Scientific Projects

Scientific Projects

Our research projects centre on the following topics:

  

ERG deletions in ALL

Intragenic ERG deletions represent one of the recurrent aberrations in childhood ALL. They occur mainly in a subtype of childhood ALL defined by a specific gene expression profile and also in B-precursor ALLs that undergo switch to myeloid lineage during the induction treatment. The presence of ERG deletions neutralizes the negative prognostic impact of the other factors frequently associated with this aberration, such as IKZF1 deletions or higher age at disease presentation (Zaliova et al., Leukemia 2014). The aim of this project is to characterize origin and repertoire of ERG deletions and to elucidate their biological role in childhood ALL.

marketa.zaliova@lfmotol.cuni.cz

 

 

NGS a SNParrays in classification of childhood ALL and in identification of therapeutic targets and novel aberrations

With the introduction of high-throughput technologies, such as high-density SNParrays and next generation sequencing (whole exome sequencing (WES) and whole transcriptome sequencing (RNAseq)) the classification of childhood ALL was further refined and the list of recurrent aberrations (both primary and secondary) occurring in childhood ALL was significantly extended. We learn more and more about the genetic background (and thus about the biology) of childhood ALL and we also find new prognostic factors and therapeutic targets among the newly discovered lesions. Moreover, novel non-recurrent aberrations (so called private aberrations) are found in a certain proportion of patients. Theses aberrations cannot be diagnosed by other means than using the above mentioned non-targeted extensive approaches. The aim of this project is to characterize and re-classify so called B-other ALL (shortly, the B-precursor ALLs that are not classified into genotype subgroups by routine diagnostics) and to identify potential therapeutic targets in resistant ALLs using the modern genetic approaches. Using in vitro experiments we also aim to study oncogenic potential of the newly discovered aberrations and their impact on cellular signaling.

marketa.zaliova@lfmotol.cuni.cz

 

 

Leukemias bearing TEL/ABL fusion

TEL/ABL fusion gene rarely occurs in human leukemias (Zuna & Zaliova et al., Genes Chromosomes & Cancer, 2010), however, its identification is very important for therapy tailoring as it represents a target of specific therapeutics – tyrosine kinase inhibitors (TKI). The aim of this project is to characterize TEL/ABL-positive leukemias at both clinical (frequency, disease manifestation, prognosis, role of TKI in treatment) and biological levels (expression and genomic profile) and to study potential mechanisms of TKI-resistance.

marketa.zaliova@lfmotol.cuni.cz

 

 

Minimal residual disease monitoring in childhood AML

While minimal residual disease (MRD) monitoring is an inherent part of modern therapeutic protocols in childhood ALL (where it represents an essential tool for risk assessment and therapy tailoring) its use in therapeutic protocols of childhood AML was not fully established so far. The aim of this 5-year observational study is 1) to assess practical applicability of MRD monitoring in childhood AML (efficiency of leukemia-specific target identification and of the establishment of highly sensitive detection systems); 2) to test use of peripheral blood instead of bone marrow samples and 3) to design an optimal sampling schedule in order to establish these techniques as a standard part of upcoming therapeutic protocols.

marketa.zaliova@lfmotol.cuni.cz

 

 

Role of homoeobox genes in leukaemogenesis

Homoeobox genes play a key role in both embryo- and organo-genesis. They are also supposed to be involved in cancer growth, namely in leukaemogenesis, considering the aberrant expression as it has been described. The aim of the project is to study the mechanisms responsible for the dysregulation of HOX genes. We have decided to study three of such mechanisms: DNA metylation, regulating the transcription of HOX genes during the embryonic development; mutations in the core promoter of HOX genes and alternative signalling caused by mutations of tyrosine kinases. 

julia.starkova@lfmotol.cuni.cz

 

 

Next generation sequencing for analysis of antigen receptor rearrangements and its use for assessment of immune system pathologies

Next generation sequencing is a novel method resulting in hundreds of thousands to millions of sequences that becomes accessible in common laboratory practice. NGS offers an ideal tool for assessment of immunoglobulin (Ig) and T-cell receptor gene (TCR) rearrangement spectrum that would give complex information on representation and kinetics of lymphocyte clones with particular sequences of antigen receptors. In this project we want to validate the method for minimal residual disease (MRD) evaluation in lymphoid malignancies, assuming that NGS will outperform the current method of concrete clone quantification in every aspect, including costs. Concurrently with MRD, it will be possible to study restoring of Ig and TCR repertoire after initial chemotherapy and stem cell transplantation in childhood acute lymphoblastic leukemia and kinetics of particular non-malignant lymphocyte populations targeting infections, residual leukemic cells, or self-antigens in case of graft-versus-host disease

eva.fronkova@lfmotol.cuni.cz

 

