The use of Human Inflammatory Response and Autoimmunity RT2 lncRNA PCR Array for plasma examination in breast cancer patients prior to therapy.

Long non-coding RNAs (lncRNAs) are defined as RNA molecules longer than 200 nucleotides with poor protein-coding capacity and key functions in regulation of gene expression. Dysregulations of lncRNAs (e.g. HOTAIR and MALAT I) were detected in plasma of breast cancer (BC) patients. Plasma samples are examined as liquid biopsies for purposes of non-invasive diagnostics therefore the research of plasma lncRNAs as potential plasma biomarkers became highly topical. 84 lncRNAs were profiled in 18 plasma samples - 9 BC patients and 9 age-matched healthy - using Human Inflammatory Response & Autoimmunity RT2 lncRNA PCR Array. Total RNA from plasma samples was isolated using miRNeasy Serum/Plasma Kit. Although a pre-amplification recommended for quantification from small starting RNA amounts was used, only 3 lncRNAs (A2ML1-AS1, GAS5 and SNHG5) were detected in all plasma samples. A total of 72 lncRNAs (e.g. HOTAIR or MALAT I) were detected only in some samples and 9 lncRNAs were not detected in any samples. No significant differences were observed in levels of plasma lncRNAs between the BC patients and healthy controls despite the fact that our panel contained also the lncRNAs whose levels were previously reported as significantly different in plasma or cancer tissues (e.g. GAS5, HOTAIR, MALAT I) in BC patients. Detection of lncRNAs in plasma is due to their low concentrations quite difficult as compared with tissues. Our findings suggest that analysis of plasma lncRNAs using this technology is not suitable for use as non-invasive diagnostic tool in BC patients.

[Use of Porous Hydrogel as a 3D Scaffold for the Growth of Leukemic B Lymphocytes]. / Vyuzití porózního hydrogelu jako 3D scaffoldu pro rust leukemických B lymfocytu.

BACKGROUND: Primary human B cells chronic lymphocytic leukemia undergoes apoptosis, from which they can be rescued by contact with stromal cells or by the addition of specific soluble factor, when cultured in vitro. For research purposes of the behavior of CLL cells we created 3D in vitro model in which we simulated appropriate microenvironment for CLL cells to allow study the mechanism of survival of these cells in long-term cultivation. MATERIAL AND METHODS: Our aim was the scaffold structure to be geometrically similar to the 3D morphology of supporting bone marrow tissue in a trabecular bone; the 3D scaffold was also designed to conform to biocompatibility, sufficiently large surface area for cell attachment, high porosity for cell migration, proliferation and transport of nutrients. Another requirement was a partial transparency for inspection of cell model with optical techniques. We prepared 3D scaffolds from porous hydrogel poly (2-hydroxyethyl methacrylate) (pHEMA), poly (2-hydroxyethyl methacrylate-co-2-aminoethyl methacrylate) p (HEMA-co-AEMA) and p (HEMA-co-AEMA) modified with frequently used cell adhesion peptide Arg-Gly-Asp (RGD). All hydrogel scaffolds were manufactured in four pore diameters (125, 200, 300 and 350-450 µm). Scaffolds were tested with human bone marrow stromal cell line HS-5 and human embryonic kidney cell line HEK293. RESULTS: Hydrogel scaffold p (HEMA-co-AEMA) modified with adhesion peptide Arg-Gly-Asp (RGD) with pore diameter of 350-450 µm demonstrated that it is a convenient system for 3D cell cultivation, since it promotes interaction between the cells and also between the cells and the material. This scaffold was used for seeding of co-cultivation system of HS-5 cells with CLL-cells, which were stimulated through the CD40L signaling pathway as well as via the IL-4 pathway. Viability of B-CLL cells was higher in the presence of both stimulators than with each alone. CONCLUSIONS: We have shown that 3D scaffold technology is very useful for modeling of microsystems where the cancer cells behave like in their natural microenvironment.Key words: hematooncology - leukemia - hydrogel - stromal cells This work was supported by grant COST CZ LD15144 "Cellular and acellular grounds for regeneration of bones and teeth" awarded by the Ministry of Education, Youth and Sport of the Czech Republic. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 6. 3. 2017Accepted: 26. 3. 2017.

