Core Binding Factor Genes Research Articles

Clinical and biological impact of ATP-binding cassette transporter activity in adult acute myeloid leukemia.

Chemotherapy resistance is the main cause of treatment failure in acute myeloid leukemia (AML) and has been related to ATP-binding cassette (ABC) transporter activity. However, the links between ABC activity, immunophenotype, and molecular AML parameters have been poorly evaluated. Moreover, the prognostic value of ABC activity, when compared to new molecular markers, is unknown. Here we investigated the links between ABC activity, as evaluated by JC-1 +/- cyclosporine A assay, and immunophenotypic, cytogenetic, molecular, and targeted next-generation sequencing features in 361 AML patients. High ABC activity was found in 164 patients and was significantly associated with less proliferating disease, an immature immunophenotype (expression of CD34, HLA-DR, CD117, CD13), and gene mutations defining AML as belonging to secondary-type ontogenic groups. Low ABC activity was associated with more mature myeloid differentiation (CD34-, cyMPO+, CD15+, CD33+) or monocytic commitment (CD64+, CD4+weak, CD14+), with NPM1 mutations, KMT2A rearrangements, and core-binding factor gene fusions, hallmarks of the de novo-type AML ontogeny. ABC activity was one of the major factors we identified using a random forest model for early prediction of AML ontogeny. In the 230 patients evaluated at diagnosis and intensively treated, high ABC activity was a predictive factor for primary resistance, and in multivariate analysis including full molecular data, an independent factor for event-free survival (P=0.0370). JC-1 +/- cyclosporine A assay could be used at diagnosis to predict AML ontogeny and to complete prognosis evaluation in addition to new molecular markers.

CSF3R Mutation in Acute Leukemia and MDS Patients

Introduction: CSF3R somatic mutations has been included in the 2016 revision of the WHO classification of myeloid neoplasms and acute leukemia as a specific molecular diagnostic marker of CNL. Besides CNL, CSF3R somatic mutation have been reported in about 30% of Severe Congenital Neutropenia (SCN) and in a low frequency of other myeloid malignancies (about 4% of CMML, 1.7% of adult AML, 1.9% of children AML), but not in MDS, JMML and ET (Lavallée Et al., Blood 2016; Sano H et al., Br J Haematol 2015). Lavallée et al. reported for the first time that CSF3R mutation are associated with AML with biallelic mutated CEBPA (4/14, 29%) (Lavallée et al., Blood 2016).We aimed to assess the frequency of CSF3R mutation in MDS, AML and ALL patients and the association with other genetic abnormalities.Methods: A retrospective analysis of 974 patients with hematological malignancies, including 297 cases of de novo AML, 144 cases of relapsed AML, 268 cases of de novo ALL, 191 cases of relapsed ALL and 74 cases of MDS patients. 58 genes which common mutated in hematological malignancies were tested by amplicon-based NGS with IonTorrent platform (PGM, Life Technology) and the average sequencing depth was 1000x. 36 leukemia related fusion genes were screened by RT-PCR.Results: We identified CSF3R mutation in 19 patients, which was found in 3.0% (9/297) of de novo AML, 5.6% (8/144) of relapsed AML, 0.5% (1/191) of relapsed ALL and 1.4% (1/74) of MDS. No CSF3R mutation was detected in de novo ALL. All 19 cases had no history of MPN, MDS/MPN or SCN. A total of 8 mutations of CSF3R were detected in 19 patients, 11 cases with T618I mutation, 3 cases with T615A mutation, 1 case with Y779H, Q781*, P809Hfs*8, S810Qfs*6, S813Lfs*2 and D837* mutation respectively, among which 1 patient accompanied with CSF3R T618I and Y779H mutation.In AML cohort, a total of 17 cases with mutant CSF3R, the median age was 20 years (rang, 6-49) and the frequency of CSF3R mutation have no difference between children and adult. Cytomorphology revealed AML M2 (n=11), AML M4 (n=3), AML M5 (n=1), and 1 case have no data available. The median of CSF3R variant allele frequency (VAF) was 25% (3%-49%), 3 cases with VAF <10%, 10 cases with VAF comparable to the concomitant gene mutation with highest VAF. CSF3R mutation was more common as the predominant clone in relapsed AML (87.5% VS 33.3%, P=0.05). The median of concomitant gene mutations was 2 (range 0-3), double-mutated CEBPA (CEBPAdm) was the most frequently concomitant partner of CSF3R mutation (7/17, 41.2% VS 51/382, 13.4%, P <0.05). We also identified 9 cases with AML1-ETO fusion gene which were mutually exclusive with CEBPAdm. In totally, 16 cases of 17 (94.1%) CSF3R mutated AML patient concomitant with core-binding factor gene abnormalities (Fig.1).CSF3R mutation have not been reported in ALL, we identified CSF3R mutation in one patient with B-ALL who was 10 years old and relapsed at 43 months after diagnosed. Bone marrow morphology and immunophenotyping at relapse shows no abnormal neutrophils and myeloid lineage expression were found. No fusion genes were detected. CSF3R T618I and ASXL1 Q803* mutations were detected with VAF of 50% and 51%, respectively, which were confirmed of somatic mutation rather than germline origin. As lack of genetic data at diagnosis, we could not analyze the order of mutation acquisition and clone evolution comprehensively. At the time of reporting, the patients have received allo-HSCT for 4 months and acquired sustained remission. We also detected 1 case of MDS patients (MDS-U) carried U2AF1 S34F, CSF3R Q781* and CBL G413S mutations with VAF of 45%, 32% and 31%, respectively.Due to the limited number of CSF3R mutated cases, we did not perform survival analysis in this study.Conclusion: (1) CSF3R mutation was found in 3.9% of total AML and 3% of de novo AML cohort, rarely seen in MDS and ALL. (2) In AML, CSF3R mutation are prevalent in M2 and M4, and all concomitant with core-binding factor gene abnormalities (CEBPAdm and AML1-ETO fusion gene) and NPM1 mutation which were associated with favorable-risk prognosis. (3) The CSF3R mutation could appeared as predominant clone or subclone in AML and seemly more common as the predominant clone in relapsed AML. As CSF3R mutation also have implications for therapeutic that JAK inhibitors may be efficacious in treating CSF3R-mutant CNL. The implications of prognostic and target therapy (eg. JAK inhibitors) of CSF3R mutation in AML warranting further investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Flow cytometry reveals the nuances of clonal haematopoiesis

