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Prediction of tumor suppressors and oncogenes, also called driver genes, is an essential step in understanding cancer development and discovering potential novel treatments. We recently proposed Moonlight as a bioinformatics framework to predict driver genes and analyze them in a system-biology-oriented manner based on -omics integration. Moonlight uses gene expression as a primary data source and combines it with patterns related to cancer hallmarks and regulatory networks to identify oncogenic mediators. Once the oncogenic mediators are identified, it is important to include extra levels of evidence, called mechanistic indicators, to identify driver genes and to link the observed changes in gene expression to the underlying alteration that promotes them. Such a mechanistic indicator could be for example a mutation in the regulatory regions for the candidate gene or mutations in the regulator itself. In this work, we developed new functionalities and release Moonlight2, to provide the user with the mutation-based mechanistic indicator to streamline the analyses of this second layer of evidence. The function analyzes mutation information in a cancer cohort to classify them into driver and passenger mutations. Moreover, the function estimates the potential effect of a mutation on the transcriptional, translational, or protein structure/function level. Those oncogenic mediators with at least one driver mutation are retained as the final set of driver genes. We applied Moonlight2 and the newly developed function to a case study on Basal-like breast cancer subtype using data from The Cancer Genome Atlas. We found six oncogenes (SF3B4, EBNA1BP2, KRTCAP2, ZBTB8OS, RUNX2, andPOLR2J) and ten tumor suppressor genes (KIF26B, NR5A2, ARHGAP25, EMCN, ARL15, PCOLCE, TPK1, TEK, KIR2DL4, andGMFG) containing a driver mutation in their promoter region, possibly explaining their deregulation. The Moonlight2R source code is available athttps://github.com/ELELAB/Moonlight2R.

Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity, and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments, and proficiency testing on standardized cell line–derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas below this limit detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false-negatives) were more common than erroneous candidates (false-positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best-practice guidelines and provides a resource for precision oncology., Editorial summary: Reliable detection of mutations below 0.5% variant allele frequency remains a key challenge for circulating tumor DNA sequencing assays.

Patients with chronic lymphocytic leukemia (CLL) with TP53 mutations with a >10% variant allele frequency (VAF) are often refractory to chemotherapy and benefit from targeted therapy. Malcikova and colleagues correlated TP53 mutations with, Key Points Low-burden TP53 mutations in CLL do not significantly affect the response duration to chemo- and/or immunotherapy, but shorten OS.Clonal expansion of low-burden TP53 mutations in CLL is associated with intense chemoimmunotherapy, but not with targeted therapy., Visual Abstract, Patients with chronic lymphocytic leukemia (CLL) bearing TP53 mutations experience chemorefractory disease and are therefore candidates for targeted therapy. However, the significance of low-burden TP53 mutations with

There is an emerging body of evidence that patients with chronic myeloid leukaemia (CML) may carry not only breakpoint cluster region-Abelson murine leukaemia viral oncogene homologue 1 (BCR-ABL1) kinase domain mutations (BCR-ABL1 KD mutations), but also mutations in other genes. Their occurrence is highest during progression or at failure, but their impact at diagnosis is unclear. In the present study, we prospectively screened for mutations in 18 myeloid neoplasm-associated genes and BCR-ABL1 KD in the following populations: bulk leucocytes, CD34(+)CD38(+) progenitors and CD34(+)CD38(-) stem cells, at diagnosis and early follow-up. In our cohort of chronic phase CML patients, nine of 49 patients harboured somatic mutations in the following genes: six ASXL1 mutations, one SETBP1, one TP53, one JAK2, but no BCR-ABL1 KD mutations. In seven of the nine patients, mutations were detected in multiple hierarchical populations including bulk leucocytes at diagnosis. The mutation dynamics reflected the BCR-ABL1 transcript decline induced by treatment in eight of the nine cases, suggesting that mutations were acquired in the Philadelphia chromosome (Ph)-positive clone. In one patient, the JAK2 V617F mutation correlated with a concomitant Ph-negative myeloproliferative neoplasm and persisted despite a 5-log reduction of the BCR-ABL1 transcript. Only two of the nine patients with mutations failed first-line therapy. No correlation was found between the mutation status and survival or response outcomes.

