ONCOLOGY

1. PRACTICAL ASPECTS AND
PROCEDURE
HSCT

2. INTRODUCTION
• Haematopoietic Stem Cell Transplant (HSCT) as
“transfer of stem cells, defined as progenitor cells
with repopulating capacity and the potential to
sustain long term haematopoiesis, within one
person or from one person to another, in a dose
that is sufficient to restitute haematopoiesis in all
lineages”
• …. to repopulate the bone marrow with infused
stem cells that will produce new blood

6. Indications for Autologous HSCT
• Non-Hodgkins Lymphoma
– DLBCL
– Follicular Lymphoma
– Mantle Cell Lymphoma
• Hodgkins disease
• Waldenstroms Macroglobulinanemia
• AML
• Germ cell tumours –testicular
• Autoimmune disorders – i.e. Crohn’s disease, Multiple
Sclerosis
• Neuroblastoma
• Myelofibrosis

7. Indications for Allogeneic HSCT :
• AML
• ALL
• CML
• CLL
• Multiple Myeloma
• Hodgkins disease
• Non-hodgkins lymphoma
• Aplastic anaemia
• Myelodysplastic syndromes
• Myelofibrosis

8. Pre-Transplant Evaluation

9. General pretransplant workup
1. Review of original pathology
2. Full blood count
3. S.urea, creatinine, electrolytes, Ca, phosphate, uric acid, LFTs, CRP, and
blood sugar
4. Coagulation tests (PT, APTT, fibrinogen)
5. Serology for CMV, HSV,VZV, HIV-1,HIV-2, HBV, HCV, EBV , EBV,
toxoplasma,HTLV-I, and HTLV-II, HHV-6, HHV-8,syphilis, adenovirus,
influenza (as appropriate)
6. ABO/Rh typing with subgroups

10. 8. HLA typing (by serologic and molecular methods, for allogeneic
transplantation)
9. Determination of chimerism markers (for allogeneic transplantation)
10. Creatinine clearance
11. CXR, ECG, LVEF
12. Microbiology screening
13. Throat swab, nose swab, central venous catheter exit site swab (to detect
organisms such as MRSA, MRSE, Pseudomonas), rectal swab (VRE)
14. CSF examination: for patients with ALL, aggressive NHL, or lymphoid
transformation of CML; of doubtful value in patients with AML

11. 15. Bone marrow examination (morphology, cytogenetics, MRD
determination, immune phenotyping)
16. Determination of chemosensitivity of underlying disease (as
appropriate)
17. Cryopreservation of autologous stem cells (as appropriate)
18. Delineation of platelet support strategy (as appropriate)
19. Dental check
20. Assessment by social worker
21. Sperm banking/embryo storage/in vitro fertilization (IVF)
counseling
22. Removal of any foreign bodies (e.g., biliary stents), if possible
23. Family conference and signing of consent forms

15. FINDING STEM CELLS
• Stem cell -population of undifferentiated cells with the capacity to
divide for indefinite periods, to self-renew and to generate a
functional progeny of highly specialised cells.
• Fertilised egg (zygote) a totipotent stem cell, a cell with unrestricted
differentiation potential and the only cell with the capacity to give
rise to all cells necessary for the development of foetal and adult
organs.
• ES cells forming a cluster of cells inside the blastocyst are
pluripotent stem cells, capable of generating a variety of specialised
cell types, but limited in their differentiation potential by the
inability to support the development of a foetus.

16. • Further specialisation results in generation of multipotent
stem cells residing in adult somatic tissues.
• HSCs represent the prototype of multipotent adult tissue
stem cells.
• In humans, HSCs can be found in cord blood as a result of
stem cell migratory properties during fetal development,
• Post-natally, the only organ harbouring HSCs and pursuing
active multilineage haematopoiesis is the bone marrow.