MicroRNAs in urine supernatant as potential non-invasive markers for bladder cancer detection.

Urinary bladder carcinoma contributes to 4% of newly diagnosed oncological diseases in the Czech Republic. Biomarkers for its early non-invasive detection are therefore highly desirable. Urine seems to be an ideal source of such biomarkers due to the content of cell-free nucleic acids, especially microRNAs (miRNAs).To find potential biomarkers among miRNAs in urine supernatant, we examined in total 109 individuals (36 controls and 73 bladder cancer patients) in three phases. In the first - discovery - phase, microarray cards with 381 miRNAs were used for miRNA analysis of 13 controls and 46 bladder cancer patients. In the second - verification - phase, the results of this first phase were verified on the same groups of subjects by single-target qPCR assays for the selected miRNAs. For the third - validation - phase, new independent samples of urine supernatant (23 controls and 27 bladder cancer patients) were analyzed using single-target qPCR assays for 13 verified in the previous phase. The results of all phases were normalized to miR-191, miR-28-3p, and miR-200b, which were selected as suitable for our study by the qBase+®.We found that miR-125b, miR-30b, miR-204, miR-99a, and miR-532-3p are significantly down-regulated in patients' urine supernatant. In our experiments, the analysis of miR-125 levels provided the highest AUC (0.801) with 95.65% specificity and 59.26% sensitivity, the analysis of miR-99a lead to AUC (0.738) with 82.61% specificity and 74.07% sensitivity. We demonstrate that levels of these miRNAs could potentially serve as promising diagnostic markers for the non-invasive diagnostics of bladder cancer.

[Identification of a Family with SUFU Germline Deletion Based on a Case of Desmoplastic Medulloblastoma in an Infant]. / Identifikace rodiny s nosicstvím germinální delece genu SUFU na podklade dia-gnózy desmoplastického meduloblastomu u batolete.

BACKGROUND: Medulloblastoma, an embryonal neuroectodermal tumor of the cerebellum, is the most common malignant brain tumor in children. There are approximately 15 cases diagnosed in the Czech Republic each year. The recent World Health Organization classification recognizes several histopathological subtypes of medulloblastoma: classical, desmoplastic/ nodular with its extensive-nodularity variant, and anaplastic/ large-cell variant. Further molecular analysis identified four basic subgroups of medulloblastoma: WNT, SHH, Group 3, and Group 4. The subgroup of SHH meduloblastoma is associated with somatic mutations of SHH, PTCH1, SUFU, SMO and TP53, while the most common mutations found in infants up to three years of age were PTCH1 and SUFU. The majority of medulloblastomas are sporadic diseases, whereas only about 5- 10% of all cases occur in connection with hereditary genetic syndromes. CASE: We present a case of a 21-months old girl diagnosed with a localized posterior fossa tumor. The histopathological examination revealed a desmoplastic/ nodular medulloblastoma. The treatment comprised a radical exstirpation of the tumor followed by adjuvant chemotherapy. With the use of array-CGH, a partial biallelic deletion of the SUFU gene (locus 10q24.32) was detected in the tumor DNA, whereas a monoallelic deletion was found in the peripheral lymphocyte DNA of the patient. These findings were confirmed by an independent qPCR method. Monoallelic germline deletion of SUFU was also identified in the patients mother, who was a healthy carrier. Pedigree of the family suggested a transition of the germline deletion of SUFU, since another brain tumors (including one case diagnosed before the age of three years) were identified in previous generations. CONCLUSION: Germline mutations in SUFU gene are believed to predispose to infant desmoplastic/ nodular medulloblastomas, basal cell carcinomas and meningiomas. The susceptibility gene shows autosomal dominant inheritance with an incomplete penetrance. There is no evidence-based surveillance strategy suggested for the carriers of germline SUFU mutations/ deletions so far. Our recommendation is based both on a family history of our patient and similar cases described in the literature. Since the germinal mutations in SUFU are responsible for up to 50% of all desmoplastic medulloblastomas in children under three years of age, genetic testing of SUFU should be encouraged in this population of patients.

[Molecular Prognostic Markers and Their Clinical Relevance in Chronic Lymphocytic Leukemia]. / Molekulární prognostické markery chronické lymfocytární leukemie a jejich klinický význam.

