Nikola Tom

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.

Low-burden TP53 mutations in chronic phase of myeloproliferative neoplasms: association with age, hydroxyurea administration, disease type and JAK2 mutational status

The multistep process of TP53 mutation expansion during myeloproliferative neoplasm (MPN) transformation into acute myeloid leukemia (AML) has been documented retrospectively. It is currently unknown how common TP53 mutations with low variant allele frequency (VAF) are, whether they are linked to hydroxyurea (HU) cytoreduction, and what disease progression risk they carry. Using ultra-deep next-generation sequencing, we examined 254 MPN patients treated with HU, interferon alpha-2a or anagrelide and 85 untreated patients. We found TP53 mutations in 50 cases (0.2–16.3% VAF), regardless of disease subtype, driver gene status and cytoreduction. Both therapy and TP53 mutations were strongly associated with older age. Over-time analysis showed that the mutations may be undetectable at diagnosis and slowly increase during disease course. Although three patients with TP53 mutations progressed to TP53-mutated or TP53-wild-type AML, we did not observe a significant age-independent impact on overall survival during the follow-up. Further, we showed that complete p53 inactivation alone led to neither blast transformation nor HU resistance. Altogether, we revealed patients age as the strongest factor affecting low-burden TP53 mutation incidence in MPN and found no significant age-independent association between TP53 mutations and hydroxyurea. Mutations may persist at low levels for years without an immediate risk of progression.

Detection and Functional Analysis of TP53 Mutations in CLL

Chronic lymphocytic leukemia (CLL) represents a prototype disease in which TP53 gene defects lead to inferior prognosis. Here, we present two distinct methodologies which can be used to identify TP53 mutations in CLL patients; both protocols are primarily intended for research purposes. The functional analysis of separated alleles in yeast (FASAY) can be flexibly adapted to a variable number of samples and provides an immediate functional readout of identified mutations. Amplicon-based next-generation sequencing then allows for a high throughput and accurately detects subclonal TP53 variants (sensitivity <1% of mutated cells).

The influence of mutational status and biological characteristics of acute myeloid leukemia on xenotransplantation outcomes in NOD SCID gamma mice

PurposeThis study aimed at analyzing the association of gene mutations and other acute myeloid leukemia (AML) characteristics with engraftment outcomes in immunodeficient mice and to select the engraftment outcomes that best reflect patient survival.MethodsMutations in 19 genes as well as leukemia- and patient-related characteristics were analyzed for a group of 47 de novo AML samples with respect to three engraftment outcomes: engraftment ability, engraftment intensity (percentage of hCD45+ cells) and engraftment latency. Leukemia-related characteristics were additionally analyzed in an extended group of 68 samples that included the 47 de novo samples, and additional 21 samples from refractory and relapsed cases. Engraftment outcomes were compared with overall and event-free survival of the patients.ResultsFor the 47 de novo samples, no single mutation influenced engraftment, whereas the NPM1mut/DNMT3Amut co-mutation was associated with higher engraftment ability. NPM1mut/FLT3-ITDneg had lower engraftment intensity. Among leukemia-related characteristics, a complex karyotype was associated with higher engraftment intensity. Among patient-related characteristics, higher cytogenetic risk was associated with higher engraftment intensity, and failure to achieve clinical remission was associated with shorter engraftment latency. In the extended group of 68 samples, white blood count was associated with higher engraftment ability, and the presence of a complex karyotype was associated with higher engraftment intensity. Association with patient overall survival was seen only for engraftment intensity.ConclusionsThe engraftment of AML was influenced by mutation-interactions and other AML characteristics, rather than by single mutated genes, and engraftment intensity best reflected clinical penetrance of AML.

Transcription factor YY1 can control AID-mediated mutagenesis in mice

Activation‐induced cytidine deminase (AID) is crucial for controlling the immunoglobulin (Ig) diversification processes of somatic hypermutation (SHM) and class switch recombination (CSR). AID initiates these processes by deamination of cytosine, ultimately resulting in mutations or double strand DNA breaks needed for SHM and CSR. Levels of AID control mutation rates, and off‐target non‐Ig gene mutations can contribute to lymphomagenesis. Therefore, factors that control AID levels in the nucleus can regulate SHM and CSR, and may contribute to disease. We previously showed that transcription factor YY1 can regulate the level of AID in the nucleus and Ig CSR. Therefore, we hypothesized that conditional knock‐out of YY1 would lead to reduction in AID localization at the Ig locus, and reduced AID‐mediated mutations. Using mice that overexpress AID (IgκAID yy1f/f) or that express normal AID levels (yy1f/f), we found that conditional knock‐out of YY1 results in reduced AID nuclear levels, reduced localization of AID to the Sμ switch region, and reduced AID‐mediated mutations. We find that the mechanism of YY1 control of AID nuclear accumulation is likely due to YY1‐AID physical interaction which blocks AID ubiquitination.

