Number of RUNX1 mutations, wild-type allele loss and additional mutations impact on prognosis in adult RUNX1-mutated AML.

RUNX1-mutated acute myeloid leukemia (AML) show a distinct pattern of genetic abnormalities and an adverse prognosis. We analyzed the impact of multiple RUNX1 mutations and RUNX1 wild-type (WT) loss in 467 AML with RUNX1 mutations (mut): (1) RUNX1 WT loss (n=53), (2) >1 RUNX1mut (n=94) and (3) 1 RUNX1mut (n=323). In 1 RUNX1mut, +8 was most frequent, whereas in WT loss +13 was the most abundant trisomy (+8: 66% vs 31%, P=0.022; +13: 15% vs 62%, P<0.001). Analyses of 28 genes in 163 selected cases revealed SRSF2 (39%), ASXL1 (36%), DNMT3A (19%), IDH2 (17%) and SF3B1 (17%) as most frequently mutated genes. RUNX1 WT loss showed a higher frequency of ASXL1mut compared with the other cases (50% vs 29%, P=0.009). Median overall survival (OS) in the total cohort was 14 months. WT loss (OS: 5 months) and >1 RUNX1mut (14 months) showed an adverse impact on prognosis compared with 1 RUNX1mut (22 months; P=0.002 and 0.048, respectively). Mutations in ASXL1 and ⩾2 additional mutations correlated with shorter OS (10 vs 18 months, P=0.028; 12 vs 20 months, P=0.017). Thus, the number of RUNX1mut, RUNX1 WT loss and the number and type of additional mutations is biologically and clinically relevant.

SETD2 and histone H3 lysine 36 methylation deficiency in advanced systemic mastocytosis.

The molecular basis of advanced systemic mastocytosis (SM) is not fully understood and despite novel therapies the prognosis remains dismal. Exome sequencing of an index-patient with mast cell leukemia (MCL) uncovered biallelic loss-of-function mutations in the SETD2 histone methyltransferase gene. Copy-neutral loss-of-heterozygosity at 3p21.3 (where SETD2 maps) was subsequently found in SM patients and prompted us to undertake an in-depth analysis of SETD2 copy number, mutation status, transcript expression and methylation levels, as well as functional studies in the HMC-1 cell line and in a validation cohort of 57 additional cases with SM, including MCL, aggressive SM and indolent SM. Reduced or no SETD2 protein expression-and consequently, H3K36 trimethylation-was found in all cases and inversely correlated with disease aggressiveness. Proteasome inhibition rescued SETD2 expression and H3K36 trimethylation and resulted in marked accumulation of ubiquitinated SETD2 in SETD2-deficient patients but not in patients with near-normal SETD2 expression. Bortezomib and, to a lesser extent, AZD1775 alone or in combination with midostaurin induced apoptosis and reduced clonogenic growth of HMC-1 cells and of neoplastic mast cells from advanced SM patients. Our findings may have implications for prognostication of SM patients and for the development of improved treatment approaches in advanced SM.

Molecular characterization of EZH2 mutant patients with myelodysplastic/myeloproliferative neoplasms.

Mutations in the epigenetic regulator gene EZH2 are frequently observed in patients with myelodysplastic/myeloproliferative neoplasms (MDS/MPN; 10-13%) and are associated with a poor outcome. To gain more insight into EZH2 pathology, we sought to genetically characterize a cohort of 41 EZH2-mutated MDS/MPN patients using targeted deep next-generation sequencing (NGS), colony-forming progenitor assays and transcriptome analysis. Stable short hairpin RNA (shRNA)-mediated downregulation of EZH2 was performed in MDS-derived F-36P, MOLM-13 and OCI-M2 cells to study EZH2-specific changes. Targeted NGS revealed a complex pattern of mutations with a total of 190 individual mutations. EZH2 mutations frequently co-occur with TET2 (58%), RUNX1 (40%) and ASXL1 (34%) mutations. Colony assays indicated EZH2 mutations to be mostly early events in leukemogenesis and showed a complex mutational hierarchy. Gene expression data revealed a number of differently expressed genes between EZH2 wild-type and mutant patients including known EZH2 targets. Comparison of patient transcriptome to EZH2-downregulated cell line data revealed several genes as novel EZH2 targets, showing opposite as well as unidirectional regulation between cell lines and patients. Some genes, such as CXXC5, ETS1 and VAV3 have previously been implied to have a role in leukemogenesis. Their precise role in MDS/MPN needs to be further investigated.

Ordering of mutations in acute myeloid leukemia with partial tandem duplication of MLL (MLL-PTD).

Partial tandem duplication of MLL (MLL-PTD) characterizes acute myeloid leukemia (AML) patients often with a poor prognosis. To understand the order of occurrence of MLL-PTD in relation to other major AML mutations and to identify novel mutations that may be present in this unique AML molecular subtype, exome and targeted sequencing was performed on 85 MLL-PTD AML samples using HiSeq-2000. Genes involved in the cohesin complex (STAG2), a splicing factor (U2AF1) and a poorly studied gene, MGA were recurrently mutated, whereas NPM1, one of the most frequently mutated AML gene, was not mutated in MLL-PTD patients. Interestingly, clonality analysis suggests that IDH2/1, DNMT3A, U2AF1 and TET2 mutations are clonal and occur early, and MLL-PTD likely arises after these initial mutations. Conversely, proliferative mutations (FLT3, RAS), typically appear later, are largely subclonal and tend to be unstable. This study provides important insights for understanding the relative importance of different mutations for defining a targeted therapeutic strategy for MLL-PTD AML patients.

The impact of TP53 mutations and TP53 deletions on survival varies between AML, ALL, MDS and CLL: an analysis of 3307 cases.

Alterations in TP53 have been described in many cancer types including hematological neoplasms. We aimed at comparing TP53 mutations (mut) and deletions (del) in a large cohort of patients with hematological malignancies (n=3307), including AML (n=858), MDS (n=943), ALL (n=358), CLL (n=1148). Overall, alterations in TP53 were detected in 332/3307 cases (10%). The highest frequency was observed in ALL (total: 19%; mut+del: 6%; mut only: 8%; del only: 5%) and AML (total: 13%; mut+del: 5%; mut only: 7%; del only: 1%), whereas TP53 alterations occurred less frequently in CLL (total: 8%) and MDS (total: 7%). TP53 mutations were significantly more frequent in patients ⩾60 vs <60 years in AML (9% vs 2%, P<0.001) and ALL (12% vs 6%, P<0.001). TP53mut+del had a significant negative impact on overall survival in all entities, whereas differences were observed regarding TP53mut only or TP53del only: TP53mut only impacted survival in AML (36 vs 9 months, P<0.001) and MDS (65 vs 19 months, P<0.001), TP53del only in CLL (not reached vs 64 months, P=0.008) and MDS (65 vs 24 months, P=0.011). As substantial differences between the entities are observed regarding correlation to age and survival, we suggest evaluation of both TP53 deletion and mutation status.

Additional mutations in SRSF2, ASXL1 and/or RUNX1 identify a high-risk group of patients with KIT D816V(+) advanced systemic mastocytosis.

Most patients with KIT D816V(+) advanced systemic mastocytosis (SM) are characterized by somatic mutations in additional genes. We sought to clarify the prognostic impact of such mutations. Genotype and clinical characteristics of 70 multi-mutated KIT D816V(+) advanced SM patients were included in univariate and multivariate analyses. The most frequently identified mutated genes were TET2 (n=33 of 70 patients), SRSF2 (n=30), ASXL1 (n=20), RUNX1 (n=16) and JAK2 (n=11). In univariate analysis, overall survival (OS) was adversely influenced by mutations in SRSF2 (P<0.0001), ASXL1 (P=0.002) and RUNX1 (P=0.03), but was not influenced by mutations in TET2 or JAK2. In multivariate analysis, SRSF2 and ASXL1 remained the most predictive adverse indicators concerning OS. Furthermore, we found that inferior OS and adverse clinical characteristics were significantly influenced by the number of mutated genes in the SRSF2/ASXL1/RUNX1 (S/A/R) panel (P<0.0001). In conclusion, the presence and number of mutated genes within the S/A/R panel are adversely associated with advanced disease and poor survival in KIT D816V(+) SM. On the basis of these findings, inclusion of molecular markers should be considered in upcoming prognostic scoring systems for patients with SM.

Clinical and biological implications of ancestral and non-ancestral IDH1 and IDH2 mutations in myeloid neoplasms.

Mutations in isocitrate dehydrogenase 1/2 (IDH1/2(MT)) are drivers of a variety of myeloid neoplasms. As they yield the same oncometabolite, D-2-hydroxyglutarate, they are often treated as equivalent, and pooled. We studied the validity of this approach and found IDH1/2 mutations in 179 of 2119 myeloid neoplasms (8%). Cross-sectionally, the frequencies of these mutations increased from lower- to higher risk disease, thus suggesting a role in clinical progression. Variant allelic frequencies indicated that IDH1(MT) and IDH2(MT) are ancestral in up to 14/74 (19%) vs 34/99 (34%; P=0.027) of cases, respectively, illustrating the pathogenic role of these lesions in myeloid neoplasms. IDH1/2(MT) was associated with poor overall survival, particularly in lower risk myelodysplastic syndromes. Ancestral IDH1(MT) cases were associated with a worse prognosis than subclonal IDH1(MT) cases, whereas the position of IDH2(MT) within clonal hierarchy did not impact survival. This may relate to distinct mutational spectra with more DNMT3A and NPM1 mutations associated with IDH1(MT) cases, and more ASXL1, SRSF2, RUNX1, STAG2 mutations associated with IDH2(MT) cases. Our data demonstrate important clinical and biological differences between IDH1(MT) and IDH2(MT) myeloid neoplasms. These mutations should be considered separately as their differences could have implications for diagnosis, prognosis and treatment with IDH1/2(MT) inhibitors of IDH1/2(MT) patients.

