About the project and description of the research programme

Description of the Beneficiary University or Department

The project is intended to be grounded on the existing laboratory facilities of the National Specialized Hospital for Active Treatment of Haematologic Diseases (NSBALHZ) – Sofia, Bulgaria, i.e. the Laboratory of Cytopathology, Histopathology, Immunology and Immunochemistry and the Laboratory of Cytogenetics and Molecular Biology, in collaboration with the Hematology Clinic [designated as Partner#1]. The NSBALHZ is the assignee of a unique medical institution with a 60-years old history. It was initially founded in 1948 as a Research Institute of Hematology and Blood Transfusion with a Clinical Department. In 1972 it was included in the structures of the Medical Academy of Sofia and since 1990 it has been an independent institution, founded and regulated by a Governmental Decree. According the Decree ¹32/22th Feb 2008 the NSBALHZ was established to undertake all activities in regards to diagnosis, therapy and monitoring of blood diseases, as well as the research and education in the field of Clinical Hematology.

Laboratory of Cytopathology, Histopathology, Immunology and Immunochemistry

The Laboratory of Cytopathology, Histopathology, Immunology and Immunochemistry is a Hematopathology-oriented national-scale laboratory that has the specialised expertise in:

Cytomorphological and pathohistological examination of bone marrow, peripheral blood, body fluids (cerebrospinal fluid, pleural fluid, ascites), aspiration biopsy for accessible tumor masses, lymph nodes, skin lesions; spleen; tumors with other primary localization aiming at precise characterization of disorders of the blood and lymphoid tissues

Specialized cytolochemical invesigation for hematological disorders through investigation of bone marrow and peripheral blood cells for the detection of enzymatic activity; lipids; carbohydraytes; detection of iron stores in the bone marrow and pironinophilia for RNA synthesis.

Specialized immunohistochemical expertise for identification of more than 30 leukocyteassociated antigens for diagnosis and subcategorization of hematological neoplasms and their distinction from tumours of other histological origin (carcinomas, sarcomas, melanoma); determination of the proliferative index through immunohistochemical staining for Ki-67, PCNA, etc for prognostic purposes, identification of tumour markers (p53, NSE, NPM delocalization) in the neoplastic cell populations; or in-cell amplified molecular markers by RT-CPR at a single cell level (BCR-ABL, MDR1).

Flow cytometric analysis for immunophenotyping of

  1. lymphocytes in the blood and in the body fluids and determination of the absolute counts of the main subpopulations – Â-cells, CD4+ and CD8+ T- cells, TCRαβ and TCRγδ Ò-cells, activated HLA DR+ Ò-cells, NK-cells, etc.;
  2. leukemias from the blood and the bone marrow, incl. identification of leukemic cells in other body fluids (e.g., CSF, body cavities exudations) – determination of the leukemic cells lineage (myeloid, B-/T-/NK-lymphoid, undifferentiated, biphenotypic, biclonal), maturation, aberrant phenotypes and prognostic markers;
  3. leukemic phase lymphomas from the blood, the bone marrow or body fluids– identification, determination of the lymphoid differentiation, identification of prognostic phenotypic markers or antigens for targeted therapy.
  4. monitoring of minimal residual disease;
  5. CD34(+) HSC counts in the blood after mobilization treatment and in the final graft for HSCT; detection and counting of HSC subpopulations (CD133, CXCR4);
  6. detection of residual leukocytes in leukocyte depleted blood derived products;
  7. determination of multidrug resistance through determination of the level of MDR1/P-glycoprotein;
  8. flow cytometric beads based array for quantitation of in cell levels of apoptosis related markers bcl-2, activated Caspase-3, cPARP in cell lysates from bone marrow;
  9. flow cytometry based DNA analysis;
  10. flow cytometry based “in cell” PCR analysis.

Determination of proteins in serum and other body fluids:: âêëþ÷èòåëíî

  1. total immunoglobulins (IgG, IgM, IgA) using nephelometric analysis;
  2. Cryoglobulins;
  3. Ñ3 and Ñ4 complement fractions, RF, ÑRP and ÀST using nephelometric analysis on demand.