ToTem: a tool for variant calling pipeline optimization

BackgroundHigh-throughput bioinformatics analyses of next generation sequencing (NGS) data often require challenging pipeline optimization. The key problem is choosing appropriate tools and selecting the best parameters for optimal precision and recall.ResultsHere we introduce ToTem, a tool for automated pipeline optimization. ToTem is a stand-alone web application with a comprehensive graphical user interface (GUI). ToTem is written in Java and PHP with an underlying connection to a MySQL database. Its primary role is to automatically generate, execute and benchmark different variant calling pipeline settings. Our tool allows an analysis to be started from any level of the process and with the possibility of plugging almost any tool or code. To prevent an over-fitting of pipeline parameters, ToTem ensures the reproducibility of these by using cross validation techniques that penalize the final precision, recall and F-measure. The results are interpreted as interactive graphs and tables allowing an optimal pipeline to be selected, based on the user’s priorities. Using ToTem, we were able to optimize somatic variant calling from ultra-deep targeted gene sequencing (TGS) data and germline variant detection in whole genome sequencing (WGS) data.ConclusionsToTem is a tool for automated pipeline optimization which is freely available as a web application at https://totem.software.

Activation-induced deaminase and its splice variants associate with trisomy 12 in chronic lymphocytic leukemia

Activation-induced cytidine deaminase (AID) is a mutator enzyme essential for somatic hypermutation (SHM) and class switch recombination (CSR) during effective adaptive immune responses. Its aberrant expression and activity have been detected in lymphomas, leukemias, and solid tumors. In chronic lymphocytic leukemia (CLL) increased expression of alternatively spliced AID variants has been documented. We used real-time RT-PCR to quantify the expression of AID and its alternatively spliced transcripts (AIDΔE4a, AIDΔE4, AIDivs3, and AIDΔE3E4) in 149 CLL patients and correlated this expression to prognostic markers including recurrent chromosomal aberrations, the presence of complex karyotype, mutation status of the immunoglobulin heavy chain variable gene, and recurrent mutations. We report a previously unappreciated association between higher AID transcript levels and trisomy of chromosome 12. Functional analysis of AID splice variants revealed loss of their activity with respect to SHM, CSR, and induction of double-strand DNA breaks. In silico modeling provided insight into the molecular interactions and structural dynamics of wild-type AID and a shortened AID variant closely resembling AIDΔE4, confirming its loss-of-function phenotype.

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

613 Clonal Selection of TP53 Mutations in Chronic Lymphocytic Leukaemia Detected by Ultra-deep Pyrosequencing

patients with stage II, microsatellite stable colon cancer with clinical data and of colon mucosa samples from 50 healthy donors, obtained during routine colonoscopy using the newly developed 450,000 CpG site platform for DNA methylation studies (Illumina Infinium HumanMethylation450BeadChip). This array includes CpG and CNG sites, CpG islands/shores/shelves/isolated CpGs in the genome, non-coding RNA (microRNAs and long non-coding RNAs) and sites surrounding the transcription start sites (−200 bp to −1,500 bp, 5′-UTRs and exons 1) for coding genes, but also for the corresponding gene bodies and 3′-UTRs. This study has been developed in the context of the COLONOMICS project (www.colonomics.org) in which for those normal-tumor paired samples and controls we also have data on molecular expression, SNPs/CNVs and miRNAs. The biomarkers identified must be validated. Results and Discussion: The preliminary analysis of the methylation patterns among different groups show differences in the methylation patterns of tumor and normal mucosa. A total of 106,566 CpG sites were differentially methylated between tumor and normal tissue (at 5% significance level after Bonferroni correction). The analyses of principal component (PCA) clearly discriminate between tumor and normal tissue samples. Some tumors show a predominant tumor hypermethylation pattern while others show hypomethylation and these patterns depict tumor subgroups that are likely to have different phenotype and outcomes. Epigenetic events may contain prognostic information. Using supervised analyses we have been able to identify several potential candidates for diagnostic and prognostic biomarkers to discriminate between tumor and non-tumor tissues. Conclusion: These differences in the methylation patterns shown to be promising in predicting the diagnostic and prognosis of CRC patients based on epigenetic characteristics of the tumors and normal mucosa.