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The inversion of chromosome 16 [inv(16)] in acute myeloid leukemia (AML) is associated with a p-arm deletion in a subset of patients. The inversion results in two fusion genes: 5′-CBFB/MYH11–3′ on 16p and 5′- MYH11/CBFB-3′ on 16q. We have studied cells from 42 patients with inv(16) (38 patients) or t(16;16) (four patients) to define the frequency and characteristics of the deletion further. Using fluorescence in situ hybridization (FISH) with probes from cosmids, cosmid contigs, and yeast artificial chromosomes (YACs), we found that six patients with inv(16) had a deletion of between 150 and 350 kb centromeric to the p-arm inversion breakpoint cluster region (p-ibc). This region was shown to contain the 5′ portion of the myosin heavy chain (MYH11) gene. YACs containing the p-ibc, which had been useful as FISH probes in the diagnosis of inv(16), detected the inversion in deletion as well as nondeletion patient cells. Thus, the deleted region identified in patients is entirely contained within the human genomic content of the YACs. Southern blot experiments using probes flanking the p-ibc indicated that the deletion removes segments within 10 kb centromeric of the p-ibc. Reverse transcription-polymerase chain reaction (RT-PCR) using primers from the 5′ region of CBFB and the 3′ region of MYH11 (distal to the p-ibc) produced the 5′-CBFB/MYH11–3′ chimeric transcript in inv(16)/del patients. These data confirm that the 5′-CBFB/MYH11–3′ chimeric transcript, rather than the reciprocal 5′- MYH11/CBFB-3′, is the critical product for chromosome 16-related leukemogenesis.

We investigated whether recombinant alpha 2b interferon (r alpha 2bIFN) would reduce the proportion of bone marrow Philadelphia chromosome (Ph) cells in chronic-phase chronic myelogenous leukemia (CML) by treating 107 previously untreated patients daily with r alpha 2bIFN at 5 x 10(6)IU/m2 subcutaneously. Patients with complete remission, partial remission, or partial hematologic remission received treatment until progression; those with progressive disease were taken off study and observed for survival. Sixty-three (59%) of the patients achieved at least a partial hematologic remission (24 complete remissions and 39 partial remissions). The median time to response for the 63 responders was 3.4 months, with a median duration of remission of 52 months and with 81% of responders continuing in remission beyond 12 months. The median survival for the 107 patients was 66 months. Of 78 patients with cytogenetic follow-up data, 31 (40%) achieved a partial cytogenetic response (n = 17) or a complete cytogenetic response (n = 14). The percentage of cytogenetic responders among all patients was 29% (31 of 107 patients). The median time to first cytogenetic response was 9 months. A major dose reduction of r alpha 2bIFN (> or = 50%) was required at some time during treatment in 38% of patients, 26% required 10% to 49% dose reductions, and 36% had minor dose reductions of < or = 10%. No association was observed between dose received and the attainment of a cytogenetic response. None of the usual prognostic factors (sex, race, performance status, weight loss, time from diagnosis to treatment, hepatosplenomegaly, age, symptoms, hemoglobin, or platelet, blast, basophil, or white blood cell count) were significantly related to survival. These data provide confirmation that major cytogenetic responses to prolonged administration of subcutaneous r alpha 2bIFN occur in 20% to 38% (95% confidence interval) of chronic- phase Ph-positive patients. Although it is hypothesized that patients achieving major cytogenetic responses to r alpha 2bIFN should have prolonged remission duration and survival compared with nonresponders, analyses of the effect of cytogenetic responders by both “landmark” and time-dependent covariate techniques fail to provide statistically significant evidence for an effect of cytogenetic response on remission duration or survival. This may be due in part to an effect size insufficiently large to be detected with the number of patients treated in this study. Thus, confirmation of remission duration or survival benefit, if any, of r alpha 2bIFN therapy in Ph-positive chronic-phase CML must await the outcome of randomized trials comparing IFN with conventional agents.

The (8;21)(q22;q22) translocation is a frequent karyotypic abnormality seen in approximately 40% of patients with acute myeloid leukemia subtype M2 (AML-M2) and an abnormal karyotype. The translocation interrupts two genes, AML1 on chromosome 21 and ETO on chromosome 8, that are consequently fused in the der(8) chromosome to produce a novel chimeric gene and message. Selected genomic DNA probes from chromosome 21 and from chromosome 8 near the breakpoint junction detect rearrangements in the DNA of about 80% of the patients with the rearrangement at diagnosis and in relapse. We analyzed the DNA of 20 patients with t(8;21) AML by standard Southern blot with probes originating from chromosomes 21 and 8 near the breakpoint junction, and we identified rearranged bands in 17 of the 20 patients at diagnosis and in relapse. We also used the polymerase chain reaction (PCR) with appropriate primers from the AML1 and ETO genes to amplify the cDNAs from a cell line with the t(8;21) and from seven AML patients with the t(8;21). We detected a fused transcript in the cell line and in all of the patients analyzed, including three patients who did not show any rearrangement by Southern blot analysis and one patient in hematologic remission, who later relapsed. Combining the results from Southern blot and PCR analysis, we could detect the t(8;21) in all of the patients tested. These results indicate that, whereas several DNA probes used as genetic markers do detect the t(8;21) in most, but not all Southern blots of patients with AML, PCR amplification with primers from AML1 and ETO can be used as a more sensitive and accurate means for detecting this chromosomal abnormality, and for observing the patients' response to therapy.

The inversion of chromosome 16 [inv(16)] in acute myeloid leukemia (AML) is associated with a p-arm deletion in a subset of patients. The inversion results in two fusion genes: 5'-CBFB/MYH11-3' on 16p and 5'-MYH11/CBFB-3' on 16q. We have studied cells from 42 patients with inv(16) (38 patients) or t(16;16) (four patients) to define the frequency and characteristics of the deletion further. Using fluorescence in situ hybridization (FISH) with probes from cosmids, cosmid contigs, and yeast artificial chromosomes (YACs), we found that six patients with inv(16) had a deletion of between 150 and 350 kb centromeric to the p-arm inversion breakpoint cluster region (p-ibc). This region was shown to contain the 5' portion of the myosin heavy chain (MYH11) gene. YACs containing the p-ibc, which had been useful as FISH probes in the diagnosis of inv(16), detected the inversion in deletion as well as nondeletion patient cells. Thus, the deleted region identified in patients is entirely contained within the human genomic content of the YACs. Southern blot experiments using probes flanking the p-ibc indicated that the deletion removes segments within 10 kb centromeric of the p-ibc. Reverse transcription-polymerase chain reaction (RT-PCR) using primers from the 5' region of CBFB and the 3' region of MYH11 (distal to the p-ibc) produced the 5'-CBFB/MYH11-3' chimeric transcript in inv(16)/del patients. These data confirm that the 5'-CBFB/MYH11-3' chimeric transcript, rather than the reciprocal 5'-MYH11/CBFB-3', is the critical product for chromosome 16-related leukemogenesis.

