Haematology JC042: High White Cell Count: Acute And Chronic Leukaemia, Bone Marrow Transplantation, Immunogenetics

Question 1

Clinical approach for patients with high WBC

Answer 1

Look at:
1. ***Absolute WBC count
- mild / gross

2. WBC ***Differential count
   - manual count if in doubt
3. ***Other cell lines —> reflect BM function
   - Hb
   - Plt
4. Reactive / Underlying primary BM disease e.g. Leukaemia?

Question 2

Leukaemia classification

Answer 2

1. Acute myeloid leukaemia
2. Chronic myeloid leukaemia
3. Acute lymphoblastic leukaemia
4. Chronic lymphocytic leukaemia

Question 3

Risk factors for Leukaemia (Davidson)

Answer 3

1. Ionising radiation
   - ***RT
   - X-ray of fetus in pregnancy
   - Atomic bomb
2. Cytotoxic drugs
   - ***Chemothearpy (esp. Alkylating agents)
   - Industrial exposure to Benzene
3. ***Genetic
4. ***Immunodeficiency state (e.g. Hypogammaglobulinaemia)
5. Retroviruses

Question 4

Acute leukaemia

Answer 4

AML, ALL:
- neither is a single disease (>20 subtypes for AML, >10 subtypes for ALL —> B / T cell lineage)
—> differentiated by **Morphology, **IHC (e.g. MPO stain)
—> diagnosed by ***Flow cytometry (detecting Ag on blasts)

***Revision:
- Morphology: PB smear, BM aspiration, Trephine biopsy
- Cytochemistry: Staining (Myeloperoxidase, Sudan Black B)
- Immunophenotype: IHC, Flow cytometry
- Cytogenetics: Karyotype, FISH
- Molecular genetics: PCR

Question 5

Clinical features of Acute leukaemia

Answer 5

Bone marrow failure
—> caused by **Maturation arrest
—> accumulation of leukaemic blasts
—> **Anaemia, **Thrombocytopenia, **WBC: High / Normal / Low

Extramedullary features:
1. Hepatomegaly
2. Splenomegaly
3. Lymphadenopathy
4. Extra-nodal site involvement: CNS, Testicular (both more common in ALL)

Question 6

Workup for suspected Acute leukaemia

Answer 6

Diagnosis
1. CBP + D/C
2. **PT / APTT (including D-dimer + Fibrinogen)
3. Biochemistry (RFT, Electrolytes, LDH, Urate) (impending **TLS)
4. BM examination + Cytogenetics +/- Gene panel
5. CXR (mediastinal mass)

Pre-treatment
1. ECG, Echocardiogram (***before anthracyclines)
2. Hepatitis serology, HIV, G6PD
3. Central venous catheter insertion (for chemotherapy infusion)
(4. Lung function
5. HLA typing for HSCT)

Treatment:
1. Supportive
2. Specific

Haematological emergencies in Acute leukaemia (must monitor):
1. **Neutropenic sepsis / Septic shock
- Septic workup + Empirical **broad spectrum bactericidal antibiotics (early)
- Resuscitation if needed (BP, vitals)

2. Tumour lysis syndrome (記: LDH, Urate, K, PO4)
   —> monitor RFT, Electrolytes, LDH, Urate: ↑ LDH, ↑ Urate, ↑ K, ↑ PO4
   —> may develop renal failure / arrhythmia rapidly
   - **Hydration (IV fluid)
   - Urate lowering agents (*Allopurinol, Febuxostat, Rasburicase)
3. Leukostasis (CNS symptoms: altered mental status / ***Respiratory compromise)
   - Urgent leukapheresis / chemotherapy for cytoreduction
4. RBC + Platelet transfusion
5. Antifungal prophylaxis (for prolonged severe neutropenia)
6. Proper nursing care
   - reverse isolation, face mask, hand hygiene, low bacteria diet
   - limit no. of visitors

Question 7

Specific treatment: Chemotherapy of AML as an example

Answer 7

1. Chemotherapy
   - **Induction (1 session) —> **Daunorubicin + **Cytarabine
   - **Consolidation (when in remission) (3 sessions) —> Cytarabine