 

Predicting resistence of leukaemic cells to L-Asparaginase

Acute lymphoblastic leukaemia, the most prevalent form of childhood cancer, is treated using a combined chemotherapy involving the L-Aspariginase enzyme (L-Asp). Its cytotoxic effect is based on its ability do deplete extracellular asparagine and glutamine, to which depletion leukaemic cells are particularly sensitive; most likely due to the lowered activity of the glutamine-dependent enzyme Asparagine Synthetase (ASNS). Although the resistance to L-Asp has been associated with the heightened level of ASNS in leukaemic blasts, the hypothesis has not been corroborated by latest research. The main goal of our project is to identify the genes associated with the workings of L-Asp in leukaemic cells and to discover the pathways involved in the development of resistance.

julia.starkova@lfmotol.cuni.cz

 

The origin of childhood leukaemias

The goal of the project is a detailed analysis of preleukaemic cells, obtained for this purpose using four different approaches. A combination of the four ought to make possible a molecular-genetic, cytogenetic and functional characterisation of preleukaemic cells, as well a clarification of at least some mechanisms leading them to take the full leukaemic turn. Other then learning about the origins, genesis and development of the leukaemic clone, this approach can ultimately contribute towards discovering a successful means of affecting this transformation and finding the optimum treatment strategy in the already diagnosed cases, for it is the leukaemic cells missed by chemotherapy that in some cases lead to a later recurrence of the disease. 

 jan.zuna@lfmotol.cuni.cz

 

 

Role of WT1 gene in haematopoiesis and a leukaemogenesis

Acute myeloid leukaemia (AML) is the second most frequent type of childhood acute leukaemia, and a very aggressive one at that. Its treatment results in a successful remission in only about half of the patients. New therapeutics targets are therefore sought, such that would help improve the prognosis. Among these is the WT1, a gene involved in the regulation of haematopoietic development and one whose abnormal expression and/or mutation is found in most of leukaemias and other blood cancers. The goal of this project is a detailed knowledge about WT1’s role in haematopoiesis and laukaemogenesis, including assessing its potential significance in AML therapy.

ludmila.boublikova@lfmotol.cuni.cz

 

 

Studying lineage plasticity of leukaemic cells

It was presupposed in certain older models of haematopoiesis that the first step in the development of haemotopoietic cells is the strict separation of the lymphoid and myeloid lineages. Recent developments have indicated that the lineage plasticity is greater and that on their way towards the lymphoid/myeloid form, the cells can pass through a stage of common T/myelo and B/myelo progenitors. Possibly, the leukaemic cells have an augmented potential towards this, or their direct non-malignant counterpart is the T/myelo or B/myelo progenitor cell. Among children patients with acute leukaemias, we have identified some with T/myelo mixed-phenotype acute leukaemias (MPAL’s), and some who ‘lineage-switched’ from B-lymphoid to myeloid leukaemia during induction treatment. The lineage-switch is studied using a murine model.

ester.mejstrikova@lfmotol.cuni.cz

 

 

Differentiation plasticity of haematopoietic cells

For a long time, haematopoiesis was understood to be a strictly regulated process that, by way of gradual narrowing down the developmental choices of cell lineages, gives finally rise to fully differentiated cells. A number of recent papers, though, has documented the development of myeloid cells from myeloid precursors under certain, molecularly defined, conditions. Often seen in blood cancers is the aberrant expression of various antigens. It is not yet clear whether such promiscuousness stems from the naturally occurring developmental changes. The goal of this project is to clarify the relationship between the promiscuousness of the malignant vis-a-vis the plasticity of the normal haematopoietic cells, and to describe the regulatory pathways leading either to lineage restriction or to release from such restriction.

ondrej.hrusak@lfmotol.cuni.cz

 

 

MAP (Microsphere-based affinity protomics) 

