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Epigenetic changes are among the most common alterations observed in cancer cells, yet the mechanism by which cancer cells acquire and maintain abnormal DNA methylation patterns is not understood. Cancer cells have an altered distribution of DNA methylation and express aberrant DNA methyltransferase 3B transcripts, which encode truncated proteins, some of which lack the COOH-terminal catalytic domain. To test if a truncated DNMT3B isoform disrupts DNA methylation in vivo, we constructed two lines of transgenic mice expressing DNMT3B7, a truncated DNMT3B isoform commonly found in cancer cells. DNMT3B7 transgenic mice exhibit altered embryonic development, including lymphopenia, craniofacial abnormalities, and cardiac defects, similar to Dnmt3b-deficient animals, but rarely develop cancer. However, when DNMT3B7 transgenic mice are bred with Eμ-Myc transgenic mice, which model aggressive B-cell lymphoma, DNMT3B7 expression increases the frequency of mediastinal lymphomas in Eμ-Myc animals. Eμ-Myc/DNMT3B7 mediastinal lymphomas have more chromosomal rearrangements, increased global DNA methylation levels, and more locus-specific perturbations in DNA methylation patterns compared with Eμ-Myc lymphomas. These data represent the first in vivo modeling of cancer-associated DNA methylation changes and suggest that truncated DNMT3B isoforms contribute to the redistribution of DNA methylation characterizing virtually every human tumor. Cancer Res; 70(14); 5840–50. ©2010 AACR.

Supplementary Methods, Figures 1-10, Tables 1-8 from DNMT3B7, a Truncated DNMT3B Isoform Expressed in Human Tumors, Disrupts Embryonic Development and Accelerates Lymphomagenesis

The roles of RNA 5-methylcytosine (RNA:m5C) and RNA:m5C methyltransferases (RCMTs) in lineage-associated chromatin organization and drug response/resistance are unclear. Here we demonstrate that the RCMTs, namely NSUN3 and DNMT2, directly bind hnRNPK, a conserved RNA-binding protein. hnRNPK interacts with the lineage-determining transcription factors (TFs), GATA1 and SPI1/PU.1, and with CDK9/P-TEFb to recruit RNA-polymerase-II at nascent RNA, leading to formation of 5-Azacitidine (5-AZA)-sensitive chromatin structure. In contrast, NSUN1 binds BRD4 and RNA-polymerase-II to form an active chromatin structure that is insensitive to 5-AZA, but hypersensitive to the BRD4 inhibitor JQ1 and to the downregulation of NSUN1 by siRNAs. Both 5-AZA-resistant leukaemia cell lines and clinically 5-AZA-resistant myelodysplastic syndrome and acute myeloid leukaemia specimens have a significant increase in RNA:m5C and NSUN1-/BRD4-associated active chromatin. This study reveals novel RNA:m5C/RCMT-mediated chromatin structures that modulate 5-AZA response/resistance in leukaemia cells, and hence provides a new insight into treatment of leukaemia.

The t(4;11)(q21;q23) fuses mixed-lineage leukemia (MLL) to AF4, the most common MLL-fusion partner. Here we show that MLL fused to murine Af4, highly conserved with human AF4, produces high-titer retrovirus permitting efficient transduction of human CD34+ cells, thereby generating a model of t(4;11) pro-B acute lymphoblastic leukemia (ALL) that fully recapitulates the immunophenotypic and molecular aspects of the disease. MLL-Af4 induces a B ALL distinct from MLL-AF9 through differential genomic target binding of the fusion proteins leading to specific gene expression patterns. MLL-Af4 cells can assume a myeloid state under environmental pressure but retain lymphoid-lineage potential. Such incongruity was also observed in t(4;11) patients in whom leukemia evaded CD19-directed therapy by undergoing myeloid-lineage switch. Our model provides a valuable tool to unravel the pathogenesis of MLL-AF4 leukemogenesis.

Introduction Cutaneous composite lymphoma (CCL) is extremely rare. When 2 potentially distinct lymphoid lesions occur at one skin site, distinguishing between one neoplastic clone and a secondary reactionary lymphoid response versus a second neoplasm is difficult. In this study, we describe a unique case of CCL along with a review of reported cases in literature to identify clues and discuss issues that are relevant to the diagnosis of CCL. Design Review of a CCL case from our institution and a systematic review of reported cases of CCL in the literature. Results A total of 18 studies describing 22 cases and a case report from our institution are included. The mean age at diagnosis was 68 years. Most cases herein presented with multiple skin lesions (67%) and reported a history of immune suppression (76%). Nineteen cases (83%) had a combination of T-cell and B-cell neoplasms, whereas the remaining cases had 2 distinct B-cell clones. Clonal differentiation was confirmed based on morphology and immunohistochemistry in all cases, and by polymerase chain reaction studies in 19 cases. Complete remission was achieved in only one quarter of reported cases. Conclusion Diagnosing CCL can be challenging because accurate differentiation of 2 or more clonal populations at 1 site is tedious. A stepwise approach and integration of clinical, morphologic, immunohistochemistry, and molecular data along with an understanding of the prognosis of the lymphomas in question is essential for an accurate diagnosis and necessary because of therapeutic and prognostic implications.