Abstract 117 Heterozygous deletions of the long arm of chromosome 5 are among the most common abnormalities in de novo (∼15% of patients) and therapy-related myeloid neoplasms (t-MN) (∼40% of patients). Two minimally deleted segments have been identified - the minimally deleted segment within 5q31.2 is associated with de novo AML and t-MNs, whereas the other spans 5q33.1 and is associated with MDS with an isolated del(5q). Current studies support a haploinsufficiency model, in which loss of a single allele of more than one gene on 5q contributes to the development of myeloid neoplasms. Using mouse models, we previously showed that haploinsufficiency of Egr1 (5q31.2) or Apc (5q22-frequently deleted in t-MN) independently recapitulates some features of human myelodysplastic syndromes (MDS). To test the hypothesis that reduced levels of EGR1 and APC cooperate in the pathogenesis of MDS/AML, we generated mice expressing a single allele of Egr1 and Apc: Mx1-Cre+Apcfl/+Egr1+/−(Apcdel/+Egr1+/−). At 2 mos of age, we induced deletion of a single allele of Apc by injection of 3 doses of pI-pC. Survival curves clearly show that Egr1 and Apc haploinsufficiency cooperate in the development of disease with a median survival of 129 days for Apcdel/+Egr1+/− mice and 296 days for Apcdel/+mice (P Mutations in TP53 are commonly found in t-MNs with a del(5q) and loss of Tp53 in mouse models has been shown to promote AML by enabling aberrant self renewal. To test the hypothesis that loss of TP53 may adversely advance disease development, we crossed Tp53+/− to Egr1+/− and Apcdel/+ mice. Similar to Apcdel/+Egr1+/− mice, Apcdel/+Tp53+/− mice rapidly developed macrocytic anemia with a median survival of 144 days, suggesting that partial loss of TP53 function accelerates the Apcdel/+ -induced macrocytic anemia. Triple heterozygous mice (Apcdel/+Tp53+/−Egr1+/−) had a median survival of 178 days, but survival was not statistically different than Apcdel/+Egr1+/− mice (P=0.35) suggesting that Egr1 and Tp53 loss play redundant roles in the development of disease in Apcdel/+ mice. Thus, in the context of Apc haploinsufficiency, loss of Egr1 or Tp53 function promotes erythroid failure. These results are in contrast to the setting of ribosomal protein haploinsufficiency, as is the case in MDS with an isolated del(5q), where induction of TP53 is essential for erythroid failure. It has been proposed that inactivation of TP53 (through additional TP53 mutations) would be required for progression to AML, in the setting of a 5q deletion. To this end we transduced Egr1+/−Apcdel/+ bone marrow cells with a Tp53-specific shRNA, known to reduce Tp53 transcripts by ∼90%, and transplanted them into lethally irradiated C57BL/6 mice. Although penetrance of disease was low, 2 out of 13 mice (15%) developed an aggressive AML, as compared to 0 of 12 mice transplanted with Egr1+/−Apcdel/+ cells transduced with control shRNA. These data suggest that EGR1 and APC haploinsufficiency cooperate in the development of myeloid disorders, characterized by ineffective erythropoiesis, and that further mutations, such as that achieved by complete inactivation of TP53, are required for progression to AML. Disclosures: No relevant conflicts of interest to declare.

We investigated whether recombinant alpha 2b interferon (r alpha 2bIFN) would reduce the proportion of bone marrow Philadelphia chromosome (Ph) cells in chronic-phase chronic myelogenous leukemia (CML) by treating 107 previously untreated patients daily with r alpha 2bIFN at 5 x 10(6)IU/m2 subcutaneously. Patients with complete remission, partial remission, or partial hematologic remission received treatment until progression; those with progressive disease were taken off study and observed for survival. Sixty-three (59%) of the patients achieved at least a partial hematologic remission (24 complete remissions and 39 partial remissions). The median time to response for the 63 responders was 3.4 months, with a median duration of remission of 52 months and with 81% of responders continuing in remission beyond 12 months. The median survival for the 107 patients was 66 months. Of 78 patients with cytogenetic follow-up data, 31 (40%) achieved a partial cytogenetic response (n = 17) or a complete cytogenetic response (n = 14). The percentage of cytogenetic responders among all patients was 29% (31 of 107 patients). The median time to first cytogenetic response was 9 months. A major dose reduction of r alpha 2bIFN (or = 50%) was required at some time during treatment in 38% of patients, 26% required 10% to 49% dose reductions, and 36% had minor dose reductions ofor = 10%. No association was observed between dose received and the attainment of a cytogenetic response. None of the usual prognostic factors (sex, race, performance status, weight loss, time from diagnosis to treatment, hepatosplenomegaly, age, symptoms, hemoglobin, or platelet, blast, basophil, or white blood cell count) were significantly related to survival. These data provide confirmation that major cytogenetic responses to prolonged administration of subcutaneous r alpha 2bIFN occur in 20% to 38% (95% confidence interval) of chronic-phase Ph-positive patients. Although it is hypothesized that patients achieving major cytogenetic responses to r alpha 2bIFN should have prolonged remission duration and survival compared with nonresponders, analyses of the effect of cytogenetic responders by both "landmark" and time-dependent covariate techniques fail to provide statistically significant evidence for an effect of cytogenetic response on remission duration or survival. This may be due in part to an effect size insufficiently large to be detected with the number of patients treated in this study. Thus, confirmation of remission duration or survival benefit, if any, of r alpha 2bIFN therapy in Ph-positive chronic-phase CML must await the outcome of randomized trials comparing IFN with conventional agents.

