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The objective of our study was to evaluate high-dose cytarabine in consolidation therapy in patients with newly diagnosed acute promyelocytic leukemia (APL). Patients (age 16-60 years) received induction therapy according to the AIDA protocol (all-trans retinoic acid, idarubicin) followed by one cycle of ICE (idarubicin, cytarabine, etoposide) and two cycles of HAM (cytarabine 3 g/m(2) q12h, days 1-3; mitoxantrone 10 mg/m(2), days 2 and 3). From 1995 to 2003, 82 patients were enrolled. In total, 72 patients (88%) achieved a complete remission, and 10 patients (12%) died from early/hypoplastic death (ED/HD). A total of 71 patients received at least one cycle of HAM. Relapse-free survival (RFS) and overall survival (OS) after 46 months were 83 and 82%, respectively. White blood cell count above 10.0 x 10(9)/l at diagnosis and additional chromosomal aberrations were unfavorable prognostic markers for OS, whereas no prognostic markers for RFS were identified including FLT3 mutations. In conclusion, high-dose cytarabine in consolidation therapy for patients with newly diagnosed APL is an effective treatment approach.

Polycythemia vera (PV), essential thrombocythemia (ET), and myeloid metaplasia with myelofibrosis (MMM) are clonal disorders arising from hematopoietic progenitors. An internet-based protocol was used to collect clinical information and biological specimens from patients with these diseases. High-throughput DNA resequencing identified a recurrent somatic missense mutation JAK2V617F in granulocyte DNA samples of 121 of 164 PV patients, of which 41 had homozygous and 80 had heterozygous mutations. Molecular and cytogenetic analyses demonstrated that homozygous mutations were due to duplication of the mutant allele. JAK2V617F was also identified in granulocyte DNA samples from 37 of 115 ET and 16 of 46 MMM patients, but was not observed in 269 normal individuals. In vitro analysis demonstrated that JAK2V617F is a constitutively active tyrosine kinase.

To assess the prevalence of mutations in the CEBPA gene, which encodes the myeloid transcription factor CEBPA in specific cytogenetic subgroups, we initially studied 125 patients with acute myeloid leukemia (AML). Five of the eight patients with del(9q) as the sole aberration or in combination with a single additional abnormality other than t(8;21) had CEBPA mutations associated with loss of CEBPA function. Consequently, 41 additional del(9q) cases were analyzed; nine had CEBPA loss-of-function mutations. The overall prevalence of CEBPA loss-of-function mutations in cases with del(9q) in a noncomplex karyotype was 41% (14 of 34 patients), whereas none of the patients who had a del(9q) in a complex karyotype (n = 7) or together with a t(8;21) (n = 10) demonstrated mutant CEBPA. We have shown for the first time that AML with del(9q) in the context of a noncomplex karyotype is strongly associated with CEBPA loss-of-function mutations. Loss of a critical segment of 9q, most likely in 9q22, and disruption of CEBPA function possibly cooperate in the pathogenesis of del(9q) AML., ((c) 2005 Wiley-Liss, Inc.)

Background and Objectives: Karyotype is one of the most important prognostic factors in acute myeloid leukemia (AML)., Design and Methods: To assess the diagnostic value of molecular cytogenetics in AML patients older than 60 years, we compared the results of chromosome banding with those of fluorescence in situ hybridization (FISH) applying a comprehensive DNA-probe set for the detection of the most relevant AML-associated chromosome aberrations in a prospective series of 283 patients registered for the multicenter treatment trial AML HD98-B., Results: Four cases of inv(16)/t(16;16) and 2 cases of t(11q23) were only detected by FISH. Molecular cytogenetic analysis was also more sensitive for the detection of genomic imbalances, in particular 7q-, +11q, 17p-, and 20q-, but virtually all cases of aneuploidy or deletions that were missed on banding analysis were identified in patients without assessable metaphases, in patients with normal karyotypes but poor chromosome morphology, in patients with a leukemia-specific balanced rearrangement, or in patients with complex karyotypes., Interpretation and Conclusions: Our results support the use of FISH as a complementary method for the detection of inv(16)/t(16;16) and t(11q23) in all older AML patients eligible for intensive therapy. Molecular cytogenetics should also be considered in cases with insufficient yields of metaphase cells, poor chromosome morphology, or both. Routine screening for chromosomal imbalances by FISH does not improve cytogenetic risk assessment in patients with adequate pretreatment karyotype information.