Background Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. Results In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. Conclusion These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

Clinical laboratories routinely use formalin-fixed paraffin-embedded (FFPE) tissue or cell block cytology samples in oncology panel sequencing to identify mutations that can predict patient response to targeted therapy. To understand the technical error due to FFPE processing, a robustly characterized normal cell line was used to create FFPE samples with four different pre-tissue processing formalin fixation times. A total of 96 FFPE sections were then distributed to different laboratories for targeted sequencing analysis by four oncopanels, and variants resulting from technical error were identified. Tissue sections that failed more frequently showed low cellularity, lower than recommended library preparation DNA input, or target sequencing depth. Importantly, sections from block surfaces were more likely to show FFPE-specific errors, akin to “edge effects” seen in histology, and the depth of formalin damage was related to fixation time. To assure reliable results, we recommend avoiding the block surface portion and restricting mutation detection to genomic regions of high confidence.

Background High-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. Results Here 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. Conclusions ToTem is a tool for automated pipeline optimization which is freely available as a web application at https://totem.software. Electronic supplementary material The online version of this article (10.1186/s12859-018-2227-x) contains supplementary material, which is available to authorized users.

This 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. Mutations 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. For the 47 de novo samples, no single mutation influenced engraftment, whereas the NPM1 mut /DNMT3A mut co-mutation was associated with higher engraftment ability. NPM1 mut /FLT3-ITD neg 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. The 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.

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 (IgAID yy1(f/f)) or that express normal AID levels (yy1(f/f)), we found that conditional knock-out of YY1 results in reduced AID nuclear levels, reduced localization of AID to the S 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.

Genetic mutations in acute myeloid leukaemia (AML) are assumed to occur in a sequential order; however, the predominant hierarchical roles of specific mutated genes have not been fully described. In this study, we aimed to determine the clonal involvement of the most frequent AML-associated mutations. Using a targeted sequencing panel for 18 genes, we traced changes and relative clonal contribution of mutations in 52 patients. We analysed 35 pairs of diagnosis and relapse samples, 27 pairs of primary samples and corresponding patient-derived xenografts, and 34 pairs of total leukocytes and corresponding isolated primitive cells or blast populations. In both relapse and xenografts, we observed conservation of main leukaemic clones and variability was limited to subclones with late-acquired mutations. AML evolution thus mainly involved modification of subclones while the clonal background remained unchanged. NPM1 mutations were identified as the most probable leukaemia-transformation lesion, remaining conserved in contrast to high variation of accompanying subclonal FLT3 and NRAS mutations. DNMT3A mutations represented the most stable mutations forming a preleukaemic background in most samples. Mutations in genes IDH1/2, TET2, RUNX1, ASXL1 and U2AF1 were detected both as preleukaemic and as subclonal lesions, suggesting a non-specific order of acquisition.

The occurrence of mutations in the BCR-ABL1 kinase domain (KD) can lead to treatment resistance in chronic myeloid leukaemia patients. Nowadays, next-generation sequencing (NGS) is an alternative method for the detection of kinase domain mutations, compared to routinely used Sanger sequencing, providing a higher sensitivity of mutation detection. However, in the protocols established so far multiple rounds of amplification limit reliable mutation detection to approximately 5% variant allele frequency. Here, we present a simplified, one-round amplification NGS protocol for the Illumina platform, which offers a robust early detection of BCR-ABL1 KD mutations with a reliable detection limit of 3% variant allele frequency.

In this multi-centre study, we analysed the prognostic impact of mutations in 19 genes associated with myeloid malignancies in 258 newly diagnosed acute myeloid leukaemia patients (aged 19-70 years) undergoing intensive therapy. We identified five patient groups with different prognostic risks and different benefits from allogeneic hematopoietic stem cell transplantation (alloHSCT) within the intermediate cytogenetic risk group patients (n = 184). The most adverse prognosis was observed in patients with DNMT3A and FLT3-ITD co-mutation, whose survival could be significantly improved with alloHSCT. In contrast, the most favourable prognosis without any further benefit from alloHSCT was identified in patients with mutations in NPM1 or CEBPA, after exclusion of the unfavourable prognostic groups defined by mutations in DNMT3A, RUNX1 or genes from chromatin/spliceosome group. An additional analysis of 113 diagnosis-remission paired samples revealed that persistence of non-DNMT3A mutations (above 2% VAF) represented a further negative prognostic factor. The proposed model offers a possible molecular stratification and treatment guidance for intermediate cytogenetic risk group patients.

