COMPLEX GENETIC HETEROGENEITY INFLUENCES PROGNOSIS IN ADULT B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA NEGATIVE FOR RECURRENT FUSION GENES

Background: Although recent high-resolution genome-wide profiling analysis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) samples has contributed to the identification of many novel somatic genetic alterations, a deep molecular characterization of adult ALL is still challenging, especially for those cases lacking recurrent fusion genes. Aims: In order to better molecularly dissect this ALL subgroup, we performed an integrative molecular approach including gene-candidate high-resolution screening and genome-wide profiling analyses. Methods: We retrospectively analyzed 28 newly diagnosed BCR-ABL1- negative BCP-ALL subjects (19 males/9 females; median age 41.5 years; negative for known fusion genes) and 28 BCR-ABL1-positive BCP-ALL subjects as a comparison group. In BCR-ABL1-negative ALL karyotype was normal in 10/28 (36%). Copy number alterations (CNA) of know leukemiarelated genes, such as IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1, and genes within PAR1: CRLF2, CSF2RA, IL3RA have been assessed by the SALSA MLPA kit P335 IKZF1 (MRC Holland). In addition, sequence mutations have been investigated in TP53, CRLF2, JAK2, LEF1, PAX5 and IL7R by nextgeneration deep-sequencing (NGS) (Roche Applied Science; IRON-II study oligonucleotide primer plates). Positivity for newly described BCR-JAK2, PAX5- JAK2, ETV6-ABL1, EBF1-PDGFRB, NUP-ABL1 gene fusions occurring in BCR-ABL1-like ALL (Roberts KG et al., Cancer Cell. 2012) was assessed by PCR amplification and sequencing. Finally, SNP arrays (SNP 6.0, Affymetrix), gene expression profile analyses (GeneChip® Human Transcriptome Array 2.0) and whole exome sequencing (Illumina) were performed to more fully assess genomic complexity. Results: Overall, 76% of BCR-ABL1-negative subjects showed an abnormality of at least one of the analyzed known leukemia genes: 7 (25%) had one, 4 (14%) had two, 6 (21%) had three, and 6 (21%) had four or more alterations. In subjects showing no abnormalities, SNP arrays analysis revealed amplifications of chromosome 1q in 2/6 cases (33%). Deletions of CDKN2A/B were the most frequent (39%) and in 73%, they occurred together with other abnormalities, suggesting that multiple events are needed to induce the full leukemia phenotype. Other common CNA included: deletions of IKZF1 (25%), ETV6 (25%), PAX5 (14%), EBF1 (11%), PAR1 region (11%) and RB1 (7%). NGS showed mutations of JAK2 and CRLF2 in 7% (R683S/G) and 4% (F232C), respectively. No positivity for newly described fusion genes activating tyrosine kinase was confirmed. Importantly, subjects with no abnormalities showed better survival rates compared to those with one or more molecular alterations (p<0.01). The BCR-ABL1-positive subgroup shared the same CNA of BCRABL1- negative cases, such as deletions of IKZF1 (71%), CDKN2A/B (21%), PAX5 (14%), BTG1 (11%), EBF1 (11%), and ETV6 (4%), but they did not show mutations in the analyzed genes. Summary and Conclusion: BCP-ALL lacking recurrent fusion genes is a highly heterogeneous and complex disease. Current diagnostic procedures need to be revised to improve risk assessment and to guide therapeutic decisions.