The purpose of our study was (i) to evaluate the impact of all-trans retinoic acid (ATRA) given as adjunct to chemotherapy and (ii) to compare second consolidation vs maintenance therapy in elderly patients with acute myeloid leukemia (AML). A total of 242 patients aged >or=61 years (median, 66.6 years) with AML were randomly assigned to ATRA beginning on day +3 after the initiation of chemotherapy (ATRA-arm, n=122) or no ATRA (standard-arm, n=120) in combination with induction and first consolidation therapy. A total of 61 patients in complete remission (CR) were randomly assigned to second intense consolidation (n=31) or 1-year oral maintenance therapy (n=30). After induction therapy the intention-to-treat analysis revealed a significant difference in CR rates between the ATRA- and the standard-arm (52 vs 39%; P=0.05). Event-free (EFS) and overall survival (OS) were significantly better in the ATRA-compared to the standard-arm (P=0.03 and 0.01, respectively). OS after second randomization was significantly better for patients assigned to intensive consolidation therapy (P<0.001). The multivariate model for survival revealed lactate dehydrogenase, cytogenetic risk group, age, and first and second randomization as prognostic variables. In conclusion, the addition of ATRA to induction and consolidation therapy may improve CR rate, EFS and OS in elderly patients with AML.

Background: In patients with acute myeloid leukemia (AML), the presence or absence of recurrent cytogenetic aberrations is used to identify the appropriate therapy. However, the current classification system does not fully reflect the molecular heterogeneity of the disease, and treatment stratification is difficult, especially for patients with intermediate-risk AML with a normal karyotype., Methods: We used complementary-DNA microarrays to determine the levels of gene expression in peripheral-blood samples or bone marrow samples from 116 adults with AML (including 45 with a normal karyotype). We used unsupervised hierarchical clustering analysis to identify molecular subgroups with distinct gene-expression signatures. Using a training set of samples from 59 patients, we applied a novel supervised learning algorithm to devise a gene-expression-based clinical-outcome predictor, which we then tested using an independent validation group comprising the 57 remaining patients., Results: Unsupervised analysis identified new molecular subtypes of AML, including two prognostically relevant subgroups in AML with a normal karyotype. Using the supervised learning algorithm, we constructed an optimal 133-gene clinical-outcome predictor, which accurately predicted overall survival among patients in the independent validation group (P=0.006), including the subgroup of patients with AML with a normal karyotype (P=0.046). In multivariate analysis, the gene-expression predictor was a strong independent prognostic factor (odds ratio, 8.8; 95 percent confidence interval, 2.6 to 29.3; P<0.001)., Conclusions: The use of gene-expression profiling improves the molecular classification of adult AML., (Copyright 2004 Massachusetts Medical Society)

The family of immune associated nucleotide binding proteins (Ian) is a distinct family of GTP-binding proteins conserved in plants, mice, rats and humans that are associated with immune functions, suggesting involvement in conserved defense mechanisms. Recently, the rat Ian4 (rIan4) was cloned and it appears to be identical to the gene Iddm1/lyp responsible for severe lymphopenia and the development of insulin-dependent diabetes in the BB-DP rat. Here we describe the characterization of a new human member of the Ian family: hIan5. hIan5 is highly homologous to rIan4, has a predicted molecular weight of 35 kDa and contains distinct G motifs of GTP-binding proteins (G-1 to G-4) in the N-terminus. Human Ian5 is anchored to the mitochondria by the hydrophobic COOH-terminal domain. Human Ian5 is highly expressed in lymph node and spleen. Different blood fractions show high hIan5 expression in CD4- and CD8-positive T cells and monocytes, but not in B lymphocytes. In contrast, in B-CLL (chronic lymphocytic leukemia) and mantle cell lymphoma samples, hIan5 mRNA was upregulated. The current data underline the role of hIan5 in T-lymphocyte development and function, and for the first time suggest that upregulation of Ian proteins is associated with B-cell malignancy, possibly by inhibiting apoptosis.

