Core Binding Factor Translocations Research Articles

Hematological Neoplasms with Eosinophilia.

Eosinophils in peripheral blood account for 0.3-5% of leukocytes, which is equivalent to 0.05-0.5 × 109/L. A count above 0.5 × 109/L is considered to indicate eosinophilia, while a count equal to or above 1.5 × 109/L is defined as hypereosinophilia. In bone marrow aspirate, eosinophilia is considered when eosinophils make up more than 6% of the total nuclear cells. In daily clinical practice, the most common causes of reactive eosinophilia are non-hematologic, whether they are non-neoplastic (allergic diseases, drugs, infections, or immunological diseases) or neoplastic (solid tumors). Eosinophilia that is associated with a hematological malignancy may be reactive or secondary to the production of eosinophilopoietic cytokines, and this is mainly seen in lymphoid neoplasms (Hodgkin lymphoma, mature T-cell neoplasms, lymphocytic variant of hypereosinophilic syndrome, and B-acute lymphoblastic leukemia/lymphoma). Eosinophilia that is associated with a hematological malignancy may also be neoplastic or primary, derived from the malignant clone, usually in myeloid neoplasms or with its origin in stem cells (myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions, acute myeloid leukemia with core binding factor translocations, mastocytosis, myeloproliferative neoplasms, myelodysplastic/myeloproliferative neoplasms, and myelodysplastic neoplasms). There are no concrete data in standardized cytological and cytometric procedures that could predict whether eosinophilia is reactive or clonal. The verification is usually indirect, based on the categorization of the accompanying hematologic malignancy. This review focuses on the broad differential diagnosis of hematological malignancies with eosinophilia.

Standardization of Molecular MRD Levels in AML Using an Integral Vector Bearing ABL and the Mutation of Interest.

Quantitative PCR for specific mutation is being increasingly used in Acute Myeloid Leukemia (AML) to assess Measurable Residual Disease (MRD), allowing for more tailored clinical decisions. To date, standardized molecular MRD is limited to typical NPM1 mutations and core binding factor translocations, with clear prognostic and clinical implications. The monitoring of other identified mutations lacks standardization, limiting its use and incorporation in clinical trials. To overcome this problem, we designed a plasmid bearing both the sequence of the mutation of interest and the ABL reference gene. This allows the use of commercial standards for ABL to determine the MRD response in copy number. We provide technical aspects of this approach as well as our experience with 19 patients with atypical NPM1, RUNX1 and IDH1/2 mutations. In all cases, we demonstrate a correlation between response and copy number. We further demonstrate how copy number monitoring can modulate the clinical management. Taken together, we provide proof of concept of a novel yet simple tool, which allows in-house MRD monitoring for identified mutations, with ABL-based commercial standards. This approach would facilitate large multi-center studies assessing the clinical relevance of selected MRD monitoring.

TIM-3 Expression Level on AML Blasts Correlates With Presence of Core Binding Factor Translocations Rather Than Clinical Outcomes.

BackgroundT-cell immunoglobulin and mucin domain-containing molecule 3 (TIM-3) expresses on leukemic stem and progenitor populations of non-M3 acute myeloid leukemia (AML) as well as T lymphocytes. TIM-3 is thought to be involved in the self-renewal of leukemic stem cells and the immune escape of AML cells, however its correlation with AML prognosis is still controversial and worthy of further investigation.Methodswe simultaneously assessed TIM-3 expression levels of leukemic blasts and T lymphocytes in the bone marrow of de novo AML patients using flow cytometry. The correlations of TIM-3 expression between leukemic blasts and T lymphocytes and the correlations of TIM-3 expression with various patient parameters were analyzed. In addition, the Cancer Genome Atlas (TCGA) data of AML patients were acquired and analyzed to verify the results.ResultsTIM-3 expression of CD34+ leukemic blasts (R2 = 0.95, p<0.0001) and CD34+CD38- leukemic stem cells (R2 = 0.75, p<0.0001) were significantly and positively correlated with that of the whole population of leukemic blasts. In addition, TIM-3 expression level of leukemic blasts correlated significantly and positively with that of CD8+ (R2 = 0.44, p<0.0001) and CD4+ (R2 = 0.16, p=0.0181) lymphocytes, and higher TIM-3 expression of leukemic blasts was significantly associated with a greater proportion of peripheral CD8+ T lymphocytes (R2 = 0.24, p=0.0092), indicating that TIM-3 on leukemic blasts might alter adaptive immunity of AML patients. Regarding clinical data, the presence of core binding factor (CBF) translocations was significantly correlated with higher TIM-3 expression of leukemic blasts (CBF versus non-CBF, median 22.78% versus 1.28%, p=0.0012), while TIM-3 expression levels of leukemic blasts were not significantly associated with the remission status after induction chemotherapy (p=0.9799), overall survival (p=0.4201) or event-free survival (p=0.9873). Similar to our results, TCGA data showed that patients with CBF translocations had significantly higher mRNA expression level of HAVCR2 (the gene encoding TIM-3) (median, 9.81 versus 8.69, p<0.0001), and as all patients in the cohort were divided into two groups based on the median HAVCR2 expression level, 5-year overall survivals were not significantly different (low versus high, 24.95% versus 24.54%, p=0.6660).ConclusionTIM-3 expression level on AML blasts correlates with presence of CBF translocations rather than clinical outcomes.

