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Haematopoietic Stem/Progenitor Cell Transplantation and Transplant-related Complications Prof. Ilona Hromadníková, Ph.D. Department of Molecular Biology and Cell Pathology Third Medical Faculty, Charles University in Prague [email protected] History • • • 1891 bone marrow per os application to patients with hematopoiesis disorders 1939 intravenous infusion of bone marrow cells the 50thies – experimental trials on animals • second half of the 50thies – first clinical trials failures → due to unknown HLA system so far 1960 1st bone marrow transplant in Czech Republic (ÚVN, Prague) for acute leukemia protocol creation for bone marrow can preparation, concept of volunteer bone marrow donor registry • break-through with discovery of HLA system – „modern era“ (second half of the 60thies ) allogeneic BMT of HLA-identical siblings History • development of experimental and clinical realization: E.D. Thomas (1990 Nobel prize for medicine) - bone marrow cell infusion is safe - bone marrow can be preserved - long-term reconstitution in recipient depends on the level of histocompatibility - immunosupressive therapy can attenuate graft versus host reaction in recipient - curative action even in a very advanced stages of leukemia, more efficient in early stage of the disease • 1968 1st BMT with HLA-identical sibling in Seattle, USA • treatment not only of malignant haematologic diseases, but later also bone marrow aplasia, solid tumors, some inborn metabolic disorders and others History in Czech Republic • 1976 1st BMT with HLA-compatibility examination in Hradec Králové • 1991 1st BMT with bone marrow from unrelated donor from foreign registry, The Institute of Hematology and Blood Transfusion in Prague • 1989 – transplant initiation in the Department of Paediatrics (University Hospital Motol) • 1991 – The Bone Marrow Transplantation Unit in University Hospital Motol • 1991 – Pilsen University Hospital (allogeneic, autologous transplant) • 1993 – Hradec Králové (autologous transplant) • 1993 – I. Internal Clinic 1st Medical Faculty in Prague (autologous transplant in malignant lymphomas, lymphomas and later breast cancer) • 1994 – II. Internal Clinic in Brno-Bohunice • 1995 – Haematology Clinic in Olomouc Basic terms • hematopoietic stem cell transplantation the aim is - to settle recipient‘s bone marrow with donor‘s stem cells, which give birth to the complete hematopoiesis substitute the original term „bone marrow transplant“ • donor – person from which transplanted cells originate (= graft) • myeloablation – process before Tx in most cases necessary for „making space“ in bone marrow and destroy defective recipient‘s stem cells (by irradiation or pharmacologically) • engraftment – time when donor hematopoiesis is firstly detectable • rejection– non-acceptance of the graft, donor´s hematopoiesis stops, restoration of recipient´s hematopoiesis Basic terms • graft versus host disease (GvHD) – T lymphocytes in the donor graft recognize the tissue antigens of the host as heterogeneous and start immune reaction against them • graft versus leukemia effect (GvL) – T lymphocytes (transferred together with stem cells) react against residual leukemic cells of the recipient • relapse – return of the disease symptoms • remission – achievement symptomless period it‘s suppposed that the disease is still present, however, without obvious clinical symptoms Principle of the treatment • various lympho-hematopoietic malignancies: destruction of patologic hematopoiesis using intensive, mostly cytostatic and radiating preparation followed by stem cell transfer from healthy matched donor, after engraftment hematopoiesis reconstitution • inborn failures of host defence (immunodeficiencies), inborn and acquired failures of hematopoiesis, inherited metabolic disorders: replacement of the recipient‘s defective hematopoietic stem cells with normal donor‘s cells able to create missing cell line or cell product • transplantation itself technically simple – suspense mixture of partly purified cells is injected into peripheral vein of the recipient and stem cells find the way into bone marrow and settle there Transplantation types • • • • autologous syngeneic allogeneic xenogeneic Cell sources • • • • bone marrow peripheral blood umbilical cord blood combinations possible for one transplant Transplantation types Autologous Tx transfer of patient's own stem cells taken from patient before starting conditioning regime (before transplantation) usage only when the graft is not infiltrated with basic disease (mustn‘t contain tumor cells) or the patient is in complete remission graft versus host reaction is rare, but can occurs missing and/or decreased immune action of the graft against leukemic cells, bigger risk of relapse than in allogeneic transplantation Transplantation types Syngeneic Tx transfer of stem cells from identical twin of the patient genetic identity doesn‘t induce immunologic reaction GvL effect is missing Transplantation types Allogeneic Tx stem cell transfer from the (healthy) HLA-matched donor → the (patient) recipient - siblings - other relatives - unrelated donors Xenogeneic Tx stem cell transfer between two different animal species, no usage in routine clinical practice Graft collection • donor for allogeneic Tx donor work-up: besides tests on HLA, AB0, RhD compatibility, clinical examination to check the ability to undergo the general anaesthesia for BM