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Severe congenital neutropenia (CN) is a preleukemic bone marrow failure syndrome with a 20% risk of evolving into leukemia or myelodysplastic syndrome (MDS). Patterns of acquisition of leukemia-associated mutations were investigated using next-generation deep-sequencing in 31 CN patients who developed leukemia or MDS. Twenty (64.5%) of the 31 patients had mutations in RUNX1. A majority of patients with RUNX1mutations (80.5%) also had acquired CSF3Rmutations. In contrast to their high frequency in CN patients who developed leukemia or MDS, RUNX1mutations were found in only 9 of 307 (2.9%) patients with de novo pediatric acute myeloid leukemia. A sequential analysis at stages prior to overt leukemia revealed RUNX1mutations to be late events in leukemic transformation. Single-cell analyses in 2 patients showed that RUNX1and CSF3Rmutations were present in the same malignant clone. Functional studies demonstrated elevated granulocyte colony-stimulating factor (G-CSF)–induced proliferation with diminished myeloid differentiation of hematopoietic CD34+cells coexpressing mutated forms of RUNX1 and CSF3R. The high frequency of cooperating RUNX1and CSF3Rmutations in CN patients suggests a novel molecular pathway of leukemogenesis: mutations in the hematopoietic cytokine receptor (G-CSFR) in combination with the second mutations in the downstream hematopoietic transcription fator (RUNX1). The detection of both RUNX1and CSF3Rmutations could be used as a marker for identifying CN patients with a high risk of progressing to leukemia or MDS.

Translocations involving nucleoporin 98kD(NUP98) on chromosome 11p15 occur at relatively low frequency in acute myeloid leukemia (AML) but can be missed with routine karyotyping. In this study, high-resolution genome-wide copy number analyses revealed cryptic NUP98/NSD1translocations in 3 of 92 cytogenetically normal (CN)–AML cases. To determine their exact frequency, we screened > 1000 well-characterized pediatric and adult AML cases using a NUP98/NSD1-specific RT-PCR. Twenty-three cases harbored the NUP98/NSD1fusion, representing 16.1% of pediatric and 2.3% of adult CN-AML patients. NUP98/NSD1-positive AML cases had significantly higher white blood cell counts (median, 147 × 109/L), more frequent FAB-M4/M5 morphology (in 63%), and more CN-AML (in 78%), FLT3/internal tandem duplication (in 91%) and WT1mutations (in 45%) than NUP98/NSD1-negative cases. NUP98/NSD1was mutually exclusive with all recurrent type-II aberrations. Importantly, NUP98/NSD1was an independent predictor for poor prognosis; 4-year event-free survival was < 10% for both pediatric and adult NUP98/NSD1-positive AML patients. NUP98/NSD1-positive AML showed a characteristic HOX-gene expression pattern, distinct from, for example, MLL-rearranged AML, and the fusion protein was aberrantly localized in nuclear aggregates, providing insight into the leukemogenic pathways of these AMLs. Taken together, NUP98/NSD1identifies a previously unrecognized group of young AML patients, with distinct characteristics and dismal prognosis, for whom new treatment strategies are urgently needed.

Pediatric mixed-lineage leukemia (MLL)–rearranged acute monoblastic leukemia with t(9;11)(p22;q23) has a favorable outcome compared with other MLL-rearranged AML. The biologic background for this difference remains unknown. Therefore, we compared gene expression profiles (GEPs; Affymetrix HGU133 + 2.0) of 26 t(9;11)(p22;q23) patients with 42 other MLL-rearranged AML patients to identify differentially expressed genes. IGSF4, a cell-cell adhesion molecule, was found to be highly expressed in t(9;11)(p22;q23) patients, which was confirmed by real-time quantitative polymerase chain reaction and Western blot. IGSF4expression within t(9;11)(p22;q23) patients was 4.9 times greater in French-American-British morphology classification (FAB)–M5 versus other FAB-types (P= .001). Methylation status investigation showed that high IGSF4-expressing t(9;11)(p22;q23) patients with FAB-M5 have no promoter hypermethylation, whereas all other cases do. Cell-line incubation with demethylating agent decitabine resulted in promoter demethylation and increased expression of IGSF4. Down-regulation of IGSF4by siRNA did not affect proliferation or drug sensitivity. In a cohort of 79 MLL-rearranged AML cases, we show significant better overall survival for cases with high IGSF4expression (5-year overall survival 0.70 vs 0.37, P= .03) In conclusion, we identified IGSF4overexpression to be discriminative for t(9;11)(p22;q23) patients with FAB-M5, regulated partially by promoter methylation and resulting in survival benefit.