The impact of genetic aberrations on rituximab-based therapeutic regimens has been intensely studied in chronic lymphocytic leukemia (CLL). According to the current consensus chemoimmunotherapy consisting of rituximab and DNA-damaging drugs is not suitable for patients with TP53 defects. In our study, we focused on CLL patients with an intact TP53 gene and investigated four recurrently mutated genes in CLL, genomic aberrations by FISH, and IGHV status with the aim of analyzing their impact on progression-free survival (PFS) after front-line therapy with FCR (fludarabine, cyclophosphamide, rituximab) or BR (bendamustine, rituximab) regimens. Using next-generation sequencing, we analyzed 120 patients treated with FCR and 57 patients treated with BR at a university hospital. We used a 10% cut-off for variant allele frequency and recorded the following mutation frequencies in the pre-therapy samples: ATM 23%, SF3B1 20%, NOTCH1 19% and BIRC3 11%. The data on cytogenetic aberrations (11q22, 13q14, trisomy 12) and IGHV mutation status were also considered in PFS analyses. In univariate analyses, we observed a negative impact of BIRC3 mutations and 11q22 deletion in both regimens, while the unmutated IGHV status was associated with a significantly shorter PFS only in the FCR-treated cohort. In a multivariate analysis, only deletion 11q22 in both regimens, and the unmutated IGHV in the FCR cohort maintained an independent association with the reduced PFS. Notably, sole 11q22 deletion, without an ATM mutation on the other allele, manifested the shortest PFS of all analyzed markers. Deletion 11q22 and IGHV status predict PFS in previously untreated CLL patients.

Mantle cell lymphoma (MCL) is characterized by the hallmark t(11;14)(q13;q32) translocation, leading to cyclin D1 over-expression. Additionally, disrupting the DNA damage response pathway through ATM or TP53 defects plays an important role in MCL pathogenesis. Using deep next-generation sequencing we analyzed the mutual composition of ATM and TP53 mutations in 72 MCL patients, and assessed their impact on progression-free survival (PFS) and overall survival (OS). Mutated ATM and TP53 alleles were found in 51% (37/72) and 22% (16/72) of the cases examined, respectively, with only three patients harboring mutations in both genes. Only a mutated TP53 gene was associated with the significantly reduced PFS and OS and the same output was observed when ATM and TP53 defective groups included also sole deletions 11q and 17p, respectively. Determining the exact ATM/p53 pathway dysfunction may influence the selection of MCL patients for innovative therapies based on the targeted inhibition of selected proteins.