Molecular Profiling in Extramedullary Acute Myeloid Leukemia Reveals Distinct Subgroups Defined By Mutations in KIT and RUNX1

Background: Between 2 to 23% of patients with acute myeloid leukemia manifest extramedullary involvement including myeloid sarcoma. While the genomic landscape of AML and the respective clinical and outcome correlations are well characterized, our understanding of the molecular underpinnings of extramedullary AML remains limited. Various cytogenetic abnormalities have been described in association with extramedullary AML, including core binding factor (CBF) translocations t(8;21) and inv(16). Recent studies have also applied targeted gene sequencing approaches (21 to 39 genes) in small patient cohorts (6-18) to generate limited mutational profiles of extramedullary AML [Li et al. Leukemia 2015; Pastoret et al. Leukemia & Lymphoma 2016; Kashofer et al. Leukemia & Lymphoma]. Here we utilize a comprehensive next generation sequencing (NGS) panel interrogating 585 genes implicated in hematological malignancies in 17 patients and provide the most comprehensive mutational profile of extramedullary AML to date.Methods: 17 pathologically confirmed cases of extramedullary AML were identified. Genomic DNA extraction was performed from formalin-fixed, paraffin-embedded (FFPE) tumor samples. Isolated DNA was sent for sequencing using the HemePACT panel, a custom hybridization capture-based NGS assay encompassing 585 genes related to hematologic malignancies. All HemePACT data were compared to an unmatched normal sample. Data were passed to standard pipelines used to detect candidate substitutions and indels (Caveman and pindel respectively). Candidate variants were manually reviewed to eliminate single nucleotide polymorphisms and artifacts and to find a subset of high-confidence putative oncogenic variants. Cytogenetic data was obtained from standard clinical karyotyping and fluorescence in-situ hybridization assays from bone marrow aspirates from patients with bone marrow AML involvement.Results: Demographic data for sequenced extramedullary AML patients is provided in Table 1. We identified a total of 80 mutations. The median number of mutations /sample is 4 (range:1-11). The most frequently mutated genes are KIT found in 7/17 patients (41%) and RUNX1 found in 6/17 (35%) patients. Intriguingly, KIT and RUNX1 mutations are mutually exclusive(p<0.05), and 13/17 (76.5%) sequenced extramedullary AML samples have mutations in either KIT or RUNX1 . (Figure 1) DNMT3A mutations are found in 3/17 (17%) patients. FLT3-tyrosine kinase domain (TKD) mutations are found in 4/17 (23%) of patients. DNMT3A and FLT3-TKD mutations co-segregate with RUNX1 (2/3 and 2/4 respectively) but not with KIT mutations. As only 2/17 patients underwent cytogenetic evaluation from extramedullary AML, mutational data from extramedullary AML was largely integrated with cytogenetic data from bone marrow aspirates. 7/17 (41.1%) of sequenced patients had CBF translocations (6/7 with inv(16)/t(16;16), and 1/7 with t(8;21) present in bone marrow cytogenetic evaluation by karyotype or FISH. Out of 7 patients with CBF translocations, 6/7 (85.7%) have KIT mutations present in extramedullary AML. 1 patient with inv(16) found by karyotyping from myeloid sarcoma did not have a KIT mutation in extramedullary AML. No patients with RUNX1 mutations in extramedullary AML (0/6) have CBF translocations. Kaplan-Meier analysis from this small cohort did not show a statistically significant difference in overall survival between patients with KIT and RUNX1 mutations (p=0.57).Overall, our data suggest the potential for distinct and specific mutational profiles associated with AML extramedullary involvement.Conclusions: Our study provides the most comprehensive mutational profiling of extramedullary AML to date. Combined molecular and cytogenetic profiling demonstrate that extramedullary AML is characterized by a distinct repertoire of acquired mutations enriched for mutations in KIT and RUNX1. Importantly our data identify two distinct and non-overlapping molecular subgroups. One subgroup is defined by CBF alterations and KIT mutations, while the second most prominent subtype is defined by mutations in RUNX1. Recognition of these subtypes and their co-mutations may form the basis for genotype-clinical correlations, as well as experimental models to explore the biology of extramedullary AML. Additional extramedullary AML patient samples are being sequenced and will be presented. [Display omitted] DisclosuresGoldberg:Genentech: Research Funding; Pfizer: Research Funding; ADC Therapeutics: Research Funding. Levine:Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership; Roche: Research Funding; Celgene: Research Funding; Qiagen: Equity Ownership.

Acute Myeloid Leukemia with Isocitrate Dehydrogenases (IDH) 1 and 2 Mutations. a Real-World Study from the European IDH Research Group

Acute Myeloid Leukemia with Isocitrate Dehydrogenases (IDH) 1 and 2 Mutations. a Real-World Study from the European IDH Research Group

Engineered type 1 regulatory T cells designed for clinical use kill primary pediatric acute myeloid leukemia cells.

