Monitoring of Minimal Residual Disease Using Next-Generation Deep-Sequencing in 460 Acute Myeloid Leukemia Cases identifies RUNX1 Mutated Patients with Resistant Disease

RUNX1 (runt-related transcription factor 1) deregulations constitute a disease-defining aberration in AML. RUNX1 mutations were proposed as clinically useful biomarkers to follow disease progression from MDS to s-AML, as well as to monitor minimal residual disease (MRD). Study design: First, a next-generation amplicon deep-sequencing (NGS) assay was developed and a validation study was performed on genomic DNA obtained from mononuclear cells on a longitudinal series of 116 retrospective samples obtained from 25 patients. These samples were collected between 11/2005 and 6/2010 and were characterized for RUNX1 mutations by DHPLC and Sanger sequencing (conventional methods). In median, 3,346 reads per amplicon were generated and in all cases NGS analyses concordantly detected the mutations known from conventional methods. Furthermore, in 2/25 (8%) cases, NGS detected additional low-level mutations with 0.9% and 3.2% of reads mutated that were not observed by standard Sanger technique. Concerning MRD monitoring, in 7/25 (28%) cases an increasing clone size, i.e. mutations as low as 0.2% - 7.0%, was detectable up to 9 months earlier than by conventional methods. This established assay then was applied to characterize an unselected prospectively collected cohort during the subsequent 12-months routine diagnostics period starting 07/2010. Results: In total, 2,705 NGS RUNX1 mutation analyses were performed on a variety of hematological malignancies. We report on analyses on 460 AML cases at diagnosis including 369 de novo AML, 57 s-AML, and 34 t-AML cases (median age: 68 years; females: 204; males 256). 51% of cases presented with a normal karyotype, 38% harbored non-complex cytogenetic alterations, 10% carried a complex aberrant karyotype, and 1% of patients were characterized by favorable cytogenetics. Overall, 141 RUNX1 mutations were observed in 24.3% (112/460) of cases. At diagnosis, the clone size ranged from 2% to 95% (median: 40%). 82% (92/112) of mutated patients carried one, whereas 18% (20/112) harbored two (n=17) or more (n=3) mutations. The 141 mutations were characterized as follows: 43% (60/141) frame-shift mutations, 34% (49/141) missense, 15% (21/141) nonsense, 5% (7/141) exon-skipping, and 3% (4/141) in-frame insertion/deletion alterations, respectively. The mutations were predominantly located in the RHD domain (54%) or TAD domain (20%). In subsequent serial NGS analyses 31/112 evaluable RUNX1 mutated cases were studied and in 88 individual samples the alterations detected at diagnosis were specifically investigated with high coverage. With a median sampling interval of 50 days for the NGS analyses between 2 and 9 samples per patient were analyzed during the first year of treatment. In this cohort, three categories of patients were detectable: (i) 55% (17/31) of patients responded to therapy and were characterized by a total clearance of the mutated clone at the first time point of follow-up (804-fold median sequencing coverage; sensitivity ∼1:800). (ii) A second group consisted of 10% (3/31) of patients with refractory disease that stayed mutated, but were excluded from further analyses since they underwent transplantation. (iii) The third group comprised 35% (11/31) of patients: None of these patients demonstrated a clone size reduction below 0.7% of reads at the first follow-up analysis (reduction to a median of 21% mutated reads; range 0.7% - 41%). Also, at the second time point (in median 108 days after initial diagnosis), mutated clones were still detectable (reduction to a median of 8% mutated reads; range 4% - 15%). Most of these cases (10/11) had refractory disease as assessed by cytomorphology or molecular analyses. 10/11 cases did harbor a normal karyotype; n=1 with del(7q). Further, 6 of these 11 patients with refractory disease, as defined using NGS, were found to carry RUNX1 double mutations. Finally, in all (3/3) cases with double mutations in the same domain and refractory disease a changing antiparallel distribution of the clone size from initial diagnosis to first follow-up was observed. Conclusions: NGS accurately detects and quantifies RUNX1 mutations in AML with high sensitivity. The technique of deep-sequencing was observed to be superior to current routine methods, in particular during follow-up and in detecting MRD and thus has the potential to enable an individualized monitoring of disease progression and treatment efficacy.

Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Grossmann:MLL Munich Leukemia Laboratory: Employment. Harbich:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.