Over the past few years, several large-scale studies using next-generation sequencing (NGS) of whole-genomes (WGS) and whole-exomes (WES) have defined the mutational landscape of chronic lymphocytic leukemia (CLL) [1,2,3,4]. NGS studies have also revealed the clonal heterogeneity in CLL and showed that clonal evolution contributes to the variability in clinical course among CLL patients [3]. Clonal evolution is considered a key condition in CLL progression and relapse after treatment. Most CLL cases are diagnosed during the inactive disease phase, genetic aberrations’ underlying progress in CLL activity leading to the need for therapy are poorly understood and should be explored. A large number of frequently mutated genes have been identified and several putative driver mutations likely to confer selective growth advantage to CLL tumor cells have been proposed [1,2,3]. In addition, clonal shifts between paired treatment-naïve and relapsed CLL samples have been reported due to pre-existing subclone expansion under therapeutic pressure, demonstrating that clonal evolution likely underlies CLL relapse [3, 5]. Nevertheless, there are still a limited amount of longitudinal WES studies analyzing consecutive CLL samples before treatment intervegntion [6]. The acquisition of driver mutations accompanied by selectively neutral passenger changes during disease prior to therapy influence is therefore poorly documented. Here, WES was performed on consecutive treatment-naïve samples of CLL patients from three groups with different disease course: Active disease (AD) group: patients with an active disease before the second analyzed time-point (TP2); Stable disease (SD) group: cases with a period of stable phase after diagnosis followed by progression within 3 years after; and Indolent disease (ID) group: those with a long-term stable indolent disease. Moreover, we applied a novel integrative bioinformatics tool called “Cancer Genome Interpreter” to identify driver mutations [7]., The research leading to these results has mainly received funding from the European Union Seventh Framework Programme [FP7/2007–2013] under Grant Agreement no 306242-NGS-PTL. In addition, this work was supported by grants from the Spanish Fondo de Investigaciones Sanitarias PI15/01471, PI18/01500, Instituto de Salud Carlos III (ISCIII), European Regional Development Fund (ERDF) “Una manera de hacer Europa”, “Consejería de Educación, Junta de Castilla y León” (SA085U16), “Proyectos de Investigación del SACYL”, Spain: GRS 994/A/14, BIO/SA10/14, BIO/SA31/13, GRS 1172/A/15,“Fundación Memoria Don Samuel Solórzano Barruso”, by grants (RD12/0036/0069) from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC) CB16/12/00233 and USAL >Programa XIII>. M. Hernández-Sánchez is supported by FEHH-Janssen (“Sociedad Española de Hematología y Hemoterapia”). M Quijada-Álamo is supported by an “Ayuda Predoctoral de la Junta de Castilla y León” (JCYL-EDU/529/2017). We are grateful to I. Rodríguez, S. González, T.Prieto, M. Á. Ramos, A. Martín, A. Díaz, A. Simón, M.del Pozo, V. Gutiérrez and S. Pujante from Centro de Investigación del Cáncer, Salamanca, for their technical assistance. D. Tamborero is supported by project SAF2015–74072-JUN, which is funded by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Dearrollo Regional (FEDER). This work was supported by Seventh Framework Programme (NGS-PTL/2012–2015/no.306242) and Ministry of Education, Youth and Sports (2013–2015, no. 7E13008); by the Ministry of Education, Youth and Sports of the Czech Republic under the CEITEC 2020 project (LQ1601); by the Ministry of Health, Czech Republic - conceptual development of research organization (FNBr, 65269705); by the Specific University Research (nr. MUNI/A/0968/2017) provided by MEYS; and by the project CZ.02.1.01/0.0/0.0/16_013/0001634 National Center for Medical Genomic - modernization of infrastructure and research of genetic variation in the population, funded by OP RDE.

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 (sensitivity1% of mutated cells).

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

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.

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 patient's 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.

The clinical course in chronic lymphocytic leukaemia (CLL) patients is diverse, reflecting the heterogeneous biological background of this disease (Guieze & Wu, 2015). Among reported defects, mutations in NOTCH1 and TP53 genes represent potent CLL progression drivers and contribute to disease chemo-refractoriness (Fabbri et al, 2011; Malcikova et al, 2015). Although parallel occurrence of NOTCH1 mutations and TP53 defects has been noted in CLL patients (Weissmann et al, 2013; Stilgenbauer et al, 2014), the clonal composition of these coexisting aberrations has not been yet studied. To clarify this phenomenon, we examined CLL patients with concurrently detected hotspot NOTCH1 and TP53 mutations using single cell analysis.