Type 1 regulatory (Tr1) T cells induced by enforced expression of interleukin-10 (LV-10) are being developed as a novel treatment for chemotherapy-resistant myeloid leukemias. In vivo, LV-10 cells do not cause graft-versus-host disease while mediating graft-versus-leukemia effect against adult acute myeloid leukemia (AML). Since pediatric AML (pAML) and adult AML are different on a genetic and epigenetic level, we investigate herein whether LV-10 cells also efficiently kill pAML cells. We show that the majority of primary pAML are killed by LV-10 cells, with different levels of sensitivity to killing. Transcriptionally, pAML sensitive to LV-10 killing expressed a myeloid maturation signature. Overlaying the signatures of sensitive and resistant pAML onto the public NCI TARGET pAML dataset revealed that sensitive pAML clustered with M5 monocytic pAML and pAML with MLL rearrangement. Resistant pAML clustered with myelomonocytic leukemias and those bearing the core binding factor translocations inv(16) or t(8;21)(RUNX1- RUNX1T1). Furthermore, resistant pAML upregulated the membrane glycoprotein CD200, which binds to the inhibitory receptor CD200R1 on LV-10 cells. In order to examine if CD200 expression on target cells can impair LV-10 cell function, we overexpressed CD200 in myeloid leukemia cell lines ordinarily sensitive to LV-10 killing. Indeed, LV-10 cells degranulated less and killed fewer CD200-overexpressing cells compared to controls, indicating that pAML can utilize CD200 expression for immune evasion. Altogether, the majority of pAML are killed by LV-10 cells in vitro, supporting further LV-10 cell development as an innovative cell therapy for pAML.

Prognostic and Biologic Significance of Long Non-Coding RNA (lncRNA) Profiling in Cytogenetically Abnormal Acute Myeloid Leukemia (CA-AML)

Introduction: Aberrant expression levels of lncRNAs have been shown to independently associate with outcome of younger and older patients (pts) with cytogenetically normal AML. However, the prognostic and biologic significance of lncRNA expression in CA-AML pts have not been extensively studied.Methods: We performed whole transcriptome profiling (RNA-seq) in 469 pts with de novo CA-AML. Cytogenetic analyses were performed in institutional laboratories and the results were reviewed centrally. All pts were treated on frontline Cancer and Leukemia Group B (CALGB)/Alliance protocols.Results: To evaluate the prognostic significance of lncRNA expression in CA-AML, we analyzed RNA-seq data of 469 pts by applying a machine learning algorithm-based approach. As CA-AML pts constitute a heterogeneous group, we first determined which other clinical and molecular parameters were prognostic [i.e., associated with event-free survival (EFS)] in our dataset. Among the parameters tested, the European LeukemiaNet (ELN) Risk Group status and age group [i.e., younger than 60 years (y) or aged 60 y and older] significantly associated with clinical outcome of CA-AML pts. Next, we individually identified each lncRNA that associated with EFS while adjusting for ELN Risk Group and age group. We conducted random forest analyses to select the prognostic lncRNAs, whose combined expression levels could generate an effective outcome predictor for CA-AML pts. For each step of the random forest analyses, a bootstrap technique was applied; a simple random sample of pts was drawn which served as the training set and the out-of-sample pts were used as the independent validation set. We identified 55 prognostic lncRNAs and used their expression levels to separate our CA-AML cohort into a lncRNA low-risk (n=161) and a lncRNA high-risk (n=308) group. With regard to clinical characteristics, pts in the lncRNA low-risk group were younger (P<.001) and had lower platelet counts (P<.001) and higher white blood cell counts (P=.01) than pts in the lncRNA high-risk group. Concerning cytogenetic abnormalities, pts in the low-risk group more often had core-binding factor translocations or inversions (P<.001) and less often complex karyotypes (P<.001) than pts in the high-risk group. Pts in the lncRNA low-risk group had higher complete remission (CR) rates than pts in the high-risk group (91% vs 48%, P<.001). LncRNA low-risk group status also associated with longer disease-free survival (DFS; 5-y rates 49% vs 12%, P<.001), overall survival (OS; 5-y rates: 58% vs 14%, P<.001) and EFS (5-y rates: 45% vs 6%, P<.001). With regard to the accuracy of outcome prediction, the lncRNA risk classification had a C-index of 0.73, which compares favorably with other prognostic classifiers of AML pts. In multivariable analyses, lncRNA low-risk status was an independent marker for higher CR rates, as well as for longer DFS, OS and EFS (P<.001 in all comparisons), after adjusting for other covariates. Finally, we examined the prognostic value of the lncRNA risk classification within the Favorable and Intermediate ELN Groups of our dataset, for which lncRNA risk groups had adequate pt numbers. Among pts in ELN Favorable Group, lncRNA low-risk pts (n=128) had higher CR rates (P=.003) and longer DFS (P<.001), OS (P<.001) and EFS (P<.001) than lncRNA high-risk pts (n=32). Similarly, in the ELN Intermediate Group (n=85), lncRNA low-risk group status (n=28) associated with higher CR rates (P=.01), longer OS (P=.01) and EFS (P=.005) and a trend for longer DFS (P=.08).To gain biological insights, we examined the molecular pathways regulated by the 55 prognostic lncRNAs. To minimize the confounding effects of differences in the concurrent cytogenetic abnormalities, we restricted these analyses to the ELN Favorable Group. We identified approximately 900 transcripts that were differentially expressed between lncRNA low- and high-risk pts. DAVID pathway analyses showed enrichment of genes involved in the processes of phosphorylation, acetylation and RNA-binding. Ingenuity pathway analyses of up-stream regulators identified aberrant activity of homeobox genes such as MEIS1, HOXA9 and HOXA10 in the lncRNA low-risk group and other transcription factors such as MYC, FOSB and JUN in the high-risk group.Conclusion: We conclude that lncRNA profiling provides meaningful prognostic and biologic information in CA-AML pts. DisclosuresKolitz:Magellan Health: Consultancy, Honoraria. Powell:Rafael Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Stone:Cornerstone: Consultancy; Astellas: Consultancy; Orsenix: Consultancy; Merck: Consultancy; Celgene: Consultancy, Other: Data and Safety Monitoring Board, Steering Committee; Novartis: Consultancy, Research Funding; Arog: Consultancy, Research Funding; Fujifilm: Consultancy; Ono: Consultancy; Jazz: Consultancy; Sumitomo: Consultancy; Pfizer: Consultancy; Otsuka: Consultancy; Argenx: Other: Data and Safety Monitoring Board; Amgen: Consultancy; AbbVie: Consultancy; Agios: Consultancy, Research Funding. Uy:Curis: Consultancy; GlycoMimetics: Consultancy. Wang:Amgen: Consultancy; Amgen: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Jazz: Speakers Bureau. Stock:Jazz Pharmaceuticals: Consultancy.