Chronic lymphocytic leukemia is the most common leukemia in Western countries affecting particularly elderly adults. Despite the constantly improving therapy options, chronic lymphocytic leukemia is still an incurable disease owing to considerable clinical and bio-logical heterogeneity. Pathogenesis of chronic lymphocytic leukemia is not fully understood; however, aberrant antigenic stimulation, apoptosis deregulation and microenvironmental interactions play a crucial role in disease development. The most important molecular prognostic markers with clinical relevance include mutation status of heavychain immunoglobulin genes (IGHV), presence of cytogenetic aberrations and TP53 and ATM gene mutations. Recent implementation of next generation sequencing technologies has enabled more accurate analysis of both wellestablished and novel potential prognostic markers. The most relevant candidates are mutations in SF3B1, NOTCH1 and BIRC3 genes, which are now intensively studied with respect to their clinical importance. The other examined molecular mechanisms of chronic lympho-cytic leukemia pathogenesis include deregulation of B cell receptor signalization and abnormal regulation of gene expression by microRNA. The precise characterization of molecular abnormalities improves the risk stratification of chronic lymphocytic leukemia patients, which could possibly benefit from new treatment approaches.

[Molecular biological diagnostics of KRAS and BRAF mutations in patients with colorectal cancer - laboratory experience]. / Molekulárne biologická diagnostika mutací genu KRAS a BRAF u pacientu s kolorektálním karcinomem - zkusenosti laboratorního pracoviste

BACKGROUND: Targeted biological therapy based on blocking growth factor receptors and inhibition of cancer-inducing signaling pathways is a new treatment facility for patients with colorectal cancer (CRC). Therapeutic agents are monoclonal antibodies targeting epidermal growth factor receptor (EGFR). Gene aberrations in the EGFR-induced pathways are negative predictors of therapeutic response. Determination of -non-mutated KRAS is a requirement for the indication of targeted anti-EGFR therapy in the present time, BRAF mutation analysis is recommended. Comparison of our results with published data and verification of routine laboratory methods in relation to diagnostic kits were the purposes of this study. PATIENTS AND METHODS: In addition to routine methods based on PCR, direct sequencing as well as two diagnostic kits for KRAS (codon 12 and 13) and BRAF (codon 600) mutation analysis were used for 132 patients. RESULTS: KRAS mutations were detected in 45 patients (34%), V600E mutation of the BRAF gene in 9 patients (7%). Both mutations simultaneously were not detected. Tissues from primary tumor and metastases were available from 33 patients. KRAS mutation was detected in 13 cases of this group. KRAS mutations in tumor and metastasis were of the same type in 9 patients; types of mutation in both tissues were different in one case. KRAS mutation only in one tissue was detected in 3 cases. BRAF mutation in both tissues was detected in the 4 patients. A low percentage of tumor cells in 17 patients specimen did not allow performance of routine analysis and diagnostic kit was used. CONCLUSION: The frequency of KRAS and BRAF mutations in our cohort of patients corresponds to published data. The suitability of metastatic tissue analysis due to tumor heterogeneity was confirmed. KRAS analysis requires a comprehensive methodological approach with regard to reduced DNA quality and different percentage of tumor cells in tissue. For this reason, commercial diagnostic kits constitute a suitable supplement to standard methods.

[Chromosome banding analysis of peripheral blood lymphocytes stimulated with IL2 and CpG oligonucleotide DSP30 in patients with chronic lymphocytic leukemia]. / Cytogenetické vysetrení lymfocytu periferní krve po stimulaci IL2 a CpG oligonukleotidem DSP30 u pacientu s chronickou lymfocytární leukemií

