PB1654 COMPREHENSIVE AND SEQUENTIAL GENETIC ANALYSIS OF B‐CELL ALL REVEALS AN ARRAY OF CHANGES WITH A HIGH INCIDENCE OF TP53 MUTATIONS AND IKZF1 DELETIONS
Authors: Montaño A. , Forero‐Castro M. , Robledo C. , Coca A. García‐de , Fuster J. L. , de las Heras N. , Olivier C. , Hernández‐Sánchez M. , Corchete‐Sánchez L. A. , Martín‐Izquierdo M. , Riesco S. , González T. , Ribera J. , Ribera J. M. , Benito R. , Hernández‐Rivas J.M. 
Published: June 2019

Background:

B‐Cell Precursor Acute Lymphoblastic Leukemia (BCP‐ALL) is a disease with specific secondary genetic alterations such as mutations (eg. TP53mutations) and copy number alterations (CNAs) (eg.IKZF1deletions) some of which have been associated with drug resistance, treatment failure and relapse. The clonal basis of relapse disease is complex and not yet fully understood.

Aims:

To perform an integrated genomic analysisby combiningnext‐generation sequencing (NGS), array comparative genomic hybridization (a‐CGH), and multiplex ligation‐dependent probe amplification (MLPA) in order to identify the clonal shifts related to BCP‐ALL progression.

Methods:

NGS, a‐CGH, and MLPA were carried out in matched diagnosis‐relapse samples from thirteen BCP‐ALL patients (four children and nine adults). The mutational status of TP53(E4‐E11), JAK2(E12‐E16), PAX5(E2‐E3),LEF1(E2‐E3), CRLF2(E6) and IL7R(E5) genes was performed by applying 454 NGS technology (454 Life Sciences, USA). All samples were tested on an array‐CGH 12X135K platform (Roche NimbleGen, USA). In addition, MLPA was performed using SALSA MLPA P335‐B1 ALL‐IKZF1‐probemix (MRC‐Holland, Netherlands) which contains probes for IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1 genes, as well as genes from the PAR1 region (CRLF2, CSF2RA, IL3RAand P2RY8).

Results:

The median age of patients was 31 years (range 4–80). All patients showed at least one genetic alteration at diagnosis and relapse. A significant increase in the frequency of CNAs at the time of relapse (median, 47 alterations per sample) compared to diagnosis (median, 6 alterations) (p = 0.019) was observed. By aCGH, the most recurrent broad and focal CNAs observed at diagnosis and/or relapse were del(7p) (77%) and del(9p) (62%) following of dup(X)(31%), dup(7q)(31%), del(13q)(23%), dup(1q)(23%), dup(21)(15%), del(12p)(15%), and del(17p)(15%). Our integrative MLPA‐aCGH analysis showed that the percentages of deleted genes in the following paired diagnosis/relapse BCP‐ALL samples were:IKZF1(54% vs. 62%), CDKN2A/B(54% vs. 23%), PAX5(38% vs. 23%), EBF1(23% vs. 15%), BTG1(23% vs. 23%), ETV6(15% vs. 15%), RB1(8% vs. 15%) and PAR1(15% vs. 8%). All patients exhibited heterogeneous changes in the pattern of CNAs from diagnosis to relapse: 8% of patients acquired only new genetic lesions at relapse, 38% of patients acquired new lesions and lost lesions present at diagnosis, and the remaining 54% of the patients simultaneously retained, lost and acquired new lesions at relapse. 71% of patients with IKZF1del at diagnosis retained this alteration at relapse, while only 42.9% of patients with CDKN2A/Bdel and/or PAX5del retained these deletions at relapse. Finally, del(17p) was also identified in two patients at diagnosis, in one of which was retained at relapse.NGS analysis revealed the presence of 6 mutations (5 in TP53gene and 1 in PAX5gene) in 4/13(31%) patients at diagnosis and/or relapse (3 missense mutations, 1 splicing site mutation and 2 deletion‐insertions).Two TP53mutations were only detected at relapse whereas the remaining three showed an increase of their mutational burden at relapse (53% to 71%, 11% to 21% and 3.5% to 26% respectively). One pediatric patient with low risk disease acquired a TP53mut at relapse.

Summary/Conclusion:

The combination of NGS, a‐CGH, and MLPA analysis allowed to identify clonal patterns of genetic evolution associated with relapse BCP‐ALL. The alterations on IKZF1(7p), TP53(17p) and CDKN2A/Bgenes were mostly involved in paired diagnostic and relapse samples, being more frequent at relapse.