Next-Generation Deep-Sequencing Enables a Quantitative Monitoring of RUNX1 Mutations in 534 Patients with Myelodysplastic/Myeloproliferative Neoplasms and Myelodysplastic Syndromes

Somatic mutations of key candidate genes have gained interest as biomarkers predicting poor survival in myelodysplastic/myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS). RUNX1 (runt-related transcription factor 1) deregulations constitute such a disease-defining molecular aberration and are usually tested applying a combination of denaturing high-performance liquid chromatography and direct Sanger sequencing. Patient-specific RUNX1 mutations were proposed to represent clinically useful molecular alterations to follow disease progression from MDS to s-AML. Study design: Using genomic DNA obtained from mononuclear cells a next-generation amplicon deep-sequencing (NGS) assay targeting the complete coding region of RUNX1 was developed on a longitudinal series of 116 retrospective samples obtained from 25 patients collected between 11/2005 and 6/2010 (454 Life Sciences, Branford, CT). Subsequently, this assay was applied to characterize an unselected prospectively collected MPN/MDS patient cohort during their course of disease. Results: Here, we present analyses on a cohort of 534 patients (females: 200; males 334). The median age was 72.0 years (25.2–95.7 years). The cohort included 149 chronic myelomonocytic leukemias (CMML), 11 cases with 5q- syndrome, 10 cases with refractory cytopenia with unilineage dysplasia (RCUD), 15 cases with refractory anemia with ring sideroblasts (RARS), 105 cases with refractory cytopenia with multilineage dysplasia (RCMD), 135 cases with refractory anemia with excess blasts-1 (RAEB-1), 87 cases with refractory anemia with excess blasts-2 (RAEB-2), and 22 cases with t-MDS, respectively. In total, 130 RUNX1 mutations were observed in 17.8% (95/534) of these patients. The mutational clone size ranged from 1.7% to 94% and amounted to a median of 31%. In comparison to our data from an AML cohort, i.e. 460 patients at diagnosis with 112 (24.3%) cases mutated, the median clone size was about 10% lower in MPN/MDS. In detail, 74.7% (71/95) of patients harbored one mutation, whereas 25.3% (24/95) of cases harbored two (17.9%; 17/95) or >=3 (7.4%; 7/95) mutations. The 130 RUNX1 mutations were characterized as follows: 29% frame-shift mutations, 42% missense, 14% nonsense, 13% exon-skipping, and 2% in-frame insertion/deletion alterations, respectively. The following codons were recurrently mutated: Arg174 (8/95 patients; 9.4%), Arg177 (6/95 patients; 7.0%), and Arg135 (5/95 patients; 5.3%). The mutations were predominantly located in the RHD domain (55%) and TAD domain (13%) and in cases with 2 or more alterations only 15% (4/24) harbored mutations outside of these regions. In all cases with 3 concomitant mutations both domains were affected (4/4 patients). Further, cases with >1 RUNX1 mutation were more frequently observed in CMML (33.3%; 8/24 mutated), RAEB-1 (17.2%; 5/29 mutated) and RAEB-2 (34.5%, 10/29 mutated) as compared to other disease groups, respectively. In subsequent serial analyses including 56 samples from 22 cases the amplicons carrying the respective known alteration were analyzed with increased coverage for disease status monitoring (in median 833 reads/amplicon were sequenced; leading to a sensitivity of ∼1:800). With a median time span of 2.5 months between the molecular analyses a total of 2 to 4 samples per patient were analyzed. In 5/22 patients, this assay then allowed to monitor the treatment success of allogeneic stem cell transplantation: in 3 cases the mutations known before transplantation became undetectable; in 2 cases the same mutated clones still remained detectable at a level of 0.2% and 23%, respectively. Further, in 17 patients quantitative assessment of mutated RUNX1 read counts was used to monitor stable disease (n=12) or allowed to follow an increasing clone size in 3 patients that progressed into s-AML (39% -> 53% increase; 31% -> 42% increase; 7% -> 37% increase). Summary: Unbiased techniques such as deep-sequencing provide the required diagnostic specificity and sensitivity to enable classification and individualized monitoring of disease progression. We here demonstrate that amplicon-based NGS is a suitable method to accurately detect and quantify the broad spectrum of molecular RUNX1 aberrations with high sensitivity. It is therefore suitable for therapy guidance.

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. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.