18. Sources of Stem Cells
• Bone Marrow
Peripheral Blood
Cord Blood

22. • The most primitive human HSCs express CD34 and lack
CD38 cell surface antigens.
• The CD34+ CD38- HSC compartment, which
constitutes ±0.1% of bone marrow cells, is
heterogeneous and contains also c-kit, flt3, and CD133
expressing cells.
• Characteristically, c-kit, flt3 and tie2 function as
receptors for early-acting haematopoietic growth
factors: stem cell factor, flt3 ligand and angiopoietin,
which act as key positive regulators of haematopoiesis

23. Leukaemic stem cells
• Conventional chemotherapy-based treatment
of leukaemia, and cancer in general, is
primarily directed against the bulk of
malignant cells, and thus does not eliminate
the abnormal stem cells. These cells are the
origin of cancer recurrence and are
responsible for relapse

24. HLA Matches and Tissue type

25. • Tissue compatibility is determined by genes of the major
histocompatibility complex(MHC), known as the HLA
system in man, that are clustered on the short arm of
chromosome 6. The HLA region is a multigenic system
• Immune responses against incompatible HLA antigens
represent a major barrier to haematopoietic stem cell
transplantation (HSCT).
• The homologous HLA Class I (HLA-A, -B, -C) and Class II
(HLA-DR, -DQ, -DP)antigens are codominantly expressed
and differ in their structure

28. Looking for a related donor
• 1. HLA-A/B/DR typing (serology or low resolution DNA typing) of patient,
the sibling(s) and both parents to determine the haplotypes.
• 2. If a sibling is geno-identical the transplant can be performed after a
confirmatory typing on a second blood sample from patient and donor but
without further HLA investigations.
• 3. If one sibling is a monozygotic twin this is a syngeneic transplant, this
donor can be chosen but some of the GvL effect might be lost.
• 4. If both parents share a common haplotype or if one parent is HLA-ABDR
homozygous, this is considered to be a pheno-identical transplant. High
resolution Class I and II typing is recommended to define the exact degree
of compatibility.

29. • 5. If a family donor with only one HLA difference (5/6
match) is found, the transplant can be performed despite
the possibility of an increased risk of GvHD. This is a
related one antigen mismatched donor transplant. High
resolution Class I and II typing is recommended to define
the exact degree of compatibility.
• 6. Parents and/or siblings sharing only one haplotype
with the recipient are considered as a haploidentical-
related donor transplant.

30. Looking for an unrelated donor
• 1. One should obtain a complete Class I and Class II high resolution
typing for the patient (HLA-A, -B, -C, -DRB1 and -DQB1).
• Several levels of compatibility are defined:
– 12 out of 12 (A/B/C/DRB1/DQB1/DPB1)
– 10 out of 10 (A/B/C/DRB1/DQB1) or
– 8 out of 8 (A/B/C/DRB1).
• The level of HLA incompatibility (low or high resolution typing) accepted
can vary from centre to centre but the priority is to look for the highest
degree of compatibility at the allele level.
• 2. Look at the BMDW (Bone Marrow Donor Worldwide) database to
evaluate the probability of finding a donor, and determine which registries
have suitable donors.

31. • Search simultaneously for a cord blood unit through BMDW and
Netcord in order to find an unrelated cord blood donor.
• If several HLA identical bone marrow donors are found, choose the
male, ABO identical, and/or CMV-negative donor.
• If there is no well matched (10/10, 9/10, or possibly 8/10) unrelated
donor or if the time frame is too short, choose a cord blood unit, as
long as the number of nucleated cells is >2 x 107/kg and there are
no more than 2 HLA mismatches (4/6).

33. Peripheral Blood Stem Cell Mobilisation and
Collection
• Donors treated with G-CSF(Filgrastim) at a dose of 10
mcg/kg bd by iv for 4 days.
• The first dose should be administered under
supervision in the ward, and subsequent injections will
be given by donors (or relatives, medical centre,
community nurse) at home.
• Paracetamol 20 mg/kg every 4 to 6 hours may be given
for relief of bone pain if necessary.

34. Peripheral Blood Stem Cell Collection
• Leucopheresis performed using the appropriate cell separator.
• Cubital veins used for venous access by preference.
• Leucopheresis is carried out on day +5 (and +6 if necessary) after starting
GCSF.
• Approximately 12 litres of blood will be processed over 3 hours on each
occasion.
• TLC, CD34 + assay is obtained on each collection, as well as T lymphocyte
and NK cell numbers.
• Optimal number of CD34 + cells = > 5 x 106 cells/kg; Minimum number of
CD34 + cells = 2 x 106 cells/kg.