Objectives: The biannual Fellow In-Service Hematopathology Examination (FISHE) assesses knowledge in five content areas. We examined the relationship between taking the FISHE and performance on it with outcomes on the first attempted American Board of Pathology Hematology subspecialty certifying examination (ABP-HE). Methods: The pass rate between the ABP-HE candidates who took the spring FISHE and those who did not were compared. The likelihood of fellows passing the ABP-HE based on their percentiles on the FISHE was also assessed. Results: ABP-HE candidates who took the spring FISHE had a higher pass rate (96.4%) than those who did not (76.1%, P

An interstitial deletion of the long arm of chromosome 5, del(5q), is a recurring abnormality in myeloid disorders, including myelodysplastic syndromes (MDS), de novo acute myeloid leukemia (AML), and therapy-related myeloid neoplasms (t-MN) comprising therapy-related MDS and AML (t-MDS/t-AML).[1][1

The roles of RNA 5-methylcytosine (RNA:m5C) and RNA:m5C methyltransferases (RCMTs) in lineage-associated chromatin organization and drug response/resistance are unclear. Here we demonstrate that the RCMTs, namely NSUN3 and DNMT2, directly bind hnRNPK, a conserved RNA-binding protein. hnRNPK interacts with the lineage-determining transcription factors (TFs), GATA1 and SPI1/PU.1, and with CDK9/P-TEFb to recruit RNA-polymerase-II at nascent RNA, leading to formation of 5-Azacitidine (5-AZA)-sensitive chromatin structure. In contrast, NSUN1 binds BRD4 and RNA-polymerase-II to form an active chromatin structure that is insensitive to 5-AZA, but hypersensitive to the BRD4 inhibitor JQ1 and to the downregulation of NSUN1 by siRNAs. Both 5-AZA-resistant leukaemia cell lines and clinically 5-AZA-resistant myelodysplastic syndrome and acute myeloid leukaemia specimens have a significant increase in RNA:m5C and NSUN1-/BRD4-associated active chromatin. This study reveals novel RNA:m5C/RCMT-mediated chromatin structures that modulate 5-AZA response/resistance in leukaemia cells, and hence provides a new insight into treatment of leukaemia., Resistance to chemotherapy is a serious issue that can be influenced by RNA epigenetics and chromatin structure. Here, the authors show in leukaemia cells that RNA 5-methylcytosine (RNA:m5C) and RNA:m5C methyltransferases (RCMTs) mediate chromatin structures that can modulate 5-Azacitidine response and resistance.

This chapter covers the topics of myeloproliferative neoplasms, myelodysplastic/myeloproliferative neoplasm, and myeloid/lymphoid neoplasms associated with eosinophilia and rearrangements of PDGFRA, PDGFRB, or FGFR1 or with PCM1-JAK2. Disease entities are reviewed in accord with the 2016 revision of the World Health Organization (WHO) classification of hematopoietic neoplasms. Each topic is discussed for its definition, clinical presentations, morphologic features, ancillary testing, differential diagnosis, and a brief summary of treatment and prognosis. For each disease, there is a fact sheet to provide a quick review of the key points. A total of 41 figures are provided to illustrate the morphologic features of various entities. This updated version has incorporated new discoveries and understanding of molecular genetics of the diseases and their implications in diagnosis, classification, prognosis, and therapy.

Objectives: Session 1 of the 2013 Society for Hematopathology/European Association for Hematopathology Workshop was devoted to the cases of acute myeloid leukemia (AML) with recurrent cytogenetic abnormalities. Methods: Based on World Health Organization 2008 criteria, seven specific translocations are defined as “recurrent” in AML. Of these seven, three are considered to be AML defining regardless of blast percentage. Workshop cases provided the opportunity to consider potential new AML-defining cytogenetic mutations, as well as other unique aspects of AML with cytogenetic abnormalities. Results: Most of the 38 cases submitted were acute promyelocytic leukemia (APL) with t(15;17)(q24.1;q21.1) and so-called variants (12 cases), AML with t(8;21)(q22;q22) (seven cases), AML with inv(3)(q11q26.2) (six cases), and AML with 11q23 translocations (five cases). Conclusions: This review focuses on providing updated recommendations for the rapid diagnosis of APL, discussing the types and significance of variant RARA mutations in APL-like leukemias, and refining low-blast-count (oligoblastic) AML. In addition, the significance of unique morphologic, immunophenotypic, and genetic variations in AML defined by a recurrent cytogenetic abnormality is included.