BACKGROUND: Chromosomal aberrations play an important role as prognostic factors in chronic lymphocytic leukemia (CLL). These aberrations are mostly detected by fluorescent in situ hybridization (FISH), as chromosomal banding analysis has been scarce due to low proliferative activity of malignant B-lymphocytes in vitro. In 2006, a new method using stimulation with IL-2 and CpG oligonucleotide DSP30 for metaphase generation in CLL was published [1]. The objective of our study was to verify the efficacy of stimulation and to evaluate if the method is suitable for routine diagnostics. PATIENTS AND METHODS: In total, peripheral blood samples of 369 CLL patients were analyzed in parallel by chromosomal banding analysis and by FISH probes for 13q14, 11q22-23, CEP12 and 17p13. RESULTS: Out of 369 patients, 307 (83%) were successfully stimulated for metaphase generation. Chromosomal aberrations were detected in 243 (79%) out of 307 patients evaluated by chromosomal banding analysis. Other aberrations that are not included into standard FISH panel were detected in patients karyotypes, e.g. del(6q), del(14q), t(14;18)(q32;q21), t(11;14)(q13;q32) and t(18;22)(q21;q11). One hundred and three (42%) patients showed complex aberrant karyotype not detected by FISH analysis. CONCLUSION: Stimulation with IL-2 and oligonucleotide DSP30 is an efficient method how to induce proliferation of malignant B-lymphocytes and allows detection of a substantial number of chromosomal aberrations in addition to those detected by standard FISH panel. Using this method in routine diagnostics is helpful particularly in identification of patients with complex aberrant karyotype.

Long-term results of allogeneic hematopoietic stem cell transplantation after reduced-intensity conditioning with busulfan, fludarabine, and antithymocyte globulin.

UNLABELLED: Reduced-intensity conditioning (RIC) is widely used for allogeneic stem cell transplantation (SCT). Here we present our long-term experience with RIC regimen consisting of fludarabine (30 mg/m2/day on days -10 to -5), busulfan (4mg/kg/day on days -6 and -5) and antithymocyte globulin (ATG Fresenius, 10 mg/kg/day on days -4 to -1) (Flu-Bu-ATG) in a cohort of 71 patients with various hematological malignancies including chronic myeloid leukemia (24 patients), acute myeloid leukemia (19 patients), lymphoma (20 patients), multiple myeloma (3 patients), myelodysplastic syndrome (3 patients), and myelofibrosis (2 patients). The median age was 50 years. The overall response rate was 87%, including 83% CR and 4% PR. The incidence of acute and chronic GVHD was 35% and 52% and the cumulative incidence of non-relapse mortality at 1 year and 4 years was 8% and 14%. With the median follow-up of 55.0 months, the 2- and 4-year event-free survival (EFS) was 49.0% and 40.3%, and the overall survival (OS) was 73.2% and 62.6%, respectively. Gender, age at SCT, type of donor, disease status at SCT, previous autologous transplantation, and complete chimerism by day +100 did not significantly influence EFS and OS. In a multivariate analysis, no presence of chronic GVHD (p=0.029, HR: 2.5),and diagnosis other than CML (p=0.018, HR: 4.6), and CD34+ dose < 5x106/kg (p=0.010, HR: 2.8) were significant predictors of poor OS. Flu-Bu-ATG protocol is a RIC regimen that combines effective disease control with low non-relapse mortality and acceptable toxicity profile. KEYWORDS: reduced-intensity conditioning, fludarabine, busulfan, antithymocyte globulin.

MicroRNA isolation and stability in stored RNA samples.

MicroRNAs (miRNAs) are small RNA molecules, which act as post-transcriptional regulators of a gene expression, with important functions within the cell physiology. Whilst many authors have focused on the study of miRNA expression in physiological and pathological processes, various technical variables related to miRNA isolation have simultaneously emerged and the stability of the stored miRNA samples has been questioned. A robust method for RNA isolation is essential for reproducible results and miRNAs instability in the stored samples would make for an alarming situation for most expression studies. Here these issues are discussed and we investigate the stability of miRNAs isolated from clinical samples of B lymphocytes (chronic lymphocytic leukemia) by the most commonly utilized method based on a Trizol/TRI-Reagent solution (RNAs stored at -80 degrees C). To assess the stability of miRNAs, a Real Time-PCR analysis was performed for a panel of 29 miRNAs from a freshly isolated RNA sample and after 14 days storage at -80 degrees C. Furthermore, a Real Time-PCR analysis was repeatedly performed for a stored RNA sample over a period of approximately 10 months. We observed high stability of isolated miRNAs and respective cDNAs. The reproducibility and efficiency of the Trizol/TRI-Reagent isolation method was also tested and compared to the mirVana Isolation kit (Ambion) and RNeasy kit (Qiagen). In conclusion, Trizol/TRI-Reagent based isolation is a robust reproducible method, and obtained miRNA samples do not show any tendency to degradation when properly stored and handled.

