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Acute promyelocytic leukemia (APL) is associated with a reciprocal and balanced translocation involving the retinoic acid receptor-α(RARα).All-transretinoic acid (ATRA) is used to treat APL and is a potent morphogen that regulatesHOXgene expression in embryogenesis and organogenesis.HOXgenes are also involved in hematopoiesis and leukemogenesis. Thirty-nine mammalian HOXgenes have been identified and classified into 13 paralogous groups clustered on 4 chromosomes. They encode a complex network of transcription regulatory proteins whose precise targets remain poorly understood. The overall function of the network appears to be dictated by gene dosage. To investigate the mechanisms involved in HOXgene regulation in hematopoiesis and leukemogenesis by precise measurement of individual HOXgenes, a small-array real-timeHOX(SMART-HOX) quantitative polymerase chain reaction (PCR) platform was designed and validated. Application of SMART-HOXto 16 APL bone marrow samples revealed a global down-regulation of 26 HOXgenes compared with normal controls. HOXgene expression was also altered during differentiation induced by ATRA in thePML-RARα+NB4 cell line. PML-RARα fusion proteins have been reported to act as part of a repressor complex during myeloid cell differentiation, and a model linkingHOXgene expression to this PML-RARα repressor complex is now proposed.

Acute myeloid leukemia (AML) is a heterogeneous group of hematological malignancies with individual cases showing wide variations in response to treatment. Pre-treatment karyotype analysis may be used to stratify cases into one of three prognostic classes: favorable, intermediate or adverse. Favorable karyotypes, t(15;17), t(8;21) and inv(16), are associated with the presence PML-RARα, AML1-ETOand CBFβ-MYH11gene rearrangements respectively. The adverse cytogenetic abnormalities are monosomy 5, monosomy 7, deletion of chromosome 5q, abnormalities of chromosome 3q and a complex karyotype. All remaining abnormalities are associated with an intermediate prognosis. Approximately 5–10% of cases cannot be stratified due to failure of cytogenetic analysis. Cryptic gene rearrangements, which cannot be detected by karyotyping, may lead to assignment of cases to incorrect prognostic classes. These problems may result in sub-optimal or over-treatment of patients. Using an artificial neural network-based analysis of HOXgene expression profiles generated by real-time quantitative PCR (RT-QPCR) we have previously shown that the favorable and intermediate cytogenetic classes are characterised by low HOXA5(Ct value > 29.5) and high HOXB3(Ct value < 25) expression respectively (Blood 2006; 108: Abstract 2318). We have now measured HOXA5and HOXB3expression levels by RT-QPCR in a fresh set of 78 newly diagnosed cases of AML (31 favorable, 38 intermediate and 9 adverse karyotypes as determined by conventional cytogenetic analysis). All 31 cases with favorable cytogenetics had HOXA5Ct values > 29.5 with twenty-nine (93.5%) having Ct values > 33. Therefore, using a HOXA5Ct ≥; 33 to define membership of the favorable prognostic class and HOXA5Ct < 33 to define membership of a non-favorable class, 72 cases (92.3%) were correctly assigned (29 favorable and 43 non-favorable). Of the 4 cases with non-favorable cytogenetics, originally misclassified by HOXA5expression profiling, two were subsequently found to have cryptic rearrangements of PML-RARαand therefore should have been included within the favorable group. This increased the percentage of cases correctly assigned by HOXA5expression profiling alone to 94.9%. We also found that within the favorable group, AML with t(15;17) or t(8;21) was characterized by low expression of HOXB3(Ct range 30.9 to 37.4, median Ct 34.1) whereas AML with inv(16) had a distinct signature characterized by higher HOXB3expression (Ct range 26.4 to 30.1, median Ct 28.0). In addition, we have identified a subset of patients with intermediate cytogenetics who have high white cell counts, low HOXB3expression and an inferior response to treatment. Therefore, in AML, the measurement of expression levels of only two HOXgenes, HOXA5and HOXB3, complements cytogenetic analysis and may improve the yield of favorable gene rearrangements detected and provide additional prognostic information for cases with intermediate or failed cytogenetics.

The acute myeloid leukemias (AMLs) are a heterogeneous group of hematological malignancies with diverse clinical outcomes. Pre-treatment karyotype analysis identifies biologically distinct subgroups and is currently used as a predictor of response to induction chemotherapy and risk of relapse. Cases may be stratified into one of three prognostic groups as follows: relatively favorable prognosis [t(8;21), t(15;17) and inv(16)]; adverse prognosis [−5/del(5q), −7, abnormalities of chromosome 3q and complex karyotype]; and intermediate prognosis [remainder including normal karyotype]. HOXgenes encode master transcription factors which regulate key developmental processes including differentiation, proliferation and apoptosis. Humans have 39 HOXgenes and multiple lines of evidence implicate their deregulated expression in the pathogenesis of AML. Drabkin et al. ( Leukemia 2002; 16: 186–95) have reported that AMLs with a relatively favorable prognostic karyotype are associated with low levels of HOXgene expression whereas AMLs with an adverse prognostic karyotype have higher levels of expression. To further characterize HOXgene expression in cytogenetic prognostic groups we determined the expression profiles of 26 HOXgenes by real-time quantitative PCR (Q-PCR) in diagnostic samples, representative of the three prognostic groups, from 26 patients with de novoAML. Profiles were then analyzed using Artificial Neural Network based computational approaches to identify a subset of HOXgenes which could discriminate between prognostic groups in a predictive fashion. Predictive models were developed for each prognostic group. Predictive classification performance for prognostic groups based on blind data of 88%, 92%, and 97% (with equal sensitivity and specificity) were achieved for the three prognostic groups. The models were interrogated to determine the nature of the relationship between the key HOXgenes identified and prognostic group. The relatively favorable prognosis group was primarily defined by downregulation of HOXA5and upregulation of HOXC4. The intermediate prognosis group was characterized by upregulation of HOXB3and downregulation of HOXD10and the adverse prognosis group by downregulation of both HOXC5and HOXD3. Although the sample size is small, the results show that Artificial Neural Network based computational approaches are capable of further characterizing HOXgene expression within AML prognostic groups as determined by presenting karyotype and that measuring the expression levels of a small number of HOXgenes at diagnosis can provide useful clinical information in cases where karyotype analysis has been unsuccessful.