Genomic Heterogeneity In Pediatric Acute Lymphoblastic Leukemia

Pediatric B cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) is characterized by recurrent chromosomal aneuploidies and translocations, which are important for diagnosis and risk stratification. Classical cytogenetic and FISH are routinely used for the diagnosis of most of these alterations, particularly those involving balanced translocations, and numerical changes. Recently, genome wide analysis, such as aCGH and SNP technologies have identified copy number alterations (CNA) in genes involved in lymphoid differentiation: IKZF1, PAX5, EBF1 BTG1; and in cell cycle control: CDNK2A/B, RB1 and ETV6. Multiplex ligation–dependent probe amplification (MLPA) technique has been used for the identification of those focal genetic aberrations in a single assay but it cannot reliably detect aberrations present in less than 50% of cells. Cytoscan HD SNP array (Affymetrix) can overcome this limitation and is the only array which enables independent confirmation of CNA with SNP allelic information. Thus minor genetic subclones missed by other methods, can be detected.

The aim of this work was to compare between SNP array and MLPA platforms in a routine diagnostic setting.

Samples of 39 pediatric BCP-ALL patients well characterized by classical cytogenetic and FISH at diagnosis, were studied by MLPA P335 (MRCHolland) and SNP array (Affymetrix). In total 273 genomic loci were evaluated, 13 (5%) were not informative, of them 7 were not covered by the MLPA kit and 6 gave not evaluable MLPA results. 231/260 (89%) of the loci showed concordance, 39/260 (15%) with CNA and 192/260 (74%) normal. A total of 68 CNA were detected, 29 were discordant and 39 concordant.

IKZF1 deletion was identified by both methods in 5 cases (13%). However, a deletion was identified in 3 additional samples, only by cytoscan. In these cases, the deletion was present in a minor subpopulation (in 45% or less of the cells), while the main cytogenetic aberration was present in the majority of cells (60%-90%).

We conclude that although MLPA is a useful technique, it is not sensitive enough to detect small populations. In this small cohort, 3 IKZF1 deletions were missed since they were present only in a minor subclone. Interestingly, one of these patients relapsed, and the relapse clone harbored the IKZF1 deletion.