Identification of somatic hypermutations in the TP53 gene in B-cell chronic lymphocytic leukemia.

Abnormalities of the TP53 gene are associated with a particularly severe prognosis in patients with B-cell chronic lymphocytic leukemia (B-CLL). This tumor-suppressor is mostly inactivated by the deletion of one and point mutation of the other allele and has not been previously shown to be hypermutated in B-CLL. We identified two patients whose lymphocytes showed repeatedly an extensive proportion of TP53 mutated cells by FASAY analysis (the yeast functional assay) and harbored various TP53 mutations, mostly single-base substitutions, in individual cells. The mutation targeting exhibited characteristic traits of the somatic hypermutation process. In the first patient (harboring the unmutated IgVH locus) a significant bias to point mutations at CG pairs (21/25; P=0.009), their remarkable preference for the RGYW/WRCY motives (28%) and the highest expression of the activation-induced cytidine deaminase (AID) mRNA among the 34 tested B-CLL samples. In the second patient no CG bias was observed but the targeting of point mutations into the RGYW/WRCY motives was even more prominent here (7/16; 44%). Moreover, six out of eight point mutations affecting AT pairs were localized in the WA/TW motives, which are also characteristic for the somatic hypermutations. This patient, who was IgVH-mutated, already did not express any significant amount of the AID transcript. Our findings add a new aspect to the mosaic of the p53 mutability in B-CLL.

[Human genome sequencing--next generation technology or will the routine sequencing of human genome be possible?]. / Sekvenování lidského genomu--technologie nové generace aneb budeme rutinne sekvenovat lidské genomy?

DNA sequencing has become a standard method widely used in molecular genetic analysis of biological materials. Its use in medicine is widespread, especially in diagnostics of inherited disorders and cancer related diseases. Development of DNA diagnostics has been strongly accelerated by publication of the human genome sequence in 2001. During the last few years one can observe rapid development of novel sequencing technologies, which have led to the introduction of so called "New Generation Sequencing". These new technologies based on principles of massive parallel sequencing (e.g. Roche/454, Illumina Genome Analyzer IIx, Life Technologies SOLiD 3 and others) enable a massive increase of sequencing capacity and in parallel also a fundamental decrease of costs. This major technological breakthrough allowed development of the whole-genome sequencing including analyses of individual human genomes. It also started the era of personal genomics. The first sequenced individual human genomes belonged to famous geneticists J. C. Venter (2007) and J. D. Watson (2008), but they were rapidly followed by sequencing analyses of other individuals from various ethnic groups. These studies brought substantial information about interpersonal differences in genome structure (through characterization of nucleotide polymorphisms, DNA deletions and amplifications etc.). Sequencing of cancer cell genomes, e.g. acute myeloid leukemia has already brought first important clinically relevant results. Although currently we are still unable to interpret the relevance of all detected genome variants, it is obvious, that the possibility to sequence individual human genomes represents a fundamental breakthrough not only in DNA diagnostics but also in clinical medicine.

[Proteomic analysis of cancer cells]. / Proteomická analýza nádorových bunek.

The rapid development of analytical instrumentation and methodical approaches in the course of the last two decades has significantly extended the possibilities of studying proteins in living systems. Proteomic analysis provides ever deeper insights into the molecular nature of biological processes in terms of qualitative and quantitative changes in protein composition in connection with the physiological and pathological states of the organism. Thus, proteomic analysis contributes to a better understanding of these processes and becomes a tool for the development and validation of diagnostic and therapeutic approaches. Thanks to recent achievements, the attention of cancer specialists is more and more focused on human proteome research. In this brief review we explain the principles of widely used proteomic techniques (gel electrophoresis, liquid chromatography, mass spectrometry analysis, protein array technologies) and show examples of their application in oncology, namely hematooncological diseases.

Chromothripsis - Extensive Chromosomal Rearrangements and Their Significance in Cancer.