Acute myeloid leukemia and myelodysplastic syndrome with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) have a poor prognosis. Indeed, the inv(3)(q21q26.2)/t(3;3)(q21;q26.2) has been recognized as a poor risk karyotype in the revised International Prognostic Scoring System. However, inv(3)(q21q26.2)/t(3;3)(q21;q26.2) is not among the cytogenetic abnormalities pathognomonic for diagnosis of acute myeloid leukemia irrespective of blast percentage in the 2008 WHO classification. This multicenter study evaluated the clinico-pathological features of acute myeloid leukemia/myelodysplastic syndrome patients with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and applied the revised International Prognostic Scoring System to myelodysplastic syndrome patients with inv(3)(q21q26.2)/t(3;3)(q21;q26.2). A total of 103 inv(3)(q21q26.2)/t(3;3)(q21;q26.2) patients were reviewed and had a median bone marrow blast count of 4% in myelodysplastic syndrome (n=40) and 52% in acute myeloid leukemia (n=63) (P

The drivers of abnormal DNA methylation in human cancers include widespread aberrant splicing of the DNMT3B gene, producing abnormal transcripts that encode truncated proteins that may act as dominant negative isoforms. To test whether reduced Dnmt3b dosage can alter tumorigenesis, we bred Dnmt3b(+/-) mice to Eµ-Myc mice, a mouse model susceptible to B-cell lymphomas. Eµ-Myc/Dnmt3b(+/-) mice showed a dramatic acceleration of lymphomagenesis, greater even than that observed in Eµ-Myc mice that express a truncated DNMT3B isoform found in human tumors, DNMT3B7. This finding indicates that Dnmt3b can act as a haploinsufficient tumor suppressor gene. Although reduction in both Dnmt3b dosage and expression of DNMT3B7 within the Eµ-Myc system had similar effects on tumorigenesis and DNA hypermethylation, different molecular mechanisms appear to underlie these changes. This study offers insight into how de novo DNA methyltransferases function as tumor suppressors and the sensitivity of Myc-induced lymphomas to DNA methylation.

A 40 year old male patient with nodular lymphocyte predominance Hodgkin's disease had a nodal relapse of the mixed cellularity subtype 14 years after entering complete remission with total nodal irradiation. This case is unusual because of the very long disease-free interval and also because the histology at relapse changed to the mixed cellularity subtype. A review of the literature of late relapses in Hodgkin's disease is presented, and possible mechanisms involved in their development are discussed. Hodgkin's disease.

Anemia is the predominant clinical manifestation of myelodysplastic syndromes (MDS). Loss or deletion of chromosome 7 is commonly seen in MDS and leads to a poor prognosis. However, the identity of functionally relevant, dysplasia-causing, genes on 7q remains unclear. Dedicator of cytokinesis 4 (DOCK4) is a GTPase exchange factor, and its gene maps to the commonly deleted 7q region. We demonstrate that DOCK4 is underexpressed in MDS bone marrow samples and that the reduced expression is associated with decreased overall survival in patients. We show that depletion of DOCK4 levels leads to erythroid cells with dysplastic morphology both in vivo and in vitro. We established a novel single-cell assay to quantify disrupted F-actin filament network in erythroblasts and demonstrate that reduced expression of DOCK4 leads to disruption of the actin filaments, resulting in erythroid dysplasia that phenocopies the red blood cell (RBC) defects seen in samples from MDS patients. Reexpression of DOCK4 in -7q MDS patient erythroblasts resulted in significant erythropoietic improvements. Mechanisms underlying F-actin disruption revealed that DOCK4 knockdown reduces ras-related C3 botulinum toxin substrate 1 (RAC1) GTPase activation, leading to increased phosphorylation of the actin-stabilizing protein ADDUCIN in MDS samples. These data identify DOCK4 as a putative 7q gene whose reduced expression can lead to erythroid dysplasia.

The classification and diagnostic criteria of the myeloproliferative neoplasms have changed significantly in the 2008 World Health Organization monograph on the classification of hematologic malignancies. Many of the changes arose from the findings that the different malignancies are associated with abnormal cell signaling because of translocations or mutations in genes for protein tryosine kinases involved in the normal growth and regulation of hematopoietic cells. These include ABL1, PDGFRA, PDGFRB, FGFR1, JAK2, MPL, and KIT. The new classification attempts to reflect the related molecular pathogenesis of the different entities and incorporates the identification of the molecular defects into the diagnostic criteria for some of the individual diseases. Issues concerning the new classification are discussed, and the new diagnostic criteria are reviewed and commented upon.