Purpose: To assess the prognostic relevance of mutations in the CEBPA gene encoding CCAAT/enhancer binding protein alpha (C/EBP alpha) in a large prospective series of younger adults with acute myeloid leukemia (AML) and normal cytogenetics., Patients and Methods: The entire CEBPA coding region was sequenced in diagnostic samples from 236 AML patients 16 to 60 years of age with normal cytogenetics who were uniformly treated on two consecutive protocols of the AML Study Group Ulm, and CEBPA mutation status was correlated with clinical outcome., Results: CEBPA mutations were detected in 36 (15%) of 236 patients. Twenty-one (9%) of 236 patients had mutations predicted to result in loss of C/EBP alpha function. Remission duration and overall survival (OS) were significantly longer for the 36 patients with CEBPA mutations (P =.01 and P =.05, respectively). On multivariate analysis, wild-type CEBPA was an independent prognostic marker affecting remission duration (hazard ratio, 2.85; P =.01) and OS (hazard ratio, 1.87; P =.04). Analysis of cooperating mutations (both types of activating FLT3 mutations and MLL partial tandem duplications) showed that FLT3 mutations had no significant prognostic influence in patients with CEBPA mutations. Furthermore, there was no significant overlap between the subgroup of patients with CEBPA mutation with predicted loss of C/EBP alpha function and patients with FLT3 or MLL mutations, suggesting that CEBPA loss-of-function mutations define a distinct biologic subclass of AML with normal cytogenetics., Conclusion: Mutant CEBPA predicts favorable prognosis and may improve risk stratification in AML patients with normal cytogenetics.

One strategy to predict clinical outcome in patients with acute myeloid leukemia (AML) is detection of minimal residual disease (MRD) after achievement of hematologic complete remission (CR). We established a real-time RT-PCR assay by use of TaqMan technology for the identification of MRD by quantification of the most frequent fusion transcripts resulting from t(9;11)(p22;q23). To achieve comparable PCR efficiencies between the different PCR assays, primers were chosen to obtain amplicons of nearly identical lengths. MLL/AF9 copy numbers were normalized to the housekeeping gene porphobilinogen deaminase (PBGD). The sensitivity of the assay, as determined at the cellular level, was comparable to that of qualitative single-round RT-PCR. Samples from eight patients with t(9;11)-positive AML were analyzed. At diagnosis and relapse, normalized copy numbers were positive and ranged from 490 to 5,558. Samples from two of seven patients collected at the time of CR became negative, whereas five cases still had positive normalized copy numbers with values between 5 and 5,286. The implications of MRD detection by MLL/AF9 fusion transcript quantification for the clinical management of t(9;11)-positive AML have to be determined in further studies., (Copyright 2003 Wiley-Liss, Inc.)

