Transcript Autologous Tx

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