The (8;21)(q22;q22) translocation is a frequent karyotypic abnormality seen in approximately 40% of patients with acute myeloid leukemia subtype M2 (AML-M2) and an abnormal karyotype. The translocation interrupts two genes, AML1 on chromosome 21 and ETO on chromosome 8, that are consequently fused in the der(8) chromosome to produce a novel chimeric gene and message. Selected genomic DNA probes from chromosome 21 and from chromosome 8 near the breakpoint junction detect rearrangements in the DNA of about 80% of the patients with the rearrangement at diagnosis and in relapse. We analyzed the DNA of 20 patients with t(8;21) AML by standard Southern blot with probes originating from chromosomes 21 and 8 near the breakpoint junction, and we identified rearranged bands in 17 of the 20 patients at diagnosis and in relapse. We also used the polymerase chain reaction (PCR) with appropriate primers from the AML1 and ETO genes to amplify the cDNAs from a cell line with the t(8;21) and from seven AML patients with the t(8;21). We detected a fused transcript in the cell line and in all of the patients analyzed, including three patients who did not show any rearrangement by Southern blot analysis and one patient in hematologic remission, who later relapsed. Combining the results from Southern blot and PCR analysis, we could detect the t(8;21) in all of the patients tested. These results indicate that, whereas several DNA probes used as genetic markers do detect the t(8;21) in most, but not all Southern blots of patients with AML, PCR amplification with primers from AML1 and ETO can be used as a more sensitive and accurate means for detecting this chromosomal abnormality, and for observing the patients' response to therapy.

Abstract 2922 The myelodysplastic syndromes (MDS) are a heterogeneous group of hematological malignancies characterized by ineffective hematopoiesis and a highly variable clinical course, for which novel treatments are beginning to emerge. Conventional cytogenetic studies (CCS) of bone marrow (BM) are routinely used in clinical practice to detect abnormal clones in proliferating (metaphase) cells, identifying clonal aberrations in ∼50% of de novo MDS cases. Cytogenetics is also one of the key International Prognostic Scoring System (IPSS) components used to estimate overall survival and leukemia-free survival in MDS. Chromosome abnormalities may be quantified at presentation and during treatment by the use of fluorescence in situ hybridization (FISH) with DNA probes for specific chromosome loci (e.g., chromosomes 5, 7, 8 and 20) in non-proliferating (interphase) nuclei. However, it is not clear whether or not peripheral blood CCS will yield the same diagnostic and prognostic data as bone marrow CCS at disease presentation or whether patients without apparent chromosome abnormalities by CCS have “hidden” abnormalities that can be identified by interphase FISH. To answer these questions, 15 members of the International Working Group on MDS Cytogenetics agreed to perform CCS and FISH in parallel on both peripheral blood and bone marrow samples collected from MDS patients. To be certain that all participating sites scored and interpreted their individual FISH data in a similar fashion, a quality assurance (QA) study was completed with each site studying two identical test (proficiency) samples. Concordance among sites was very good to excellent allowing for the establishment of a standardized protocol with clear scoring criteria before patient samples were processed. In the second phase of the study, a total of 77 MDS patients were accrued to the study with 61% showing an abnormal karyotype. A FISH panel consisting of eight probe sets [-5/5q-, -7/7q-/der(1;7), +8/8q-, -11/+11/11q-/add(11q), 12p-/+21/t(12;21), -13/13q-, 17p- and 20q-/i(20q)/i(20p), Abbott Molecular, Inc.] was performed on both specimen types. While CCS was frequently unsuccessful (57.5%) in the PB specimens, FISH was informative (concordant with BM/PB CCS) in 92% of cases, with 49% of PB FISH demonstrating an abnormal clone. FISH was discordant in 4 of 77 BM and PB samples (5%), while CCS and FISH on BM and PB were discordant in 6 of 77 BM specimens (8%) and 6 of 73 PB specimens (8%). The data suggest that evaluation of interphase nuclei from PB on follow-up (non-diagnostic) FISH studies on MDS patients will be equally informative (and less costly and stressful) as a BM sample. Disclosures: Slovak: PerkinElmer: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding. Ohyashiki:Nippon Shinyaku Co., Ltd.: Research Funding.