We evaluated stem cell mobilization in 195 consecutive sibling donors who underwent a uniform mobilization regimen of granulocyte colony-stimulating factor (G-CSF) at 10 microg/kg/day divided into twice daily dosing. On day 5, peripheral blood (PB) CD34 cells/microL were measured immediately prior to peripheral blood stem cell (PBSC) apheresis. Failed mobilization was defined as20 CD34 cells/microL on day 5. The median age was 52 years and 73 (37%) were 55 years or greater. Comorbid conditions by the Charlson Comorbidity Index (CCI) occurred in 13%, but only 3% had Karnofsky performance status (PS)100%. Eight (4%) failed mobilization, defined as20 CD34 cells/microL on day 5. Older age was associated with fewer CD34 cells/microL (P=.002). In addition, 6/73 (8.2%) older donors failed mobilization compared to 2/122 (1.6%) younger donors (P=.054). Comorbidity, sex, race, and donor weight did not influence mobilization. Although low PS was very uncommon, it was associated with reduced mobilization (P=.021), but not mobilization failure. A small fraction of older donors mobilize poorly, and this is not explained by standard measures of comorbidity or PS.

In the 2008 World Health Organization-sponsored classification of hematopoietic and lymphoid malignancies, changes were made in the classification and criteria for the diagnosis of some of the myeloproliferative disorders. These changes were initiated by recent insights into the molecular pathogenesis of these disorders. In this article, the changes made to the myeloproliferative neoplasm and the basis of the new classification and altered diagnostic criteria are summarized and discussed.

Context.—Diffuse large B-cell lymphoma is a heterogenous group of lymphomas. In this review, we present a brief description of the large number of entities recognized in the recently published (2008) World Health Organization classification of tumors of hematopoietic and lymphoid tissues. Objective.—We highlight the unique clinicopathologic and molecular genetic features of these new and previously recognized entities, to illustrate the rational for the development of this classification. To help simplify the understanding of this now large and complex group of diseases, we have attempted to create broader subgroups of related entities. We discuss large B-cell lymphoma that are not otherwise specified, those that are based on anatomic site, those that have unique histology or phenotype or genotype, those that are associated with Epstein-Barr virus or Kaposi sarcoma–associated herpesvirus and herpesvirus 8, and those that are unclassifiable. Data Sources.—World Health Organization classification of tumors of hematopoietic and lymphoid tissues (2008), published literature from PubMed (National Library of Medicine), and primary material from the authors' institution were reviewed. Conclusions.—Recognition of the different subtypes of diffuse large B-cell lymphoma as described in the World Health Organization classification scheme will lead to improved understanding of the unique clinicopathologic and genetic features associated with these subtypes of lymphoma.

FHL2 is a multifunctional LIM domain protein that acts as a transcriptional modulator mediating proliferation and apoptosis in a tissue-specific manner. Upregulation of FHL2 has been detected in a variety of cancers. We demonstrate that upregulation of FHL2 is associated with a subset of acute myeloid leukemia with a characteristic gene-expression signature, and abnormalities of chromosome 5. In mice, expression of endogenous Fhl2 is downregulated coordinately during the differentiation of hematopoietic cells. Upregulation of FHL2 enhances proliferation of myeloid progenitor cells, and serial-replating efficiency of hematopoietic cells in vitro. Chimeric mice with enforced expression of FHL2 in bone marrow cells, are characterized by an expanded pool of myeloid progenitor cells, enhanced granulopoi esis and megakaryocytopoiesis. In addition, enhanced expression of FHL2 promotes cell-cycle entry of myeloid progenitor cells and increases the frequency of apoptosis of bone marrow cells in vivo. These results raise the possibility that deregulation of FHL2 contributes to the development of human myeloid disorders. Leukemia (2009) 23, 1650-1657; doi:10.1038/leu.2009.78; published online 16 April 2009

