High Resolution Genomic Profiling of Primary “Ultra High Risk” and Refractory Chronic Lymphocytic Leukemia: Results from the CLL2O Trial
Authors: Edelmann Jennifer , Saub Jenny , Ibach Stefan , Holzmann Karlheinz , Tausch Eugen , Bloehdorn Johannes , Mertens Daniel , Zenz Thorsten , Cymbalista Florence , Leblond Veronique , Delmer Alain , Gribben John G. , Hallek Michael , Döhner Hartmut , Stilgenbauer Stephan 
Published: November 2019

Abstract
Introduction:
“Ultra high-risk” chronic lymphocytic leukemia (CLL) can be defined by TP53 loss and/or mutation, and refractory or early relapsing disease after intense chemo(immuno)therapy. Since its pathogenic mechanisms are not fully understood and TP53 alteration is only found in approx. half of the cases, it was our aim to identify novel genetic alterations that might contribute to the pathogenesis of “Ultra high-risk CLL”.
Methods:
We screened 112 samples [36 treatment naïve patients with del(17p), 26 with del(17p) at relapse, and 50 refractory to purine analogue-based therapy] from the CLL2O trial of the GCLLSG by Affymetrix® 6.0 single nucleotide polymorphism (SNP) arrays [N=79 paired to intraindividual reference DNA, N=33 unpaired].
Results:
In total, 663 copy number alterations (CNA) were detected corresponding to a mean of 5.9 CNAs per case [6.0 paired, 5.7 unpaired]. Compared to an unselected CLL cohort at time of first treatment this number was strikingly high [CLL8 trial: 1.8 CNAs per case].
The most frequent aberrations, apart from the well established ones, were: +(8)(q24.21) [15%], del(8p) [14%], del(18p) [14%], del(10)(q24.32) [12%], del(15)(q15.1) [11%], del(6)(q21) [10%], del(3)(p26.1-p25.3) [10%], del(9)(p21.3) [10%], del(3)(p21.31) [9%], +(2)(p16.1-p15) [8%], del(4)(p15.2-p15.1), del(9)(q13 q21) [8%].
The high number of CNAs per case was associated with alterations in DNA damage response (DDR) genes. Patients with both del(17p) and TP53 mutation [N=80] had a mean of 6.4 CNAs, whilst del(17p) cases without TP53 mutation had 4.7 [N=7]. Cases without TP53 alteration [N=20] had a CNA score of 4.4.
Apart from TP53, we found that loss of three other components of the DDR pathway, ATM, RAD18 and SMC5, had impact on the CNA score. In particular, in del(17p) / TP53 mutated cases [N=80], the number of CNAs per case was further increased by a loss of one of the genes: ATM 8.8 CNAs per case [del(11)(q22.3), N=8]; RAD18 9.8 CNAs per case [del(3)(p26.1-p25.3), N=9]; SMC5 10.0 CNAs per case [del(9)(q13 q21), N=7].
Within the group with no TP53 alteration [N=20], cases without ATM, RAD18 or SMC5 loss [N=9] had a mean value of only 2.0 CNAs per case.
Frequent occurrence of discontinuous lesions [N=168 in 71 cases] implicated a high frequency of chromothripsis. Referring to its definition of 10 switches between 2 or more copy number states, chromothripsis was found in 11 cases [10%]. With a cut point of ≥ 5 switches, 37 cases were affected [33%]. Chromothripsis further increased the mean number of CNAs per case [≥ 10 switches: 9.6 vs. 5.5 CNAs per case; ≥5 switches: 8.8 vs. 4.7 CNAs per case].
Interestingly, prior therapy was not associated with an increased number of CNAs in this cohort [7.3 CNAs per case in treatment-naïve cases, 5.4 in refractory and 6.0 in relapsed cases]. The number of prior chemotherapy lines did not have impact on genomic complexity [1 line (N=23): 5.8 CNAs; 2 (N=16): 4.6 CNAs; 3 (N=15): 4.4 CNAs; ≥ 4 (N=22): 5.7 CNAs). This did also apply to the group of del(17)(p13) / TP53 mutated cases without loss of ATM, RAD18 or SMC5 [N=62] [no previous therapy (N=22): 7.4 CNAs; 1 or 2 previous therapy lines (N=20): 4.7 CNAs; ≥ 3 (N=20): 4.6].
Neither genomic complexity (> 3 CNAs per case) nor chromothripsis had any impact on the clinical outcome. We hypothesised whether the recurrent lesions mentioned above are associated with the refractory nature of the disease. Clinical significance tests revealed that only del(9)(p21.3) encompassing the CDKN2A/CDKN2B gene locus and +(8)(q24.21) had significant impact on the clinical outcome [del(9)(p21.3): shorter OS (p=0.038), tendency to shorter PFS (p=0.12); +(8)(q24.21): shorter PFS (p=0.0016), tendency to shorter OS (p=0.064), inferior response (p=0.0079)]. Results for +(8)(q24.21) were in line with an accumulation of cases with 8q gain in the group of refractory [16%, N=8 of 50] and relapsed cases [23%, N=6 of 26]. The minimally amplified region on 8q24.21 [16.6 kb] could be delineated to a region previously linked to the enhancement of MYC expression.
Conclusion:
The majority of “ultra high-risk” CLL showed high genomic complexity. Alteration of genes involved in DNA damage response [TP53, ATM, RAD18, SMC5] and chromothripsis increased the number of aberrations per case, while, somewhat surprisingly, prior therapy did not. Prognostic impact of additional genomic lesions can be suggested for +(8)(q24.21) [MYC] and del(9)(p21.3) [CDKN2A/CDKN2B].

Disclosures
Leblond: Roche: Honoraria, Speakers Bureau. Gribben:Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria. Stilgenbauer:Pharmacyclics, Janssen: Honoraria, Research Funding.