The objective of the AML HD93 treatment trial was to evaluate the outcome in young adults with acute myeloid leukemia (AML) after postremission therapy was stratified according to cytogenetically defined risk. The rationales for the study design were based (i) on previous favorable results with high-dose cytarabine in AML with t(8;21), inv/t(16q22) and in AML with normal karyotype, and ii) on encouraging results obtained in several phase II trials using autologous stem cell transplantation (SCT). Between July 1993 and January 1998, 223 eligible patients, 16-60 years of age with newly diagnosed AML other than French-American-British type M3/M3v, were entered into the trial. Risk groups were defined as follows: low risk: t(8;21) or inv/t(16q22); intermediate risk: normal karyotype; high risk: all other chromosomal abnormalities. Following intensive double induction therapy with idarubicin, cytarabine and etoposide, all patients in complete remission (CR) received a first consolidation therapy with high-dose cytarabine and mitoxantrone (HAM). A second consolidation therapy was stratified according to the risk group: low risk: HAM; intermediate risk: related allogeneic SCT or sequential HAM; high risk: related allogeneic or autologous SCT. Double induction therapy resulted in a high CR rate of 74.5%, and 90% of the responding patients were eligible for consolidation therapy. Survival for all 223 trial entrants was 40%, and for the 166 patients who entered CR, disease-free (DFS) and overall survival were 40 and 51% after 5 years, respectively. Within the low-, intermediate- and high-risk groups, DFS and survival after 5 years were 62.5 and 87, 40 and 49 and 17 and 26% respectively, without advantage for allogeneic transplantation in the intermediate- and high-risk groups. Postremission therapy-related mortality was 0, 7 and 14%, respectively. This study demonstrates the feasibility of cytogenetically defined risk-adapted consolidation therapy. The overall trial results are at least equivalent to those of published trials supporting the risk-adapted treatment strategy.

To assess the prognostic relevance of activating mutations of the FLT3 gene in homogeneously treated adults 16 to 60 years of age with acute myeloid leukemia (AML) and normal cytogenetics, pretreatment samples from 224 patients entered into 2 consecutive multicenter treatment trials were analyzed for FLT3 internal tandem duplications (ITDs) and Asp835 mutations. Treatment included intensive double-induction therapy and postremission therapy with high cumulative doses of high-dose cytarabine. ITDs were detected in 32% of the patients and were related to de novo AML and to high white blood cell (WBC) counts, percentages of peripheral blood (PB) and bone marrow (BM) blasts, and serum lactate dehydrogenase levels. Asp835 mutations were present in 14% of the patients and were associated with WBC counts and percentages of PB and BM blasts that were higher than those of patients without FLT3 mutations. With a median follow-up of 34 months, remission duration and overall survival (OS) were significantly shorter for patients with Asp835 mutations or an ITD than for those without FLT3 mutations (P =.03 and P =.0004, respectively). These results were attributable mainly to the negative prognostic effect of FLT3 ITDs. On multivariate analysis, mutant FLT3 was an independent marker affecting remission duration and OS (hazard ratio, 2.35 and 2.11, respectively). Fluorescence in situ hybridization did not detect monoallelic FLT3 deletions in ITD-positive patients. FLT3 mutations identify a subset of young AML patients with normal cytogenetics who do not benefit from intensive chemotherapy, including double-induction and postremission therapy with high-dose cytarabine.

Purpose: To evaluate the incidence and clinical significance of partial tandem duplications (PTDs) of the mixed lineage leukemia (MLL) gene in a large series of newly diagnosed adult patients (16 to 60 years old) with acute myeloid leukemia (AML) intensively treated within the multicenter treatment trials AML-HD93 and AML-HD98A., Patients and Methods: Identification of MLL PTD was performed centrally using Southern blot analysis in pretreatment samples from 525 of 683 assessable patients. PTD was confirmed by polymerase chain reaction (PCR) and sequencing of the PCR products., Results: MLL PTD was identified in none of the 129 patients with t(8;21), inv(16), and t(15;17); in 19 (7.7%) of 247 patients with normal karyotype; and in 10 (8.5%) of 119 patients with all other abnormalities, with 30 cases of t(11q23) excluded. In the group of patients with a normal karyotype, there was no difference in the presenting clinical features between the PTD-positive and the PTD-negative cases. Sixteen (89%) of the 18 assessable PTD-positive patients and 158 (78%) of the 203 PTD-negative patients achieved a complete remission. After a median follow-up time of 19 months, 11 of the 16 PTD-positive patients relapsed compared with 54 of the 158 PTD-negative patients; the median remission durations of the PTD-positive and the PTD-negative groups were 7.75 months and 19 months, respectively (P <.001). Multivariate analysis identified the MLL PTD status as the single prognostic factor for remission duration., Conclusion: Within the subgroup of young adult AML patients with normal karyotype, MLL PTD is associated with short remission duration.

