John Anastasi

Acquired mutations in GATA1 in the megakaryoblastic leukemia of Down syndrome.

Children with Down syndrome have a 10–20-fold elevated risk of developing leukemia, particularly acute megakaryoblastic leukemia (AMKL). While a subset of pediatric AMKLs is associated with the 1;22 translocation and expression of a mutant fusion protein, 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) 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 hematopoiesis, one study has shown that it is required for normal development in vivo. Together, these findings indicate that loss of wildtype GATA1 constitutes one step in the pathogenesis of AMKL in Down syndrome.

Cooperative and Antagonistic Interplay between PU.1 and GATA-2 in the Specification of Myeloid Cell Fates

PU.1 and GATA transcription factors appear to antagonize each others 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 human programmed cell death-2 (PDCD2) gene is a target of BCL6 repression: Implications for a role of BCL6 in the down-regulation of apoptosis

BCL6, a gene on chromosome 3 band q27, encodes a Kruppel-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 of Rp8, 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.

Low-Dose Interleukin-2 Immunotherapy Does Not Improve Outcome of Patients Age 60 Years and Older With Acute Myeloid Leukemia in First Complete Remission: Cancer and Leukemia Group B Study 9720

PURPOSE Cancer 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 METHODS AML 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. RESULTS A 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. CONCLUSION Low-dose rIL-2 maintenance immunotherapy is not a successful strategy in older AML patients.

Abnormal Cytogenetics at Date of Morphologic Complete Remission Predicts Short Overall and Disease-Free Survival, and Higher Relapse Rate in Adult Acute Myeloid Leukemia: Results From Cancer and Leukemia Group B Study 8461

PURPOSE As most patients with acute myeloid leukemia (AML) with morphologic complete remission (CR) ultimately relapse, better predictors for outcome are needed. Recently, Cheson et al suggested using cytogenetic remission (CRc) as part of the criteria for CR. To our knowledge, ours is the first relatively large study evaluating the usefulness of CRc attained immediately following induction chemotherapy. PATIENTS AND METHODS We included AML patients treated on Cancer and Leukemia Group B front-line studies with cytogenetic samples obtained at diagnosis and at the first day of documented CR following induction. Patients with abnormal cytogenetics at diagnosis, and normal cytogenetics at CR (NCR; n = 103) were compared with those with abnormal cytogenetics both at diagnosis and at CR (ACR; n = 15) for overall survival (OS), disease-free survival (DFS), and cumulative incidence of relapse (CIR). Cox proportional hazards models determined the prognostic significance of cytogenetics at CR, adjusting for other covariates. RESULTS Clinical features were similar for both groups, with the exception of favorable cytogenetics [t(8;21), inv(16)/t(16;16), t(15;17)] at diagnosis, which was more frequent (P =.03) in the NCR group. Median follow-up was 3.1 years (range, 1.0 to 11.4 years). ACR patients had significantly shorter OS (P =.006) and DFS (P =.0001), and higher CIR (P =.0001). In multivariable models, the NCR and ACR groups were predictors for OS (P =.03), DFS (P =.02), and CIR (P =.05). The relative risk of relapse or death was 2.1 times higher for ACR patients than for NCR patients (95% CI, 1.1 to 3.9). CONCLUSION Our data suggest that converting to normal karyotype at the time of first CR is an important prognostic indicator and support the use of CRc as a criterion of CR in AML.

CUX1 is a haploinsufficient tumor suppressor gene on chromosome 7 frequently inactivated in acute myeloid leukemia.

