Claudia Erpelinck

A Single Oncogenic Enhancer Rearrangement Causes Concomitant EVI1 and GATA2 Deregulation in Leukemia

Chromosomal rearrangements without gene fusions have been implicated in leukemogenesis by causing deregulation of proto-oncogenes via relocation of cryptic regulatory DNA elements. AML with inv(3)/t(3;3) is associated with aberrant expression of the stem-cell regulator EVI1. Applying functional genomics and genome-engineering, we demonstrate that both 3q rearrangements reposition a distal GATA2 enhancer to ectopically activate EVI1 and simultaneously confer GATA2 functional haploinsufficiency, previously identified as the cause of sporadic familial AML/MDS and MonoMac/Emberger syndromes. Genomic excision of the ectopic enhancer restored EVI1 silencing and led to growth inhibition and differentiation of AML cells, which could be replicated by pharmacologic BET inhibition. Our data show that structural rearrangements involving the chromosomal repositioning of a single enhancer can cause deregulation of two unrelated distal genes, with cancer as the outcome.

Prognostic impact, concurrent genetic mutations, and gene expression features of AML with CEBPA mutations in a cohort of 1182 cytogenetically normal AML patients: further evidence for CEBPA double mutant AML as a distinctive disease entity.

We evaluated concurrent gene mutations, clinical outcome, and gene expression signatures of CCAAT/enhancer binding protein alpha (CEBPA) double mutations (CEBPA(dm)) versus single mutations (CEBPA(sm)) in 1182 cytogenetically normal acute myeloid leukemia (AML) patients (16-60 years of age). We identified 151 (12.8%) patients with CEBPA mutations (91 CEBPA(dm) and 60 CEBPA(sm)). The incidence of germline mutations was 7% (5 of 71), including 3 C-terminal mutations. CEBPA(dm) patients had a lower frequency of concurrent mutations than CEBPA(sm) patients (P < .0001). Both, groups were associated with a favorable outcome compared with CEBPA(wt) (5-year overall survival [OS] 63% and 56% vs 39%; P < .0001 and P = .05, respectively). However, in multivariable analysis only CEBPA(dm) was a prognostic factor for favorable OS outcome (hazard ratio [HR] 0.36, P < .0001; event-free survival, HR 0.41, P < .0001; relapse-free survival, HR 0.55, P = .001). Outcome in CEBPA(sm) is dominated by concurrent NPM1 and/or FLT3 internal tandem duplication mutations. Unsupervised and supervised GEP analyses showed that CEBPA(dm) AML (n = 42), but not CEBPA(sm) AML (n = 18), expressed a unique gene signature. A 25-probe set prediction signature for CEBPA(dm) AML showed 100% sensitivity and specificity. Based on these findings, we propose that CEBPA(dm) should be clearly defined from CEBPA(sm) AML and considered as a separate entity in the classification of AML.

High EVI1 levels predict adverse outcome in acute myeloid leukemia: prevalence of EVI1 overexpression and chromosome 3q26 abnormalities underestimated

Inappropriate expression of EVI1 (ecotropic virus integration-1), in particular splice form EVI1-1D, through chromosome 3q26 lesions or other mechanisms has been implicated in the development of high-risk acute myeloid leukemia (AML). To validate the clinical relevance of EVI1-1D, as well as of the other EVI1 splice forms and the related MDS1/EVI1 (ME) gene, real-time quantitative polymerase chain reaction was performed in 534 untreated adults with de novo AML. EVI1-1D was highly expressed in 6% of cases (n = 32), whereas 7.8% were EVI1(+) (n = 41) when all splice variants were taken into account. High EVI1 predicted a distinctly worse event-free survival (HR = 1.9; P = .002) and disease-free survival (HR = 2.1, P = .006) following multivariate analysis. Importantly, we distinguished a subset of EVI1(+) cases that lacked expression of ME (EVI1(+)ME(-); n = 17) from cases that were ME(+) (EVI1(+)ME(+); n = 24). The atypical EVI1(+)ME(-) expression pattern exhibited cytogenetically detectable chromosomal 3q26 breakpoints in 8 cases. Fluorescence in situ hybridization revealed 7 more EVI1(+)ME(-) cases that carried cryptic 3q26 breakpoints, which were not found in the EVI1(+)ME(+) group. EVI1(+)ME(-) expression predicts an extremely poor prognosis distinguishable from the general EVI1(+) AML patients (overall survival [OS]: P < .001 and event-free survival [EFS]: P = .002). We argue that EVI1/ME quantitative expression analysis should be implemented in the molecular diagnostic procedures of AML.

Prediction of molecular subtypes in acute myeloid leukemia based on gene expression profiling

This study shows that gene expression profiling allows accurate prediction of certain acute myeloid leukemia subtypes, e.g. those characterized by expression of chimeric transcription factors. We examined the gene expression profiles of two independent cohorts of patients with acute myeloid leukemia [n=247 and n=214 (younger than or equal to 60 years)] to study the applicability of gene expression profiling as a single assay in prediction of acute myeloid leukemia-specific molecular subtypes. The favorable cytogenetic acute myeloid leukemia subtypes, i.e., acute myeloid leukemia with t(8;21), t(15;17) or inv(16), were predicted with maximum accuracy (positive and negative predictive value: 100%). Mutations in NPM1 and CEBPA were predicted less accurately (positive predictive value: 66% and 100%, and negative predictive value: 99% and 97% respectively). Various other characteristic molecular acute myeloid leukemia subtypes, i.e., mutant FLT3 and RAS, abnormalities involving 11q23, −5/5q-, −7/7q-, abnormalities involving 3q (abn3q) and t(9;22), could not be correctly predicted using gene expression profiling. In conclusion, gene expression profiling allows accurate prediction of certain acute myeloid leukemia subtypes, e.g. those characterized by expression of chimeric transcription factors. However, detection of mutations affecting signaling molecules and numerical abnormalities still requires alternative molecular methods.

Risk stratification of intermediate-risk acute myeloid leukemia: integrative analysis of a multitude of gene mutation and gene expression markers

Numerous molecular markers have been recently discovered as potential prognostic factors in acute myeloid leukemia (AML). It has become of critical importance to thoroughly evaluate their interrelationships and relative prognostic importance. Gene expression profiling was conducted in a well-characterized cohort of 439 AML patients (age < 60 years) to determine expression levels of EVI1, WT1, BCL2, ABCB1, BAALC, FLT3, CD34, INDO, ERG and MN1. A variety of AML-specific mutations were evaluated, that is, FLT3, NPM1, N-RAS, K-RAS, IDH1, IDH2, and CEBPA(DM/SM) (double/single). Univariable survival analysis shows that (1) patients with FLT3(ITD) mutations have inferior overall survival (OS) and event-free survival (EFS), whereas CEBPA(DM) and NPM1 mutations indicate favorable OS and EFS in intermediate-risk AML, and (2) high transcript levels of BAALC, CD34, MN1, EVl1, and ERG predict inferior OS and EFS. In multivariable survival analysis, CD34, ERG, and CEBPA(DM) remain significant. Using survival tree and regression methodologies, we show that CEBPA(DM), CD34, and IDH2 mutations are capable of separating the intermediate group into 2 AML subgroups with highly distinctive survival characteristics (OS at 60 months: 51.9% vs 14.9%). The integrated statistical approach demonstrates that from the multitude of biomarkers a greatly condensed subset can be selected for improved stratification of intermediate-risk AML.

Deregulated expression of EVI1 defines a poor prognostic subset of MLL-rearranged acute myeloid leukemias: a study of the German-Austrian Acute Myeloid Leukemia Study Group and the Dutch-Belgian-Swiss HOVON/SAKK Cooperative Group

PURPOSE To evaluate the prognostic value of ecotropic viral integration 1 gene (EVI1) overexpression in acute myeloid leukemia (AML) with MLL gene rearrangements. PATIENTS AND METHODS We identified 286 patients with AML with t(11q23) enrolled onto German-Austrian Acute Myeloid Leukemia Study Group and Dutch-Belgian-Swiss Hemato-Oncology Cooperative Group prospective treatment trials. Material was available from 177 AML patients for EVI1 expression analysis. RESULTS We divided 286 MLL-rearranged AMLs into three subgroups: t(9;11)(p22;q23) (44.8%), t(6;11)(q27;q23) (14.7%), and t(v;11q23) (40.5%). EVI1 overexpression (EVI1(+)) was found in 45.8% of all patients with t(11q23), with t(6;11) showing the highest frequency (83.9%), followed by t(9;11) at 40.0%, and t(v;11q23) at 34.8%. Concurrent gene mutations were rare or absent in all three subgroups. Within all t(11q23) AMLs, EVI1(+) was the sole prognostic factor, predicting for inferior overall survival (OS; hazard ratio [HR], 2.06; P = .003), relapse-free survival (HR, 2.28; P = .002), and event-free survival (HR, 1.79; P = .009). EVI1(+) AMLs with t(11q23) in first complete remission (CR) had a significantly better outcome after allogeneic transplantation compared with other consolidation therapies (5-year OS, 54.7% v 0%; Mantel-Byar, P = .0006). EVI1(-) t(9;11) AMLs had lower WBC counts, more commonly FAB M5 morphology, and frequently had additional trisomy 8 (39.6%; P < .001). Among t(9;11) AMLs, EVI1(+) again was the sole independent adverse prognostic factor for survival. CONCLUSION Deregulated EVI1 expression defines poor prognostic subsets among AML with t(11q23) and AML with t(9;11)(p22;q23). Patients with EVI1(+) MLL-rearranged AML seem to benefit from allogeneic transplantation in first CR.