Flow cytometry reveals the nuances of clonal haematopoiesis

CSF3R Mutations are frequently associated with abnormalities of RUNX1, CBFB, CEBPA, and NPM1 genes in acute myeloid leukemia

Mutations in the colony-stimulating factor 3 receptor (CSF3R) gene occur frequently in chronic neutrophilic leukemia and are rare in de novo acute leukemia. The objective of this study was to assess the incidence of CSF3R mutations in acute leukemia and their association with other genetic abnormalities. Amplicon-targeted, next-generation sequencing of 58 genes was performed retrospectively on 1152 patients (acute myeloid leukemia [AML], n = 587; acute lymphoid leukemia [ALL], n = 565). Reverse transcriptase-polymerase chain reaction analysis was used to detect 35 leukemia-specific gene fusions. CSF3R mutations (26 patients) were detected in 3.6% (13 of 364 patients), 4.6% (8 of 175 patients), and 8.3% (4 of 48 patients) of those with de novo, relapsed, and secondary AML, respectively, and in 0.2% (1 of 565 patients) of those with ALL. In total, 9 distinct CSF3R mutations were detected. Membrane-proximal missense mutations and cytoplasmic truncations were identified as mutually exclusive. The proportion of patients who had French-American-British subtypes M2 and M4 in the CSF3R-mutated group was significantly greater than that in the CSF3R wild-type group for both the de novo AML cohort (P = .001) and the relapsed AML cohort (P = .024). All de novo and relapsed AMLs with CSF3R mutations were associated with genetic alterations in transcription factors, including RUNX1-RUNX1T1, CBFB-MYH11, double-mutated CCAAT/enhancer binding protein α (CEBPAdm), and NPM1 mutations; and core-binding factor gene abnormalities and CEBPAdm accounted for 90.5% (19 of 21 patients). CSF3R mutations are uncommon in AML; however, when they occur, they are often associated with core-binding factor gene abnormalities and CEBPAdm. An in-depth understanding of the interaction between these genetic alterations could facilitate a clearer understanding of the role of CSF3R mutations in AML development and may be used for disease classification, prognosis, and the development of targeted therapy.

