RUNX1 Deletions Are a Novel Mechanism of Loss of Function in AML and Are Associated with Adverse Cytogenetics.
Authors: Haferlach Claudia , Grossmann Vera , Zenger Melanie , Kern Wolfgang , Haferlach Torsten , Kohlmann Alexander , Schnittger Susanne 
Published: October 2019

Abstract
Abstract 2517

Background:
RUNX1 is a crucial hematopoietic transcription factor located on the long arm of chromosome 21 (21q22). Three types of acquired alterations of the RUNX1 gene have been described in AML so far: 1. translocations leading to fusion genes such as t(8;21)(q22;q22)/RUNX1-RUNX1T1 2. molecular mutations, which usually lead to loss of normal RUNX1 function, and 3. amplifications, which are predominately found in AML with complex karyotype. Another possible mechanism that causes loss of function is a partial or complete deletion of the gene.

Aim:
To clarify whether RUNX1 is also affected by partial or complete deletions in AML.

Patients and Methods:
We screened 623 AML patients (pts) for RUNX1 deletions (del) by interphase fluorescence in situ hybridization using probes spanning the complete RUNX1 gene (MetaSystems, Altlussheim, Germany). The cohort comprised 472 de novo, 85 secondary (s-AML), and 66 therapy-related AML (t-AML). Median age was 67.3 yrs (range: 18 to 91.5 yrs). For all pts cytogenetics was available and categorized according to refined MRC criteria (Grimwade et al. Blood 2010). Cytogenetics were favorable in 164, intermediate in 210 and adverse in 249 pts, respectively. RUNX1 mutation analysis was performed in 252 cases. In addition, 19 pts with RUNX1 del were analyzed by genomic arrays (Human CGH 12×270K Whole-Genome Tiling Array, Roche NimbleGen, Madison, WI (n=12); SNP 6.0 arrays, Affymetrix, Santa Clara, CA (n=7)).

Results:

Frequency and clinical characteristics:
In 57/623 (9.1%) cases deletions affecting RUNX1 were identified. The frequency of RUNX1 del did not vary significantly between de novo (8.9%), s-AML (11.8%) and t-AML (7.6%). However, RUNX1 del were more frequent in pts with adverse cytogenetics (18.1%) compared to intermediate (5.7%) or favorable cytogenetics (0%, p<0.0001). In contrast, RUNX1 mutations (mut) were more frequent in intermediate (26.1%) as compared to favorable (0%) and unfavorable cytogenetics (10.5%, p=0.003). Pts harboring RUNX1 del were significantly older than those with 2 RUNX1 copies (mean: 70.0 vs 61.6 yrs, p<0.0001) and showed lower WBC count (mean: 13,225 vs 21,419/μl, p=0.010), while no difference was observed with respect to hemoglobin level and platelet count.

Cytogenetics:
38/57 (66.7%) pts with RUNX1 del harbored a complex karyotype, one case showed a normal karyotype while a variety of different abnormalities were found in the remaining 18 pts.

Type of cytogenetic alteration:
In 5 cases one RUNX1 allele was lost due to monosomy 21. In two cases a deletion of the long arm of chromosome 21 was cytogenetically visible. 29 pts showed derivative chromosomes 21 resulting from unbalanced translocations or duplications of the long arm of chromosome 21. Ring chromosomes 21 were found in 3 cases. In 7 pts a translocation involving 21q22 was identified. 11 cases with a RUNX1 del showed cytogenetically normal chromosomes 21. Remarkably, 6 of these 11 pts showed a trisomy 21.
RUNX1 mutations: 36 pts with RUNX1 del were also evaluated for RUNX1 mut. In 7 pts (19.4%) a RUNX1 mut in the remaining allele was detected. Thus, the mutation frequency did not differ from pts without RUNX1 del (40/216, 18.5%).

Genomic array data:
The size of the deletion on chromosome 21 varied between 258 kb and 11,792 kb (median: 1,987 kb). In one case a homozygous RUNX1 deletion was detected. Based on array data pts with RUNX1 del were subdivided into two groups: The first group comprised 7 pts with a non-complex karyotype and an interstitial deletion on chromosome 21 encompassing RUNX1. Secondly, in 12 pts with RUNX1 del and a complex karyotype array data revealed between 2–16 changes in copy number state on chromosome 21. Interestingly, in 4/12 (30%) cases the ERG gene, located 2.6 Mb telomeric to RUNX1, was amplified.

Conclusions:
1. Partial or complete deletions of RUNX1 are frequent recurrent genetic events in AML. 2. They are associated with adverse cytogenetics, lower WBC count and are more frequent in elderly patients. 3. Loss of one RUNX1 copy results either from clear-cut interstitial deletions on chromosome 21 or occurs based on highly rearranged chromosomes 21 showing several changes in copy number states accompanied by gains and losses of several regions on chromosome 21. Whether the pathogenetic impact of RUNX1 del is comparable to RUNX1 mut and if they have prognostic impact independent of cytogenetics remains to be studied.

Disclosures:
Haferlach: MLL Munich Leukemia Laboratory: Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment. Zenger:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership.