Abstract
Abstract 3440
The t(10;11)(p13;q14)/PICALM-MLLT10 (CALM-AF10) rearrangement is most frequently associated with T-lineage acute lymphoblastic leukemia/lymphoma (T-ALL), and is rarely observed in AML. The EZH2 gene, located on 7q36.1, is a highly conserved histone H3 lysine 27 methyltransferase that influences stem cell renewal. EZH2 mutations were observed in 10% of patients with myelofibrosis, myelodysplastic/myeloproliferative neoplasms, or chronic myelomonocytic leukemia. In a previous study, we had investigated AML patients for EZH2 deletions using FISH. 6/20 (30%) of cases had been detected to carry a deletion. Additionally, we had screened these 6 cases for molecular mutations in EZH2 (transcript-ID: ENST00000320356) using an amplicon-based deep-sequencing assay, and one of the 6 patients was harboring both an EZH2 deletion and an EZH2 mutation. More interestingly, this double-mutated case was carrying a PICALM-MLLT10 rearrangement. Therefore, in this study, we were interested to investigate an expanded cohort of 13 cases (T-lineage ALL and AML) harboring a PICALM-MLLT10 rearrangement. Our cohort comprised 12 adults and one pediatric patient (7 males, 6 females) and was characterized by a predominant T-cell origin: 11 patients had T-ALL, 1 patient had mixed phenotype T/myeloid acute leukemia, and 1 patient had AML. EZH2 alterations were detected in 3/13 (including the index case). In more detail, the EZH2 mutation carriers were characterized as follows: Patient #1 (male, 26 years, AML) had a splice site mutation in exon 14 with a mutation load of 13% in a cysteine-rich region. Patient #2 (male, 19 years, T-ALL) harbored a missense mutation (Phe136Leu) with a mutation load of 93%. Patient #3 (female, 53 years, T-ALL) showed three concomitant EZH2 missense mutations in exon 5: His120Gln, Tyr124His, and Gly150Arg. The mutation load detected was 17% for each alteration. A fourth patient had a 1459G>A base substitution (corresponding to Ala487Thr) which to our knowledge had not been described before. However, this alteration had to be interpreted as germline as it was still detectable in the remission state. In contrast, in an independent cohort of 12 patients with PICALM-MLLT10 negative T-ALL (7 females, 5 males) analyzed for comparison no EZH2 mutation was detected. Interestingly, in patients #2 and #3, the mutations were located in exon 5 in the region which interacts with the DNMT1, DNMT3A, and DNMT3B DNA methyltransferase genes (D1). Moreover, DNMT3A mutations were recently identified in patients with AML and MDS in association with poor outcomes. Therefore, we additionally performed investigation for DNMT3A mutations in all 13 patients with PICALM-MLLT10 positive leukemias but detected no mutation. To investigate further molecular associations, we analyzed these cases also for RUNX1 mutations and FLT3-ITD, but we did not detect any mutation in these molecular genes. Further, we compared the gene expression profiles of 8 patients with PICALM-MLLT10 positive T-ALL to the profiles of 21 PICALM-MLLT10 negative T-ALL patients. Hierarchical clustering revealed a distinct gene expression signature of the PICALM-MLLT10 positive cases. Significant upregulation was found for HOXA5 and HOXA9 genes. Other differentially overexpressed HOX were HOXA3, A4, A6, A7, A10. Genes with a function for cell differentiation and regulation of apoptosis (ZAK) as well as for signal transduction (AKT3) were significantly underexpressed. Subsequently, we compared the gene expression profiles of 2 EZH2 mutated patients to 6 EZH2 wild-type patients in the PICALM-MLLT10 positive cohort. By hierarchical clustering, both EZH2 mutated cases showed a distinct gene expression signature. Increased expression was observed for genes with a role for the regulation of transcription (ZNF207, KDM5B, or CASZ1) or for intracellular transport (SARB1). In summary, we detected EZH2 mutations in 3/13 cases in this series of PICALM-MLLT10 positive malignancies, comprising mostly T-ALL, but also AML or mixed phenotype acute leukemia. This further emphasizes a cooperative effect of EZH2 mutations with the PICALM-MLLT10 fusion in acute leukemias of different lineages.
Disclosures:
Grossmann: MLL Munich Leukemia Laboratory: Employment. Artusi:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.
Discovery of agents that eradicate leukemia stem cells using an in silico screen of public gene expression data