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.

Molecular Profiling of High-Risk Pediatric Acute Myeloid Leukemia

Background: Several landmark genomic profiling studies have dramatically advanced our understanding of the origin, progression and clonal evolution of adult acute myeloid leukemia (AML) and directly impacted clinical care. However, very little is known about the mutational landscape of pediatric AML, a distinct entity that shares few genetic and clinical characteristics with adult AML. To investigate potential drivers of high-risk pediatric AML, comprehensive genomic profiling was performed on high-risk AML samples as part of a prospective clinical next-generation sequencing program.Methods:Samples obtained from patients with known high-risk features at diagnosis or with refractory or relapsed AML were selected for molecular profiling. Comprehensive testing included whole-exome sequencing (WES) of matched tumor (bone marrow or chloroma tissue) and normal tissue (peripheral blood or buccal swab) samples and transcriptome analysis (RNAseq). Targeted sequencing of 467 cancer-associated genes was used when tumor tissue was limited. Sequencing was performed on Illumina's HiSeq 2500 with 150X and 500X average coverage for WES and targeted sequencing, respectively. Variants were filtered to select alterations in cancer-related genes or genes relevant for patient care.Results:Fifteen patients with AML (mean age 7.7 yrs; range 0.75-19 yrs) met high-risk criteria (high-risk features at diagnosis = 4, relapsed disease = 8, refractory disease = 3) and were selected for profiling. WES and RNAseq were performed on 11 samples, WES only on 3 samples and targeted DNA sequencing on 1 sample. The median number of variants was 60 (range 14- 5950) per case. After filtering, 54 mutations were identified in 35 genes with a mean of 3.6 mutated genes per patient sample (range 0-14); two samples only carried a fusion gene with no other genetic alterations. At least one driver genetic alteration was detected in each patient sample. Thirteen samples carried mutations in at least one gene known to be altered in AML (e.g. IDH1, WT1, TP53, NRAS) (mean, 2; range, 1-6) and 5 samples carried novel mutations in 15 genes not previously implicated in AML (e.g. CARD9, CHD9, Axin1). Mutations in 11 AML related genes were detected in more than one sample including NRAS in 4, TP53 in 3 and KRAS, PTPN11 PHF6, JAK3 in 2 samples each; genes not previously implicated in AML were only mutated in single patients. Of note, mutations in genes encoding members of the RAS pathway occurred in 60% of cases (9/15 samples). RNAseq identified gene fusions in 7/11 samples (63%). Four fusions involving KMT2A and core binding factor genes were also detected by FISH while three fusions were detected by RNAseq only: NUP98-NSD1 in two patients and CBFA2T3-GLIS2 in one patient. Samples carrying driver gene fusions had the lowest number of mutated genes (0-1) compared to samples lacking a gene fusion (1-5 mutated genes), with one exception of a patient with history of infant ALL who later developed KMT2A-AFF1AML with the highest number of mutated genes (n=14). There was no correlation between the number of mutated genes and age, clinical characteristics, initial risk classification at diagnosis or intensity of therapy prior to sequencing.Conclusion:Our study provides an initial overview of the genetic alterations that characterize high-risk, chemo-resistant pediatric AML. Analysis of the data highlights the overall low genetic complexity of high-risk AML despite the aggressive clinical behavior and exposure to intense chemotherapy, including stem cell transplant. Of interest, similar to adult AML, we found that mutations leading to aberrant activation of the RAS pathway were also very frequent in our cohort of pediatric high-risk AML, while genes typically mutated early in the process of leukemogenesis in adult AML, such as NPM1, DNMT3A, FLT3, IDH1, IDH2 were not affected. Such findings suggest that distinct, age-specific mechanisms of leukemogenesis might exist. Furthermore, our data also highlights the important role of RNA sequencing in complementing current standard diagnostic tools, allowing the identification of driver fusion genes in samples for which no other driver event is detected. Larger studies, preferably including diagnostic samples and utilizing broader approaches, are needed to better understand the mechanisms responsible for the initiation and progression of childhood AML. DisclosuresNo relevant conflicts of interest to declare.

