Gene expression profiling of acute myeloid leukemia (AML) allows the discovery of previously unrecognized molecular entities. Here, we identified a specific subgroup of AML, defined by an expression profile resembling that of AMLs with mutations in the myeloid transcription factor CCAAT/enhancer-binding protein alpha (C/EBP␣), while lacking such mutations. We found that in these leukemias, the CEBPA gene was silenced, which was associated with frequent promoter hypermethylation. The leukemias phenotypically showed aberrant expression of Tcell genes, of which CD7 was most consistent. We identified 2 mechanisms that may contribute to this phenotype. First, absence of Cebpa led to up-regulation of specific T-cell transcripts (ie, Cd7 and Lck) in hematopoietic stem cells isolated from conditional Cebpa knockout mice. Second, the enhanced expression of TRIB2, which we identify here as a direct target of the T-cell commitment factor NOTCH1, suggested aberrantly activated Notch signaling. Putatively activating NOTCH1 mutations were found in several specimens of the newly identified subgroup, while a large set of control AMLs was mutation negative. A gene expression prediction signature allowed the detection of similar cases of leukemia in independent series of AML. IntroductionAcute myeloid leukemia (AML) is a heterogeneous disease with respect to its underlying genetic abnormalities and clinical biology. 1 It has therefore been postulated that AML, although always characterized by a malignant accumulation of myeloid progenitor cells, in fact represents not a single disease, but a group of neoplasms. Cytogenetic and molecular analyses, studying specific chromosomal translocations and mutations, are used to identify subgroups of AML with distinct biologic and clinical behavior. Recent developments in microarray research have resulted in further improvements in the characterization of the molecular heterogeneity of AML, 2 and gene expression profiling (GEP) studies have demonstrated that specific chromosomal aberrations, such as the common translocations t(8;21), t(15;17), and inv(16), correlate with unique expression signatures. [3][4][5] In a GEP study of 285 cases of de novo AML, we found that specific expression profiles are associated with other recurrent genetic aberrations such as mutations in NPM1 and alterations involving MLL, and with overexpression of the proto-oncogene EVI1 as well. 4,6 Some AMLs lacking these particular abnormalities were found to express the same characteristic profiles, suggesting that deregulation of the same pathways had occurred through yet-unknown mechanisms. This study also showed that 2 distinct expression clusters (no. 4 and no. 15) consisted primarily of AML cases with mutations in CEBPA, the gene encoding the basic leucine zipper transcription factor CCAAT/enhancer-binding protein alpha (C/EBP␣). C/EBP␣ is a critical regulator of hematopoietic stem cell (HSC) homeostasis and myeloid differentiation, [7][8][9][10] and multiple studies have shown that C/EBP␣ function is frequently ...

The transfusion of random platelets did result in a rise in platelet count (mean increments: 68 ϫ 10 9 /L and 87 ϫ 10 9 /L at day 0 and day 1, respectively), but this was significantly and consistently less over the lifespan of the transfused platelets than the mean increment seen with AC-Tx (116 ϫ 10 9 /L and 146 ϫ 10 9 /L on days 0 and 1, respectively). More importantly, the survival of the random platelets was only approximately half that of antigen-compatible platelets (T 1/2 of random platelets, 1.0 days; antigen-compatible platelets, 1.9 days) ( Figure 1).The range of day-1 increments varied widely, both with R-Tx (range: 10-217 ϫ 10 9 /L) and AC-Tx (range: 62-216 ϫ 10 9 /L). Unexpectedly large increments may occur with R-Tx when a proportion of the platelets is coincidentally HPA-1a/5b negative (estimated likelihood, 1 in 13 where the platelet pool is from 4 "random" donors) 2 ; unexpectedly low increments may be seen with AC-Tx due to the presence in the fetal circulation of maternal HLA class I antibodies 3 or when platelet transfusions are inappropriately administered. Babies with NAIT should therefore have their platelet counts monitored regularly whatever treatment is used and appropriate changes to treatment instigated when necessary.In conclusion, we agree with Kiefel et al that R-Tx is an acceptable initial treatment for NAIT where HPA-1a/5b-negative platelets are not immediately available. However, our data show that HPA-1a/5b-negative platelets give larger increments, have a longer half-life, and only occasionally fail to provide therapeutically significant platelet increments. We therefore encourage blood services in other countries to establish panels of HPA-1a/5b-negative donors to provide HPA-1a/5b-negative platelets for immediate use in cases of suspected NAIT and for intrauterine or neonatal therapy of cases of known NAIT. 4

Recently, gene expression profiling has allowed the identification of specific molecular signatures of established as well as novel subgroups of acute myeloid leukemia (AML). CEBPA mutations are observed in ~10% of AML. In a gene expression profiling study of 285 AML cases, we previously found two distinct clusters consisting of almost all specimens with CEBPA mutations, i.e. cluster #15 and cluster #4. In cluster #4, only 9 out of 15 cases were CEBPA mutant. Thus, this cluster contained 6 patients with a highly similar expression signature, but without CEBPA mutations. We wished to investigate whether these cases represent a previously unidentified subgroup of leukemias. All 6 cases appeared morphologically immature (FAB-M0 or M1) and did not carry any of the common genetic aberrations. Supervised array expression analysis revealed CEBPA to be among the most highly downregulated genes in these 6 cases, which was associated with CEBPA CpG promoter hypermethylation in 4/6. None of the CEBPA mutant cases or a random selection of 10 AML cases from other clusters exhibited CEBPA promoter methylation. Strikingly, several T-cell related genes, e.g. CD7, CD3D, IL7R and T-cell receptor delta locus, appeared significantly overexpressed in these cases. Multicolor flowcytometry demonstrated high expression of CD34 and combinations of myeloid (e.g. CD13, CD33, CD117 and/or myeloperoxidase) as well as T-lymphoid (CD7, CD3, TdT) lineage markers on all six. Expression of T-cell markers correlated with T-cell receptor rearrangements in 4/6 cases. Other highly expressed genes in these biphenotypic acute leukemias included NOTCH1 and its recently identified target gene TRIB2. Sequencing analysis demonstrated putatively activating NOTCH1 mutations in three out of six samples, as described previously in T-cell acute lymphoblastic leukemia (T-ALL). The remaining 3 cases and 25 control AMLs, including all CEBPA mutant cases, did not demonstrate any NOTCH1 mutation. Finally, based on this first cohort of patients, we determined the minimal number of probe sets accurately predicting cluster #4 biphenotypic cases, and identified eight probe sets, including those for T-cell receptor delta/alpha locus. Applying this predictor set to an independent new cohort of 272 AML cases, an identical subgroup of another 6 AML patients with frequent NOTCH1 mutations and CEBPA promoter hypermethylation could be identified. Available clinical data suggested an adverse prognosis of this type of leukemia. In conclusion, we identified a subgroup of biphenotypic acute leukemias characterized by a CEBPA mutant gene expression profile, frequent CEBPA promoter hypermethylation, and frequent NOTCH1 mutations. To our knowledge, this is the first report to describe NOTCH1 mutations in leukemia other than T-ALL.