EVI1 is critical for the pathogenesis of a subset of MLL-AF9–rearranged AMLs

The proto-oncogene EVI1 (ecotropic viral integration site-1), located on chromosome band 3q26, is aberrantly expressed in human acute myeloid leukemia (AML) with 3q26 rearrangements. In the current study, we showed, in a large AML cohort carrying 11q23 translocations, that ∼ 43% of all mixed lineage leukemia (MLL)-rearranged leukemias are EVI1(pos). High EVI1 expression occurs in AMLs expressing the MLL-AF6, -AF9, -AF10, -ENL, or -ELL fusion genes. In addition, we present evidence that EVI1(pos) MLL-rearranged AMLs differ molecularly, morphologically, and immunophenotypically from EVI1(neg) MLL-rearranged leukemias. In mouse bone marrow cells transduced with MLL-AF9, we show that MLL-AF9 fusion protein maintains Evi1 expression on transformation of Evi1(pos) HSCs. MLL-AF9 does not activate Evi1 expression in MLL-AF9-transformed granulocyte macrophage progenitors (GMPs) that were initially Evi1(neg). Moreover, shRNA-mediated knockdown of Evi1 in an Evi1(pos) MLL-AF9 mouse model inhibits leukemia growth both in vitro and in vivo, suggesting that Evi1 provides a growth-promoting signal. Using the Evi1(pos) MLL-AF9 mouse leukemia model, we demonstrate increased sensitivity to chemotherapeutic agents on reduction of Evi1 expression. We conclude that EVI1 is a critical player in tumor growth in a subset of MLL-rearranged AMLs.

Mutational spectrum of myeloid malignancies with inv(3)/t(3;3) reveals a predominant involvement of RAS/RTK signaling pathways

Myeloid malignancies bearing chromosomal inv(3)/t(3;3) abnormalities are among the most therapy-resistant leukemias. Deregulated expression of EVI1 is the molecular hallmark of this disease; however, the genome-wide spectrum of cooperating mutations in this disease subset has not been systematically elucidated. Here, we show that 98% of inv(3)/t(3;3) myeloid malignancies harbor mutations in genes activating RAS/receptor tyrosine kinase (RTK) signaling pathways. In addition, hemizygous mutations in GATA2, as well as heterozygous alterations in RUNX1, SF3B1, and genes encoding epigenetic modifiers, frequently co-occur with the inv(3)/t(3;3) aberration. Notably, neither mutational patterns nor gene expression profiles differ across inv(3)/t(3;3) acute myeloid leukemia, chronic myeloid leukemia, and myelodysplastic syndrome cases, suggesting recognition of inv(3)/t(3;3) myeloid malignancies as a single disease entity irrespective of blast count. The high incidence of activating RAS/RTK signaling mutations may provide a target for a rational treatment strategy in this high-risk patient group.

An autonomous CEBPA enhancer specific for myeloid-lineage priming and neutrophilic differentiation

Neutrophilic differentiation is dependent on CCAAT enhancer-binding protein α (C/EBPα), a transcription factor expressed in multiple organs including the bone marrow. Using functional genomic technologies in combination with clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 genome editing and in vivo mouse modeling, we show that CEBPA is located in a 170-kb topological-associated domain that contains 14 potential enhancers. Of these, 1 enhancer located +42 kb from CEBPA is active and engages with the CEBPA promoter in myeloid cells only. Germ line deletion of the homologous enhancer in mice in vivo reduces Cebpa levels exclusively in hematopoietic stem cells (HSCs) and myeloid-primed progenitor cells leading to severe defects in the granulocytic lineage, without affecting any other Cebpa-expressing organ studied. The enhancer-deleted progenitor cells lose their myeloid transcription program and are blocked in differentiation. Deletion of the enhancer also causes loss of HSC maintenance. We conclude that a single +42-kb enhancer is essential for CEBPA expression in myeloid cells only.

Congenital thrombocytopenia in a neonate with an interstitial microdeletion of 3q26.2q26.31.

Interstitial deletions encompassing the 3q26.2 region are rare. Only one case‐report was published this far describing a patient with an interstitial deletion of 3q26.2 (involving the MDS1‐EVI1 complex (MECOM)) and congenital thrombocytopenia. In this report we describe a case of a neonate with congenital thrombocytopenia and a constitutional 4.52 Mb deletion of 3q26.2q26.31 including TERC and the first 2 exons of MECOM, involving MDS1 but not EVI1. The deletion was demonstrated by array‐CGH on lymphocytes. Our report confirms that congenital thrombocytopenia can be due to a constitutional deletion of 3q26.2 involving MECOM. We suggest that in case of unexplained neonatal thrombocytopenia, with even just slight facial dysmorphism, DNA microarray on peripheral blood should be considered early in the diagnostic work‐up.