PurposeCancer and Leukemia Group B (CALGB) 9720 evaluated subcutaneous low-dose recombinant interleukin-2 (rIL-2) maintenance immunotherapy as a strategy for prolonging remission in older patients with acute myeloid leukemia (AML).Patients and MethodsAML patients age 60 years and older in first complete remission after induction and consolidation chemotherapy were randomly assigned to no further therapy or a 90-day regimen of 14-day cycles of low-dose rIL-2, aimed at expanding natural killer (NK) cells, followed by 3-day higher doses aimed at activating cytotoxicity of expanded NK cells to lyse residual AML cells. All randomly assigned patients were included in an intention-to-treat analysis.ResultsA total of 163 (64%) of 254 patients who completed induction and consolidation chemotherapy on CALGB 9720 were randomly assigned to rIL-2 (n = 81) or no further therapy (n = 82); the most common reasons for lack of random assignment were patient refusal and relapse. Fifteen patients randomly assigned to rIL-2 never initiated it because of refusal, intercurrent medical problems, or relapse, and 24 patients initiated rIL-2 but stopped early because of toxicity or relapse. Grade 4 toxicities during rIL-2 therapy included thrombocytopenia (65%) and neutropenia (64%), and grade 3 toxicities included anemia (33%), infection (24%) and malaise/fatigue (14%). Forty-two patients (52%) randomly assigned to rIL-2 completed the full 90-day course. Patients in both arms had similar distributions of both disease-free (combined median = 6.1 months; P = .47) and overall survival (combined median = 14.7 months; P = .61) after random assignment. Moreover, the 42 patients who completed all planned therapy did not show prolongation of disease-free or overall survival.ConclusionLow-dose rIL-2 maintenance immunotherapy is not a successful strategy in older AML patients.

KIT mutations have been identified in several malignancies, including acute myeloid leukemia (AML) and systemic mastocytosis (SM). Mast cell leukemia (MCL) is the most aggressive mast cell neoplasm, but has not been well studied due to its rarity. We identified novel KIT transcripts in two patients with MCL and two patients with SM with an associated hematological disorder, but not from two patients with SM. Similar novel KIT transcripts were also observed in normal CD34+ cells from bone marrow and umbilical cord blood, suggesting that altered KIT isoforms may be specific to the blast stage of hematopoietic precursors. The novel KIT proteins lack several domains including the ATP binding site, and one was inactive in a functional test for autophosphorylation. Our discovery of novel KIT transcripts underscores the importance of analysing entire protein encoding regions when studying genes of interest.

In practice, cases of therapy-related myelodysplastic syndrome (t-MDS) are often classified according to morphologic schemes used for de novo MDS. However, there are few data addressing the appropriateness of such classification. We studied 155 patients with therapy-related acute myeloid leukemia (t-AML)/t-MDS to determine whether subclassification by the World Health Organization (WHO) criteria for de novo MDS provides prognostic information in t-MDS. In addition, we assessed whether cytogenetic stratification by the International Prognostic Scoring System (IPSS) guidelines or karyotypic complexity was prognostically important. We found no differences in median survival times among patients classified into the different WHO subgroup of MDS or according to their bone marrow blast percentage; our results indicate a uniformly poor outcome in t-MDS regardless of morphologic classification. However, significant survival differences correlated with cytogenetic stratification according to IPSS guidelines and/or karyotypic complexity. We found only a borderline difference in median survival of patients with an initial t-MDS diagnosis compared with patients with an initial t-AML diagnosis.

Epigenetic alterations play a central role in the control of normal and malignant blood cell development. We demonstrate here that expression of a truncated DNA methyltransferase 3B isoform DNMT3B7, which has been shown to alter cellular epigenetic patterns, decreases the overall number of hematopoietic stem and progenitor cells (HSPCs), and markedly diminishes blood cell reconstitution within the female hormonal microenvironment. Gene expression profiling of HSPCs isolated from DNMT3B7 transgenic embryos identified Apolipoprotein E (Apoe) as overexpressed. The CpG island controlling Apoe expression had lower levels of modified cytosines in DNMT3B7 transgenic HSPCs, corresponding with the observed increase in gene expression. Furthermore, we observed that spleens and bone marrows of female mice transplanted with DNMT3B7 transgenic HSPCs express very high levels of Apoe. Finally, the introduction of Apoe-overexpressing HSPCs into male recipients decreased bone marrow engraftment, recapitulating our original observations in female recipients. Our work reveals a dynamic interplay between the intrinsic epigenetic changes in HSPCs and extrinsic endocrine factors acting on these cells to regulate the efficiency of HSPC engraftment and reconstitution. We have identified a novel mechanism by which gender-specific hormones modulate HSPC function, which could serve as a target for augmenting hematopoiesis in cases with limited HSC functionality. Stem Cells 2015;33:3643–3654

