Filip Pardy

Detailed analysis of therapy-driven clonal evolution of TP53 mutations in chronic lymphocytic leukemia

In chronic lymphocytic leukemia (CLL), the worst prognosis is associated with TP53 defects with the affected patients being potentially directed to alternative treatment. Therapy administration was shown to drive the selection of new TP53 mutations in CLL. Using ultra-deep next-generation sequencing (NGS), we performed a detailed analysis of TP53 mutations’ clonal evolution. We retrospectively analyzed samples that were assessed as TP53-wild-type (wt) by FASAY from 20 patients with a new TP53 mutation detected in relapse and 40 patients remaining TP53-wt in relapse. Minor TP53-mutated subclones were disclosed in 18/20 patients experiencing later mutation selection, while only one minor-clone mutation was observed in those patients remaining TP53-wt (n=40). We documented that (i) minor TP53 mutations may be present before therapy and may occur in any relapse; (ii) the majority of TP53-mutated minor clones expand to dominant clone under the selective pressure of chemotherapy, while persistence of minor-clone mutations is rare; (iii) multiple minor-clone TP53 mutations are common and may simultaneously expand. In conclusion, patients with minor-clone TP53 mutations carry a high risk of mutation selection by therapy. Deep sequencing can shift TP53 mutation identification to a period before therapy administration, which might be of particular importance for clinical trials.

Investigation of next-generation sequencing data of Klebsiella pneumoniae using web-based tools

Purpose. Rapid identification and characterization of multidrug‐resistant Klebsiella pneumoniae strains is necessary due to the increasing frequency of severe infections in patients. The decreasing cost of next‐generation sequencing enables us to obtain a comprehensive overview of genetic information in one step. The aim of this study is to demonstrate and evaluate the utility and scope of the application of web‐based databases to next‐generation sequenced (NGS) data. Methodology. The whole genomes of 11 clinical Klebsiella pneumoniae isolates were sequenced using Illumina MiSeq. Selected web‐based tools were used to identify a variety of genetic characteristics, such as acquired antimicrobial resistance genes, multilocus sequence types, plasmid replicons, and identify virulence factors, such as virulence genes, cps clusters, urease‐nickel clusters and efflux systems. Results. Using web‐based tools hosted by the Center for Genomic Epidemiology, we detected resistance to 8 main antimicrobial groups with at least 11 acquired resistance genes. The isolates were divided into eight sequence types (ST11, 23, 37, 323, 433, 495 and 562, and a new one, ST1646). All of the isolates carried replicons of large plasmids. Capsular types, virulence factors and genes coding AcrAB and OqxAB efflux pumps were detected using BIGSdb‐Kp, whereas the selected virulence genes, identified in almost all of the isolates, were detected using CLC Genomic Workbench software. Conclusion. Applying appropriate web‐based online tools to NGS data enables the rapid extraction of comprehensive information that can be used for more efficient diagnosis and treatment of patients, while data processing is free of charge, easy and time‐efficient.

Abstract 3084: MicroRNA involvement in DNA damage response and BCR signaling in malignant B cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The biology of B cell Non-Hodgkin lymphomas (NHLs) is largely influenced by (de)regulation of B cell receptor signaling (BCR sig.) and DNA damage response pathway (DDR). We and others have shown that in NHLs, miR-34a is involved in DDR, and miR-150/miR-155 are involved in BCR sig. (Mraz et al., 2009, 2014; Cui et al., 2014). To identify miRNAs involved in DDR and BCR sig. we used NGS technology (HiSeq) in chronic lymphocytic leukemia (CLL) B cells treated with BCR inhibitor or DNA damaging drugs. To investigate the DDR-related miRNAs, primary CLL cells were treated with fludarabine in vitro and paired samples (before/after treatment, n = 20) were analyzed for miRNAs’ profile. This identified 6 differentially expressed miRNAs (FDR 1.5, P<0.05); according to our knowledge this is the first analysis of miRNA profiles during therapy administration in NHLs. Importantly, the miR-34a level was a significant predictor (p<0.05) of patients’ response to FCR therapy (complete response vs. others) and the progression free survival (19.9 vs. 26.4 mo.; HR: 2.29). A similar trend was observed for miR-1246, however, this was not statistically significant (P = 0.11). Additionally, low miR-34a is an independent predictor (in a multivariate analysis) of a shorter overall survival (16.7 mo. vs. not reached; P = 0.0002; HR: 3.30). This suggests that CLL cells with low levels of miR-34a fail to down-modulate genes that are crucial for DDR. Several pro-survival genes targeted by miR-34a were recently identified in various cell types (BIRC3, BCL2, FOXP1, YY1, Survivin). Some of these proteins were down-modulated in CLL B cells that up-regulate miR-34a during DDR or we have transfected with synthetic miR-34a, but not in cells that fail to induce miR-34a. Surprisingly, miR-34a, miR-1246 and miR-1248 share numerous validated and predicted targets with miR-150, a known negative regulator of BCR sig. (Mraz et al., 2014). This suggests possible convergence in the mechanism of action of DNA damaging drugs and BCR inhibitors recently approved for treatment of NHLs (such as ibrutinib). To compare the effects of FCR administration with the administration of ibrutinib, we analyzed miRNA and gene expression (HiSeq) in samples from ibrutinib treated CLL patients (n = 9) before and during the therapy. The convergence of pathways targeted by DDR and BCR inhibition through changes in miRNAs’ expression are currently being interrogated. Supported by: SoMoPro II-no. 4SGA8684; NGS-PTL (306242); EHA Fellowship award; IGA MZ CR NT11218-6/2010; CZ.1.07/2.4.00/17.0042; MUNI/A/0830/2013; MH CZ-DRO (FNBr, 65269705), CZ.1.05/1.1.00/02.0068, CZ.1.07/2.3.00/30.0009. Citation Format: Katerina Cerna, Jan Oppelt, Lenka Radova, Katerina Musilova, Vaclav Seda, Gabriela Pavlasova, Michal Jez, Nikola Tom, Filip Pardy, Jitka Malcikova, Karla Plevova, Boris Tichy, Yvona Brychtova, Michael Doubek, Martin Trbusek, Jiri Mayer, Jaroslav Koca, Raffaele Calogero, Sarka Pospisilova, Marek Mraz. MicroRNA involvement in DNA damage response and BCR signaling in malignant B cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3084. doi:10.1158/1538-7445.AM2015-3084