35. Temperature
• Cryopresevation : - 196, -156, or -80⁰C, reflecting the storage temperatures in
liquid and vapor phase nitrogen and in cryopreservation mechanical freezers,
respectively.
• Spread of infectious agents (i.e., aspergillus as well as viral spread), through the
liquid phase of the nitrogen tanks, the currently recommended optimal storage
conditions are in the vapor nitrogen phase, at -156⁰C. Mechanical freezers might
represent a viable alternative.
• Several studies examined the possibility to store HSCs at suprafreezing
temperature, at -4 C. A preclinical study that examined PBSCs mobilized in
autologous plasma with post-storage clonogenic and viability assays suggested
that a storage up to five days is safe. A small case series by Ruiz-Arguelle et al.
successfully used PBPCs after 96 hr storage at --4 C for rescue after high dose
chemotherapy.

36. FREEZING RATE
• The controlled rate freezing technique is considered standard
• concentrated stem cells are frozen down at a rate of 1–2 ͦC/min up to a
temperature point of about -40ͦ ͦC. Then, the freezing process down to a target of -
120 C is performed at a faster pace, about 3–5ͦ C/min.
• For umbilical cord stem cells, bone marrow, and PBSCs the controlled rate freezing
process is considered standard and found to be superior to uncontrolled freezing
approaches.
• Hence, the use of uncontrolled rate freezing in which the specimen is first cooled
down to -4 C and then directly deposited into a freezer at -80C or put into liquid
phase nitrogen has been evaluated. The uncontrolled method is safe and reveals
comparable results to the controlled rate process for BM and PBSCs

37. DURABILITY
• The actual durability, defined as the time that stem cells can be preserved,
is still unclear. The viability of the stem cells in cryopreservation has been
questioned in different studies after a time course of cryopreservation of 6
months.
• Clinical validity of preclinical studies was documented in anecdotal reports
when successful trilineage engraftment was achieved with BM, stored for
7 years.
• A systematic review evaluating the combined experience of the Brigham
and Women’s Hospital and the EBMT Group noticed that HSC can be
effectively cryopreserved for up to 11 years.
• A retrospective study from Seattle revealed full trilineage recovery in
patients receiving HSC, stored for up to 7.8 years without consistent
detrimental effects

38. CRYOPRESERVATIVES
• Cryopreservatives are necessary additives to stem cell concentrates, since they
inhibit the formation of intra and extracellular crystals and hence cell death.
• The standard cryoprotectant is DMSO, which prevents freezing damage to living
cells.
• Used at concentrations of 10% combined with normal saline and serum albumin .
This was established to be a safe and non-stem cell toxic agent.
• DMSO has clinically significant side effect profile. Nausea, vomiting, and abdominal
cramps occur in about half of all the cases. Other side effects -cardiovascular,
respiratory ,CNS, renal, hemolytic, and hepatotoxic presentations.
• Alternative preservation methods -propylene glycol, a combination of alpha
tocopherol, atalase, and ascorbic acid and the glucose dimer trehalose as intra and
extracellular cryoprotectant

39. THAWING
• The standard method is warming in a water
bath at 37˚C until all ice crystals disappear.

40. WASHING PROCEDURE
• Since the DMSO is assumed to have toxic effect on the
stem cells ,the process of washing out the
cryopreservative after the thawing can still be
considered standard.
• The wash out of the most popular cryopreservative has
conceivable benefits for the recipient, i.e.
– reduction of toxicity, since the degree of DMSO toxicity is
proportional to the amount of DMSO contained in the
infused stem cell solution .
– also suggested that wash out of DMSO can enhance
engraftment

41. • The current standard washing protocol follows the New York
Blood Center protocol
two step process
• dilution of the thawed stem cell unit with 2.5% human serum
albumin and 5% dextran is followed by centrifugation at 10˚ C
for 10 min.
• The supernatant is then removed and HSA and dextran
solution is again added twice to a final DMSO concentration of
less then 1.7%. The washed solution is infused as soon as
possible.

43. Transplant Procedure

44. Conditioning Therapy
• The term “conditioning” in HSCT means to “condition”, e.g. to prepare the patient
for its transplant.
• the conditioning or preparative regimen; it is essential for disease eradication and
the creation of “space” within the marrow cavity to allow engraftment of
allogeneic stem cells
• The first stage of the transplant. May be given in one dose or over several days.
• Necessary for:
– Suppressing the patients immune system to lessen the chance of graft rejection
– Destroying remaining cancer cells
– "creation of space“
• Conditioning regimen is dependent on the type of disease, the type of transplant,
co-morbidities and age.