BACKGROUND Acute myeloid leukemia (AML) with t(6;11)(q27;q23) is a well established but rare entity, and few studies have reported the full clinical, hematologic, and outcome data of patients with this disease. METHODS To characterize the features of t(6;11) AML, the authors searched the Cancer and Leukemia Group B (CALGB) cytogenetic database comprising 2667 adults with newly diagnosed, de novo AML and identified 16 patients (0.6%) with t(6;11). A review of the literature identified 33 adults with de novo t(6;11) AML for whom survival data were available. RESULTS CALGB patients had a median age of 45 years (range, 22–65 years) and commonly presented with French–American–British (FAB) subtype M4 or M5 (81%). Gingival involvement at presentation was common (31%). All patients with gingival involvement had FAB M4. Compared with other patients with M4 AML in the CALGB database (n = 429), patients with M4 and t(6;11) (n = 7) had a higher frequency of gingival hypertrophy at presentation (71% vs. 17%, P = 0.003). Patients with t(6;11) were more likely to be African American (P = 0.02) and to die during induction (P = 0.03) than those without t(6;11). The complete response (CR) rate was 69% (11 of 16 patients), and CR duration was short (median, 9 months). The estimated probability of 2-year survival was 13%. Both long-term survivors received allogeneic stem cell transplantation. The estimated probability of 2-year survival of patients reported in the literature was 15%. CONCLUSIONS Although the patient sample was small, the authors suggested that investigational approaches, including allogeneic transplantation, be considered for adults with t(6;11) AML. Cancer 2004. © 2004 American Cancer Society.

Genetic—particularly cytogenetic—analysis is almost routine now in the evaluation of patients with myeloid diseases. Although the findings from such studies are not contributory in all cases, in many instances they can be integrated with morphology, cytochemical features, and immunophenotypic studies to confirm a diagnosis, help resolve a difficult differential diagnosis, recognize disease subsets, or identify pathways involved in the pathogeneses of the disease process. The first series of cases in the Workshop illustrated the use of genetic information in the diagnosis of the myeloid disorders. The cases demonstrated how the results of genetic and molecular-genetic testing can be integrated into patient evaluation, and also aptly illustrated that the findings have varying degrees of significance, ranging from important and sometimes critical to the diagnosis to noncontributory and of little significance. The cases also illustrated some advantages and limitations of the different techniques for acquiring genetic information, including banded chromosome analysis, metaphase fluorescence in situ hybridization (FISH), interphase FISH (Figs 1, 2, and 3), and polymerase chain reaction (PCR)-based studies. Finally, these cases served to raise some specific issues regarding how the molecular-genetic information is obtained and incorporated into the final pathologic diagnosis.

The translocation t(8;14)(q24;q11) has been described at the cytogenetic and molecular level in pediatric cases of T-cell leukemia/lymphoma [1]. Although infrequent, the cases reported show a very ...

PU.1 and GATA transcription factors appear to antagonize each other's function in the development of distinct lineages of the hematopoietic system. In contrast, we demonstrate that PU.1, like GATA-2, is essential for the generation of mast cells. PU.1−/− hematopoietic progenitors can be propagated in IL-3 and differentiate into mast cells or macrophages upon restoration of PU.1 activity. Using these progenitors and a conditionally activatable PU.1 protein, we show that PU.1 can negatively regulate expression of the GATA-2 gene. In the absence of GATA-2, PU.1 promotes macrophage but not mast cell differentiation. Reexpression of GATA-2 in such progenitors enables the generation of mast cells. We propose a developmental model in which cooperative function or antagonistic crossregulation by PU.1 of GATA-2 promotes distinct myeloid cell fates.

The Cancer and Leukemia Group B conducted a phase 3 trial of the P-glycoprotein modulator PSC-833 in untreated acute myeloid leukemia patients aged 60 years and older. Patients were randomized to 1 of 2 regimens, with doses determined in a prior phase 1 study, consisting of cytarabine 100 mg/m2/d by 7-day infusion, with daunorubicin 60 mg/m2 and etoposide 100 mg/m2 daily for 3 days (ADE), or daunorubicin 40 mg/m2 and etoposide 60 mg/m2 for 3 days with PSC-833, 2.8 mg/kg over 2 hours, and then 10 mg/kg/d by 3-day infusion (ADEP). The ADEP arm was closed after randomization of 120 patients (61 to ADE and 59 to ADEP) because of excessive early mortality. Rates of complete remission, nonresponse, and death were 46%, 34%, and 20% for ADE, versus 39%, 17%, and 44% for ADEP (P = .008). Nevertheless, disease-free survival (median 7 vs 8 months; P = .38) and overall survival (approximately 33% alive at 1 year) did not differ and were similar to historical results. Although the number of patients was limited, ADE patients whose pretreatment cells exhibited PSC-833–modulated dye efflux in vitro (n = 22) had worse outcomes than those without efflux (n = 11) (complete remission, nonresponse, and death rates of 41%, 41%, and 18%, compared with 91%, 9%, and 0%; P = .03), but with ADEP outcomes were nearly identical. Moreover, for patients with PSC-833–modulated efflux, median disease-free survival was 5 months with ADE and 14 months with ADEP (P = .07). Further modulation trials in older patients must await the design of less-toxic regimens.

