Assessment of the Genomic Landscape of Intermediate Risk Acute Myeloid Leukemia As Defined By 2010 ELN Risk Classification
Authors: Panina Ekaterina , Rücker Frank G. , Bullinger Lars , Dolnik Anna , Herzig Julia , Blätte Tamara J. , Gaidzik Verena I. , Paschka Peter , Teleanu Maria-Veronica , Jahn Nikolaus , Schmalbrock Laura K. , Weber Daniela , Kubanek Lena , Kindler Thomas , Westermann Jörg , Lubbert Michael , Salih Helmut R. , Thol Felicitas , Heuser Michael , Ganser Arnold , Döhner Hartmut , Döhner Konstanze 
Published: October 2019

Abstract
Background: In 2010 an international expert working group (European LeukemiaNet, ELN) has published recommendations for the diagnosis and management of acute myeloid leukemia (AML) including a risk stratification by cyto- and molecular genetics, subdividing AML into four risk groups. Emerging data on molecular markers in AML has led to an update of stratification criteria by ELN in 2017 including the recommendation for screening of the high-risk (HR) molecular markers ASXL1, RUNX1, and TP53 that have been shown to confer poor prognosis. The identification of HR markers results in a shift of the prognostic stratification towards adverse risk.
Aim: To investigate the mutational landscape and to assess the prevalence of HR markers in patients (pts) with newly diagnosed AML classified as intermediate-I or -II risk (inter-I/-II) based on the 2010 ELN criteria in a prospective real-world application.
Methods: Using a next-generation targeted sequencing approach [HaloPlex HS (Agilent) on a Miseq (Illumina)], we performed a prospective analysis of all coding regions of 42 target genes including the HR marker ASXL1, RUNX1, and TP53 in 329 newly diagnosed intermediate risk AML pts all enrolled in the AMLSG Biology and Outcome (BiO)-Registry [NCT01252485] of the German-Austrian AML Study Group (AMLSG). Pt genetic features obtained at diagnosis were as follows: inter-I: normal karyotype, n=198 (60%); inter-II: trisomy 8, n=28 (9%), nullisomy Y, n=12 (4%), t(9;11)(p21.3;q23.3), n=7 (2%), others, n=83 (25%); FLT3-internal tandem duplication (FLT3-ITD+), n=75 (23%), mutations (mut) in tyrosine kinase domain of FLT3 (FLT3-TKDmut), n=12 (3.6%), NPM1mut, n=59 (18%); median age was 67 years (range: 21-89 yrs); 60% of pts were male.
Results: Overall, 1253 mut in 315 pts (96%) were identified. Mut in at least one of the HR markers were identified in 50% (n=166) of the pts. Mut in ASXL1 occurred in 32% (105/329), followed by RUNX1 in 26% (87/329), and TP53 in 4% (13/329) of the pts, respectively. Pts with mut in one of the three HR markers showed lower WBC (median 7.63 vs 24.25 109/L, P=.003), lower hemoglobin value (median 8.8 vs 9.3 g/dl, P=.01), lower LDH serum level (median 330 vs 559 U/l, P<.0001) and were associated with older age at diagnosis (median 69.4 vs 66.8 yrs, P=.01). Furthermore, HR markers correlated with male gender (67% HR+ vs 51% HR-, P=.003). Mut in HR markers showed an inverse correlation with FLT3-ITD+ (6% vs 39%, P<.0001), NPM1mut (1% vs 34%, P<.0001), the genotype NPM1mut/FLT3-ITD+ (1% vs 37%, P<.0001), normal karyotype (54% vs 66%, P=.025) and KMT2A rearrangement (0% vs 5%, P=.0035). Further, pts with HR markers exhibited more mut per case (mean 4.6 vs 2.8, P=.0001) and more frequently had co-mut in splicing genes such as SRSF2 (37% vs 14%, P<0.0001) and SF3B1 (3% vs 0%, P=.029) and chromatin-cohesin genes such as EZH2 (9% vs 1%, P=.0009) and STAG2 (21% vs 4%, P<0.0001); here, co-mut with SRSF2 and SF3B1 were mutually exclusive. Pts with HR wildtype showed a significant association with NPM1 (34% vs 1%, P<.0001), DNMT3A (36% vs 20%, P=.0002), WT1 (8% vs 0.6 %, P=.0015) and GATA2 mut (7% vs 1.8%, P=.03) with significant co-mutational patterns such as WT1 with NPM1 (P=.03). Interestingly, we found BRAF mut in 3% (9/329) of the pt cohort with 5/9 pts harboring the p.V600E hotspot mut. Further analyses of survival data in these genotypes are currently ongoing.
Conclusion: In this prospective study we could obtain real world mut data showing a high prevalence of HR marker mut in intermediate-I or -II risk AML pts as defined by 2010 ELN risk classification, thus revealing a significant proportion of pts (50%) harboring HR mut that confer an inferior survival. This high prevalence clearly demonstrates the clinical importance of HR marker screening as recommended by 2017 ELN criteria to identify pts, whose prognosis may be improved by more intensive therapy. Further, we could show that pts with HR marker mut differ significantly from HR wildtype pts with regard to clinical and molecular characteristics indicating a distinct biological subgroup which could potentially benefit from novel treatment approaches beyond conventional chemotherapy. Finally, novel genotypes such as NPM1mut/WT1mut will be further investigated with regard to their prognostic impact.

Disclosures
Bullinger: Bristol-Myers Squibb: Speakers Bureau; Pfizer: Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Research Funding, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer Oncology: Research Funding; Janssen: Speakers Bureau. Paschka:Sunesis: Membership on an entity's Board of Directors or advisory committees; Otsuka: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Speakers Bureau; Astex: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Travel support; Jazz: Speakers Bureau; Bristol-Meyers Squibb: Other: Travel support, Speakers Bureau; Janssen: Other: Travel support; Agios: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees, Travel support; Takeda: Other: Travel support. Lubbert:Celgene: Other: Travel Grant; Janssen: Honoraria, Research Funding; Teva: Other: Study drug. Salih:Several patent applications: Patents & Royalties: e.g. EP3064507A1. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Döhner:Sunesis: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Research Funding; Janssen: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celator: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; Astellas: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Astex Pharmaceuticals: Consultancy, Honoraria; Pfizer: Research Funding; Celator: Consultancy, Honoraria; Sunesis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Agios: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; AROG Pharmaceuticals: Research Funding; AbbVie: Consultancy, Honoraria; Agios: Consultancy, Honoraria; Pfizer: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Bristol Myers Squibb: Research Funding; Seattle Genetics: Consultancy, Honoraria; Astellas: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.