45. Conditioning regimens
• Autologous SCT –
– no genetic difference between the transplanted stem
cells and the patient
– only role of the conditioning regimen is tumor
eradication.
• Allogeneic stem -serves two purposes :
– host immunosuppression to prevent graft rejection;
– host myeloablation in order to eradicate malignant
hematopoiesis

46. Conditioning Regimens
Myeolablative conditioning
• Irreversibly destroys the haemopoietic function of the bone marrow
with high doses of chemotherapy +/- TBI.
• Higher level of disease control
• Younger patients with a good performance status
• Quicker engraftment of donor cells
• Higher toxicities associated with higher transplant related mortality

47. Myeloablative conditioning
• This approach depends on a steep dose–response relationship to
achieve high cytotoxicity against malignant cells, but at the cost of
considerable hematopoietic and systemic toxicity.
• It has also been employed in many nonmalignant conditions,
especially those associated with increased risk of rejection.
• Regimens incorporating either TBI or busulfan (Bu) alongside
cyclophosphamide [Cy], as CyTBI or BuCy, respectively remain the
most commonly used,
• Other high dose cytotoxic drugs (most commonly alkylating agents,
especially melphalan) are sometimes used for myeloablative effect.

48. Non-myeloablative conditioning
• These regimens were developed following observation of the sometimes powerful graft-vs.-
leukemia (GVL) effect following donor lymphocyte infusions (DLI) even in the absence of
conventional cytotoxic therapy.
• GVL following DLI is mediated at least in part by allogeneic T cells, although the precise
mechanism remains unclear.
• Nevertheless, it is believed that GVL can occur even in the absence of overt GVHD.
• Underlying principle of nonmyeloablative HSCT is that less intensive conditioning may reduce
regimen-related toxicity (RRT) by reducing direct toxicity and limiting inflammatory cytokine
release.
• Most typically incorporate fludarabine in combination with low-dose TBI or chemotherapy
(e.g. melphalan or cyclophosphamide).
• Divided into “reduced-intensity” and “minimal-intensity” regimens

49. Reduced intensity conditioning
• Regimens that have been developed to reduce the
morbidity and mortality of allogeneic transplant.
• It aims to use enough immunosuppression to allow donor
cells to engraft without completely eradicating the
recipients bone marrow.
• Can be given to older patients
• Less regimen related toxicities
• Reduction in morbidity and transplant related mortality

51. Conditioning regimens Autologous
transplantation
Lymphoma

52. • Leukemia

53. • Myeloma

54. Stem Cell Re-infusion
• At least 24 hour after the conditioning will be given on Day 0. These are
generally given through a central line and takes approximately 30
minutes.
• Stem cells are either:
Cryopreserved
• Usually for autologous transplants
• Most common side effects are reactions to DMSO
Fresh
• Usually for allogeneic transplants
• Administered much like a blood transfusion
• Generally better tolerated than frozen cells

55. Complications of Stem Cell Re-infusions
Immediate Immunologic
complications
• Acute Haemolytic Reaction
• Allergic / anaphylactic
reactions
• Transfusion-related acute lung
injury
Delayed Immunologic
complications
• Delayed haemolytic reactions
Non-immunologic complications
• DMSO Toxicity
• Septic infusion reaction
• Fat emboli
• Bleeding due to excessive
anticoagulation
• Circulatory overload
• Hypothermia
• Non-immunologic haemolysis
Other issues
• Rupture of cellular therapy
product bag.

56. Reactions and side effects of infused stem cells
• Patients receiving fresh stem cell infusions should be observed in
the same way as they would during any other fresh blood product
infusion.
• Reactions and side effects for patients receiving thawed stem cells
are the same as for fresh stem cells, plus the following:
– • flushing of the face often occurs if given too quickly;
– • altered taste due to the preservative (DMSO);
– • hematuria post-infusion;
– • pain around the kidney area as red blood cells are passed through
the kidney;
– • excretion of the DMSO through the skin and breath for several days.
Patient, family and visitors should be warmed of this.

