

Use of 5-azacitidine for therapy-related myeloid neoplasms in patients with concomitant active neoplastic disease

BackgroundPatients diagnosed with therapy-related myeloid neoplasms (TRMN) with concomitant active neoplastic disorder (CAND) are usually proposed for best supportive care (BSC). We evaluated the feasibility of using 5-azacytidine (AZA) in this setting. MethodsAll patients referred to Gustave Roussy between 2010 and 2015 for TRMN diagnosis (less than 30% blast) and eligible for AZA treatment were included. Patients with CAND proposed for BSC were also described. Patient's outcomes were analyzed based on the presence or not of a CAND. ResultsFifty-two patients with TRMN were analyzed, including 19 patients with CAND (14 eligible for AZA) and 33 without CAND eligible for AZA. The 5 patients with CAND ineligible for AZA had a worst performance status (p=0.016) at diagnosis and a shorter overall survival (OS) (0.62 months). Baseline characteristics of patients eligible for AZA were similar in the 2 groups except a trend for best performance status in patients with CAND (p=0.06). Overall response rate (71.4% vs 60.3%), transfusion independence (50.0% vs 45.5%) and OS (12.7 months vs 10.8 months) were similar between patients with and without CAND respectively (p=ns). ConclusionHere we report the feasibility and efficacy of AZA for selected patients with TRMN and a CAND.

A Novel Somatic Mutation in IKZF1 Is Common in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplant for Myelofibrosis

Background:Myeloproliferative neoplasms (MPN) including polycythemia vera, essential thrombocytosis, and primary and secondary myelofibrosis (MF) are characterized by driver mutations in JAK2, CALR, and MPL. Additional somatic variants can be seen. Currently allogeneic hematopoietic stem cell transplant (HCT) is the only treatment with curative potential, but it is associated with a high risk of mortality, exceeding 50% in some patient subsets. The risk of disease progression usually outweighs the risk of transplant in patients with advanced MF. Here, we have evaluated the mutational profile using a targeted next-generation sequencing panel of 54 genes involved in myeloid neoplasms in patients with MF treated with HCT.Patients and Methods:48 patients with a diagnosis of primary (n=18) or secondary (n=30) MF who received a HCT at our center and from whom genomic DNA was available from pre-HCT marrow aspirates were evaluated. DNA was isolated from whole marrow aspirates obtained prior to HCT. Paired-end indexed libraries were sequenced at an average read depth of 1000x on Illumina MiSeq platform. Sequences were aligned to hg19 using the Burrows-Wheeler Aligner, and nucleotide variants were identified using the Genome Analysis Toolkit and Picard tools. Variants were annotated with ANNOVAR. Variants present at >5% variant allele frequency (VAF) with at a minimum of 25 variant reads were selected for further analysis. Variants were called "mutations" if they resulted in (1) non-synonymous changes in amino acid sequence, including nonsense and missense mutations, (2) the variant was known to be associated with hematologic malignancies in COSMIC database, or (3) the variant was present at < 0.5% in 1000 genomes and ExAC databases. Comparisons between clinical variables and mutations were analyzed with chi-square tests, adjusted for multiple testing.Results:The average age of the 48 patients was 55 years (range 23-72) and 52% of patients were female. Disease risk was estimated with DIPSS-Plus (High 33%, INT-2 53%, INT-1 7%, Low 7%). Unfavorable cytogenetics were present in 27% of patients. Donors included a matched sibling donor in 30% (n=14), a matched unrelated donor in 60% (n=29), and an alternative donor in 10% (n=5). At 3 years, overall survival was 52%, 17% of patients had relapsed disease, and 31% had non-relapse mortality.In our cohort, 38% had a mutation in CALR (n=15 type-I and n=3 type-II), 29% in JAK2 (n=14), 0% in MPL, and 33% were triple-negative (n=16; no evidence of CALR, JAK2, or MPL mutation). The average VAF of CALR mutations was 50% (range 20-82%) and JAK2 mutations was 58% (range 25-81%). In addition to driver mutations, 94% of patients had at least one other mutation in 26 of the 54 genes sequenced. On average, patients had 2.1 other mutations (range 0-5 mutations in addition to driver mutation). The most common other mutations were in ASXL1 (31%), IKZF1 (23%), ETV6 (17%), and U2AF1 (17%). The VAF of these other mutations spanned 9-76%.Patients with triple-negative MF were more likely to have primary MF (p=0.019) and platelets <100 (p<0.001), than patients with JAK2 and CALR mutations. Patients with ≥3 other mutations were more likely to have hemoglobin <10 (p=0.02) and a higher rate of relapse (p<0.01) than patients with <3 other mutations. Patients with ASXL1 mutations were more likely to have peripheral blood blasts (p<0.001).Mutations in ASXL1, ETV6, and U2AF1 have been previously reported in myeloid malignancies. Like other groups, we found ASXL1 mutations associated with worse prognosis. Here we report a novel, recurrent mutation in the gene IKZF1. All patients with IKZF1 mutation carried the same insertion of 11 nucleotides leading to an early frameshift and ultimate nonsense mutation (L92fs), which would lead to deletion of the zinc-finger domain. Such a mutation likely leads to loss of IKZF1 tumor suppressor function.Conclusions:Somatic mutations, in addition to driver mutations, are common in patients with MF treated with HCT. Mutation profiling in this patient subset may improve relapse risk prediction. Loss of heterozygosity on chromosome 7 mapping to the IKZF1 locus has been found by others to be associated with leukemic transformation in patients with MPN (Jaeger et al. 2010. Leukemia. 24: 1290). Given their frequency in this high risk MF population, further study of the role of IKZF1 mutations in MPN is warranted. DisclosuresRadich:Novartis: Consultancy, Other: laboratory contract; Bristol-MyersSquibb: Consultancy; TwinStrand: Consultancy; ARIAD: Consultancy; Pfizer: Consultancy.

UTX mutations in Myeloid Neoplasms

Genetic studies in myeloid neoplasms have revealed that somatic mutations and deletions frequently affect epigenetic regulation via DNA methylation and histone modification. One frequently affected epigenetic complex is polycomb repressive complex 2 (PRC2), which trimethylate Histone3Lysine27 (H3K27) to create a repression mark for the expression of a variety of genes that includes essential homeobox genes. Members of this complex include EZH2, EED and SUZ12, which exert methyltransferase activity, and perhaps indirectly also DNTM3A and ASXL1. The histone demethylase ubiquitously transcribed X (UTX) counters the enzymatic activity of PRC2 by removing di- and trimethyl groups from H3K27. It thus removes repressive marks. We were the first to report UTX mutations in a small portion of CMML and MDS cases. In another cohort, frequent somatic loss-of-function mutations in UTX were found in ALL. The goal of the present study was to understand the functional role of UTX and the consequences of its mutations on H3K27 methylation status, specifically with respect to their relevance to myeloid neoplasia.A total of 1,979 patients with various myeloid neoplasms (n = 1,686) and other hematologic disorders (n = 293) were screened for gene mutations in UTXand other reported driver genes relevant to myeloid neoplasms using whole exome sequencing and/or targeted deep-sequencing. Expression array analyses were performed on 200 samples. In addition, we also used sequencing and expression data from the de novo AML TCGA repository.UTXwas mutated in 55 (2.8%) cases out of 1,979 cases. The mutations were found in 2.5% of MDS, 3.1% de novo AML (including 8.3% CBF AML), 4.8% MDS/MPN, and 1.4% secondary AML (sAML). Of those, 77% were missense mutations and 23% nonsense, splice site and frameshift mutations, without an evident hot spot. The male-to-female ratio was 1.2 (55% vs. 45%). UTX gene expression was significantly higher in females than in males (p<.001). After gender adjustment and dichotomized using a threshold of expression of 2 standard deviations from the mean, low UTX expression levels were identified in 13/183 (7%) individuals. When we focused on clonal burden using variant allele frequencies (VAFs) to investigate clonal architecture within the tumor population, in 37 cases UTX constituted subclonal events and in 18 they were dominant.We then examined the molecular context of UTX lesions. Average mutation burden in patients with UTX mutations was higher than in WT UTX carriers (4 vs. 1.5, p<.001). UTX mutations co-occurred with other driver genetic mutations such as ASXL1, ZRSR2, CUX1, NF1. Surprisingly, EZH2 mutations are also enriched in UTX mutated cases although they function in the opposite direction of H3K27 trimethylation. Focusing on dominant clone and subclonal events in cases with these two mutations, EZH2mutations were enriched in cases with dominant UTX clone. This suggests that they play important roles in the cancer’s pathogenesis.To clarify the role of UTX in the maintenance of leukemia, genomic knockouts of UTX were developed in human leukemic cell lines using the CRISPR-Cas9 system. RNA sequencing revealed that knockout cell enrichment for developmental regulators such as Hox genes. In addition, we made knockdowns of human CD34+ cells using short hairpin RNAs against UTX. The cells showed enhanced colony formation and increased replating efficiency consistent with retained clonogenicity.The truncating pattern of UTX mutations in myeloid neoplasms suggests that they are loss-of-function hits. Missense mutations thus need to be confirmed. Functional analysis in vitro shows that low expression of UTX may have functionally equivalent consequences. If so, an additional 7% of patients may have low UTX expression, and may thus phenocopy patients with UTX mutations. Combined, a total of ~10% out of myeloid neoplasm patients may harbor UTX deficiency. Epigenetic modifying drugs related to H3K27 such as inhibitors of EZH2and histone deacetylases are in development. It is controversial to which patients they should be applied. Our findings could be key to a deeper understanding of epigenetic alterations, drug function, and response. DisclosuresMakishima:The Yasuda Medical Foundation: Research Funding. Mukherjee:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Carraway:Celgene: Research Funding, Speakers Bureau; Baxalta: Speakers Bureau; Incyte: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Sekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