Determination of monoclonal proteins IgG, IgA, IgM, IgD, IgE, kappa and lambda free light chains in serum, urine and CSF through:

  1. Immunoelectrophoresis;
  2. Immunofixation.

Relevant equipment is available such as general laboratory and histological tools and microscopes, bench-top flowcytometers [a 3-colour 5-parameters FACSCalibur, and an 8- colour 10-parameters FACSCantoII]; cytocentrifuge, nephelometer, automatic capillary electrophoresis system.

The laboratory has hosted and participated in several national scientific projects funded by the National Research Fund focused on Cytologic, genotypic, immunomorphologic and flowcytometric study of Disease Free Survival in patients with leukaemias and lymphomas; New iImmunophenotypic approaches for Haematopoietic Stem Cells, Studies of apoptotic and regulatory markers in myelodysplastic syndromes (MDS) and early diagnosis of leukaemic evolution; Modern diagnostic approaches for staging and prognosis in patients with early breast cancer; Ìîrphological, molecular and immunological analysis of heterogeneity and clonal evolution of Ph-positivie leukemia; as well as in international projects on Studies of apoptosis in MDS and AML; p53, MDM2 isoforms & WAF1/cip1 proteins in Hodgkin’s lymphomas” (with the Department of Histology, University of Athens, Greece, funded by the International Union Against Cancer (UICC)).(Supplementary material - list of projects)

Laboratory of Cytogenetics and Molecular Biology

Laboratory of Cytogenetics and Molecular Biology is a unique laboratory in our country specialized on haematological disorders which has the expertise to perform the following genetic and molecular studies:

Cytogenetic tests - Karyotyping using chromosome banding techniques.

Fluorescent in situ hybridization (FISH) - BCR-ABL/t(9;22); MLL rearrangements involving 11q23; 5q31 anomalies.

Molecular genetic analysis by PCR based methods including Nested Primers PCR, in cell RT-PCR, PCR Restriction Fragment Length Polymorphism (RFLP), PCR-Enzyme-Linked Immuno Sorbent Assay (ELISA), Quantitative Real-Time PCR, ARMS-PCR (Amplification Refractory Mutation System) of:

  • Fusion genes expression of: (major) BCR-ABL/t(9;22); (minor) BCR-ABL/t(9;22); (micro) BCR-ABL/t(9;22); AML1-ETO/t(8;21); CÂFb-MYH11/inv(16)/t(16;16); PMLRARA/ t(15;17); MLL-AF9/t(9;11); MLL-AF6/t(6;11); MLL-AF4/t(4;11); TELAML1/ t(12;21); E2A-PBX1/t(1;19); SIL-TAL1; FLT3-ITD.
  • Aberrant gene expression of: MDR1; MRP; BCRP1; LRP; EVI1; BAALC; PRAME; Survivin genes.
  • Gene rearrangements for: IgH; BCL2-IgH/t(14;18)
  • Other markers: Cytokeratin 19; Mamoglobin; JAK2 mutations
  • Quantitative PCR for: (major) BCR-ABL/t(9;22); (minor) BCR-ABL/t(9;22); WT1; TEL-AML1/t(12;21)

Relevant equipment is available such as conventional thermocyclers (MastercyclerGradient, Techne TC3000. Techne TC 412); Real-Time Thermocyclers (Bioer, RotorGene); CO2 incubator Haereus; Hybridizer Haereus; UV cross linker; Photodocumentation System; a fluorescent microscope Olympus with image documentation system for the cytogenetic studies.

The laboratory has hosted and participated in 6 national scientific projects funded by the National Research Fund on the Prognostic significance of BCR-ABL transcripts in patients with leukemia; the activity of Wilms' tumor (WT1) and survivin genes and their prognositic significance for induction of complete clinical remission in patients with acute myeloid leukemia; Minimal residual disease in patients after high-dose and myeloablative therapy; as well as in 4 international projects on the Application of PCR in hematological malignancies (with Institute Paoli-Calmettes, Marseille, France; funded by Centre International des Étudiants et Stagiaires (CIES)); Molecular screening for gene rearrangements in haematological malignancies (with Necker-Enfans Malades, Paris, France, supported by TEMPUS Programme); Development of plasmid standards of the main fusion transcripts involved in leukemias for Real Time quantitative PCR detection [a component of Biomed-I, with Institute Paoli-Calmettes Marseille, France, Supported by International Union Against Cancer (UICC) and EU - Europe Against Cancer (EAC)]; Molecular genetics, immunological and Functional study of the multidrug resistance (MDR) in patients with leukemias, particularly the analysis of MDR1 gene expression in Ph-positive chronic and acute leukaemias (Bulgarian-Indian Co-operative Project).