Mutational and Transcriptomic Landscape of AML with Core-Binding Factor Rearrangements

Background Acute myeloid leukemias (AML) with t(8;21)(q22;q22);RUNX1-RUNX1T1 and inv(16)(p13.1q22) or t(16;16)(p13.1;q22);CBFB-MYH11 are recurrent genetic entities commonly designated as core binding factor (CBF) AML. Both subgroups have distinct gene expression signatures and are characterized by recurrent mutations in KIT, FLT3, and RAS pathway genes. More recently, ASXL1 and ASXL2 mutations have been identified in t(8;21) AML. The TCGA study has analyzed a limited number of AML with CBF rearrangements, but to date more comprehensive CBF AML cohorts have not been unbiasedly explored by next-generation sequencing. Therefore, we have performed RNA sequencing of 415 genetically diverse AML specimens, including 48 CBF AML samples. In this analysis, we compared the mutational profile and transcriptomic landscape of both CBF subgroups to that of non-CBF AML.Methods Analysis of mutations and gene expression was performed as previously described (Lavallée et al, Nature Genetics, doi:10.1038/ng.3371). Mutations in all genes that are recurrently mutated in hematological malignancies are reported. In order to identify novel acquired recurrent mutations in CBF subgroups, genes with variants in ≥ 3 samples were systematically confirmed by Sanger sequencing of non-tumoral DNA.Results Genes mutated in the t(8;21) cohort are: KIT (8/20, 40%), FLT3 and ASXL2 (4/20 each, 20%), ASXL1, NRAS, ZBTB7A, TET2, SMC1A (3/20 each, 15%), DNMT3A (2/20), and JAK2, SMC3, STAG2, WT1, and CSF3R (1/20 each). Mutations in inv(16) AML are found in the following genes: KIT (14/28, 50%), NRAS (12/28, 43%), FLT3 (8/28, 29%), PRRC2B (3/28, 11%), KRAS (2/28, 7%) and BCORL1, DNMT3A, GATA2 and NF1 (1/28). The most frequent mutations were found in activated signaling genes (KIT, NRAS, KRAS, FLT3, JAK2, CSF3R), identified in 14/20 (70%) and 25/28 (89%) of t(8;21) and inv(16) AML samples respectively. 38% of mutated samples contained 2 to 5 such mutations, and the sum of their variant allele frequencies never exceeded ~50%, suggesting that each mutation occurs in a different subclone. This result supports the hypothesis that these mutations and CBFfusion genes are strong collaborators in AML. Several novel observations emerged from these analyses. First, we identified 2 frameshift and 1 missense novel acquired mutations in ZBTB7A, which are specific to the t(8;21) subgroup (3/20 vs 1/395, p = 0.0004). ZBTB7A encodes a transcription factor of the POK/ZBTB family and other genes encoding this family of transcription factors, such as BCL6 and PLZF, are rearranged in hematological malignancies. Second, we established that ASXL2 mutations are very rare (2/395) in t(8;21) negative samples and thus specifically associated to RUNX1 -RUNX1T1 fusions (p < 0.0001). Third, mutually exclusive mutations in cohesin complex genes (SMC1A, SMC3 and STAG2) are frequent in t(8;21) AML (5/20, 25%). Lastly, a novel acquired PRRC2B A1506S missense mutation was identified in 3 inv(16) AML samples. PRRC2B, a gene with poorly described functions, was the only non-activated signaling gene recurrently mutated in this subgroup.Using the most significantly and differentially expressed genes, we identified signatures of 145 and 127 genes specific to t(8;21) and inv(16) groups, respectively. 78% and 81% of these genes have not been previously described in gene set enrichment analyses of CBF AML, and are potential novel CBF diagnostic markers or genes that are functionally related to CBF fusions. Using gene signatures and principal component analyses (PCA), CBF subgroups homogeneously clustered together with one sole exception: a sample harboring a t(16;21);RUNX1-CBFA2T3 unambiguouslygrouped with t(8;21) specimens. The rare but recurrent RUNX1-CBFA2T3 chimeric proteins are known to share similar structural characteristics with RUNX1-RUNX1T1, and our observations now unify the transcriptomic networks of these 2 genetic entities. We also identified and characterized 8 additional RUNX1 fusions in our AML cohort, including 6 novel fusions, which share a different transcriptomic profile compared to RUNX1-RUNX1T1 positive samples, thereby suggesting that they might have distinct functional consequences.Conclusion Our comprehensive RNA sequencing analysis substantially contributes to a better understanding of mutations and gene expression profiles in CBF AML, and reports a unity between RUNX1 -RUNX1T1 and RUNX1-CBFA2T3 genetic networks. DisclosuresNo relevant conflicts of interest to declare.