BACKGROUND: Chromosome rearrangements play an important role in cancer pathophysiology. Recently, chromothripsis has been proposed among the mechanisms leading to their formation. Chromothripsis leads to fragmentation of chromosomes and their reconstitution with tens to hundreds of rearrangements clustered in small genome regions. In contrast to the traditional concept of malignant transformation, abnormalities caused by chromothripsis are not accumulated gradually but arise during a single event. The resulting structural variants are extensive and often cause oncogene activation or tumor suppressor inactivation. Chromothripsis is associated with many tumor types, especially with brain and bone tumors. Besides that, it has been described also in congenital disorders. The exact mechanism of chromothripsis origin has not been clarified yet; however, several hypotheses have been prosed, among which DNA damage in micronucleus seems to be most likely. Similarly, an impact of chromothripsis on cellular processes has not been fully understood, yet its association with unfavorable prognosis has been observed. PURPOSE: The purpose of this article is to summarize the current knowledge about chromothripsis and to present gathered pieces of information in a structured way. We focused on describing the basic features of chromothripsis, potential mechanisms of its origin, its impact on cellular processes and providing an overview of diseases where chromothripsis has been noted, with particular attention to cancer. Finally, we suggest a potential use of current knowledge about chromothripsis in the optimization of personalized treatment. Supported by Ministry of Health of the Czech Republic, grant no. 15-31834A. All rights reserved. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 31. 12. 2018 Accepted: 19. 3. 2019.

Encyclopedia of CLL Subsets - a Unique Bioinformatics Tool and Database for Analysis of Subsets of Stereotypical B-Cell Receptors in CLL.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is clinically and biologically highly variable disease which is closely related with multiple cellular and molecular markers, including sequence motifs of B-cell receptors. These motifs are highly similar (stereotyped) within one third of CLL patients and create homogeneous groups called stereotyped CLL subsets. The homogeneity is reflected also in clinical and biological characteristics of the disease. To facilitate access to the information about individual subsets, we have created a publicly available web-based tool Encyclopedia of CLL Subsets. MATERIALS AND METHODS: The Encyclopedia of CLL subsets belongs to our bioinformatics platform Antigen Receptor Research Tool (ARResT) developed for analysis, clustering, and annotation of immunoglobulin sequences. To gather primary knowledge about the subsets, we have analyzed a dataset of 7,500 CLL patients published by Agathangelidis et al in 2012 [1]. We have created an overview of major stereotyped subsets and their characteristics. Additional clinical and cytogenomic information about individual subsets has been obtained by machine text processing of available literature from server PubMed and is regularly updated. RESULTS: We have created a unique web-based application Encyclopedia of CLL Subsets available from http: //arrest.tools/subsets for an interactive access to the information about stereotyped CLL subsets. A user can obtain and compare basic information about the major subsets including their clinical and cytogenomic characteristics. These have been manually curated from machine processed results from PubMed database by experts in CLL research. Through the Encyclopedias user interface, user can also directly use our published tool ARResT/AssignSubsets to assign new immunoglobulin sequences to the major subsets. CONCLUSION: The Encyclopedia of CLL Subsets is a publicly available online tool facilitating access to the most recent research knowledge about stereotyped CLL subsets and enabling analysis of own data and interpretation of the results. This gives the Encyclopedia a great potential for its use in clinical routine. This work was supported by Czech Ministry of Health grant No. 34272A. All rights reserved. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 1. 3. 2019 Accepted: 4. 3. 2019.

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 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.

ERIC recommendations for TP53 mutation analysis in chronic lymphocytic leukemia-update on methodological approaches and results interpretation.

In chronic lymphocytic leukemia (CLL), TP53 gene defects, due to deletion of the 17p13 locus and/or mutation(s) within the TP53 gene, are associated with resistance to chemoimmunotherapy and a particularly dismal clinical outcome. On these grounds, analysis of TP53 aberrations has been incorporated into routine clinical diagnostics to improve patient stratification and optimize therapeutic decisions. The predictive implications of TP53 aberrations have increasing significance in the era of novel targeted therapies, i.e., inhibitors of B-cell receptor (BcR) signaling and anti-apoptotic BCL2 family members, owing to their efficacy in patients with TP53 defects. In this report, the TP53 Network of the European Research Initiative on Chronic Lymphocytic Leukemia (ERIC) presents updated recommendations on the methodological approaches for TP53 mutation analysis. Moreover, it provides guidance to ensure that the analysis is performed in a timely manner for all patients requiring treatment and that the data is interpreted and reported in a consistent, standardized, and accurate way. Since next-generation sequencing technologies are gaining prominence within diagnostic laboratories, this report also offers advice and recommendations for the interpretation of TP53 mutation data generated by this methodology.