Purpose: To prospectively compare cytogenetic and molecular cytogenetic analysis for the detection of the most relevant chromosome abnormalities in a large series of patients with acute myeloid leukemia (AML)., Patients and Methods: Two hundred forty consecutive adult patients with AML entered onto the multicenter treatment trial AML HD93 were studied. Chromosome banding and fluorescence in situ hybridization (FISH) applying a comprehensive set of genomic DNA probes were performed in a single reference laboratory., Results: Two cases of inv(16), three cases of t(11q23), and three cases of t(8;21)var were only detected by molecular cytogenetics. By FISH, aberrations were identified in three cases with normal karyotypes: inv(16), -Y (in a patient with low metaphase yield on chromosome banding) and a 12p microdeletion. Additional aneuploidies, in particular +8q and +11q, were diagnosed by FISH; however, virtually all these aberrations occurred in patients with complex karyotypes or as an additional abnormality in leukemias with an AML-specific translocation. Finally, aberrations were detected by FISH in eight of 14 patients with no assessable metaphases., Conclusion: In most cases of AML, conventional cytogenetic study reliably detects chromosomal abnormalities, and this method should not be replaced by FISH. FISH should be used as a complementary method for the detection of more subtle abnormalities, such as inv(16) and t(11q23), in all patients with newly diagnosed AML and for suspected t(8;21)var. Furthermore, molecular cytogenetics using this comprehensive set of DNA probes provides a valuable diagnostic tool for patients with poor chromosome morphology, low or no yields of metaphase cells, or both.

The ORC5L gene encoding a subunit of the human origin recognition complex (ORC) maps to chromosome band 7q22, a region frequently deleted in adult acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Because of its localization within a region that is commonly deleted in patients with myeloid malignancies and because of the implication of its protein product in cell cycle control (DNA replication) and regulation of gene expression (transcriptional silencing), ORC5L appeared to be a candidate tumor suppressor gene for myeloid disorders associated with 7q22 deletions. Polymerase chain reaction amplification and sequencing analysis of the coding region of the remaining ORC5L allele has not revealed any mutations in nine patients with AML or MDS exhibiting 7q22 deletions. Allelic expression analysis indicates that ORC5L is not imprinted. These data suggest that ORC5L does not function as a tumor suppressor in patients with myeloid neoplasms.

Loss of chromosome 7 (-7) or deletion of the long arm (7q-) are recurring chromosome abnormalities in myeloid leukemias. The association of -7/7q- with myeloid leukemia suggests that these regions contain novel tumor suppressor gene(s), whose loss of function contribute to leukemic transformation or tumor progression. Based on chromosome banding analysis, two critical regions have been identified, one in band q22 and another in bands q32-q35. Presently there are no data available on the molecular delineation of the distal critical region. In this study we analyzed bone marrow and blood samples from 13 patients with myeloid leukemia (de novo myelodysplastic syndrome [MDS], n = 3; de novo acute myeloid leukemia [AML], n = 9; therapy-related (t-) AML, n = 1) which, on chromosome banding analysis, exhibited deletions (n = 12) or in one case a balanced translocation involving bands 7q31-qter using fluorescence in situ hybridization (FISH). As probes we used representative clones from a contig map of yeast artificial chromosome (YAC) clones that spans chromosome bands 7q31.1-qter. In the 12 cases with loss of 7q material, we identified a commonly deleted region of approximately 4 to 5 megabasepairs in size encompassing the distal part of 7q35 and the proximal part of 7q36. Furthermore, the breakpoint of the reciprocal translocation from the patient with t-AML was localized to a 1,300-kb sized YAC clone that maps to the proximal boundary of the commonly deleted region. Interestingly, in this case both homologs of chromosome 7 were affected: one was lost (-7) and the second exhibited the t(7q35). The identification and delineation of translocation and deletion breakpoints provides the first step toward the identification of the gene(s) involved in the pathogenesis of 7q35-q36 aberrations in myeloid disorders.