Abstract 580 Therapy-related acute myeloid leukemia/myelodysplasia (t-AML/t-MDS) accounts for 10–20% of all cases of AML, and its incidence is rising. Treatment options are limited and the prognosis very poor, highlighting the need for new therapies in t-AML/t-MDS. However, the genetic mutations contributing to transformation in t-AML/t-MDS are largely unknown, limiting the development of novel targeted therapeutics. Our group previously reported the sequence of the first two cancer genomes, both in patients with de novo AML (Nature 456:66, 2008; NEJM 361:1058, 2009). Herein, we report the sequence of the cancer genome of a patient with t-AML. The patient presented with early-onset breast, then ovarian cancer (age 96% of heterozygous SNPs were detected. Similar data were obtained for the skin sample. A total of 27 validated somatic single nucleotide variants or indels were detected in coding sequences. None of these mutations have been previously reported in de novo AML. Eight novel chromosomal translocations were identified and the breakpoints defined. One translocation, t(3;4)(q27.3;p15.32), resulted in the production of an in frame fusion transcript of DGKG (diacylglycerol kinase gamma) with BST1 (bone marrow cell stromal antigen 1). Studies are underway to characterize the effect of this fusion gene on hematopoietic cell growth and differentiation. In addition to -7 and del(5q), somatic copy number alterations on chromosome 3 and 12 were identified. There is controversy whether haploinsufficiency of genes on chromosomes 7 and 5q is sufficient to contribute to transformation, or whether further mutations lead to loss of heterozygosity of one or more genes in these regions. In the present case, careful review of the sequence and array data revealed no ‘homozygous' somatic single nucleotide variants, indels, or copy number alterations of coding genes on the remaining copy of chromosome 5 or 7. The patient's clinical presentation strongly suggested genetic cancer susceptibility. Analysis of the skin genome of this patient identified a heterozygous deletion of exons 7–9 of TP53, likely contributing to the early onset of her breast cancer; a uniparental disomy event resulted in the deletion being homozygous in the leukemia sample. Interestingly, the mutant TP53 allele is expressed, and it is predicted to produce a truncated p53 protein lacking most of its DNA binding domain. Functional studies of the mutant p53 protein are underway. Of note, based on a detailed family history and genotyping of the patient's mother, we suspect that the TP53 deletion occurred spontaneously. Ongoing whole genome sequencing studies in a large number of t-AML samples should identify novel somatic mutations and germline variants that contribute to t-AML. Disclosures: No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is the most common type of acute leukemia in adults. It is estimated that 13,410 cases will be diagnosed and 8,990 will die of AML in the United States in 2007 (http://seer.cancer.gov). AML is a genetically diverse hematopoietic malignancy with variable response to treatment. Expression profiling of protein-coding genes using DNA microarray in AML has resulted in inconsistent data from different laboratories. Therefore, further validation of these observations in large cohorts and in independent studies is definitely required before clinical application becomes feasible. Recently, Golub and colleagues described a new, bead-based flow cytometric microRNA (miRNAs, miRs) expression profiling method that could successfully classify tumors. MiRNAs are endogenous ∼22 nucleotide non-coding RNAs, which can function as oncogenes and tumor suppressors. To provide new insights into the complex genetic alterations in leukemogenesis and to identify novel markers for diagnosis and treatment of AML, we performed a genome-wide analysis of miRNA expression profiles using the bead-based method on 54 AML samples with common translocations including t(15;17), t(8;21), inv(16), and 11q23 rearrangement, along with normal controls. In both unsupervised and supervised hierarchical cluster analyses, we observed that t(15;17) samples grouped together as one cluster, as do the 11q23 rearrangement samples. Interestingly, t(8;21) and inv(16), both CBF (core-binding factor) AMLs, grouped together as a unique cluster. Forty-one miRNAs exhibited significantly differential expression between different subtypes of AMLs, and/or between AMLs and normal controls. Notably, expression signature of a minimal number of two, three, and seven miRNAs could be used for class prediction of CBF, t(15;17), and 11q23 rearrangement AMLs, respectively, with an overall diagnostic accuracy of 94–96%. We further showed that overexpression of the two discriminatory miRNAs in CBF AML is associated with epigenetic regulation, rather than DNA copy number amplification. Moreover, several important target genes of these discriminatory miRNAs have also been validated. We are currently exploring the role of these discriminatory miRNAs and their critical target genes in the development of AML using in vitro and in vivo models. This work will enhance our understanding of the biological role of these miRNAs and their targets in leukemogenesis.

Therapy-related myelodysplastic syndrome (t-MDS) and acute myeloid leukemia (t-AML) are late complications of cytotoxic therapy used in the treatment of both malignant and non-malignant diseases. The most common subtype of t-MDS/t-AML (~75% of cases) develops after exposure to alkylating agents, and is characterized by loss of a whole chromosome 5 and/or 7, or a deletion of the long arms of these chromosomes [-5/del(5q), -7/del(7q)]. Patients who develop t-MDS/t-AML in this setting typically show a latency of 5 years from alkylating agent exposure, and present with cytopenias and myelodysplasia. In the University of Chicago’s series of 306 patients with t-MDS/t-AML, 64 (21%) patients had abnormalities of chromosome 5, 85 (28%) patients had abnormalities of chromosome 7, and 65 (21%) patients had abnormalities of both chromosomes 5 and 7 (Smith et al. Blood102:43, 2003). Survival times of these t-AML patients are short (median 8 mos), and new therapeutic approaches are needed. Genetic pathways in t-MDS/t-AML are poorly-understood. Monosomy 7/del(7q) has been associated with activating mutations of the RAS pathway and methylation silencing of the CDKN2B (p15INK4B) gene, whereas -5/del(5q) is associated with TP53 mutations and a complex karyotype. To identify novel molecular pathways involved in the pathogenesis of t-MDS/t-AML, we previously performed gene expression profiling of CD34+ hematopoietic progenitor cells from t-AML patients, and identified two major groups, characterized by a -7/del(7q) or normal karyotype (Group A) or -5/del(5q) (Group B) (Qian et al., Proc Natl Acad Sci USA99:14925, 2002). Patients with a -5/del(5q) have a higher expression of genes involved in cell cycle control (CCNA2, CCNE2, CDC2), checkpoints (BUB1), or growth (MYC), and loss of expression of ICSBP. In addition, FHL2 is upregulated in this subgroup. FHL2 is a multi-functional LIM domain protein that has been implicated as a transcriptional co-activator or co-repressor mediating proliferation and apoptosis in a tissue-specific fashion. Interacting partners of FHL2 include WT1, b-catenin, PLZF, and the androgen receptor. In subsequent studies, we determined that hematopoietic cells express a novel isoform of FHL2 (termed B-FHL2), that contains a unique 5′-UTR, but encodes the same peptide, suggesting that FHL2 is regulated by a different promoter in hematopoietic cells. To examine the effects of enhanced expression of FHL2 on hematopoiesis, we transduced primary bone marrow cells from mice treated with 5-FU with retroviral vectors expressing FHL2 (MIGR1-FHL2, MSCV-FHL2). Up-regulation of FHL2 enhanced proliferation of myeloid progenitor cells, and self-renewal capacity of hematopoietic cells in vitro. To determine if up-regulation of FHL2 in vivo leads to alterations in lineage-specific differentiation, we used retroviral transduction/transplantation techniques. Expression of FHL2 resulted in increased numbers of colony-forming cells and GM colonies, but not BFU-E or GEMM colonies. Recipient mice followed for up to 12 months were characterized by enhanced myelopoiesis and partially blocked B lymphopoiesis, with no detectable effect on T cell development. Expression of FHL2 did not induce leukemia in transplanted mice. These results suggest that FHL2 is involved in hematopoiesis. Furthermore, upregulation of FHL2 results in increased stem cell and progenitor cell self-renewal; however, leukemogenesis may require the acquisition of secondary cooperating mutations.