collection blood counts, blood group, atypical antibody screening infectious disease markers: hepatitis B, C, syphilis, HIV 1+2, CMV, herpex simplex, herpes zoster, toxoplasmosis blood collection (7 – 21 days prior to Tx) • patient - autologous Tx patient is treated with homologous blood preparate (the blood is irradiated to minimize the risk of GvHD development) • bone marrow collection in general or epidural anaesthesia from dorsal and lateral hip bones, cca 1 – 5 ml Recommended minimal amount of stem cell collection expressed as the number of nucleated cells per weight kilogram of the recipient • for autologous Tx min. 1,5x108 cells/kg • for HLA-identical sibling Tx for leukemias, hemoglobinopathy and inherited metabolic disorders min. 1,5 – 2x108/kg for aplastic anemia min. 3x108/kg • for unrelated allogeneic Tx min. 2-3x108/kg if the graft must be cryopreserved or else processed – increase the number at least by 30% each transplant centre has its own protocols for the minimal sufficient cell numbers for certain conditions Risk of bone marrow collection • minimal, from analyzed 1270 collections more serious complications in 0.27% from that the half of life threatening complications were caused by the general anaesthesia, the other were caused mainly by infection Processing of bone marrow sample • if AB0 incompatibility occurs – removal of erythrocytes or plasma • Concentration of stem cells prior to cryopreservation (buffy coat separation) • Removal of T lymphocytes to minimize the risk of GvHD (monoAb separation), used only in case of HLA non-identical Tx • Removal of tumor cells (marrow purification) most often using monoAb or 4hydroperoxycyclophosphamide (i.e. negative selection) – used for auto Tx • Stem cell positive selection – isolation of hematopoietic cells (monoAb separation using CD34 as a marker) Bone marrow cryopreservation • untreated BM is possible to store at 2-4°C for 72 hrs, but the lifetime of leukocytes and progenitor cells continuously decreases • necessity for long-term storage: in autoTx for shortening of aplastic phase after chemotherapy of hematological malignancies, marrow collection in remission in case of future relapse freezing in liquid nitrogen (-196°C), its vapours (-140°C) or in freezer at -80°C in bags from special material (5-10% DMSO - cryoprotective effect) transport in special containers let thaw in water at 37-40°C cca 15 min prior to the usage Peripheral blood progenitor cells • pluripotent stem cell – precursor for development of blood cell lineages, gives rise to progenitor cells programmed for the main cell lineages of hematopoiesis • expresses CD34, forms cca 0,2% of mononuclear cells (1/10 of bone marrow) of healthy individual • enhancement of concentration in blood (so-called mobilisation) by: - myelosuppressive chemotherapy (cyclophosphamide) – 10-100x higher c (only in patients) cytokine administration (G-CSF, GM-CSF, often in combination with IL-3) – lower yields (in healthy donors only this way) cytostatic and cytokine combination – the highest yield • collection using separators: determination of time collection by monitoring of CD34+ cell count, increase of neutrophiles the yield influenced by the number and intensity of previous cytostatic treatment, previous radiotherapy, patient‘s age, etc. • for autologous Tx – planned collections prior to chemotherapy Hematopoiesis scheme CFU – colony forming unit BFU – burst forming unit Advantages of Tx with PBPC • Comparing to autologous BMT: faster engraftment, smaller demand on supportive treatment with blood derivates and antibiotics • application in allogeneic Tx (on the base of good tolerance of growth factor stimulation and leukapheresis) mobilisation of PBPC from healthy donor: G-CSF usually more CD34+ cells than from BM, negative yield influence by donor‘s age advantages for donors – outpatient collection without general anaesthesia G-CSF small side effects advantages for recipient – faster hematopoiesis reconstitution Tx from unrelated donors – higher risk of GvHD due to high amount of T-cells in graft (exact relationship not proved) Usage of umbilical cord blood for Tx • source of pluripotent hematopoietic stem cells • contains cells without markers of mature hematopoietic lineages and coexpresses CD34 • usually gained post labor prior to placenta delivery • 1 cord blood contains such amount of progenitor and stem cells comparable with the cell amount acquired from bone marrow needed for auto or allo Tx of 1 adult individual • possibility of cryopreservation, storage in blood banks (small amounts due to separation), approved unchanged repopulation potencial after 7 years of the storage • immune immaturity of the cells advantage: less Tx-related complications (GvHD) disadvantage: weaker effect against the tumor (GvL effect), longer engraftment and reconstitution of immune system Conditioning regimens Main objectives: • disease eradication by the tumorablation effect • immunosuppression to prevent rejection (host versus graft reaction) of the incoming donor cells by residual host hematopoiesis • „space-making“ in niches within the marrow stroma in bones for donor engraftment Conditioning regimens • diversity and multitude of regimens in practice • final protocol – torelable toxicity, small risk of secondary malignant diseases Total body irradiation (TBI) • the base of conditioning schemes, better results in combination with cyclophosphamide • not possible to completely eradicate leukemic