BAALC expression: a suitable marker for prognostic risk stratification and detection of residual disease in cytogenetically normal acute myeloid leukemia.

High brain and acute leukemia, cytoplasmic (BAALC) expression defines an important risk factor in cytogenetically normal acute myeloid leukemia (CN-AML). The prognostic value of BAALC expression in relation to other molecular prognosticators was analyzed in 326 CN-AML patients (<65 years). At diagnosis, high BAALC expression was associated with prognostically adverse mutations: FLT3 internal tandem duplication (FLT3-ITD) with an FLT3-ITD/FLT3 wild-type (wt) ratio of 0.5 (P=0.001), partial tandem duplications within the MLL gene (MLL-PTD) (P=0.002), RUNX1 mutations (mut) (P<0.001) and WT1mut (P=0.001), while it was negatively associated with NPM1mut (P<0.001). However, high BAALC expression was also associated with prognostically favorable biallelic CEBPA (P=0.001). Survival analysis revealed an independent adverse prognostic impact of high BAALC expression on overall survival (OS) and event-free survival (EFS), and also on OS when eliminating the effect of allogeneic stem cell transplantation (SCT) (OS(TXcens)). Furthermore, we analyzed BAALC expression in 416 diagnostic and follow-up samples of 66 patients. During follow-up, BAALC expression correlated with mutational load or expression levels, respectively, of other minimal residual disease markers: FLT3-ITD (r=0.650, P<0.001), MLL-PTD (r=0.728, P<0.001), NPM1mut (r=0.599, P<0.001) and RUNX1mut (r=0.889, P<0.001). Moreover, a reduction in BAALC expression after the second cycle of induction chemotherapy was associated with improved EFS. Thus, our data underline the utility of BAALC expression as a marker for prognostic risk stratification and detection of residual disease in CN-AML.

SETBP1 mutations occur in 9% of MDS/MPN and in 4% of MPN cases and are strongly associated with atypical CML, monosomy 7, isochromosome i(17)(q10), ASXL1 and CBL mutations.

Chronic myeloid malignancies are categorized to the three main categories myeloproliferative neoplasms (MPNs), myelodysplastic syndromes (MDSs) and MDS/MPN overlap. So far, no specific genetic alteration profiles have been identified in the MDS/MPN overlap category. Recent studies identified mutations in SET-binding protein 1 (SETBP1) as novel marker in myeloid malignancies, especially in atypical chronic myeloid leukemia (aCML) and related diseases. We analyzed SETBP1 in 1 130 patients with MPN and MDS/MPN overlap and found mutation frequencies of 3.8% and 9.4%, respectively. In particular, there was a high frequency of SETBP1 mutation in aCML (19/60; 31.7%) and MDS/MPN unclassifiable (MDS/MPN, U; 20/240; 9.3%). SETBP1 mutated (SETBP1mut) patients showed significantly higher white blood cell counts and lower platelet counts and hemoglobin levels than SETBP1 wild-type patients. Cytomorphologic evaluation revealed a more dysplastic phenotype in SETBP1mut cases as compared with wild-type cases. We confirm a significant association of SETBP1mut with -7 and isochromosome i(17)(q10). Moreover, SETBP1mut were strongly associated with ASXL1 and CBL mutations (P<0.001 for both) and were mutually exclusive of JAK2 and TET2 mutations. In conclusion, SETBP1mut add an important new diagnostic marker for MDS/MPN and in particular for aCML.

Transduction of human neural progenitor cells with foamy virus vectors for differentiation-dependent gene expression.

Neural progenitor cells are potential vehicles for delivery of therapeutic agents into the brain. Differentiation-dependent promoters may be useful to target the therapeutic transgene expression to specific neural cell types. Here we explored the potential of vectors based on the foamy virus (FV) for genetic engineering of neural progenitor cells. We demonstrate that FV vectors can mediate stable long-term constitutive expression of the enhanced green fluorescent protein (EGFP) in neural progenitor cells. For differentiation-dependent gene expression, we constructed a FV vector with an internal expression cassette containing the human 2.2 kb promoter (Gfa2) of the astrocyte-specific glial fibrillary acidic protein (GFAP) and sequences encoding EGFP. We show FV-vector-mediated delivery of the Gfa2-egfp transgene into the human neural stem cell line HNSC.100 and differentiation-dependent expression in stably transduced cell populations. Differentiation of the FV-transduced HNSC.100 cells to astrocytes upregulated expression of both the Gfa2-egfp transgene and the native gfap gene, confirming differentiation-dependent activation of the transduced Gfa2 promoter. These results demonstrate that differentiation-dependent gene expression can be achieved by FV-vector-mediated gene transfer to neural progenitor cells. Our findings support the use of FV vectors for the genetic engineering of neural progenitor cells for therapeutic and research applications.