Restrictions in the T-cell repertoire of chronic lymphocytic leukemia: high-throughput immunoprofiling supports selection by shared antigenic elements.

Immunoglobulin (IG) gene repertoire restrictions strongly support antigen selection in the pathogenesis of chronic lymphocytic leukemia (CLL). Given the emerging multifarious interactions between CLL and bystander T cells, we sought to determine whether antigen(s) are also selecting T cells in CLL. We performed a large-scale, next-generation sequencing (NGS) study of the T-cell repertoire, focusing on major stereotyped subsets representing CLL subgroups with undisputed antigenic drive, but also included patients carrying non-subset IG rearrangements to seek for T-cell immunogenetic signatures ubiquitous in CLL. Considering the inherent limitations of NGS, we deployed bioinformatics algorithms for qualitative curation of T-cell receptor rearrangements, and included multiple types of controls. Overall, we document the clonal architecture of the T-cell repertoire in CLL. These T-cell clones persist and further expand overtime, and can be shared by different patients, most especially patients belonging to the same stereotyped subset. Notably, these shared clonotypes appear to be disease-specific, as they are found in neither public databases nor healthy controls. Altogether, these findings indicate that antigen drive likely underlies T-cell expansions in CLL and may be acting in a CLL subset-specific context. Whether these are the same antigens interacting with the malignant clone or tumor-derived antigens remains to be elucidated.

EGR2 mutations define a new clinically aggressive subgroup of chronic lymphocytic leukemia.

Recurrent mutations within EGR2 were recently reported in advanced-stage chronic lymphocytic leukemia (CLL) patients and associated with a worse outcome. To study their prognostic impact, 2403 CLL patients were examined for mutations in the EGR2 hotspot region including a screening (n=1283) and two validation cohorts (UK CLL4 trial patients, n=366; CLL Research Consortium (CRC) patients, n=490). Targeted deep-sequencing of 27 known/postulated CLL driver genes was also performed in 38 EGR2-mutated patients to assess concurrent mutations. EGR2 mutations were detected in 91/2403 (3.8%) investigated cases, and associated with younger age at diagnosis, advanced clinical stage, high CD38 expression and unmutated IGHV genes. EGR2-mutated patients frequently carried ATM lesions (42%), TP53 aberrations (18%) and NOTCH1/FBXW7 mutations (16%). EGR2 mutations independently predicted shorter time-to-first-treatment (TTFT) and overall survival (OS) in the screening cohort; they were confirmed associated with reduced TTFT and OS in the CRC cohort and independently predicted short OS from randomization in the UK CLL4 cohort. A particularly dismal outcome was observed among EGR2-mutated patients who also carried TP53 aberrations. In summary, EGR2 mutations were independently associated with an unfavorable prognosis, comparable to CLL patients carrying TP53 aberrations, suggesting that EGR2-mutated patients represent a new patient subgroup with very poor outcome.

High-quality full-length immunoglobulin profiling with unique molecular barcoding.

High-throughput sequencing analysis of hypermutating immunoglobulin (IG) repertoires remains a challenging task. Here we present a robust protocol for the full-length profiling of human and mouse IG repertoires. This protocol uses unique molecular identifiers (UMIs) introduced in the course of cDNA synthesis to control bottlenecks and to eliminate PCR and sequencing errors. Using asymmetric 400+100-nt paired-end Illumina sequencing and UMI-based assembly with the new version of the MIGEC software, the protocol allows up to 750-nt lengths to be sequenced in an almost error-free manner. This sequencing approach should also be applicable to various tasks beyond immune repertoire studies. In IG profiling, the achieved length of high-quality sequence covers the variable region of even the longest chains, along with the fragment of a constant region carrying information on the antibody isotype. The whole protocol, including preparation of cells and libraries, sequencing and data analysis, takes 5 to 6 d.

A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study.

In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10(-5)). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10(-4)) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10(-6)). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.