cells with conventional doses • important for cells in G0-phase and advantage of having access to so-called „sanctuary sites“ of malignancies - poorly available for cytostatics (CNS, gonads) • most efficient to prevent rejection • total dose varies: 5-14 Gy (most often around 10 Gy) – depands on comparison of advantages and disadvantages in chosen regimen combination more often usage of fractionated dose: 6 fractions at 2 Gy over 3 days Consequences of TBI • early consequences – vomiting, diarrhea (2nd – 10th day) – mucositis, maximum of skin symptom development around 10th day > 4 Gy alopecia occurs (growth recovery after 3 months) – lethargy, headache (6th – 8th week) • late radiation consequences most important in children – no TBI if possible (usage mainly in high-risk leukemia) • if relative contraindication for TBI (previous radiotherapy, respiratory functional disorders) or severe organism damage risk (growth failure in children, severe toxicity in old patients) exists → chemotherapeutical conditioning regimes: busulphan with cyclophosphamide, BCNU, cyclophosphamide with etoposide and others Continuous regimen improvement: ↑ tumorablative efficiency and ↓ toxicity Examples of conditioning regimens TBI containing regimens (day 0 = day of Tx) Protocol Cy/TBI cyclophosphamide 60mg/kg in days -7 and -6, TBI 2 Gy in days -5, -4, -3, -2, -1 and 0 Used especially in case of hematologic malignancies, fractionated TBI in higher doses (12-14 Gy) Protocol Mel/TBI melphalan 140mg/m2 in day -1 i.v. after 12h, TBI 9.5-11.5 Gy in one fraction Used in case of hematologic malignancies, e.g. myeloma Examples of conditioning regimens TBI-free regimens Protocol Bu/Cy busulfan 4mg/kg in days -9, -8, -7, -6; cyclophosphamide 50mg/kg -5, -4, -3, -2 Used in case of various types of hematologic malignancies Protocol CCB cyclophosphamide 5625 mg/m2 (total dose for 4 days) on days -6, -5, -4, -3; cisplatinum 165 mg/m2 (total dose for 4 days) in continual infusion on days -6, 5, -4, -3; BCNU (carmustine) 600 mg/m2 in day -3 Used in autologous Tx in case of solid tumors Clinical course after Tx Early stage after Tx High toxicity effects of conditioning regimen causes: • total aplasia of hematopoietic and lymphatic system → intensive supportive treatment • acute non-hematologic toxicity: mucositis and necrosis of various intensity • veno-occlusive disease – manifestation: hepatomegalia, hyperbilirubinemia, thrombocytopenia, weight gain due to oedema formation • early pulmonary toxicity • capillary leak syndrom – injury of vascular endothelium clinical manifestation: organ or generalised injury of capillars Further late-effects of high toxicity of conditioning regimen • gonadal failure (both testicular and ovarian) in males – normal testosterone levels or normalization after some time (preventive sperm cryopreservation) in females – estrogen deficiency (substitution treatment) • growth failure in children • thyroid dysfunction - the most frequent late-effect • cataracts within several years post Tx - incidence up to 80% • ? risk of secondary malignancy occurrence Reconstitution of immune system • depands on time post Tx and Tx type (autoTx - faster reconstitution), conditioning regimen, GvHD development and treatment, infection • immunity ontogenesis hematopoiesis regeneration within 2 – 3 weeks post Tx period of the most severe immunodeficiency granulocytes NK cells lymphocytes 1st engrafted cells 1st lymphoid engrafted cells (10th – 20th day post Tx) 1 – 3 months post Tx number normalization cell population reconstitution at different tempos NK cells – within 20 days post Tx total function recovery T, B lymphocytes – total function recovery after 1 year post Tx or longer • normalization of all cell populations by the 2nd year post Tx w/o complications Graft versus host disease (GvHD) • major complication after allogeneic transplantation • consequence of incomplete match in HLA system or mismatch in other antigens outside major histocompatibility complex • acute GvHD still in 30-60% cases of HLA identical Tx despite of immunosuppressive prophylaxis (develops within the first 100 days post Tx) clinical manifestation: skin, liver, GIT intensive immunosuppressive treatment → threat of severe infection • chronic GvHD – develops de novo or following aGvHD (occurence after day 100 post Tx) Toxic or infectious lung involvement • appears as intersticial pneumonia • more often in allogeneic Tx, during aGvHD, in older patients, in single dose of TBI and in CMV+ patients (in 20-40% of patients - major cause of death after allogeneic Tx in mid 80thies ) Risk of severe infection and trombocytopenia bleeding Major clinical problems of transplantation Organ system Side effects acute bone marrow bone marrow aplasia, infection, bleeding GIT nausea, vomiting, diarrhea, orofaryngeal and intestinal changes alopecia, nails growth failure, acute skin GvHD lungs alveolar hemorraghe, intersticial pneumonia veno-occlusive disease, acute liver failure, liver acute GvHD vascular system capillary leak syndrome urinary tract, hemorrhagic cystitis, functional failures, kidney acute failure lymphatic system cell and humoral immunity failure skin chronic chronic GIT GvHD chronic skin GvHD, mucosa dryness lung fibrosis, bronchiolitis obliterans chronic liver GvHD immune defect in chronic GvHD eyes conjuctival dryness, cataracts endocrine failures endocrine and gonadal failure, hypothyroidism hypothyroidism, estrogen deficiency, sterility