Hematology Clinic

The Hematology Clinic is a highly specialized unit that concentrates patients from the whole country due to the unique opportunity to provide a broad range of activities in the area of prophylaxis, diagnostics and integrated treatment of patients with inborn and acquired, benign and malignant hematological diseases. In total the Clinic has 60 beds, including 8 boxes for HSCT and an out-patients department. The number of hospitalized patients for 2007 is 5094, and of out-patients – 5007, with a total number of patient-days of 42 454.

Project Collaborators

Partner #2
Prof. Elias Campo
Hematopathology Unit, Hospital Clinic, University of Barcelona, Barcelona, Spain

The Hematopathology Unit of the Hospital Clinic, University of Barcelona has a long experience in all aspects of diagnostic Haematopathology including morphology, immunophenotyping, cytogenetics, and molecular biology. The research activity of the group during the last 15 years has concentrated on the genetic and molecular mechanisms involved in the pathogenesis of malignant lymphomas. In these fields, the group pioneered the recognition of Cyclin D1 as a specific alteration of MCL as well as the involvement of genetic alterations in different cell cycle regulators and DNA-damage response genes in the progression of this disease. The group participates actively in different international research networks including the European MCL Research Network since 1997 and the Leukemia/Lymphoma Molecular Profiling Project.

Partner #3
Assoc. Prof. Dr. Spiro Konstantinov, Georgi Momekov, PhD, Dejan Josifov
Laboratory of Experimental Chemotherapy, Faculty of Pharmacy, Medical University of Sofia

The laboratory is fully equipped and highly experienced in isolation and establishing of primary cultures from hematopoietic cells, and routine quantitative estimation of cytotoxicity. A long-term fully functional collaboration has already been established in research projects #VU-L-203/06; #VU-L-705/07.

Partner#4
Head: Assoc.Prof. Dr. Eugenui Hadjiev, Dr Kamelia Alexandrova
Haematology Clinic, University Hospital “Alexandrovska” Medical University – Sofia

The Haematology Clinic is an integral part of the oldest Medical University in Bulgaria. It was established in 1975 as an independent clinic within the frames of the Chair of Internal Medicine. The research interests of the clinic are aimed at prognostic factors in chronic lymphocytic leukemia, lymphoproliferative disorders, and novel regimes for myeloma treatment.

Partner#5
Head: Assoc.Prof. Dr Stefcho Goranov, Dr. Vesselina Goranova-Marinova, PhD
Haematology Clinic, University Hospital “St.George”, Medical University – Plovdiv

The Medical University – Plovdiv was founded in 1945. It includes the Faculties of Medicine, Dental medicine, and Health Care, a Medical College and a hospital of its own with a capacity of 2000 beds. The Medical University issues its own journal called Folia Medica indexed in the Medline data-base. The Haematology Clinic is the largest department in South Bulgaria with 50 beds. Molecular aspects and treatment of bone disease of multiple myeloma are one of the major topics of interest, as well as myeloproliferative diseases.

Partner#6
Head: Assoc.Prof. Dr. Liana Gercheva; Dr Antonina Zhelyazkova, MD,PhD
Department of Clinical Haematology, Varna University Hospital “St.Marina”, Head

Medical University-Varna was founded in 1961 and is the third largest Medical University in the country. The Department of Clinical haematology Specialized unit aims to provide adequate haematological healthcare for people living in this Northeastern Bulgaria. In total it has 37 beds and a separate unit for the out-patients’ care. The number of the hospitalized patients per year is 2450.The department works in tight collaboration with the departments of Pathology, Immunology and Cytogenetics which assure the contemporary diagnostic approaches.