A project conceived in cooperation with F. Lund-Johansen,RikshospitaletUniversityHospital,Oslo,Norway, the goal of which is to design and validate antibody arrays able to uncover new prognostic features of childhood leukaemias. The method utilises differential detergent lysis to separate the subcellular compartments of cell lysates; size-exclusion chromatography to differentiate between protein sizes; and flow cytometry to sort the colour-coded microspheres and signal detection. It enables the simultaneous detection of up to 1700 proteins, thus being one of the ‘high-content’ methodologies. The key problem to be addressed is finding the candidate targets, analysing the primary leukaemic cells at diagnosis and to cessing ormatically  ukaemic cells during diagnosiss. ttween the promiscuousness of the malignant vs. s  conditions. gine-synbioinformatically process the resulting data. The project has been awarded financial support fromIceland,LiechtensteinandNorwayby way of the EEA andNorwaygrants mechanism.

tomas.kalina@lfmotol.cuni.cz

 

 

Reconstitution of effective CMV-specific immunity

In this project, we monitor the reconstitution of CMV-specific immunity in childhood patients having undergone haematopoietic stem cells transplantation. For the detection of CMV-specific subpopulations of CD4+ and CD8+ cells, we used the method of intracellular cytokine detection based on polychromatic flow cytometry. The obtained data is then compared with the levels of viral load determined by RQ-PCR and with the clinical progression of the CMV infection at given time-points. The results will help set the reference values that will be then used as a basis for a more efficient preemptive therapy protocol, limiting the scope of adverse side effects.

tomas.kalina@lfmotol.cuni.cz

 

Standardization of 8-colour Flow Cytometric diagnosis in haematology 

Cytometry is a key method for blood cancer diagnosis. The demanding methodology and the complexity of diagnostics has so far hampered any attempts to compare data directly between laboratories. CLIP’s cytometry department, as a founding member of the international EuroFlow consortium (www.euroflow.org), actively pursues the development of standardised practice, designing panels for measuring minimal residual disease and currently works to pass EuroFlow’s know-how to other Czech labs.

tomas.kalina@lfmotol.cuni.cz

 

Analysis of tumor cells and microenvironment using mass cytometry and protein arrays

Proposed project will develop a technological platform for investigation of complex suspension tissues (peripheral blood, bone marrow and CUSA aspirates) that contain a tumor infiltration. The approach would combine and complement two innovative high-content approaches: a single cell mass cytometry (“cancer cytomics”) to determine changes in cellular composition of tumor affected tissue and SEC-MAP array (“proteomics”) to determine changes in proteome of tumor cells. First, a reference of normal cellular development will be established for bone marrow resident B-cells to which abnormal (leukemic) cells could be compared in search for biomarkers. Next, SEC-MAP will be used to determine tumor specific proteome changes (in leukemia and in brain tumors as a model). Last, useful biomarkers will be built into single cells analysis by mass cytometry panels for “cancer cytomics” investigation. Bioinformatic tools will be developed to enable understanding the complex data. 

tomas.kalina@lfmotol.cuni.cz

 

Dysregulation of immune system: characteristics of lymphocytes in patients with immunodeficiency and autoimmunity

Immune defects are often a combination of inadequate infection control and autoimmune phenomena caused by overreaction to autologous antigens. Underlying cause may be found at impaired function of T and B cells and improper regulation of their cooperation. In the proposed project, we will characterize composition, abnormalities and systemic relationships of normal and abnormal subsets of lymphocytes using cutting edge techniques of multi-color flow cytometry (MC-FACS) and molecular genetics in all primary immunodeficiency (PID) patients in both centers and in patients with Common Variable Immunodeficiency (CVID) and selective IgA deficiency. Bioinformatic analysis of standardized MC-FACS data from 150 patients will serve us to develop a diagnostic scheme together with EuroFlow PID international group for PIDs with established molecular genetic lesion. Furthermore, cases with commonalities in MC-FACS and clinical presentation will be investigated by whole exome sequencing to elucidate new genetic variations leading to PID. 

tomas.kalina@lfmotol.cuni.cz


Immunopathological mechanisms of bone marrow failure 

The bone marrow and its microenvironment is a specific organ, one whose functioning is influenced by many factors. Its failure may come about as a result of inner pathological states, such as acute leukaemia, or it may be caused indirectly, e.g. by the cytokines produced by leukaemic and activated cells of the immune system. In aplastic anaemia, the cytotoxic T-lymphocytes have been proven to be involved. Still, the general mechanisms leading to bone marrow failure have not been entirely clarified yet. Our project focuses on both congenital and acquired blood marrow failures and its primary goal is to identify the T-lyphocyte subpopulation involved in the failure. We then intend to further study the subpopulation with respect to its proliferation history and cytokine profile; we also want to take a closer look at the haematopietic stem cells under such altered conditions. 

ester.mejstrikova@lfmotol.cuni.cz