Therapy-Related Myelodysplasia/Acute Myeloid Leukemia (t-MDS/AML) After Conventionally-Treated Breast Cancer: Impact of Cytogenetics on Outcome After Allogeneic Hematopoietic Cell Transplantation (HCT),

Abstract 4149 Background: The outcome of early stage breast cancer has improved in recent years with use of effective combination chemotherapy, including cyclophosphamide, anthracyclines or taxanes. Patients who undergo lumpectomy also receive radiation therapy for locoregional control. t-MDS/AML are rare but serious consequences of these therapeutic exposures; historically with an overall poor prognosis. Translocations involving the mixed-lineage leukemia (MLL) gene on chromosome band 11q23, and core binding factor genes on 21q22 and 16q22, are hallmarks of t-MDS/AML resulting from treatment with topoisomerase II inhibitors (anthracyclines). Loss of part or whole of chromosomes 5 and/or 7 (5-/7- abnormality) are associated with t-MDS/AML after treatment with alkylating agents and radiation. Allogeneic HCT (sibling-related, unrelated or double-cord) offers the only curative option for some of these patients. However, the long-term outcome of post-breast cancer t-MDS/AML after allogeneic HCT has not been described, and subgroups with better or worse prognosis not identified. Methods: From 1997 to 2011, 28 patients with t-MDS/AML after treatment of breast cancer with conventional chemotherapy and/or radiation underwent allogeneic HCT at City of Hope. Overall survival defined from the time of allogeneic HCT to the last date of follow up or date of death, was estimated using Kaplan-Meier method. Cox regression techniques were utilized to understand predictors of overall survival. Results: Of the 28 patients, 16 had been treated for early-stage breast cancer (I/IIA), 11 had received treatment for locally advanced disease (IIB/ III) and 1 patient had metastatic disease. Twenty-five patients received combination chemotherapy as adjuvant therapy or for metastatic disease; this included cyclophosphamide (100%), doxorubicin (88%) and taxanes (52%). Details of chemotherapy are unknown for one patient. Twenty-one patients (75%) also received radiation (2 received radiation alone). Median age at time of diagnosis of t-MDS/AML was 56 years (range: 34–79); median time from diagnosis of breast cancer to diagnosis of t-MDS/AML was 2.4 years (range 1–12.3); median time from t-MDS/AML to allogeneic HCT was 0.55 year (range: 0.15–4.1). Ten patients presented with MDS and 18 with AML. Cytogenetic abnormalities included MLL - 11q23 abnormality (n=12), 5-/7- and/or complex abnormalities (n=9), t(15;17)(q22;q21.1) (n=1), t(8;21) (n=1), inv(16)(p13.1;q22) (n=2). Of the 28 patients, 27 were treated with induction therapy and 74% were in complete remission (CR) at the time of HCT. Fourteen patients (50%) received sibling-related HCT, 13 received matched unrelated donor (MUD) transplant and 1 received a double cord transplant. Two patients received second allogeneic HCT and one patient received donor lymphocyte infusion (DLI) for relapsed t-MDS/AML. Sixty-one percent received reduced-intensity conditioning with fludarabine/melphalan and others received full-intensity conditioning. Sixteen patients developed cGvHD after transplant. After a median follow up of 2.2 years, 8 patients had died (cause of death: t-MDS/AML [n=5], sepsis [n=1], GvHD [n=1], breast cancer [n=1]), with an overall survival of 78.2% at two years post-HCT for the entire cohort. The 2-year overall survival was 71.4% for sibling-related HCT and 84.4% for MUD (log-rank p=0.14). There was a difference in overall survival by the type of cytogenetic abnormalities. Thus, the 2-year survival rate for patients with 11q23 abnormality was 90.9% while that for patients with chromosome 5-/7- abnormalities or complex abnormalities was 62.5% (log rank p=0.27). Patients with cGvHD had better outcome compared with those without cGvHD (2 year survival 87.5% vs. 64.3%, log-rank p value=0.03). These findings were confirmed on multivariate regression analysis, which revealed chromosome 5-/7- or complex abnormalities to be associated with a significantly worse outcome (HR=6.9, p=0.035), while cGvHD was associated with a better outcome (HR=0.14, p=0.02) after adjustment for age at HCT. Conclusions: Although t-MDS/AML after conventional treatment of breast cancer has historically been shown to have a poor prognosis, allogeneic HCT in patients with available donors provides a chance of cure. Among patients undergoing allogeneic HCT, a superior outcome was observed in patients with 11q23 abnormalities and among those with cGvHD. Disclosures: No relevant conflicts of interest to declare.