57. Early Complications of HSCT

59. Blood Count Recovery

60. Phases of Immune Recovery
Neutropenic Phase
• From conditioning to engraftment
Intermediate phase
• Neutrophil engraftment until D100
Late phase
• D100 +

61. Discharge planning
• Planning for discharge should begin at the time of a
patient’s admission.
• A patient can be discharged from the hospital environment
when the following criteria are met:
– A neutrophil count of 109/L
– No obvious signs of infection or bleeding
– Sustained oral nutrition intake of >4000 kJ (1000 kcal) daily
– Reasonable exercise tolerance
– Mastery of the dressing technique for the central venous
catheter
– No new evidence of GVHD
– No other clinical problems

62. • Education in the use and care of the central venous catheter for the
patient and the principal family member or friend who will be providing
assistance should begin as soon as possible after insertion and continued
throughout hospitalization. Patients should care for their own catheters,
whenever they are well enough, under the supervision of a registered
nurse.
• Several days before discharge, the patient should be encouraged to read
the discharge information and to ask questions.
• On the day of discharge, the patient must be given discharge medications
by the nursing staff, who will check the patient’s understanding of doses
and administration.
• The patient should be given a 5-day starter pack for care of the central
venous catheter.
• The physician must go through the discharge instructions with the patient
and ascertain that the patient understands how to identify problems that
can arise after discharge and whom to contact for help

63. Late Effects

64. THANK YOU

65. • Principles of donor choice
• Potential donors:
– • Autologous (the patient)
– • Allogeneic (another person)
– • HLA-identical (matched) sibling
– • Other related donor (RD)
– • HLA-matched
– • HLA-mismatched, including haploidentical (half matched,
usually a parent)
– • Unrelated donor (URD)
– • HLA-matched (often termed matched unrelated donor, MUD)
– • HLA-mismatched

66. • Total Body Irradiation
• Machine: Linear accelerator (6 MeV).
• Total Dose: Maximum midplane dose will be 12 Gy, delivered as 6 x 2 Gy fractions delivered over 3
days
• Or
• 13.2 Gy delivered as eight fractions of 1.65 Gy
• Rate: 10-12 cGy/min.
• Premedication: Dexamethasone 4 mg bd IVI 1 hour before each fraction
• Dosimetry: Test dosage procedure and dosimetry to be determined by Radiotherapy Department.
• Toxicity: nausea and vomiting
• lethargy
• alopecia and/or skin pigmentation
• interstitial pneumonitis
• parotitis, onset 6-12 hours duration 24-48 hours
• pancreatitis (rarely symptomatic)
• fever in first 12 hours
• erythema 1248 hours
• mucositis and diarrhoea
• Late effects: sterility , growth retardation in children, cataracts

67. Molecular Monitoring Post Allogeneic
Transplant
• Monitoring of CHIMERISM
• Monitoring the state of chimerism of recipients of blood or marrow transplant can be valuable for
establishing engraftment or for detecting minimal residual disease.
• This can be done using microsatellite analysis in gendermatched transplants or by FISH in gender
mismatched transplants.
• Pretransplant samples should be sent for EVERY patient donor pair for DNA extraction and storage
for future potential analysis of chimerism.
• Posttransplant assessment of chimersim will be undertaken in the following circumstances:
– Recipients of transplants using CD34+ selected cells.
– All patients receiving reduced intensity SCT
– Selected myeloablative transplants where DLI is proposed for incipient or
– overt reIapse.
– Patients receiving DLI.
• Frequency of chimerism analysis:
– Patients undergoing nonmyeloablative transplants should have microsatellite studies done on blood
according to trial or, if not on trials, on days 30, 60, and 100.
– Patients receiving DLI for CML should have chimerism anlysis performed every 6 to 12 weeks.
– Patients receiving DLI for indications other than CML should have chimerism studies performed according to
disease kinetics

68. • The MHC comprises 12 classical HLA genes located on a 3.6 Mb segment of the
short arm of chromosome 6.
• Three HLA Class I genes (A, B, C) encode for the heavy chains of HLA-A, B and -C
antigens.
• Polymorphic residues are essentially located in the a1- and a2-domains encoded
by exons 2 and 3, respectively, which form the peptide binding site. HLA Class II
antigens (DR, DQ, DP) are heterodimers encoded by an a-chain and a b-chain gene
(e.g. DRA/DRB1 or DQA1/DQB1) that co-localise at the centromeric part of the
MHC .
• Essentially all of the polymorphism is located on exon 2 of b-chain genes, whereas
the DRA gene is non polymorphic, and DQA1 and DPA1 loci exhibit a lower level of
polymorphism .
• The HLA-DR sub-region presents an additional level of complexity since a second
polymorphic DRB gene may be present, i.e. DRB3 in
DR11/DR12/DR13/DR14/DR17/DR18 haplotypes, DRB4 in DR4/DR7/DR9
haplotypes, and DRB5 in DR15/DR16 haplotypes.
• Because of the codominant expression of HLA genes, a heterozygous individual
may therefore express up to 12 different HLA antigens.