The tumor suppressor INK4B(p15) gene is silenced by CpG island hypermethylation in a majority of acute myeloid leukemias (AML). This silencing can be reversed by the treatment with hypomethylating agents, and these agents are currently being tested for therapeutic intervention. So far, it was not investigated whether or not the INK4B is hypermethylated in all cytogenetic subtypes of AML. Our experiments, which compare levels of INK4B methylation in AML with inv(16), t(8:21) and t(15;17) reveal a strikingly low level of methylation in all leukemias with inv(16). This contrasts with significant levels of DNA methylation in a high proportion of the AML from the other two groups. Surprisingly, even though there is a lack of INK4B methylation in samples from patients with inv(16), expression of the gene is very low when compared to that of PBL from healthy individuals or HL60 cells. Subsequent experiments uncovered a novel mechanism to explain the low level of INK4B expression in the inv(16) AMLs. Overexpression of the aberrant chromosome 16-associated gene CBFb-MYH11 in U937 cells results in failure to induce INK4B in response to vitamin D3. Furthermore, CBFb-SMMHC, encoded by CBFb-MYH11 directly represses transcription from an INK4B promoter in a reporter assay. Electromobility shift assays in the AML-derived cell line ME-1 and U937 cells expressing the fusion gene demonstrate that the repression is due to a change in the composition of the complexes recognizing the binding sites for the transcription regulator CBF. In conclusion, we have found that methylation is not the only way to induce silencing of the tumor suppressor p15INK4B in AML. In inv(16)-containing AML, loss of gene expression is accomplished by the direct transcriptional repressor activity of CBFβ-SMMHC.

Northern blot analysis was used to assess the level of expression of five protooncogenes and histone H3 in the bone marrow cells of patients with acute nonlymphocytic leukemia (ANLL). The relationship between the level of gene expression and the clinical characteristics of the disease and response to therapy was studied. The levels of expression of c-myc and c-myb are weakly correlated and are unrelated to French- American-British (FAB) type of ANLL. The levels of expression of c-fms, c-fes, and c-fos are highly correlated with each other and are highest in leukemia with a monocytic component (c-fms v FAB = .71, c-fes v FAB = .75). High levels of c-myc expression are associated with a high probability of not responding to remission induction therapy (P = .004). The converse is true for c-fms expression levels. High levels of expression of c-myc or c-myb are associated with short remissions (P = .059 and .065, respectively), perhaps because they are associated with a high capacity for leukemic cell self-renewal and/or an inability of leukemic cells to differentiate in response to chemotherapy.

Northern blot analysis was used to assess the level of expression of five protooncogenes and histone H3 in the bone marrow cells of patients with acute nonlymphocytic leukemia (ANLL). The relationship between the level of gene expression and the clinical characteristics of the disease and response to therapy was studied. The levels of expression of c-myc and c-myb are weakly correlated and are unrelated to French-American-British (FAB) type of ANLL. The levels of expression of c-fms, c-fes, and c-fos are highly correlated with each other and are highest in leukemia with a monocytic component (c-fms v FAB = .71, c-fes v FAB = .75). High levels of c-myc expression are associated with a high probability of not responding to remission induction therapy (P = .004). The converse is true for c-fms expression levels. High levels of expression of c-myc or c-myb are associated with short remissions (P = .059 and .065, respectively), perhaps because they are associated with a high capacity for leukemic cell self-renewal and/or an inability of leukemic cells to differentiate in response to chemotherapy.