Updated Results from a Phase 1 Study of Gilteritinib in Combination with Induction and Consolidation Chemotherapy in Subjects with Newly Diagnosed Acute Myeloid Leukemia (AML)

Updated Results from a Phase 1 Study of Gilteritinib in Combination with Induction and Consolidation Chemotherapy in Subjects with Newly Diagnosed Acute Myeloid Leukemia (AML)

Clinical Relevance of RUNX1 and CBFB Alterations in Acute Myeloid Leukemia and Other Hematological Disorders.

The translocation t(8;21), leading to a fusion between the RUNX1 gene and the RUNX1T1 locus, was the first chromosomal translocation identified in cancer. Since the first description of this balanced rearrangement in a patient with acute myeloid leukemia (AML) in 1973, RUNX1 translocations and point mutations have been found in various myeloid and lymphoid neoplasms. In this chapter, we summarize the currently available data on the clinical relevance of core binding factor gene alterations in hematological disorders. In the first section, we discuss the prognostic implications of the core binding factor translocations RUNX1-RUNX1T1 and CBFB-MYH11 in AML patients. We provide an overview of the cooperating genetic events in patients with CBF-rearranged AML and their clinical implications, and review current treatment approaches for CBF AML and the utility of minimal residual disease monitoring. In the next sections, we summarize the available data on rare RUNX1 rearrangements in various hematologic neoplasms and the role of RUNX1 translocations in therapy-related myeloid neoplasia. The final three sections of the chapter cover the spectrum and clinical significance of RUNX1 point mutations in AML and myelodysplastic syndromes, in familial platelet disorder with associated myeloid malignancy, and in acute lymphoblastic leukemia.

High EVI1 expression Is Associated with MLL rearrangements and Predicts Decreased Survival in Pediatric AML: A Report From the Children's Oncology Group.

