Co-Existing Gene Mutations at Diagnosis and at Relapse in De Novo Acute Myeloid Leukemia with MLL Translocations

Background and purpose: The Mixed-lineage leukemia (MLL) gene located at 11q23 can be fused to a variety of partner genes through chromosomal translocations (MLL-T) in acute leukemia. The co-existence of commonly known mutated genes has not been comprehensively studied in MLL-T AML. We aimed to determine (1) the prevalence and clinical relevance of gene mutations at diagnosis, and (2) the genetic evolution profile at relapse in de novo AML with MLL-T.

Materials and methods: Bone marrow samples from 80 de novo AML with MLL-T patients were analyzed on 20 gene mutations involving activating signaling pathway (class I), transcription pathway (class II), epigenetic regulators (class III), and tumor suppressors, TP53 and WT1 (class IV). MLL-T was all confirmed by FISH analysis. The common MLL fusion transcripts were detected by RT-PCR, including 26 MLLT3, 13 MLLT10, 12 MLLT4, 11 ELL, 3 MLLT1, 3 AFF1, and 2 EPS1. Another 6 infrequent or rare partner genes (one each of MLLT6,CBL, ARHGEF12, TET1, SEPT6, and SEPT9) were identified by cDNA panhandle PCR. In the remaining 4 cases, the partner genes were not identified. Mutational analyses were performed with PCR-based assays followed by direct sequencing. Twenty-two of 26 patients who relapsed had relapse samples for comparative analysis.

Results: The frequencies of gene mutations of class I, II, III, and IV, in 80 de novo MLL-T AML patients were 49.4%, 3.8%, 10.3% and 1.3%, respectively. Together, 53.8% of patients with MLL-T had at least one mutated gene. KRAS (17.7%), FLT3-TKD (11.5%), and NRAS (11.4%) mutations occurred most frequently. Among the epigenetic regulators, 7.9% of patients had DNMT3A, 2.7% TET2, 1.4% ASXL1, and none had IDH1/2 or EZH2 mutations. Three patients had 3 co-existing mutations and 9 patients had two mutations, with 6 of the 12 carrying FLT3-TKD mutations. Gene mutations of transcription pathway and tumor suppressors rarely occurred, only involving NPM1 in two cases and one each for RUNX1 and WT1 mutation. The patients with DNMT3A mutations were significantly associated with older age (P=0.005), FAB AML-M4 (P<0.0001), and higher circulating monocytes (P=0.022). Patients with NRAS mutations had lower circulating monocytes (P<0.0001). KRAS-mutated patients had a higher percentage of marrow leukemic cells (P=0.002). No differences were observed between clinico-hematological features and mutation status of other genetic subtypes. Of the 65 MLL-T AML patients who received standard chemotherapy, 53 achieved complete remission. There were no differences in the outcomes between AML patients with MLLT3-MLL and MLL-MLLT10; patients with both subtypes had a superior event-free survival compared to those with other MLL-fusion transcripts (P=0.036) and a trend towards favorable overall survival (P=0.066). We failed to find significant differences between outcomes and the mutation status of each functional class. All the 3 patients carrying FLT3-ITD had an overall survival less than 6 months. Of the 22 paired diagnosis and relapse samples, 3 patients retained the same NRAS mutations at relapse whereas another one changed from G12D to G12C at relapse. Another 3 relapsed with identical KRAS mutations; while one acquired KRAS mutation at relapse. Two patients had stable DNMT3A mutations at both phases. All the 4 patients who harbored FLT3-TKD mutations at diagnosis lost the mutations at relapse. PTPN11 mutation was found in only one case at diagnosis, while none at relapse. One each had identical mutated gene of CBL and WT1 at both diagnosis and relapse. One each acquired TP53 and WT1 at relapse.

Conclusions: Our results showed that co-existing mutations of RAS and FLT3-TKD were most frequently detected at diagnosis in de novo MLL-T AML. Gene mutations, except FLT3-ITD, had no impact on outcomes in MLL-T AML. FLT3-TKD mutations were absent in all relapse samples while clonal evolution of WT1, TP53 or KRAS mutations might contribute to the relapse of leukemia in a subset of MLL-T AML patients.

Grant support: NHRI-EX93-9011SL, NHRI-EX96-9434SI, NSC95-2314-B-195-001, NSC96-2314-B-195-006-MY3, NSC97-2314-B-182-011-MY3, MMH-E-101-09 and OMRPG3C0021.