AN EXTENSIVE MOLECULAR CYTOGENETIC CHARACTERIZATION IN HIGH-RISK CHRONIC LYMPHOCYTIC LEUKEMIA IDENTIFIES KARYOTYPE ABERRATIONS AND TP53 DISRUPTION AS PREDICTORS OF OUTCOME AND CHEMOREFRACTORINESS

Background:Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, running an indolent course in some patients and a clinically aggressive course in others. Risk assessment is important in clinical practice and prediction of outcome and response to treatment is very useful in an era in which several chemoimmunotherapy combinations and effective mechanism-driven treat- ments are available. Aims:We investigated whether an extended genetic characterization including mutational screening by next generation sequencing (NGS) and karyotype analysis could allow for a refinement of our capability to predict outcome in newly diagnosed CLL patients with high-risk features, as defined by the pres- ence of unmutated IGHV gene and/or 11q22/17p13 deletion by FISH and/or TP53 mutations. Methods:101 patients were included in this study. TP53 disruption was defined by the presence of 17p13 deletion by FISH and/or TP53 mutation by NGS. Cytogenetic analysis was performed using CpG-oligonucleotide DSP30. Each patient was categorized according to the following classification: favorable group (isolated 13q14 deletion or normal karyotype), unfavorable group (dele- tions of 11q22 or 17p13, or complex karyotype, ie, at least 3 chromosome aber- rations); intermediate group (all other karyotypic abnormalities). A cut-off of 98% homology to the germline sequence to discriminate between IGHV mutat- ed and unmutated cases. Mutational screening was performed with Ion Torrent PGM NGS platform on 20 CLL-related genes by using a 5% cut off. Results:Cytogenetic analysis showed favorable findings in 30 patients, unfa- vorable in 34 cases and intermediate in 36 cases. A complex karyotype was present in 21 patients. By NGS, 95 somatic mutations were observed in 56/101 (55.4%) cases; 80 missense mutations, 5 nonsense mutations and 10 frameshit deletions. 16 cases (15.8%) showed mutations in the TP53 gene, 11 (10.9%) in the NOTCH1 gene, 11 (10.9%) in the SF3B1 gene, 8 (7.9%) in the ATM gene, 5 (4.9%) in the BIRC3 gene, 5 (4.9%) in the PTEN gene, 4 (4.0%) in the MYD88 gene, 4 (4.0%) in the BRAF gene, 4 (4.0%) in the POT1 gene, and 18 (17.8%) cases in the remaining 11 genes. 26/56 (46.4%) mutated patients pre- sented two or more mutations. The presence of mutations was associated with unmutated IGHV status (p=0.040) and the complex karyotype (p=0.047). TP53 disruption correlated with the presence of ≥2 mutations by NGS (p=0.001) and a complex karyotype (p=0.012). By multivariate analysis an advanced Binet stage (p<0.001) and an unfavorable karyotype (p=0.001) predicted for a shorter time to first treatment (TTFT), while TP53 disruption (p=0.019) and the unfa- vorable karyotype (p=0.028) predicted for a worse overall survival (OS). A short- er time to chemorefractoriness (TTCR) was associated with TP53 disruption (p=0.001) and unfavorable karyotype (p=0.025). Patients with both unfavorable karyotype and TP53 disruption presented a dismal outcome (median OS and TTCR of 28.7 and 15.0 months respectively). Summary/Conclusions: A comprehensive analysis of chromosomal aberra- tions and gene somatic mutations in high-risk CLL showed that the cytogenetic profile was independently associated with a shorter TTFT, OS and TTCR. Since karyotyping using novel mitogens may contribute to the refinement of prognosis in high-risk CLL patients, the introduction of this technique in future CLL trials seems warranted to identify those patients that could be ideal candidates for consolidation treatment or novel treatment combinations.