AbstractAbstract 2530▪▪This icon denotes a clinically relevant abstract Background:The EVI1 gene is highly expressed in a subset of adult acute myeloid leukemia (AML) patients, and has been associated with inferior treatment outcome and specific cytogenetic abnormalities, including 11q23 rearrangements. Although translocations involving the MLL gene on chromosome 11q23 are common in pediatric AML, this group of patients as a whole carries neither favorable or adverse prognosis. We sought to examine the prevalence and clinical associations of high EVI1 expression in pediatric AML. Methods:EVI1 mRNA expression was measured via qRT-PCR in diagnostic specimens obtained from 206 patients enrolled on the pediatric trial COG-AAML03P1; expression levels were normalized against pooled normal blood controls, and correlated with clinical features and outcome. The Kaplan-Meier method was used to estimate overall survival (OS), event-free survival (EFS), and disease-free survival (DFS) in patients with high or low / absent EVI1 expression. Relapse risk (RR) was defined as time from remission at end of course 1 to relapse; estimates of RR were obtained using methods accounting for competing events such as toxic deaths. Results:EVI1 expression (range: 0 to 6660.88 fold normal peripheral blood, median 0.09) was detectable in the majority of patients (141/206, 68%), although expression higher than normal controls was present in only 57/206 patients (28%). A subset of patients had significant overexpression of EVI1; high EVI1 expression (EVI1high) was defined as EVI1 expression > 100 fold normal and was detectable in 33/206 (16%) of patients, with a median expression of 388.61 fold normal. By comparison, median expression in the remaining patients with low / absent EVI was 0.035 fold normal. EVI1high was significantly more common in African-American patients, who accounted for 31% of the EVI1high cohort as opposed to 13% of the remaining patients (p=0.016). MLL rearrangements were significantly more common in EVI1high patients (41% of EVI1high patients had 11q23 abnormalities, as opposed to 16% of patients with low / absent EVI1 expression, p=0.002). FAB class M7 was also enriched in the EVI1high subset of patients, accounting for 21%. Although EVI1 expression has been linked to 3q26 rearrangements in adult AML, no chromosome 3 abnormalities at the level of conventional cytogenetics were detected in EVI1high patients in our study. EVI1high was mutually exclusive of NPM1 mutations, CEBPA mutations, or core-binding factor translocations. Thus, the majority of EVI1high patients (79%) belonged to the “standard risk” group based on cytogenetic / molecular risk stratification. However, 4 of the 5 patients with high risk cytogenetics (monosomy 7 or del 5q), and 3 additional patients with FLT3/ITD, had high EVI1 expression. EVI1high patients had significantly lower 5 year OS from study entry (47% vs. 67%, p=0.032) compared to the remaining patients; EFS was also significantly decreased (31% vs. 52%, p=0.007). Corresponding DFS from complete remission was 43% with a RR of 43% for EVI1high patients, as compared to 59% with a RR of 31% for the patients with low / absent EVI1 expression (DFS: p=0.057, RR: p=0.119). This study was not powered to detect outcome differences based on EVI expression in the 11q23-rearranged subset; for MLL- rearranged patients, 5 year OS for EVI1high patients (n=12) was 51%, compared to 65% (p=0.705) for patients with low / absent EVI1 expression (n=26). Conclusions:High EVI1 expression was detectable in 16% of our pediatric AML study population; EVI1high was significantly associated with decreased OS and EFS. As the majority of these patients lacked additional cytogenetic/molecular prognostic markers, EVI1high has potential utility in the further risk stratification of “standard risk” patients. A larger cohort of patients from the AAML03P1 successor Phase III trial COG-AAML0531 is currently being evaluated for EVI1 expression, in order to allow for analysis of outcome based on EVI expression in the 11q23-rearranged cohort. As the MLL gene has numerous fusion partners, the study of additional patients will also allow analysis of the correlation between EVI1high and specific MLL translocations, as some MLL fusions have recently been shown to be individually prognostic and biologically distinct. Disclosures:No relevant conflicts of interest to declare.

KIT Mutations Are Rare Among Elderly AML Patients: A SWOG Report

Abstract 2510 Background:KIT receptor tyrosine kinase mutations are recurrent genetic alteration found in acute myeloid leukemia (AML) with higher prevalence in those with core binding factor (CBF) AML. Different prognostic significance of these mutations has been found in pediatric and adult AML studies. However, prevalence and prognostic impact of KIT mutations in elderly patients with AML has not been established. In this study, we evaluated the prevalence of KIT mutation and its correlation with cytogenetic, molecular subtypes and clinical outcome in elderly AML patients. Methods:Diagnostic specimens from for 207 patients with AML registered on SWOG trials S-9031 and S-9333 were available for KIT mutation analysis. Both of these clinical protocols enrolled AML patients > 55 years. The samples were analyzed for the presence of KIT mutations via direct sequencing of exons 8 and 17. Results:In the cohort of 207 patients tested, the median age, WBC, and blast count at diagnosis were 68 years (range, 56–88 years), 29,6 × 109/L (range, 7–289 × 109/L) and 67% (0–99%), respectively. Favorable, intermediate and unfavorable cytogenetics were present in 8%, 57% and 26% of the patients, respectively. Nine percent of the patients had cytogenetics abnormalities of unknown significance. Normal cytogenetics was present in 48% of the patients. Fifty-three patients (26%) harbored mutations for FLT3-ITD, 37 (19%) for DNMT3A, 52 (31%) for NPM1 and 50 (24%) for IDH1/2. Only 3 patients were positive for CEBPA mutation. Of the 207 patient specimens tested, KIT exon 17 mutations were detected in 4 patients (2%). Exon 8 mutations were not identified. The characteristics of the 4 patients with KIT mutations are described in the table. Of the 4 patients who harbored the KIT mutation, 2 had intermediate, 1 had unfavorable risk cytogenetics and 1 patient did not have available cytogenetic data. None of the patients with CBF had KIT mutations. In terms of other molecular markers, all 4 patients were wild-type for NPM1, CEBPA, DNMT3A, IDH1/2 and FLT3-ITD. The very small number of KIT mutations in this group of patient precluded correlation between KIT mutations and clinical outcome. Conclusion:The prognostic impact of KIT mutations has been shown to vary according to cytogenetics subgroups. Most studies report a negative prognostic impact of these mutations in patients with CBF AML. The prognostic implications of KIT mutations also seem to vary among different age groups, with pediatric studies indicating they have no prognostic significance and adult studies indicating a negative prognostic impact. To date, there is no large study reporting on the incidence and prognostic impact of KIT mutations in elderly patients with AML. The very low incidence of KIT mutations in our study highlights the age-specific characteristics of AML and may correlate with lower prevalence of CBF translocations in older AML. Further studies investigating the relationship among different somatic mutations in elderly AML patients may help to clarify the pathogenetic mechanism of certain AML subtypes in this patient population.TablePatient characteristics of the 4 patients with KIT mutationsPatient1234KIT mutationsExon 17 (D816V)Exon 17 (D816V)Exon 17 (D816Y)Exon 17 (D816V)SexFFMMAge at diagnosis (years)56786572WBC x109/L68.3215.629.912.8BM blast (%)72405130Cytogenetics46,XX,t(6;11) (q27;q23)t(6;14) (q11;q11)[20]/ 46,XX,t(6;14) (q11;q11)[1]46,XX[20]No data46,XY[19]/47,XY,+2[1]NPM1WTWTWTWTFLT3-ITDWTWTWTWTCEBPAWTWTWTWTDNMT3AWTWTWTWTIDH1/2WTWTWTWTResponse to induction chemotherapyCRRDCRRDRelapse-free survival (days)270–386–Overall survival (days)646264978575Abbreviations: WBC, white blood count; BM, bone marrow; CR, complete remission; RD, refractory disease; WT, wild-type; F, female; M, male. Disclosures:No relevant conflicts of interest to declare.