Introduction: The current prognostic stratification of acute myeloid leukemia (AML) patients recognizes several recurrently mutated genes in AML as prognostic markers at the time of diagnosis. However, previous studies indicate that additional prognostic information can be further obtained by detection of pre-leukemic mutations that persist in clinical remission and represent clonal hematopoiesis. Aim: To analyze persistence of pre-leukemic mutations in AML patients at the time of disease remission and assess their prognostic relevance. Methods: Paired diagnosis and remission samples were analyzed for 114 AML patients; all carried at least 1 pre-treatment mutation at the time of diagnosis. Remission samples were collected 1 - 7 months from the start of conventional induction therapy (median 3 months, none of the patients received transplantation therapy prior to the sampling). All patients achieved at least hematological remission or deeper molecular remission that was identified by standard minimal residual disease monitoring for NPM1 or FLT3-ITD mutations, KMT2A-rearragements, or RUNX1-RUNX1T1 and CBFB-MYH11 fusion genes. Sequencing of 47 exons in 19 genes was performed by targeted amplicon sequencing with ClearSeq AML Haloplex panel (Agilent) on MiSeq and NextSeq instruments (Illumina); MPL gene was excluded for inadequate coverage. Sequencing data were processed using VarDict, VarScan and Pindel algorithms. Mutation detection threshold was set to 2% variant allele frequency (VAF). Detection of CEBPA mutations was confirmed by sanger sequencing. Results: Pre-leukemic mutations persisting in remission were detected in 38/114 patients (33%). In 31/38 (82%) patients, the persistence was accompanied by loss of other mutation/s. Persisting mutations were most frequently detected in genes: DNMT3A (mutation persistence in 24 of 41 patients with pre-treatment DNMT3A mutation, 58%), IDH2 (5/19 patients, 26%), TET2 (4/12 patients, 33%) and ASXL1 (5/11 patients, 45%). On the contrary, complete clearance of all mutations in remission below 2% VAF was observed for the following genes: NPM1, FLT3, NRAS, CEBPA and IDH1, except for a single persisting FLT3-TKD mutation. The median VAF of persisting mutations was 18% (range 2 - 94%). Moreover, in almost half of the patients (17/38, 45%), VAF of persisting mutation exceeded 25% - thus more than half of the hematopoiesis was clonal at remission. Persistence of any mutation in remission was associated with impaired overall survival (OS) and event-free survival (EFS), compared to patients with no persisting mutations (2-year OS: 50% vs 81%, p=0.001; 2-year EFS: 28% vs 57%, p=0.0007). Persistence of 2 or more mutations at remission did not show further effect on OS or EFS compared to patients with exactly one persisting mutation. DNMT3A was the single persisting gene in remission in 20/38 (53%) patients, and was associated with shorter EFS but not OS compared to patients without any persisting mutations (2-year EFS: 26% vs 57%, p=0.03). Persistence of mutations in other genes than DNMT3A was detected in 14/38 (37%) patients and was associated with shorter OS but not EFS compared to patients without any persisting mutations (2-year OS: 37% vs 81%, p=0.001). Conclusions: One third of AML patients in our cohort carried pre-leukemic mutations in remission. These persisting mutations were retained at high VAFs and were limited to specific genes, while at the same time mutations in other genes were cleared by AML therapy. This supports the evidence that persisting mutations represented surviving clonal hematopoiesis, not minimal residual disease. The persistence of mutations in remission showed prognostic impact, however analysis of a larger cohort of patients is required to assess how to combine results from the mutational screening at diagnosis and remission to provide the best prognostic stratification. Supported by Ministry of Health of the Czech Republic, grant nr. 15-25809A. All rights reserved. This report was written with the support of the Specific University Research (nr. MUNI/A/0968/2017) provided by MEYS. Disclosures Mayer: Novartis: Research Funding; Eisai: Research Funding; Johnson & Johnson: Research Funding.

TP53 gene defects represent a strong adverse prognostic factor for patient survival and treatment resistance in chronic lymphocytic leukemia (CLL). Although various methods for TP53 mutation analysis have been reported, none of them allow the identification of all occurring sequence variants, and the most suitable methodology is still being discussed. The aim of this study was to determine the limitations of commonly used methods for TP53 mutation examination in CLL and propose an optimal approach for their detection. We examined 182 CLL patients enriched for high-risk cases using denaturing high-performance liquid chromatography (DHPLC), functional analysis of separated alleles in yeast (FASAY), and the AmpliChip p53 Research Test in parallel. The presence of T53 gene mutations was also evaluated using ultra-deep next generation sequencing (NGS) in 69 patients. In total, 79 TP53 mutations in 57 (31 %) patients were found; among them, missense substitutions predominated (68 % of detected mutations). Comparing the efficacy of the methods used, DHPLC and FASAY both combined with direct Sanger sequencing achieved the best results, identifying 95 % and 93 % of TP53-mutated patients. Nevertheless, we showed that in CLL patients carrying low-proportion TP53 mutation, the more sensitive approach, e.g., ultra-deep NGS, might be more appropriate. TP53 gene analysis using DHPLC or FASAY is a suitable approach for mutation detection. Ultra-deep NGS has the potential to overcome shortcomings of methods currently used, allows the detection of minor proportion mutations, and represents thus a promising methodology for near future.