Children with Down syndrome have a 10–20-fold elevated risk of developing leukemia, particularly acute megakaryoblastic leukemia (AMKL)1. While a subset of pediatric AMKLs is associated with the 1;22 translocation and expression of a mutant fusion protein2,3, the genetic alterations that promote Down syndrome–related AMKL (DS-AMKL) have remained elusive. Here we show that leukemic cells from every individual with DS-AMKL that we examined contain mutations in GATA1, encoding the essential hematopoietic transcription factor GATA1 (GATA binding protein 1 or globin transcription factor 1). Each mutation results in the introduction of a premature stop codon in the gene sequence that encodes the amino-terminal activation domain. These mutations prevent synthesis of full-length GATA1, but not synthesis of a shorter variant that is initiated downstream. We show that the shorter GATA1 protein, which lacks the N-terminal activation domain, binds DNA and interacts with its essential cofactor Friend of GATA1 (FOG1; encoded by ZFPM1)4 to the same extent as does full-length GATA1, but has a reduced transactivation potential. Although some reports suggest that the activation domain is dispensable in cell-culture models of hematopoiesis5,6, one study has shown that it is required for normal development in vivo7. Together, these findings indicate that loss of wildtype GATA1 constitutes one step in the pathogenesis of AMKL in Down syndrome.

BCL6, a gene on chromosome 3 band q27, encodes aKruppel-type zinc finger transcriptional repressor. Rearrangements of this gene are frequent in various kinds of lymphomas, particularly of the large-cell B-cell type. The BCL6 nuclear phosphoprotein is expressed in a variety of tissues and is up-regulated particularly in lymph node germinal centers. The zinc fingers of BCL6 bind DNA in a sequence-specific manner. To identify targets of the BCL6 repressive effects, we used a VP16-BCL6 fusion protein containing the zinc fingers but devoid of the repressor domains to compete with the binding of endogenous BCL6 in a transiently transfected B-cell line and then performed subtractive hybridization by using a method to selectively amplify sequences that are differentially expressed. We found that the programmed cell death-2 (PDCD2) gene is a target of BCL6 repression. This gene is the human homolog ofRp8, a rat gene associated with programmed cell death in thymocytes. Immunohistochemistry reveals the anticipated inverse relationship between BCL6 and PDCD2 expression in human tonsil. PDCD2 is detectable in cells of the germinal center in areas where there is less BCL6 expression as well as in the mantle zone, where there is little or no BCL6 expression. These results raise the possibility that BCL6 may regulate apoptosis by means of its repressive effects on PDCD2. BCL6 deregulation may lead to persistent down-regulation of PDCD2, reduced apoptosis, and, as a consequence, accumulation of BCL6-containing lymphoma cells.

We recently identified PIM1 as a gene that cooperates with BCL6 to promote the development of a variety of lymphomas, both B and T cell, and suggested that the serine/threonine kinase encoded by PI...

An interstitial deletion of chromosome 5, del(5q), is the most common structural abnormality in primary myelodysplastic syndromes (MDS) and therapy-related myeloid neoplasms (t-MNs) after cytotoxic therapy. Loss of TP53 activity, through mutation or deletion, is highly associated with t-MNs with a del(5q). We previously demonstrated that haploinsufficiency of Egr1 and Apc, 2 genes lost in the 5q deletion, are key players in the progression of MDS with a del(5q). Using genetically engineered mice, we now show that reduction or loss of Tp53 expression, in combination with Egr1 haploinsufficiency, increased the rate of development of hematologic neoplasms and influenced the disease spectrum, but did not lead to overt myeloid leukemia, suggesting that altered function of additional gene(s) on 5q are likely required for myeloid leukemia development. Next, we demonstrated that cell intrinsic loss of Tp53 in hematopoietic stem and progenitor cells haploinsufficient for both Egr1 and Apc led to the development of acute myeloid leukemia (AML) in 17% of mice. The long latency (234-299 days) and clonal chromosomal abnormalities in the AMLs suggest that additional genetic changes may be required for full transformation. Thus, loss of Tp53 activity in cooperation with Egr1 and Apc haploinsufficiency creates an environment that is permissive for malignant transformation and the development of AML.