Prospective separation of normal and leukemic stem cells based on differential expression of TIM3, a human acute myeloid leukemia stem cell marker

Hematopoietic tissues in acute myeloid leukemia (AML) patients contain both leukemia stem cells (LSC) and residual normal hematopoietic stem cells (HSC). The ability to prospectively separate residual HSC from LSC would enable important scientific and clinical investigation including the possibility of purged autologous hematopoietic cell transplants. We report here the identification of TIM3 as an AML stem cell surface marker more highly expressed on multiple specimens of AML LSC than on normal bone marrow HSC. TIM3 expression was detected in all cytogenetic subgroups of AML, but was significantly higher in AML-associated with core binding factor translocations or mutations in CEBPA. By assessing engraftment in NOD/SCID/IL2Rγ-null mice, we determined that HSC function resides predominantly in the TIM3-negative fraction of normal bone marrow, whereas LSC function from multiple AML specimens resides predominantly in the TIM3-positive compartment. Significantly, differential TIM3 expression enabled the prospective separation of HSC from LSC in the majority of AML specimens with detectable residual HSC function.

Sequential Acquisition of Somatic Mutations In Progenitors with Differential Proliferative Potential In Human Acute Myeloid Leukemia

AbstractAbstract 755 Background:Evidence suggests that leukemic transformation is a multistep process in acute myeloid leukemia (AML). However, the cell of origin for human leukemogenesis remains ill defined. Experimental systems with murine hematopoetic cells indicate that not only immature multipotent hematopoietic stem cells but also more mature, committed myeloid precursors can be a target of murine AML transformation. These findings suggest that requisite mutations for leukemogenesis may be acquired sequentially in myeloid progenitors with differential proliferative potential. To investigate this possibility in human AML, we studied primary specimens from pediatric patients who had previously been identified as having 2 distinct, disease-relevant somatic mutations. Methods:Diagnostic bone marrow or peripheral blood specimens from pediatric AML patients with two distinct genomic alterations were obtained from Children's Oncology Group (COG) AML Reference Laboratory. Early myeloid CD34+/CD33- progenitors and more differentiated CD34+/CD33+ progenitors were isolated using fluorescence-activated cell sorting (FACS). Twenty-eight of 43 patient specimens had FACS-isolated CD34+/CD33- progenitor cells and molecular data available for both somatic alterations, and were included in this study. The genomic alterations included those with core-binding factor (CBF) translocations and FLT3/ITD (n=5), CBF translocations and KIT abnormalities (n=19), or nucleophosmin (NPM1) mutations and FLT3/ITD (n=4). Aliquots of sorted subpopulations were subjected to direct colony forming cell (CFC) assays. Freshly sorted cells and resulting colony forming units-granulocyte and/or monocyte (CFU-GM) were evaluated for the presence of the patient-specific genomic alterations. Results:In 20/28 specimens (71.4%), both mutations were detected in CD34+/CD33- cells. By comparison, in 6 (21.4%) patient specimens, the receptor tyrosine kinase (FLT3/ITD or KIT) alteration was not detected in CD34+/CD33- cells, whereas in 2 (7.1%) specimens, CD34+/CD33- cells did not harbor either of the two patient-specific alterations. In contrast to these less mature CD34+/CD33- progenitor cells, analysis of more differentiated CD34+/CD33+ progenitors revealed both genomic alterations in 26/28 samples. For the remaining 2 samples, only the CBF mutation was detected. FACS-isolated CD34+/CD33- cells from 22 specimens were subjected to CFC assay. There was sufficient colony growth and evaluable molecular data for the paired mutations for 7/22 samples. Within these specimens, the majority (43 of 60) of the evaluable CFU-GM demonstrated the CBF or NPM1 abnormality but lacked the RTK mutation; in the remaining colonies, both somatic alterations were detected (10 of 60), or neither mutation was detected (7 of 60). This finding suggests that the majority of CFU-GM analyzed were derived from an abnormal cell that had not yet acquired the requisite RTK mutation necessary for frank leukemic transformation. Conclusion:These findings suggest the potential for sequential acquisition of RTK mutations in some cases of pediatric CBF or NPM1+ AML. Specifically, the presumptive class I mutation confering a proliferative advantage (e.g. the receptor tyrosine kinase mutation) appears to arise, in some instances, in a more committed myeloid progenitor with limited proliferative potential. Disclosures:No relevant conflicts of interest to declare.