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 (FDR1.5, P 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

Introduction A presence of activating mutations in NOTCH1 gene has been recently associated with reduced survival and chemo-immunotherapy resistance in chronic lymphocytic leukemia (CLL). However, a prognostic significance of the NOTCH1 mutations with respect to TP53mutation status has not been fully explained yet. Methods An examined cohort included 409 patients with CLL enriched for high risk cases; in 121 patients consecutive samples were investigated. To determine the TP53 mutation status, a functional analysis of separated alleles in yeast (FASAY, exons 4-10) combined with direct sequencing was performed; the ambiguous cases were retested using an ultra-deep next generation sequencing (MiSeq platform; Illumina). The presence of NOTCH1 hotspot mutation (c.7544_7545delCT) was analyzed using direct sequencing complemented by allele-specific PCR in the selected samples. In several patients harboring concurrent NOTCH1 and TP53 mutations, single separated cancer cells were examined using multiplex PCR followed by direct sequencing. A correlation between mutation presence and patient overall survival, time to first treatment and other molecular and cytogenetic prognostic markers was assessed using Log-rank (Mantel-cox) test and Fisher's exact test, respectively. Results The NOTCH1 and TP53 mutations were detected in 16% (65/409) and 27% (110/409) of the examined patients, respectively; a coexistence of these mutations in the same blood samples was observed in 11% (19/175) of the mutated patients. The detected increased mutation frequency attributes to more unfavorable profile of the analyzed cohort; in the TP53-mutated patients missense substitutions predominated (75% of TP53 mutations). As expected, a significantly reduced overall survival in comparison to the wild-type cases (147 months) was observed in the NOTCH1-mutated (115 months; P = 0.0018), TP53-mutated (79 months; P < 0.0001) and NOTCH1-TP53-mutated patients (101 months; P = 0.0282). Since both NOTCH1 and TP53 mutations were strongly associated with an unmutated IGHV gene status (P < 0.0001 and P = 0.0007), we reanalyzed the IGHV-unmutated patients only and interestingly, the impact of simultaneous NOTCH1 and TP53 mutation presence on patient survival was missed in this case (P = 0.1478). On the other hand, in the NOTCH1 and/or TP53-mutated patients significantly reduced time to first treatment was identified as compared to the wild-type cases (41 months vs. 25 months in NOTCH1-mutated, P = 0.0075; 17 months in TP53-mutated, P < 0.0001; and 18 months in NOTCH1-TP53-mutated patients, P = 0.0003). The similar results were observed also in the subgroup of the IGHV-unmutated patients, with the exception of patients carrying sole NOTCH1 mutation (P = 0.2969). Moreover, in the NOTCH1-TP53-mutated patients an increased frequency of del(17p)(13.1) was found in comparison to the TP53-mutated patients only (72% vs. 56%); this cytogenetic defect was not detected in the patients with sole NOTCH1 mutation. Our results might indicate, that NOTCH1 mutation could preferentially co-selected with particular, less prognostic negative type of TP53 defects. Notably, in our cohort the NOTCH1 mutation predominated in the patients harboring truncating TP53 mutations localized in a C-terminal part of the TP53 gene behind the DNA-binding domain (P = 0.0128). Moreover, in one of the NOTCH1-TP53-mutated patients the analysis of separated cancer cells revealed a simultaneous presence of NOTCH1 mutation and TP53 in-frame deletion in the same CLL cell. In contrast, in the other examined NOTCH1-TP53-mutated patient the concurrent NOTCH1 mutation and TP53 missense substitution (with presumed negative impact on patient prognosis) were found in different CLL cells. Conclusions The parallel presence of NOTCH1 hotspot mutation might be detected in a significant proportion of TP53-mutated patients and it seems to be associated with less prognostic unfavorable TP53 mutations. Nevertheless, these preliminary data should be further confirmed in a large cohort of patients. This study was supported by projects VaVPI MSMT CR CZ.1.05/1.1.00/02.0068 of CEITEC, IGA MZ CR NT13493-4/2012, NT13519-4/2012 and CZ.1.07/2.3.00/30.0009. Disclosures Brychtova: Roche: Travel grants Other. Doubek:Roche: Travel grants Other.