Making a Meal of Multiple Mutations in Acute Myeloid Malignancies

In the mid-1970s, seven renowned French, American, and British hematologists met to review more than 200 cases of acute leukemia. Although disagreements likely arose during these meetings concerning the relative gastronomic merits of croissants, apple pie, and bangers and mash, the group was able to determine criteria for the subclassification of acute myeloid and lymphoid leukemias. Their report, published in 1976 in the British Journal of Hematology (but without supplemental material in Gourmet), is the basis for the French-American-British (FAB) classification of acute leukemia. This system, used for distinguishing subtypes of acute myeloid leukemia (AML) for a quarter of a century, defines various flavors of AML based on the type and extent of tumor-cell differentiation and is quite savory for pathologists, who tend to categorize neoplasms in terms of their differentiation state. Despite being limited by the available utensils, this system was quite prescient, serving up a delectable hors d’oeuvre for what was to follow. Numerous subsequent studies have shown, not surprisingly, that the clinical course of patients with AML correlates with mutations present in specific subsets of AML. For instance, those leukemias with translocations involving the core binding factor genes RUNX1 and CBFB are associated with a relatively good prognosis, whereas tumors with complex karyotypic abnormalities and monosomies, especially of chromosome 7, are associated with poor patient outcome. The realization that clinical outcome of patients with AML is associated with specific mutations led to the notion that AML is not a single disease entity, but is rather a smorgasbord of myeloid neoplasms, each with distinct pathogenesis and clinical behavior.

RUNX1 Mutations in Clonal Myeloid Disorders: From Conventional Cytogenetics to Next Generation Sequencing, A Story 40 Years in the Making

Translocations and mutations in the core binding factor genes, RUNX1 or CBFB, are found in acute myeloid and lymphocytic leukemia, therapy-related myeloid leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, and in familial platelet disorder with predisposition to acute myeloid leukemia. Here we review the biochemical and biological properties of the normal Runx1 protein, discuss the nature of RUNX1 mutations in myeloid leukemia, their prognostic significance, and the mutations that cooperate or co-exist with them in these various diseases.

Derepression in the Desert: The Third Workshop on Clinical Translation of Epigenetics in Cancer Therapeutics

In the past decade, the scientific and medical community has witnessed dramatic progress in epigenetics and has begun to understand some of the epigenetic mechanisms that contribute to human disease, particularly cancer. In January 2007, the third biannual workshop on Clinical Translation of

Retroviral Mediated Transfer of MLL Fusion Genes into Human CD34+ Cord Blood Cells Supports the Establishment of Long-Term Myeloid Cultures with Growth Rates, Immunophenotypic and Transcriptional Features Distinct from Those of AML1-ETO Transduced Cultures.