High Expression of Neutrophil Elastase Predicts Improved Survival In Pediatric Acute Myeloid Leukemia: A Report From the Children's Oncology Group

AbstractAbstract 2762 Background:Recent studies indicated that high expression of neutrophil elastase, a primary granule serine protease encoded by ELANE (ELA2), predicts longer survival of patients with chronic-phase chronic myeloid leukemia (CML) and improved outcome following allogeneic hematopoietic stem cell transplantation for advanced-phase CML. In contrast, the prognostic role of neutrophil elastase in acute myeloid leukemia (AML) is unknown. Therefore, we evaluated its expression in participants in a recent Children's Oncology Group (COG) AML pilot protocol. Methods:COG-AAML03P1 enrolled 340 newly diagnosed children (aged 1 month to 21 years) with de novo AML, excluding those with acute promyelocytic leukemia and Down syndrome, and tested the feasibility of combining gemtuzumab ozogamicin (GO) with intensive induction chemotherapy followed by GO-containing intensification therapy or matched related donor stem cell transplantation. Diagnostic marrow specimens from 113 randomly selected patients were used for ELANE mRNA quantification using a sandwich nucleic acid hybridization method employing branched DNA molecules to amplify the signal from captured target RNA. Expression levels were normalized using beta glucuronidase (GUSB) and then correlated with patient demographics, laboratory characteristics, and clinical outcome. Cytogenetics and molecular prognostic markers were used for risk classification as follows: low risk (mutation in core-binding factor, NPM1, or CEBPa; n=43), high risk (−5/5q−, monosomy 7, or FLT3-ITD with high allelic ratio; n=16), or standard risk (all other patients with cytogenetic/molecular data; n=54). Results:Among the 113 diagnostic specimens, ELANE mRNA expression levels were undetectable in 3 samples and varied more than 70,000-fold relative to GUSB in the rest, from 0.0005 to 36.32 (median, 0.89). Patients with high ELANE expression (>median expression) were more likely to have myelomonocytic cell characteristics (FAB M4, p<0.001) and more likely to have core-binding factor translocations, t(8;21) (p=0.05) or inv(16) (p<0.003). They had a lower prevalence of megakaryocytic leukemia (p<0.02). There was no difference in the prevalence of FLT3-ITD or NPM1 mutations. As a result of these characteristics, patients with high ELANE expression were more likely to have low-risk disease (p<0.001) at the expense of standard-risk disease, while the prevalence of high-risk disease was similar. We subsequently determined the clinical outcome per median ELANE expression for the entire cohort as well as the specific risk groups. In the entire cohort, patients with high ELANE expression had a significantly superior 3-year overall survival (OS; 80±11% vs 61±13%, p=0.045) and tended to have better disease-free survival (66±14% vs 51±16%, p=0.118). Subgroup analyses were limited by the small sample size. Nevertheless, in both high- and low-risk disease, patients with high ELANE expression tended to have a better 3-year OS than patients with low ELANE expression (67±38% vs 30±29%, p=0.308 for high risk; 90±11% vs 75±25%, p=0.361 for low risk). By comparison, OS in standard-risk patients appeared similar for patients with high and low ELANE expression (70±21% vs 66±17%, p=0.849). In multivariate Cox regression analyses that included age, cytogenetics, and molecular prognostic factors, high ELANE expression was associated with reduced overall mortality (hazard ratio: 0.66); however, this result did not reach statistical significance (95% confidence interval: 0.33–1.35, p=0.260), possibly because of the small sample size. Conclusion:This study revealed significant heterogeneity of ELANE expression in pediatric AML and its association with improved OS. This association is at least partly due to the high proportion of AMLs with core-binding factor aberrations in patients with high ELANE expression. Nevertheless, high ELANE expression may predict improved OS even within specific risk groups, in particular high-risk disease, although further studies with a larger patient cohort will be necessary to confirm this finding. Disclosures:No relevant conflicts of interest to declare.

The Prognostic Impact and Stability of IDH2 Mutation and the Insufficiency of the Mutation Alone for Leukemogenesis In Adult Patients with Acute Myeloid Leukemia

