Hidden DNA Copy Number Alterations and Mutations in IKZF1, TP53, CRLF2 and JAK2 Genes Are Associated with a Poor Prognosis in B-Progenitor Acute Lymphoblastic Leukemia
Authors: Hernández-Rivas Jesús María , Forero Maribel , Robledo Cristina , Benito Rocio , Hernández María , Abáigar María , Rodríguez Ana , Corchete Luis A. , Martín Ana , Riesco-Riesco Susana , García-de-Coca A , Fuster José L. , De-las-Heras Natalia , Rodríguez Juan N. , De-la-Fuente I , Ribera Josep-Maria , Ribera Jordi , Labrador Jorge , Alonso Jose M. , García Juan L. , Del CaÃ±izo Consuelo 
Published: October 2019

Abstract
Background: In B-progenitor acute lymphoblastic leukemia (B-ALL) the identification of additional genetic alterations associated with treatment failure is still a challenge.
Aims: 1.To identify genomic gains and losses in B-ALL at the time of diagnosis and to correlate these abnormalities with the genetic characteristics and the patients outcome. 2.To assess the prevalence and prognostic impact of genetic lesions in IKZF1, TP53, CRLF2, IL7R, PAX5, JAK2 and LEF1 genes in B-ALL.
Methods: A total of 215 B-ALL patients were eligible for this study. 115 (53.5%) had less than &lt;18 years. In all cases oligonucleotide microarrays (aCGH) were carried out. For genetic gain and losses analysis, the NimbleGen CGH 12x135K array (Roche) was used. The data analysis was performed with GISTIC and aCGHweb software. In 118 of these cases (65 children and 53 adults), an integrative analysis of aCGH and NGS was performed to identify genetic lesions in genes associated with B-ALL: JAK2 (Exon 12-Exon 16), PAX5 (E2-E3), LEF1 (E2-E3), CRLF2 (E6), IL7R (E5), IKZF1 (E2-E8) and TP53 (E4-E11). The 454 GS Junior system (Roche) was used. The variant analysis was performed using Amplicon Variant Analyzer (AVA-Roche 454) and Sequence Pilot (JSI Medical Systems) software.
Results: DNA copy number aberrations (CNAs) were observed in 96.5% of cases. Gains on chromosomes 4 (10%), 6 (10%), 10 (12%), 14 (13%), 21 (31%) and X (18%) were more common in children, whereas losses in 7p (13%) and 9p21 (24%) were frequent in adults. In children, gains on chromosomes 6, 10, 14q, 17, 18, 21 and X were associated with longer overall survival (OS) while loss on 7p and 17p were associated with shorter OS in adults (Table 1).
The integrative analysis of aCGH and NGS showed that 60% of patients carried at least one alteration (deletion and/or mutation) in the seven genes analyzed. Focal deletions were common in IKZF1, CRLF2, PAX5, and LEF1 genes while broad deletions were more frequent in TP53, JAK2 and IL7R. Forty-one percent of patients harbored IKZF1 alterations (IKZF1+), 22.9% in PAX5, 18.6% in JAK2 and 11% had TP53 or CRLF2 alterations. LEF1 and IL7R genomic lesions were only present in 4.2% and 2.5% of the B-ALL, respectively. JAK2 and CRLF2 mutations were associated (p=0.01). Moreover abnormalities in IKZF1+ were associated with alterations in JAK2 (p=0.004), TP53 (p=0.04) and PAX5 (p=0.03).
The presence of alterations was most frequent in high-risk (HR) B-ALL (38% vs 18%, p=0.02), although 8% low-risk (LR) childhood patients showed genetic alterations. Fourteen B-ALL patients carried mutations in TP53, CRLF2 and/or JAK2 genes: all of them were Ph-, and 11 were classified as HR. Of note, 3 cases were children. In the HR group the presence of mutation in TP53, CRLF2 and/or JAK2 was related to a shorter OS (3-year OS: 56% vs. 36%; P=0.034) and event-free survival (3-year EFS: 56% vs. 11%; P&lt;0.002) (Figure 1), due to an increased cumulative incidence of relapse (3-year CIR: 38% vs. 88%; P&lt;0.004).
The presence of IKZF1 alterations stratified HR-Ph- B-ALL cases. Thus HR-Ph- patients with normal IKZF1 were associated with longer EFS compared to HR-Ph- B-ALL IKZF1+ (3-year EFS: 60% vs. 33%; P=0.005) (Figure 2). The presence of IKZF1 deletion was associated with shorter EFS (89% vs. 67%, P= 0.04) and increased CIR (3-year CIR: 12% vs. 33%; P=0.05) in children.
Summary/Conclusions: CNAs are frequent in B-ALL and are associated with genetic subtype, age and overall survival. The integrative analysis by aCGH and NGS techniques demonstrated that alterations in IKZF1 gene and mutations in TP53, CRLF2 and/or JAK2 genes could stratify high-risk Ph- B-ALL patients.
Subvention:FP7/2007-13, Nº306242-NGS-PTL; HUS272U13, JCyL,Consejería de Educación; BIO/SA31/13 Gerencia Regional de Salud, SACYL, Spain
Table 1. DNA copy number aberrations (CNAs) associated with overall survival in 215 B-ALL patients Association CNA n Median (months) P value Short overall survival 7p-ChildrenAdults 19613 9615 0.05 17p-ChildrenAdults 1257 694 0.029 +19ChildrenAdults 351421 Not reached10 0.001 22q+ChildrenAdults 261214 Not reached7 &lt;0.0001 Longoverall survival +6ChildrenAdults 26197 Not reached7 &lt;0.0001 +10ChildrenAdults 25196 Not reached7 0.001 +14qChildrenAdults 26188 Not reached4 &lt;0.0001 +17ChildrenAdults 24177 Not reached60 0.012 +18ChildrenAdults 25187 Not reachedNot reached &lt;0.0001 21q+ChildrenAdults 473116 Not reached8 &lt;0.0001 XChildrenAdults 522725 Not reached9 &lt;0.0001
Figure 1 Figure 1.
Figure 2 Figure 2.

Disclosures
No relevant conflicts of interest to declare.