Incidence and Characteristics of Therapy-Related Myeloid Neoplasms in Patients with Non-Hodgkins Lymphoma Treated with Radioimmunotherapy

AbstractAbstract 4924 Purpose:Radioimmunotherapy (RIT) delivers targeted radiation to tumor cells using cancer-specific monoclonal antibodies. Two agents, 90Y-ibritumomab tiuxetan (Zevalin®) and 131I-tositumomab (Bexxar) have been approved for the treatment of relapsed or refractory CD20 positive low-grade, follicular, or transformed NHL. With increasing use of RIT, it is important to elucidate short and long-term toxicities of treatment, particularly the development of therapy-related myelodysplastic syndrome and/or acute myelogenous leukemia (t-MDS/AML). We are reporting on the incidence and characteristics of t-MDS/AML in 149 patients with NHL treated at a single institution. Methods:We conducted a retrospective chart review of 149 patients with NHL treated with radioimmunotherapy at our institution. Incidences of t-MDS/AML, along with patient and disease characteristics were investigated. Results:149 patients with NHL were included in this analysis: All patients had received at least one cytotoxic chemotherapy regimen prior to RIT. Regimens most commonly included purine analogues, anthracyclines, alkylating agents, and topoisomerase II inhibitors. Seven out of 149 patients developed t-MDS/AML with an estimated incidence of 4. 69%, (t-MDS: 5, t-AML: 2). Five patients had follicular lymphoma, 1 with mantle cell lymphoma, and 1 with diffuse large B-cell lymphoma. Three patients received 131I-tositumomab and 4 patients received 90Y-ibritumomab tiuxetan. The median age at RIT, and at diagnosis of t-MDS/AML, was 71. 3 and 73. 1 years respectively. The median time from RIT to diagnosis of t-MDS/AML was 1266 days (3. 47 years). The median time from initial NHL treatment to t-MDS/AML was 1680 days (4. 60 years). Two patients developed t-AML without a preceding t-MDS; one of whom had received topoisomerase II inhibitors. Six patients had cytogenetic abnormalities, with complex karyotype present in 4 patients. The majority of the karyotypic abnormalities involved chromosomes 5, 7, 11, and 20. The median time from diagnosis of t-MDS/AML to death was 278 days (0. 76 years). Conclusion:Therapy –related myeloid neoplasms develop after exposure to cytotoxic agents and carry a worse prognosis than de novo disease as is shown in our series. Incidence varies according to underlying disease, specific agents received, timing of exposure, and dose. The incidence rate of 4. 69% observed in this study is within the range previously described for NHL patients treated with conventional-dose chemotherapy (Armitage J. O. et al., 2003). Most patients received fludarabine, an agent linked to a high incidence of t-MDS/AML (Carney D. A. et al., 2010). Roboz et al., 2007, noted that the median time to t-MDS/AML after standard initial therapy is approximately 6 years, which is slightly longer than our study (4. 60 years). This difference may be attributed to differences in chemotherapeutic agents utilized in that analysis for NHL therapy. T-AML without antecedent t-MDS was likely related to etoposide as has been observed in previous studies (Ratain M. J. et al., 1987, 1992). Chromosome 5 and 7 abnormalities were seen with the greatest frequency, a finding typical of t-MDS/AML (Armitage J. O. et al., 2003). This study provides an estimate of 4. 69% for the development of t-MDS/AML after NHL therapy which included chemotherapy/RIT, and suggests that RIT may not contribute to an increased risk of developing t-MDS/AML beyond what is expected with chemotherapy alone. Longer follow-up of NHL patients treated with RIT alone is necessary to determine the precise role of RIT in the development of t-MDS/AML. Disclosures:No relevant conflicts of interest to declare.

Therapy-related myeloid neoplasms following fludarabine, cyclophosphamide, and rituximab (FCR) treatment in patients with chronic lymphocytic leukemia/small lymphocytic lymphoma

This study is focused on therapy-related myeloid neoplasms after the most promising frontline FCR (fludarabine, cyclophosphamide, and rituximab) therapy in previously untreated chronic lymphocytic leukemia patients. A total of 28 therapy-related myeloid neoplasm patients were identified, including 19 patients from 3 well-controlled FCR frontline trials (n=426 patients), giving an estimated frequency of 4.5% (1.9–8.3%) in a follow-up period of 44 months (range 5–122 months). Clinically, therapy-related myeloid neoplasms could emerge directly from ‘prolonged myelosuppression' after FCR (10 patients), or after achieving complete hematological recovery (n=18). The overall latency was 35 months (range 3–118 months), with the former group of 23 months and the latter 42 months (P<0.001). In all, 10 cases presented as therapy-related acute myeloid leukemia and 18 as therapy-related myelodysplastic syndromes. Abnormal cytogenetics was present in 26 of 27 (96%) patients, with frequent chromosomes 5 and 7 abnormalities. The median survival was 7 months after therapy-related myeloid neoplasms. Our results indicate that the risk of therapy-related myeloid neoplasms secondary to frontline FCR therapy may not be as high as previously reported after removing the confounding factor of previous cytotoxic exposure, but this risk increased with older age and likely growth factor co-administration. Therapy-related myeloid neoplasms after FCR therapy shares clinicopathological features with therapy-related myeloid neoplasms secondary to other alkylating agents, but has a shorter latency interval indicating possible synergetic effects of the nucleotide analog fludarabine. The fact that therapy-related myeloid neoplasms can directly emerge from ‘prolonged myelosuppression' warrants a bone marrow examination to rule out therapy-related myeloid neoplasms in this clinical setting.

Therapy-Related Myeloid Neoplasms in Patients With Acute Promyelocytic Leukemia Treated With All-Trans-Retinoic Acid and Anthracycline-Based Chemotherapy

We analyzed the incidence, risk factors, and outcome of therapy-related myeloid neoplasms (t-MNs) in patients with acute promyelocytic leukemia (APL) in first complete remission (CR). From 1996 to 2008, 1,025 patients with APL were enrolled onto three sequential trials (LPA96, LPA99, and LPA2005) of the Programa Español para el Tratamiento de Enfermedades Hematológicas and received induction and consolidation therapy with all-trans-retinoic acid (ATRA) and anthracycline-based chemotherapy. Seventeen of 918 patients who achieved CR developed t-MN (10 with < 20% and seven with > or = 20% of bone marrow blasts) after a median of 43 months from CR. Partial and complete deletions of chromosomes 5 and 7 (nine patients) and 11q23 rearrangements (three patients) were the most common cytogenetic abnormalities. Overall, the 6-year cumulative incidence of t-MN was 2.2%, whereas in low-, intermediate-, and high-risk patients, the 6-year incidence was 5.2%, 2.1%, and 0%, respectively. Multivariate analysis identified age more than 35 years and lower relapse risk score as independent prognostic factors for t-MN. The median overall survival time after t-MN was 10 months. t-MN is a relatively infrequent, long-term, and severe complication after first-line treatment for APL with ATRA and anthracycline-based regimens. Therapeutic strategies to reduce the incidence of t-MN are warranted.