SUMMARY The majority of paediatric Burkitt lymphoma (pBL) patients that relapse will die of disease, but markers for this high-risk subset are unknown. MYC translocations characterize pBL, but additional genetic changes may relate to prognosis and serve as potential biomarkers. We utilized a molecular inversion probe single nucleotide polymorphism assay to perform high resolution, genome-wide copy number analysis on archival formalin-fixed, paraffin-embedded pBL and germline tissues. We identified copy number abnormalities (CNAs) in 18/28 patients (64%) with a total of 62 CNAs that included 32 gains and 30 copy number losses. We identified 7 recurrent CNAs including 1q gain (7/28, 25%), 13q gain (3/28, 11%), and 17p loss (4/28, 14%). The minimum common amplified region on 13q was at 13q31 and included the MIR17HG (MIR17-92) locus. Samples with this gain had higher levels of MIR17 RNA and showed a tendency for early relapse. Tumour-specific uniparental disomy was identified in 32% of cases and usually was recurrent. These results demonstrate that high-resolution copy number analysis can be performed on archival lymphoma tissue specimens, which has significance for the study of rare diseases.
Our study demonstrates that the copy number profile of B-LBL is distinct from B-ALL, suggesting possible differences in pathogenesis between these closely related diseases.
Summary The TP53 (p53) pathway can be inhibited by TP53 mutation or deletion or by MDM2 overexpression. Both occur in Burkitt lymphoma (BL), but many cases lack either abnormality. Expression patterns of the TP53 inhibitor MDM4 have not been reported in BL, and increased MDM4 could deregulate the TP53 pathway in cases without TP53 or MDM2 abnormalities. We investigated TP53 pathway disruption in paediatric BL patient samples (n = 30) by studying MDM4, MDM2, and CDKN1A (p21) protein and mRNA expression; TP53 mutations; TP53 protein expression; and gene copy number abnormalities. MDM4 protein was expressed in 30/30 tumours, and MDM2 protein was weakly expressed in 7/30 (23%). All cases were negative for CDKN1A protein, and CDKN1A mRNA levels were decreased. TP53 mutations were detected in 5/28 (18%) cases and confirmed by sequencing. TP53 protein was expressed in 15/30 (50%) cases, including 7/8 with TP53 genetic alterations. MDM2 protein and mRNA expression levels did not correlate with lack of TP53 genetic changes or TP53 protein expression; however, there was an inverse relationship between detectable TP53 protein expression and MDM4 copy number gains and mRNA expression. The TP53 pathway is deregulated in paediatric BL cases, and increased MDM4 expression may be the primary mechanism in some cases.
Background Burkitt lymphoma (BL) is curable, but patients with relapse or refractory disease have very poor outcomes. The underlying biological differences leading to relapse/refractory disease are unknown. We previously identified a potential association between microRNA (miR)-17-92 and relapse in patient samples. MiRs encoded by this gene have been implicated in therapy resistance, so we are seeking relationships between miRNA expression and therapy resistance in BL cells. Methods The miR-17-92 locus was targeted for deletion in Raji BL cells using custom CRISPR-Cas9 lentiviral vectors. Single cell-derived clones were established and locus deletion was determined by genomic DNA PCR. Inducible miR-17-92 expression in Raji cells was established by transduction of a cumate switch inducible lentivector system containing the miR-17-92 gene along with a CymR repressor expression vector (Raji/CymR/miR-17-92). MiR expression was assessed by TaqMan assay, and protein expression and caspase-3 cleavage were assessed by western blotting. Cells were treated with 4HC, the active metabolite of cyclophosphamide, or doxorubicin, and viability was assessed by AlamarBlue assay. Results Genomic DNA PCR verified hemizygous deletion of the miR-17-92 gene in several Raji BL clones; no homozygous deleted clones were identified. In two hemizygous clones R5 and R24, miR-17 expression was decreased to 0.24-fold and 0.37-fold, respectively, relative to the parental Raji line. The miR-17-92 target proteins PTEN and Bim were increased in the R5 and R24 clones by western blotting. Compared to Raji, R5 and R24 showed increased basal caspase-3 cleavage and much greater induction of caspase-3 cleavage with cytotoxic chemotherapy (10 µM 4HC). In addition, 0.5 and 1 µM doxorubicin showed a dose dependent induction of cleaved caspase in R5 and R24 cells that was much greater than in parental Raji cells. The IC50 for doxorubicin in R5 and R24 miR-17-92 hemizygous cells was markedly decreased (0.00025 and 0.00032 µM, respectively) compared to parental Raji cells (0.064 µM). Finally, miR-17 expression was inducible by treating cumate repressor vector transduced Raji/CymR/miR-17-92 cells with 30 µg/mL cumate (+2.8-fold at 3 days, +7.5-fold at 5 days). Conclusion We established miR-17-92 hemizygous-deleted cell lines that show increased PTEN and pro-apoptotic Bim, increased basal caspase-3 cleavage, and increased chemosensitivity. We have established Raji BL cell lines with inducible miR-17-92 expression to extend these experiments by determining the effect of increased miR-17-92 on chemosensitivity. Our findings show that high miR-17-92 expression in BL cells can contribute to therapy resistance, which may be important for understanding refractory BL in patients and could lead to more effective targeted therapies. Disclosures Cairo: Celgene: Research Funding.
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