Abstract 5198: Identification of microRNAs involved in DNA damage response in malignant B cells and their biological and clinical relevance

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA We and others have shown that expression of miRNAs influences the biology of B cell malignancies (Calin et al., 2005; Mraz et al., 2009, 2012, 2013). The aim of this study was to identify miRNAs involved in the apoptotic response of malignant B cells. Purified primary B cells of chronic lymphocytic leukemia (CLL) patients (pts.) were treated in vitro with fludarabine (F) (LC50 dose of 3.5 μg/ml; 48 h). Five paired (n=10) samples (with and without F) were analyzed using 2 NGS platforms-SOLiD (ABI) and HiSeq (Illumina). The obtained reads were mapped to miRBase using 3 tools (CLC Genomic Workbench, SHRiMP2, miRanalyzer) and data analyzed by a pair-wise comparison with edgeR and baySeq packages (Bioconductor 2.13). The overlap of these 3+2 bioinformatic approaches identified 6 miRNAs significantly changed with DNA damage. RNA-Seq validation on 5 additional paired samples (n=10) confirmed the changed expression of all 6 previously identified miRNAs (3 down-, 3 up-regulated). The most constantly up-regulated was miR-34a, which was previously shown to be regulated by p53 (He et al., 2005; Mraz et al., 2009). To test the importance of miR-34a in vivo, we collected samples from CLL pts. (n=51) treated with F, cyclophosphamide and rituximab (FCR) regimen. miR-34a was induced in all samples after F administration (day 2, p<0.001). Surprisingly, the lower basal and induced levels of miR-34a correlated with significantly (p<0.05) shorter time to treatment failure, suggesting its strong prognostic potential. We further determined the expression of miR-34a in a large cohort of CLL pts. (n=158) using an in-house designed assay for its copy-number quantification. We defined a cut-point (number of miR-34a copies) that segregates pts. with extremely unfavorable prognosis (overall survival [OS] 1.37 yrs. vs. not reached; p=0.0001; HR=3.89; CI=2.05-7.39) and this was independent of routinely used prognostic markers (FISH, IgHV, age, sex) in a multivariate analysis. We have previously described that low levels of miR-34a associate with TP53 abnormalities, so we limited the analysis to wt-TP53 samples (n=116). In this multivariate analysis miR-34a was the strongest predictor of OS (1.29 yrs. vs. not reached; p=0.002; HR=9.82; CI=2.30-42.05). The molecular pathways affected by miR-34a levels in B cells are largely unknown. However, miR-34a shares 51 predicted target mRNAs (evolutionary conserved) with at least 1 other F induced miRNA, suggesting that they might cooperate in the regulation of these genes. The pathways regulated by these miRNAs are currently being investigated using integrated analysis of miRNA and transcriptome profiling of CLL samples (n=100). Supported by: NGS-PTL (FP7-HEALTH-2012-INNOVATION-1, no. 306242); EHA Research Fellowship award; Grant Agency of Czech Rep.; IGA MZ CR NT11218-6/2010; CZ.1.07/2.4.00/17.0042; MUNI/A/0723/2012; CZ.1.07/2.3.00/30.0009, co-financed from EU and Czech Rep. Citation Format: Katerina Cerna, Jan Oppelt, Lenka Radova, Katerina Musilova, Nikola Tom, Filip Pardy, Jitka Malcikova, Karla Plevova, Boris Tichy, Yvona Brychtova, Michael Doubek, Martin Trbusek, Jiri Mayer, Jaroslav Koca, Raffaele Calogero, Sarka Pospisilova, Marek Mraz. Identification of microRNAs involved in DNA damage response in malignant B cells and their biological and clinical relevance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5198. doi:10.1158/1538-7445.AM2014-5198