PS1137 COMPLEX STRUCTURAL VARIANTS LEAD TO GENE INACTIVATION RATHER THAN EXPRESSION OF DE NOVO FUSION GENES IN CHRONIC LYMPHOCYTIC LEUKEMIA (2019)

Background: Complex structural variants (cSVs) represented by chromothripsis have recently been identified in various tumor types, including chronic lymphocytic leukemia (CLL). They lead to extensive chromosomal rearrangements due to chromosome shattering and consequent random re‐joining of scattered fragments. Substantial parts of DNA are typically lost, and other chromosomal parts potentially containing oncogenes are amplified. Importantly, albeit not common in CLL, de novo fusion gene variants with novel functions can be formed. Overall, chromothripsis is associated with unfavorable prognosis, which can be in part attributed to the frequent co‐occurrence with TP53 gene defects. Aims: To perform a detailed study of cSVs in CLL and to identify putative de novo fusion genes and their possible functions. Methods: cSVs were screened among TP53 ‐aberrant CLL cases using CytoScanHD arrays (ThermoFisher Scientific). In parallel, conventional cytogenetics, mFISH, iFISH and chromosome painting methods were used to supplement array findings. Transcriptome analysis was performed using TruSeq Stranded Total RNA Library Prep Kit on HiSeq 2000–1T (Illumina). Obtained data were analyzed with in‐house bioinformatic pipelines developed for specific detection of fusion gene variants in transcriptomic data. Results: cSVs suggestive of chromothripsis were screened among 93 CLL patients (111 samples in total) with various types of wild‐type TP53 gene inactivation using CytoScan assay. Out of them, cSVs were identified in 20 patients (21.5%; 26 samples) and were associated with defect of both TP53 alleles (p = 0.0006). In 13/20 patients we predominantly observed clustered deletions; in the remaining 7/20 patients, deletions and also amplifications were present in localized regions. The number of breakpoints ranged from 30 to 154 per case (median 54); 10 to 65 coding genes (median 24) were present in the breakpoints. cSVs were clustered in one chromosome in 9/20 cases, whereas in the rest they were detected in ≥ 2 chromosomes. Comparing the array results to cytogenetic findings, typically a higher number of chromosomes (up to 8) was involved in cSVs within the genome. It can be explained by balanced abnormalities undetectable by the array. Several chromosomes were affected recurrently with the most frequent being chr6 and chr13 in 8 cases, and chr8 and chr9 in 6 cases, respectively. Ten cases with cSVs were subjected to the transcriptome analysis. Although a high number of genomic breakpoints was detected in these cases (555 breakpoints in total affecting 236 genes), they mostly did not lead to formation of expressed fusion genes. Altogether 15 fusion gene transcripts were noted in 8/10 cases (range 1 to 4 per case); 10 fusions were validated by PCR. They mostly consisted of two fusion gene partners, however, in 2 instances, fusions of 3 and 5 genes were detected, respectively. Using online tools, we tested whether the 10 validated fusion transcripts could be translated in proteins and found out that 4 of them encoded abnormal proteins containing domains with predicted functions. Summary/Conclusion: Using our comprehensive approach we showed that chromothripsis affects over a fifth of CLL cases with aberrant TP53 and mostly leads to inactivation of genes present in genomic breakpoints. De novo fusion transcripts supposedly encoding abnormal proteins are expressed only to a limited extent. Supported by CEITEC2020 LQ1601, MZCR‐RVO 65269705, MUNI/A/1105/2018, MZCR‐AZV 16–34272A, GACR 19–11299S, H2020 116026 and 825872.