Therapy-related myeloid neoplasms (t-MN) are a late complication of the successful use of cytotoxic therapy for patients with cancer. A heterozygous deletions of the long arm of chromosome 5 [del(5q)], observed in 40% of patients, is associated with prior exposure to alkylating agents, and a high frequency of TP53 loss or mutation. In previous studies, we demonstrated that haploinsufficiency of 2 del(5q) genes, Egr1, and Apc, individually play a role in the pathogenesis of hematologic disease in mice. We now show that loss of one copy of Egr1 or Tp53 in an Apc haploinsufficient background (Apc (del/+)) accelerated the development of a macrocytic anemia with monocytosis, early features of t-MN. The development of anemia was significantly accelerated by treatment of mice with the alkylating agent, N-ethyl-N-nitrosourea (ENU), regardless of the levels of expression of Egr1 and Tp53. Transplantation of either wild type; Egr1(+/-); Tp53(+/-); Apc(del/+); or Egr1(+/-), Apc(del/+) bone marrow cells into lethally irradiated Apc(del/+) recipients resulted in rapid development of anemia that was further accelerated by administration of ENU to recipients, demonstrating that the Apc(del/+)-induced anemia was cell extrinsic and potentiated by ENU mutagenesis. These data emphasize the synergistic role of cell intrinsic and cell extrinsic (microenvironment) factors in the pathogenesis of t-MN, and raise awareness of the deleterious effects of cytotoxic therapy on the stromal microenvironment.

Therapy-related myelodysplastic syndrome (t-MDS)/acute myeloid leukemia (t-AML) has been reported after autologous bone marrow or peripheral blood stem cell transplantation (ABMT/PBSCT) for various malignancies. We retrospectively reviewed all adult ABMT/PBSCT cases performed at the University of Chicago Medical Center from 1985 to 1997 in order to determine the incidence of therapy-related leukemia. Among 649 patients, seven (1.1%) developed therapy-related acute lymphoblastic leukemia (one patient) or t-MDS/t-AML (six patients). Of these seven, primary malignancies included one case of breast carcinoma, five cases of Hodgkin's disease (HD) and one case of non-Hodgkin's lymphoma (NHL). Disease-specific incidences for therapy-related leukemia occurring after ABMT/PBSCT were one in 354 (0.3%) for breast carcinoma, five in 79 (6.3%) for HD and one in 103 (1%) for NHL. The median latency periods for the development of therapy-related leukemia from the time of initial diagnosis and of ABMT/PBSCT were 5.5 and 1.5 years, respectively, for the combined HD and NHL group of patients and 4.4 and 2.8 years, respectively, for the one breast carcinoma patient. All seven patients had clonal cytogenetic abnormalities, and five had recurring abnormalities typical of myeloid disorders. Given the similar latency period observed in patients treated with conventional chemotherapy alone, our findings support the hypothesis that therapy-related leukemia after ABMT/PBSCT likely results from pre-transplant therapy. Early detection of therapy-related leukemia is therefore critical to exclude these patients from undergoing ABMT/PBSCT.

Cyclic amegakaryocytic thrombocytopenic purpura is a rare syndrome characterized by periodic failure of megakaryocytopoiesis. In this report we describe a patient with cyclic amegakaryocytic thrombocytopenic purpura associated with a megakaryocyte specific serum IgG antibody, who responded to cyclosporin A therapy. Serial serum thrombopoietin assays during an episode of platelet cycling demonstrated a reciprocal relationship between serum thrombopoietin level and megakaryocyte mass, suggesting that megakaryocytes have an important role in the regulation of thrombopoietin metabolism.

The candidate proto-oncogene BCL3 was isolated through its involvement in the t(14;19) found in chronic lymphocytic leukemia and other B-cell neoplasms. As a member of the I kappaB family, BCL3 plays a role in the immune response by interactions with the NF-kappaB family of transcription factors. In order to study the role of BCL3 overexpression in lymphoid malignancies, we generated five lines of E mu-BCL3 transgenic mice. Transgenic animals develop normally but show splenomegaly and an accumulation of mature B cells in lymph nodes, bone marrow and peritoneal cavity. A hyperresponsive immune system is suggested by the follicular hyperplasia and plasmacytosis in lymph nodes of unimmunized animals, increased incidence of antibodies to self-antigens, and a heightened response to cross-linking of surface IgM. Statistically significant decreases in serum IgM and IgG3, but an increase in IgG1 and IgA were also observed. No lymphoid neoplasms have been identified in transgenic animals. The expansion of B cells in vivo is consistent with the overexpression of BCL3 as being one step in the multi-step process of leukemogenesis. The phenotype also suggests that BCL3 plays a part in B cell proliferation and isotype switching.