Translocations involving the MLL gene, primarily t(9;11) and t(10;11) together with rearrangements affecting the core binding factor (CBF) genes, t(8;21) and inv(16) comprise the most frequent cytogenetic abnormalities in acute myeloid leukemia (AML). Although all of these rearrangements generate chimeric transcription factors (MLL-AF9, MLL-AF10, AML1-ETO and CBFβ-MYH11) clinicopathologic features and transcriptional profiles clearly distinguish MLL-rearranged from CBF-rearranged AML. To understand how these distinct subgroups of AML arise, we have developed a model for studying the effects of MLL and CBF fusion proteins on the growth, survival and differentiation human myeloid progenitors in vitro. Using retroviral mediated gene transfer, we transduced CD34 selected normal human cord blood (CB) cells with vector (MIEG3) alone or with vectors expressing MLL-AF9, MLL-AF10 or AML1-ETO fusion genes. Whereas CB transduced with MIEG3 proliferated in liquid culture for 6 to 8 weeks, MLL-AF9, MLL-AF10 and AML1-ETO transduced cells have continued to proliferate continuously in culture for more than 16 weeks without any sign of crisis. At any point after transduction, CB expressing MLL-AF9 or MLL-AF10 exhibited a faster rate of growth as compared with AML1-ETO or MIEG3 transduced CB. This difference in growth rate was associated with a reduced frequency of spontaneous apoptosis by annexin staining in the MLL cultures, as compared with the AML1-ETO cultures, but no difference in the fraction of cells in S-phase. MLL-AF9 and MLL-AF10 transduced CB also exhibited evidence of early myeloid maturation arrest based on morphology and surface antigen expression. However, while AML1-ETO transduced cells continue to express CD34 throughout their time in culture, MLL cultures lose expression of this stem cell-associated antigen and acquire expression of c-Kit and CD33, neither of which is expressed in AML1-ETO cultures. Also, unlike AML1-ETO transduced cells, CB transduced with with MLL fusions retain serial clonogenicity for 3 or more rounds of plating in methylcellulose assays. We used quantitative realtime RT-PCR to measure expression of 3 genes that are differentially expressed in patients with MLL or CBF gene fusions based on published microarray data. While expression of SPARC increased over time in MIEG3 cultures or remained stable in CB transduced with AML1-ETO, it decreased to nearly undetectable levels in MLL-AF9 transduced cultures. In contrast, expression of both BMI-1 and HOXA9 increased in the MLL-AF9 cultures and decreased in the MIEG3 and AML1-ETO cultures. The transcriptional changes in our long-term cultures mirror the gene expression differences that have been observed in AML associated with MLL or CBF fusions and suggest that this will be a useful model to study how chimeric transcription factors contribute to myeloid leukemogenesis. Interestingly, CB transduced with a mutated MLL-AF10 (MA10ΔLZ) lacking the leucine zipper domain required for transformation of primary murine myeloid progenitors did not differ, in terms of growth or differentation, from MIEG3 transduced cells. This suggests that the effects of MLL-AF9 and MLL-AF10 on normal CB may reflect early events in myeloid leukemogenesis. The in vivo leukemogenic potential of MLL fusion transduced CB is currently being evaluated in NOD/SCID mice.

Human CD34+ Cells Expressing CBFβ-MYH11 Exhibit a Myelomonocytic Phenotype with Greatly Enhanced Proliferative Ability.

The Core Binding Factor (CBF) transcription factor complex is required for normal hematopoiesis. Knockout of either component of the complex, AML1 or CBFβ, results in embryonic lethality in mice due to a lack of definitive hematopoiesis. Chromosomal rearrangements involving the CBF genes are commonly associated with leukemia. The t(8:21) fuses the AML1 gene to ETO (AE) and is predominantly observed in leukemias of the FAB M2 subtype of acute myeloid leukemia (AML). The t(16:16) and inv(16) are associated with FAB M4Eo leukemias and result in fusion of the CBFβ gene to MYH11 (CM). While knockin of either AE or CM is not sufficient to cause disease, expression of either protein increases the incidence of leukemia in mice when combined with mutagens or specific secondary genetic manipulations. We have previously shown that retroviral transduction of primary human CD34+ cells with AE causes a dramatic accumulation of CD34+ progenitor cells with increased CFU-C and CAFC activity during an increased lifespan in vitro. These cultures retain the ability to differentiate to myeloid, lymphoid, and erythroid lineages and the ability to engraft NOD/SCID mice. Consistent with mouse studies, these cells fail to cause disease in mice or become transformed in vitro. These cultures serve as a preleukemic model for human AML associated with AML1-ETO expression. We show here that retroviral transduction of CM into human CD34+ hematopoietic cells similarly mimics a putative pre-leukemic condition initiated by inv(16). Transduced cells proliferate for up to 7 months in culture with retention of CD34 expression on a subset of cells. Cells from CM cultures display a more mature morphology than AE cells, consistent with the association with M4Eo AMLs, and have a greatly reduced ability to differentiate to the erythroid lineage in methylcellulose assays. Nevertheless, long-term CM cultures retain CFU-C activity, the ability to engraft NOD/SCID-B2M mice, and the potential for B-cell development in vitro, implicating a primitive progenitor as the target cell rather than a committed granulocyte/monocyte progenitor. Microarray analysis of AML leukemias has identified specific genes with altered expression in inv(16) patient samples. We have found that a number of these inv(16)-associated transcripts are likewise altered in CD34+ cells from our CM cultures when compared to control CD34+ cells, while others are not affected. These data suggest that our system will be useful in examining the effects of CBFβ-MYH11 on gene expression in the human preleukemic cell, in characterizing the effect of this oncogene on human stem cell biology and in defining its contribution to the development of leukemia.

Leukemic transformation in myelofibrosis with myeloid metaplasia: a single-institution experience with 91 cases

AbstractAmong 2333 consecutive patients with myelofibrosis with myeloid metaplasia (MMM) seen at our institution, 91 fulfilled the World Health Organization (WHO) criteria for leukemic transformation (LT). All episodes of LT were myeloid in origin (acute myeloid leukemia [AML]) with all French-American-British (FAB) subtypes represented except M3; the most frequent subtypes were M7 (25.4%), M0 (22.4%), and M2 (17.9%). Cytogenetic studies during LT were available in 56 patients and revealed a clonal abnormality in 51 (91%): 30 patients had complex karyotype, 2 had core-binding factor gene lesions, and 18 had abnormalities of chromosome 5 or 7. Karyotypic evolution was documented in the majority of the patients in whom serial analysis was possible. In general, LT was fatal in 98% of the cases after a median of 2.6 months (range, 0-24.2 months). Twenty-four patients received AML-like induction chemotherapy that resulted in no complete remission: 41% reverted into chronic-phase disease and the incidence of treatment-related mortality was 33%. The remaining 67 patients received either supportive care alone (48 patients) or low-intensity chemotherapy (19 patients). Overall, survival was similarly poor in all 3 treatment categories. The outcome of LT in MMM with current therapies is dismal and either supportive care alone or appropriate clinical trials should be considered.

Neonatal Lethal Osteochondrodysplasia with Low Serum Levels of Alkaline Phosphatase and Osteocalcin

Neonatal lethal skeletal dysplasias are rare and typically involve thoracic malformations and severe limb shortening. We report on a newborn boy manifesting an osteochondrodysplasia associated with fatal respiratory insufficiency who had normal lung volumes and extremity lengths. His disorder featured aberrant skeletal patterning and defective ossification including a severely osteopenic skull, apparent absence of clavicles, and clefting of the mandible and vertebrae. Serum alkaline phosphatase and osteocalcin levels were markedly low. Biochemical studies suggested parathyroid insufficiency probably from critical illness. Histopathology at autopsy excluded impaired mineralization of skeletal matrix, but endochondral bone formation appeared disorganized with growth plate clustering of chondrocytes in hypertrophic zones and in zones of provisional calcification. Parathyroid glands were not found. Despite features of two distinctive heritable entities, hypophosphatasia and cleidocranial dysplasia, the cumulative findings did not match either condition, and no mutations were found in either the tissue nonspecific ALP isoenzyme or core-binding factor genes, respectively, or in the genes encoding osteocalcin or the osteoblast transcription factor osterix. This patient could represent the extreme of cleidocranial dysplasia (a disorder not always associated with structural mutation in core-binding factor A1), but more likely he defines a unique osteochondrodysplasia disrupting both intramembranous and endochondral bone formation.

Screening for core binding factor gene rearrangements in acute myeloid leukemia

Screening for core binding factor gene rearrangements in acute myeloid leukemia

The management of patients with leukaemia: the role of cytogenetics in this molecular era.

The management of patients with leukaemia: the role of cytogenetics in this molecular era.