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 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

Abstract 3911 Background The adverse prognostic significance of p53 aberrations (gene deletion at locus 17p13.1 and/or TP53 mutations) has been already proven in chronic lymphocytic leukemia (CLL). In contrast to the standardized examination of the gene deletion by interphase FISH, various methodologies with different detection efficiency are applied for mutation analysis. To reduce inter-laboratory variability, the European Research Initiative on CLL (ERIC) has recently released recommendations for p53 mutational testing (Pospisilova et al., 2012). However, the optimal detection methodology has not been established yet. Aim To compare molecular-biological methods for exact determination of TP53 mutational status in CLL patients. Methodology The analyzed cohort included 100 high-risk CLL patients with unfavorable disease prognosis represented by unmutated IgVH gene status, 17p and 11q deletions and/or chemotherapy resistence. Mutational screening of TP53 gene was performed in all patients by the combination of the following methods: (1) direct Sanger sequencing (DNA and/or cDNA), (2) denaturing high-performance liquid chromatography (DHPLC; Varian), (3) functional analysis (FASAY), (4) CLL custom resequencing microarray (Affymetrix), (5) Roche AmpliChip p53 Test (Roche Molecular Systems). In the selected samples, the presence of mutations was confirmed by ultra-deep next generation sequencing (NGS; GS Junior System, Roche). Results The parallel p53 analysis using all five above mentioned detection techniques revealed totally 66 mutations in 47/100 patients. The predominant proportion of the identified alterations was represented by prognostically adverse missense substitutions (67%), mainly localized in p53 DNA-binding domain (5–8 exons). Other clinically relevant sequencing variants included frameshift mutations (15%), splice-site mutations (8%), nonsense mutations (6%) and in-frame deletions (4%). Although the used detection methods reached comparable sensitivity (with the exception of direct sequencing), some inconsistent results were observed. In comparison with DNA-based methodologies, the FASAY failed in recognition of nonsense mutations leading to RNA degradation (nonsense-mediated decay phenomenon). On the other hand, the technical aspects of chip arrays have not facilitated the proper determination of deletions and insertions. From this perspective, DHPLC in connection with direct sequencing enabled the most specific recognition of the present gene alterations. Using this methodic combination, 57/66 mutations covering all mutation types were clearly identified. Nevertheless, for the correct evaluation of the biological importance and the clinical consequences of the detected mutations, the DNA screening should be supplemented with functional analysis. Conclusion The heterogeneous biological properties of TP53 mutations require sensitive and specific detection methodology. Although many different methods are currently used for mutation analysis, each of them has some advantages and shortcommings. The combination of DNA testing with functional analysis offers the most efficient tool for improved prediction of the disease course and the response of patients to therapy. Disclosures: No relevant conflicts of interest to declare.

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.

fixed cell pellet) were re-scored for % abnormal nuclei among 200 consecutive round nuclei. Scores were compared to the original clinical FISH analyses (200 consecutive nuclei not selected for nuclear morphology). Results: After re-scoring by the round cell approach, 12 originally normal samples remained normal while 16 (57%) were abnormal. The average number of days between the diagnostic and follow-up sample was 826 for those that remained normal and 601 for those abnormal by scoring round cells only. In one case, 12 had originally been scored in 0.5% but round cell approach showed 10% 12. Another 2 previously normal cases with the unusual combination of 11qand 17pbecame abnormal by the round cell approach. Among previously normal samples, 3 were abnormal for multiple FISH probes by the round cell approach and 13 were abnormal for 1 FISH probe. Discussion: The present experiment shows, as in our past study, that abnormal CLL FISH patterns are confined to round nuclei. Thus, utilization of the round cell approach for scoring FISH in CLL increases the sensitivity to detect a genetic defect. We believe restricting CLL FISH panel scoring to the leukemic B-cell population will be a powerful adjunct to cytogenetic analysis for CLL patients with low lymphocyte counts, including those in clinical remission or with minimal residual disease.