Partner#7
Head: Assoc.Prof. Dr. Nikolay Tzvetkov, Dr Vanya Slavcheva
Haematology Clinic, University Hospital, Medical University – Pleven

The Pleven Medical University, one of the five medical Universities in Bulgaria, was founded in 1974. It incorporates educational and therapeutic facilities, contemporary pre-clinical base, Institute Hospital with more than 1000 beds with clinics in all major medical fields, including Hematology. Particular problems related to thrombophylia and myeloproliferative disorders, as well as chronic lymphoproliferative diseases are in the focus of research studies in collaboration with the Pathology, Immunology and Cytogenetic departments.

Partner#8
Head: Assoc.Prof.Julian Raynov, Assistant Professor Dr. Ivan Nikolov
Department of Haematology and Oncology, Military Medical Academy – Sofia

The Military Medical Academy was founded in 1989, at the Ministry of Defense of Republic of Bulgaria, as an integrated complex for medical care, education and scientific research. The Department of Hematology and Oncology is one of the most sophisticated health units in the country. It has 30 standard beds and a unit with 2 beds for intensive treatment and management of aplastic patients, as well as 20 beds for palliative treatment. In the field of Hematology, the department is focused to novel therapies of lymphoma and myeloma patients.

Partner#9
Assoc.Prof.Dr.Julian Raynov - President
Bulgarian Society of Clinical and Transfusion Haematology (BSCTH)

BSCTH is a non-profit organization of professionals in the field of Hematology in Bulgaria (www.bscth.med-bg.info). Scope of work:

  1. the promotion of medical practice and research in Hematology on the national level;
  2. the promotion of the position of Hematology as a medical discipline;
  3. accumulation, dissemination and analysis of information concerning hematological practice in the country;
  4. harmonization with the European and world-wide standards;
  5. the promotion of exchange and dissemination of knowledge and novel information; education and training in Hematology.

Areas of specific expertise:

  1. organizing scientific meetings, workshops, etc.;
  2. training courses and continuous medical education;
  3. suggesting legislative measures or programs for improvement of Hematology practice in Bulgaria;
  4. dissemination of information; publishing an official journal, guidelines, etc.;
  5. development of national standards and guidelines;
  6. establishing contacts with national and international organisations.

Competence in relation to the project proposed: BSCTH was a partner in the development of the European Haematology Curriculum and H-Net Project (the national co-ordinator being Assoc.Prof. Dr.Margarita Guenova).

Partner#10
Prof. Ivan Dimov, Assoc.Prof. Mihail Nedjalkov, Assoc.Prof.Rumjana Papancheva
Institute of Parallel Processing at the Bulgarian Academy of Science (IPP-BAS)

The Institute for Parallel Processing at Bulgarian Academy of Science (IPP-BAS) has a leading position among the scientific institutions in Bulgaria in the fields of Computer Science, Scientific Computations, Supercomputing, Natural Language Processing Networks, Communications and Control. The scientific staff includes 62 scientific (two members of the Bulgarian Academy of Sciences, five full professors, 23 associate professors, 32 research assistants) and 25 university educated specialists. Annually are published about 100 papers in refereed journals and proceedings of international conferences. Senior scientists supervise MSc theses of students from Sofia University and the Technical University as well as PhD students, preparing their theses in IPP-BAS. The institute consists of six Departments - Distributed Computing Systems and Networks, Parallel Algorithms, Scientific Computing, High-Performance Computer Architectures, Linguistic Modeling, Mathematical Methods for Sensor Data Processing and C4I Research and Development. Stochastic methods and their applications are the main research topic of the scientists from the Department of Parallel Algorithms. IPP-BAS was funded by two grants as FP5 BIS-21 ’Centre of Excellence’ and FP6 BIS-21++ ’Centre of Competence’. In 2007 IPP-BAS was awarded by the Ministry of Education and Sciences as the most successful institute of the Bulgarian Academy of Sciences for its participation in FP6. IPP-BAS coordinates the Bulgarian National Grid Initiative and is involved in the pan-European (EGEE, CoreGrid) and regional (SEE-GRID) grid project under FP6.