Loss of chromosome 7 (-7) or deletion of its long arm (7q-) are recurring chromosome abnormalities in myeloid disorders, especially in therapy-related myelodysplastic syndrome (t-MDS) and acute myeloid leukemia (t-AML). The association of -7/7q- with myeloid leukemia suggests that these regions contain a novel tumor suppressor gene(s) whose loss of function contributes to leukemic transformation or tumor progression. Based on chromosome banding analysis, two critical regions have been identified: one in band 7q22 and a second in bands 7q32-q35. We analyzed bone marrow and blood samples from 21 patients with myeloid leukemia (chronic myeloid leukemia, n = 2; de novo MDS, n = 4; de novo AML, n = 13; t-AML, n = 2) that on chromosome banding analysis exhibited deletions (n = 19) or reciprocal translocations (n = 2) of band 7q22 using fluorescence in situ hybridization. As probes, we used Alu-polymerase chain reaction products from 22 yeast artificial chromosome (YAC) clones that span chromosome bands 7q21.1-q32, including representative clones from a panel of YACs recognizing a contiguous genomic DNA fragment of 5 to 6 Mb in band 7q22. In the 19 cases with deletions, we identified two distinct commonly deleted regions: one region within band 7q22 was defined by the two CML cases; the second region encompassed a distal part of band 7q22 and the entire band 7q31 and was defined by the MDS/AML cases. The breakpoint of one of the reciprocal translocations was mapped to 7q21.3, which is centromeric to both of the commonly deleted regions. The breakpoint of the second translocation, which was present in unstimulated bone marrow and phytohemagglutinin-stimulated blood of an MDS patient, was localized to a 400-kb genomic segment in 7q22 within the deletion cluster of the MDS/AML cases. In conclusion, our data show marked heterogeneity of 7q22 deletion and translocation breakpoints in myeloid leukemias, suggesting the existence of more than one pathogenetically relevant gene.

The objective of this study was to design DNA probe sets that enable the detection of chromosome aberrations in acute myeloid leukemia (AML) by interphase cytogenetics using fluorescence in situ hybridization (FISH) and to compare the results of interphase cytogenetics with those of conventional chromosome banding analysis. One hundred five consecutive patients with adult AML entered on a multicenter treatment trial were studied with a comprehensive set of DNA probes recognizing the most relevant AML-associated structural and numerical chromosome aberrations: translocations t(8;21), t(15;17), and t(11q23); inversion inv(16);chromosomal deletions (5q-, 7q-, 9q-, 12p-, 13q-, 17p-, and 20q-); and chromosomal aneuploidies. Interphase cytogenetics was particularly sensitive for detecting the AML-specific gene fusions: 3 additional cases of inv(16) and 1 additional case of t(8;21) were identified by FISH that were missed by banding analysis, whereas equal numbers of t(11q23) and t(15;17) were detected. Five additional cases of trisomy 8q, 3 more cases of trisomy 11q, and 2 more cases of trisomies 21q and 22q were shown by FISH. These aberrations were either masked in complex karyo-types or identified in cases in which conventional banding analysis failed. On the other hand, the DNA probes selected were not informative to detect 1 case of 5q-, 9q-, and 20q-. In 5 cases, clonal aberrations were detected on banding analysis for which no FISH probes were selected. In conclusion, interphase cytogenetics proved to be more sensitive for detecting AML-specific chimeric gene fusions and some partial trisomies. Interphase cytogenetics provides a powerful technique complementary and, with further development of diagnostic DNA probes, even an alternative to chromosome banding studies for the cytogenetic analysis of AML.