Loss of chromosome 7 and del(7q) [-7/del(7q)] are recurring cytogenetic abnormalities in hematologic malignancies, including acute myeloid leukemia and therapy-related myeloid neoplasms, and associated with an adverse prognosis. Despite intensive effort by many laboratories, the putative myeloid tumor suppressor(s) on chromosome 7 has not yet been identified.We performed transcriptome sequencing and SNP array analysis on de novo and therapy-related myeloid neoplasms, half with -7/del(7q). We identified a 2.17-Mb commonly deleted segment on chromosome band 7q22.1 containing CUX1, a gene encoding a homeodomain-containing transcription factor. In 1 case, CUX1 was disrupted by a translocation, resulting in a loss-of-function RNA fusion transcript. CUX1 was the most significantly differentially expressed gene within the commonly deleted segment and was expressed at haploinsufficient levels in -7/del(7q) leukemias. Haploinsufficiency of the highly conserved ortholog, cut, led to hemocyte overgrowth and tumor formation in Drosophila melanogaster. Similarly, haploinsufficiency of CUX1 gave human hematopoietic cells a significant engraftment advantage on transplantation into immunodeficient mice. Within the RNA-sequencing data, we identified a CUX1-associated cell cycle transcriptional gene signature, suggesting that CUX1 exerts tumor suppressor activity by regulating proliferative genes. These data identify CUX1 as a conserved, haploinsufficient tumor suppressor frequently deleted in myeloid neoplasms.

Lymphadenopathy, splenomegaly, and altered immunoglobulin production in BCL3 transgenic mice

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 IκB family, BCL3 plays a role in the immune response by interactions with the NF-κB family of transcription factors. In order to study the role of BCL3 overexpression in lymphoid malignancies, we generated five lines of Eμ-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.

Therapy-Related Myelodysplastic Syndrome Morphologic Subclassification May Not Be Clinically Relevant

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.

Post‐transplant lymphoproliferative disorder subtypes correlate with different recurring chromosomal abnormalities

Although cytogenetic analysis advanced the understanding of the pathogenesis of primary non‐Hodgkin lymphoma and led to improved clinical management, there have been no large cytogenetic studies of post‐transplant lymphoproliferative disorder (PTLD). We examined the karyotypes of 36 PTLD cases and correlated them with clinical, laboratory, and pathologic findings. The cases included 2 early lesions, 13 polymorphic PTLDs, and 21 monomorphic PTLDs (18 B‐cell and 3 T‐cell proliferations). Cytogenetic abnormalities were identified in 72% of monomorphic B‐cell PTLDs and in all T‐cell PTLDs, but in only 15% of polymorphic PTLDs and in no early lesions. The most frequent clonal abnormalities in monomorphic PTLD were trisomies 9 and/or 11 (5 cases), followed by rearrangements of 8q24.1 (4 cases), 3q27 (2 cases), and 14q32 (2 cases). MYC rearrangement (8q24.1) and T‐cell‐associated chromosomal abnormalities correlated with poor outcome and short survival. PTLD with trisomy 9 and/or 11 developed early after transplant, presenting as Epstein–Barr virus–positive large B‐cell lymphoma with prolonged survival.

Haploinsufficiency of Apc leads to ineffective hematopoiesis

Loss of a whole chromosome 5 or a deletion of the long arm of chromosome 5, -5/del(5q), is a recurring abnormality in myeloid neoplasms. The APC gene is located at chromosome band 5q23, and is deleted in more than 95% of patients with a -5/del(5q), raising the question of whether haploinsufficiency of APC contributes to the development of myeloid neoplasms with loss of 5q. We show that conditional inactivation of a single allele of Apc in mice leads to the development of severe anemia with macrocytosis and monocytosis. Further characterization of the erythroid lineage revealed that erythropoiesis is blocked at the early stages of differentiation. The long-term hematopoietic stem cell (LT-HSC) and short-term HSC (ST-HSC) populations are expanded in Apc-heterozygous mice compared with the control littermates; however, the HSCs have a reduced capacity to regenerate hematopoiesis in vivo in the absence of a single allele of Apc. Apc heterozygous myeloid progenitor cells display an increased frequency of apoptosis, and decreased in vitro colony-forming capacity, recapitulating several characteristic features of myeloid neoplasms with a -5/del(5q). Our results indicate that haploinsufficiency of Apc impairs hematopoiesis, and raise the possibility that loss of function of APC contributes to the development of myelodysplasia.