AbstractAbstract 2697 Purpose:Although the clinical and biological features of Isocitrate dehydrogenase (IDH) mutations in acute myeloid leukemia (AML) have been characterized, its stability and in vivo sufficiency of the mutation alone for leukemogenesis remain uninvestigated. Patients and Methods:Mutations of IDH and other clinically relevant genes were analyzed in the bone marrow from 446 adult patients with de novo non-M3 AML. IDH2 mutations were examined serially in 140 patients at diagnosis and after chemotherapy. Results:Among the 446 adults with de novo non-M3 AML, IDH2 R172, R140, and IDH1 R132 mutations occurred at a frequency of 2.9%, 9.2%, and 6.1%, respectively. IDH2 mutation was associated with higher platelet counts (p=0.046), intermediate-risk (p=0.002) or normal karyotype (p=0.023), and isolated +8 (p=0.014), but was inversely correlated with expression of HLA-DR (p=0.002), CD34 (p=0.039), CD15 (p=0.003), CD7 (p=0.010), and CD56 (p=0.048), and was mutually exclusive with WT1 mutation (p=0.037) and core-binding factor translocations (p=0.001). All these correlations became stronger when IDH1 and IDH2 mutations were considered together, suggesting similarity of biological roles between these 2 mutations. However, IDH2 but not IDH1 mutation conferred a better prognosis (Fig 1), especially in those with normal karyotype or intermediate cytogenetics (median overall survival: not reached vs. 58 months, p=0.044 and not reached vs. 19 months, p=0.027 for normal and intermediate karyotype group, respectively). Importantly, IDH2 but not IDH1 mutation was an independent favorable prognostic factor (HR: 0.332, 95% CI: 0.159–0.694; p=0.003). Patients with IDH2−/FLT3-ITD+ genotype had especially worse prognosis (median OS of IDH2−/FLT3-ITD+ vs. IDH2+/FLT3-ITD− group: 12 months vs. not reached; p=0.003; median OS of IDH2−/FLT3-ITD+ vs. IDH2+/FLT3-ITD+ or IDH2−/FLT3-ITD− group : 12 months vs. 35 months; p<.0001) (Fig 2A). The worse prognosis was also seen in patients with IDH−/FLT3-ITD+ genotype (Fig 2B). Serial analyses of IDH2 mutations during the clinical course of 140 patients confirmed the stability of this mutation; all the patients with IDH2 mutations at diagnosis harbored the same mutation at relapse with the exception of one patient who had extramedullary but not bone marrow relapse, while none of the IDH2-wild patients acquired this mutation at relapse. Importantly, sequential samples from two patients in long-term remission retained the original R140Q mutation while other accompanied mutations, FLT3-ITD in the first patient and NPM1 in the second, respectively, disappeared. In the first patient, the skin tissue was absent of the mutation and in the second, the mutation was restricted in myeloid cells but spared in lymphocytes indicating the mutation was acquired in these two patients. Conclusion:IDH2 mutation is a stable marker during disease evolution and confers favorable prognosis. FLT3-ITD combined with wild type IDH2 exerted synergistic negative impact on survival. IDH2 mutation alone is insufficient for leukemogenesis. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The clinical spectrum of adult acute myeloid leukaemia associated with core binding factor translocations

To better understand the spectrum of adult acute myeloid leukaemia (AML) associated with core binding factor (CBF) translocations, 370 patients with newly diagnosed CBF-associated AML were analysed. Patients' age ranged from 16-83 years (median 39 years) with a slight male predominance (55%); 53% had inv(16); 47% had t(8;21). Patients with t(8;21) tended to be younger (P = 0.056), have lower peripheral blood white cell counts (P < 0.0001) and were more likely to have additional cytogenetic abnormalities (P < 0.0001). Loss of sex chromosome, del(9q) and complex abnormalities were more common among patients with t(8;21), while +22 and +21 were more common with inv(16). Overall, 87% [95% confidence interval (CI) 83-90%] of patients achieved complete response (CR) with no difference between t(8;21) and inv(16); however, the CR rate was lower in older patients due to increased resistant disease and early deaths. Ten-year overall survival (OS) was 44% (95% CI 39-50%) and, in multivariate analysis, was shorter with increasing age (P < 0.0001), increased peripheral blast percentage (P = 0.0006), in patients with complex cytogenetic abnormalities in addition to the CBF translocation (P = 0.021), and in patients with t(8;21) (P = 0.025). OS was superior in patients who received regimens with high-dose cytarabine, a combination of fludarabine and intermediate-dose cytarabine, or haematopoietic cell transplantation.

The clinical spectrum of adult acute myeloid leukemia (AML) associated with core binding factor (CBF) translocations

6513 Background: Although adults with AML containing CBF translocations [t(8;21) or inv(16)/t(16;16)] have a high complete response (CR) rate and improved overall survival (OS) compared to other AML patients (pts), not everyone does well. Methods: In order to better understand the spectrum of CBF-associated AML, we analyzed 373 pts with newly diagnosed CBF-related AML treated on SWOG, ECOG or MDA protocols. Results: Pts ranged in age from 16–83 (median 39) with a slight male predominance (56%). Overall, 53% of pts had inv(16) or t(16;16) while 47% had t(8;21); 58% had chromosomal abnormalities in addition to the CBF translocation. Pts with t(8;21) tended to be younger (p=0.03), have lower white counts (p<0.0001) and more often have additional chromosomal abnormalities (p<0.0001). CR was achieved in 86% of pts (95% confidence interval [CI] 82–89%) and the estimated resistant disease (RD) rate was 8% (CI 5–12%). Increasing age was the major factor associated with a decreased CR rate (p=0.0029) and increased incidence of RD (p=0.0014). OS at 10 years was 43% (CI 38–49%), and was significantly lower in older pts (p<0.0001) and those with higher peripheral blast percentage (p=0.0001). Relapse-free survival (RFS) was 40% (CI 34–46%) at 10 years and likewise was associated with age and peripheral blast percent. The treatment results did not differ significantly between pts with t(8:21) versus inv(16) and were not significantly influenced by the presence of additional chromosomal abnormalities. Conclusions: Although pts with CBF-associated AML do relatively well with 43% 10-year OS, considerable improvement is needed, particularly in older patients and those presenting with a higher percent of blasts in their peripheral blood. No significant financial relationships to disclose.