A Comprehensive Deep-Sequencing Study of Blast Crisis Chronic Myeloid Leukemia (CML) Reveals New Insights Into Molecular Heterogeneity and Detects Mutations In 12 Different Genes In 82.5% of Cases
Authors: Grossmann Vera , Eder Christiane , Schindela Sonja , Kohlmann Alexander , Wille Sandra , Schnittger Susanne , Kern Wolfgang , Müller Martin C. , Hochhaus Andreas , Haferlach Torsten , Haferlach Claudia 
Published: October 2019

Abstract
Abstract 884
Blast crisis is the terminal phase of chronic myeloid leukemia (CML) with a short median survival of approximately six months. At present, little is known about molecular mechanisms underlying disease progression. We hypothesized that mutations occurring in other myeloid and lymphatic malignancies are acquired during disease progression from chronic phase to blast crisis. Here, in total 40 blast crisis CML cases (n=25 myeloid, n=10 lymphoid, n=5 not specified) were analyzed, all diagnosed between 9/2005 and 7/2009. First, all cases were investigated for IKZF1 deletions by PCR using specific primer pairs for the common intragenic deletions spanning from exon 2–7, or exon 4–7 as published by Iacobucci et al. (Blood, 114:2159-67, 2009). In total, in 17.5% (7/40) of cases intragenic IKZF1 deletions were detected. Secondly, next-generation deep-sequencing (454 Life Sciences, Branford, CT) was used to investigate 11 candidate genes in all 40 patients for a broad molecular screening. Known hotspot regions were sequenced for CBL (exons 8 and 9), NRAS (exons 2 and 3), KRAS (exons 2 and 3), IDH1 (exon 4), IDH2 (exon 4), and NPM1 (exon 12). Complete coding regions were analyzed for RUNX1, TET2, WT1, and TP53. To perform this comprehensive study, amplicon-based deep-sequencing was applied using the small volume Titanium chemistry assay. To cope with the great number of amplicons, in total 59, 48.48 Access Arrays were applied (Fluidigm, South San Francisco, CA), amplifying and barcode-tagging 48 amplicons across 48 samples in one single array (2,304 reactions). In median, 430 reads per amplicon were obtained, thus yielding sufficient coverage for detection of mutations with high sensitivity. Further, ASXL1 exon 12 aberrations were investigated by Sanger sequencing. In summary, after excluding known polymorphisms and silent mutations in 33/40 patients 53 mutations were identified: RUNX1 (16/40; 40.0%), ASXL1 (12/40; 30.0%), WT1 (6/40; 15.0%), NRAS (2/40; 5.0%), KRAS (2/40; 5.0%), TET2 (3/40; 7.5%), CBL (1/40; 2.5%), TP53 (1/40; 2.5%), IDH1 (3/40; 7.5%), IDH2 (0/40), and NPM1 (0/40). Thus, 82.5% of blast crisis CML patients harbored at least one molecular aberration. In median, one affected gene per patient was observed (range 1–5). In detail, RUNX1 was associated with additional mutations in other genes, i.e. 9/16 cases were harboring additional mutations in combination with RUNX1. Similarly, in 8/12 patients with ASXL1 mutations additional aberrations were detected. With respect to myeloid or lymphoid features ASXL1 mutations (n=11) were exclusively observed in patients with myeloid blast crisis (n=1 not specified), in contrast 5/7 IKZF1 cases were detected in cases with lymphoid features (n=1 myeloid, n=1 not specified). Interestingly, besides IKZF1 (n=5) and RUNX1 (n=3) alterations there was no other mutated gene occurring in lymphoid blast crisis CML. In addition, no aberration was detected in NPM1, and in contrast to published data, in our cohort only one patient harbored a mutation in TP53. Moreover, for 8 patients with mutations in IKZF1 (n=3), RUNX1 (n=3), ASXL1 (n=1), WT1 (n=2), and IDH1 (n=2), matched DNA from the initial diagnosis at chronic state was available. In these specimens respective IKZF1 deletions, RUNX1, and ASXL1 mutations were not detectable indicating that IKZF1, RUNX1, and ASXL1 mutations had been developed during disease progression and act as driver mutations in these cases. WT1 and IDH1 mutations occurred at first diagnosis in one case each, indicating these genes would constitute passenger mutations. In conclusion, this comprehensive study on 12 molecular markers enabled to characterize for the first time that 82.5% of blast crisis CML cases harbor specific molecular mutations. IKZF1 and RUNX1 alterations were identified as important markers of disease progression from chronic state to blast crisis. Moreover, technically, a novel combination of a high-throughput sample preparation assay for targeted PCR-based next-generation deep-sequencing was developed and allowed to broaden our molecular understanding in blast crisis CML.

Disclosures:
Grossmann: MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Schindela:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Wille:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership, Research Funding.