The Mutational Patterns Associated with Cytogenetic Subsets of De Novo Acute Myeloid Leukemia (AML): A Study of 1603 Adult Patients (Pts)
Authors: Eisfeld Ann-Kathrin , Mrózek Krzysztof , Kohlschmidt Jessica , Nicolet Deedra , Orwick Shelley , Kroll Karl , Blachly James S. , Carroll Andrew J. , Kolitz Jonathan E. , Powell Bayard L , Wang Eunice S. , Stone Richard M. , de la Chapelle Albert , Byrd John C. , Bloomfield Clara D. 
Published: October 2019

Abstract
The classification of AML is based on the morphology of leukemic blasts, their immunophenotypes, and the presence of specific chromosomal abnormalities and selected gene mutations. It is widely acknowledged that some gene mutations are enriched in certain cytogenetic subsets, in which they may serve as therapeutic targets. We created a comprehensive oncoprint of mutations associated with centrally reviewed cytogenetic findings in 1603 adults (1080 aged <60 years, and 523 aged ≥60 years) with de novo AML (excluding acute promyelocytic leukemia) who were treated on CALGB/ALLIANCE protocols. The pts were classified into 5 cytogenetic subgroups: (1) cytogenetically normal (CN); (2) with complex karyotype (CK); (3) with t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22), i.e., core-binding factor (CBF)-AML; (4) with balanced rearrangements other than t(8;21) and inv(16)/t(16;16); (5) with unbalanced chromosomal abnormalities in non-complex karyotype.
Using a targeted next-generation sequencing panel, we analyzed the mutational status of 80 cancer- and leukemia-associated genes. The mutated genes were assigned to 9 previously described by the Cancer Genome Atlas Research Network (N Engl J Med. 2013;368:2059) functional groups: chromatin remodeling, cohesin complex, kinases, methylation-related, NPM1, RAS pathway, spliceosome, transcription factors, and tumor suppressors. The mutational oncoprint revealed major differences in gene mutation frequencies among the 5 major cytogenetic groups.
CN-AML pts had a broad mutational spectrum, involving all functional groups. Mutations in methylation-related genes (61% of pts), NPM1 (57%), and kinases (46%) dominated the mutation pattern.
TP53 mutations were most common in pts with CK (38%). The second most common mutation, TET2, was detected only half as often (17%). The mutation patterns associated with "typical" (containing abnormalities resulting in loss of chromosome material from 5q, 7q and/or 17p), and "atypical" (without the aforementioned abnormalities) CKs differed. TP53 mutations were present in 52% of pts with typical but only in 5% of pts with atypical CKs (P<0.001). Conversely, the latter pts harbored mutations in another tumor suppressor gene, PHF6, more often (15% vs. 2%, P=0.03). Furthermore, atypical CKs had a broader spectrum of recurrent mutations, with 6 mutations occurring in ≥10% of pts (IDH2, NPM1, PHF6, TET2 and ZRSR2 mutations and FLT3-TKD), compared with only 2 genes, TP53 and TET2, mutated in ≥10% of pts with typical CKs, suggesting major biological differences between typical and atypical CKs.
CBF-AML pts with had few detectable mutations, with a median of 1 (range, 0-5) versus 3 mutations (range, 0-9) detected in the remaining AML pts (P<0.001).
In AML with balanced rearrangements, AML pts with MLL-rearrangements often had mutations in RAS pathway genes, whereas they rarely harbored mutations in other functional groups compared with pts with non-MLL-rearranged balanced translocations [MLL-rearranged vs. non-MLL-rearranged: chromatin remodeling, 9% vs. 26%; methylation-related, 12% vs. 23%; spliceosome, 12% vs. 24%]. Some cytogenetic subgroups with particular recurrent chromosome abnormalities presented with specific molecular features, supporting claims that they represent separate AML entities. For instance, 51% of pts with t(6;11)(q27;q23) harbored KRAS mutations, compared with only 3% of all remaining pts. Among t(9;22)(q34;q11.2)-positive pts, 47% had mutations in RUNX1,and 27% mutations in ZRSR2. Pts with inv(3)(q21q26.2/t(3;3)(q21q26.2) had frequent mutations in SF3B1 and BCOR (both 38%).
Pts with gains or losses of chromosome material in non-complex karyotypes (unbalanced chromosomal abnormalities) were characterized by high mutation frequencies in methylation-related (55%) and spliceosome genes (34%). Strikingly, spliceosome mutations were enriched in pts with chromosome gains, with almost half of these pts harboring one or more mutations. Except for pts with sole +11, who most often harbored U2AF1 mutations (43%), SRSF2 was the most frequently mutated spliceosome gene in pts with unbalanced chromosomal abnormalities.
Our comprehensive depiction of frequencies of single gene mutations and functional groups in recurrent cytogenetic subsets may be useful in guiding mutation testing in AML pts, and result in more focused application of targeted therapy of AML.

Disclosures
Wang: Immunogen: Research Funding; Incyte: Speakers Bureau. Stone:Pfizer: Consultancy; Sunesis Pharmaceuticals: Consultancy; Novartis: Consultancy; Merck: Consultancy; Karyopharm: Consultancy; Agios: Consultancy; Juno Therapeutics: Consultancy; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Consultancy; ONO: Consultancy; Celator: Consultancy; Amgen: Consultancy; Jansen: Consultancy; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Xenetic Biosciences: Consultancy; Roche: Consultancy.