Other therapy:
2. **Targeted therapy (e.g. Midostaurin for FLT3 ITD mutated cases)
3. **Ab-drug conjugate (ADC) (e.g. Gemtuzumab ozogamicin (Mylotarg): Anti-CD33)
4. ***Hypo-methylating agents + BCL2 inhibitor (Azacitidine, Decitabine, Venetoclax)

High risk (Adverse risk) diseases, classified by:
- Cytogenetics
- Gene mutation profile
- Relapsed after chemotherapy
—> ***Allogeneic HSCT

(ELN-2017 risk stratification:
- classify patients with AML into
1. Favourable
2. Intermediate
3. Adverse)

Question 8

Treatment for Acute Promyelocytic leukaemia

Answer 8

Subtype of AML but not treated the same

Underlying gene abnormalities:
- **t(15;17)(q22;q21)
—> **PML-RARA (oncogenic fusion protein)
—> blocks RAR-mediated differentiation of Promyelocytes
—> accumulation of abnormal Promyelocytes

Clinical features:
1. **Pancytopenia
2. **Prominent Bleeding symptoms
3. ***DIC

DIC:
Thrombin generation
—> Fibrin generation (Widespread IV coagulation) + **Secondary fibrinolysis
—> Consumption of clotting factors, natural anticoagulants, platelets (*結血, 溶血都無曬)

Lab results:
- Prolong PT + APTT + TT + ↓ Fibrinogen (∵ consumed)
- ↑ Fibrin degradation products (***D-dimer) from Secondary fibrinolysis
- ↓ Platelet (Thrombocytopenia)

Treatment:
1. Bleeding complications
- Supportive transfusion
- Fibrinogen replacement

2. **ATRA, **Arsenic trioxide
   - binds PML-RARA —> degradation of PML-RARA

Question 9

Haematopoietic stem cell transplantation (HSCT)

Answer 9

formerly known as Bone Marrow Transplantation (BMT) (骨髓移植)

Aim:
- Replace the diseased involved BM with a normal health BM

2 types of HSCT:
1. Autologous (ASCT)
- high dose chemotherapy + ***autologous stem cell rescue (patient’s own pre-collected stem cells to reconstitute BM)

2. Allogeneic (Allo)
   - stem cells from another individual
   - ***Syngeneic: stem cells from identical twin (HLA-identical)

Question 10

Rationale of ***Autologous transplantation

Answer 10

• Allow **high dose (supra-lethal) chemotherapy necessary for disease **eradication
• Autologous stem cells “rescue” patient from high dose therapy

Indications:
1. Myeloma
2. Lymphoma
(X Leukaemia: ∵ already have BM involvement —> can only use Allogeneic HSCT)

Question 11

Allogeneic BMT

Answer 11

Difficulty:
- Self vs Non-self —> recognised via TCR + HLA-system (MHC molecules)

HLA system:
- encoded by MHC gene on chromosome 6 short arm
- HLA antigens expressed on cell surface —> involved in recognition of self (tolerate) vs non-self (eliminate)
—> **Class 1 MHC (A, B, C): All nucleated cells
—> **Class 2 MHC (DP, DQ, DR): Immune cells
- ***Co-dominant expression of maternal + paternal epitopes (alleles) of ALL leukocytes
—> siblings: 25% identical HLA, 50% haploidentical, 25% incompatible

HLA matching:
- crucial in HSCT to avoid graft rejection / graft versus host disease (GVHD)

***Full HLA matching:
- 6/6: A, B, DR-B1 (in siblings transplant)
- 8/8: A, B, C, DR-B1 (in unrelated transplant)
- 10/10: A, B, C, DR-B1, DQ-B1 (in unrelated transplant)

Haplo-identical donor transplantation:
- broaden donor pool
- father, mother (must be), 50% siblings

Question 12

ABO incompatibility in HSCT

Answer 12

***ABO incompatibility is NOT excluded in HSCT (vs solid organ transplant)

Major ABO incompatibility:
- recipients having pre-formed Ab against donor RBC Ag
- HSCT: ***RBC removed from donor graft

Minor ABO incompatibility:
- donors having pre-formed Ab against recipient RBC Ag
- HSCT: ***plasma (containing donor Ab) removed from donor graft

Question 13

Indications for HSCT

Answer 13

1. ***Acute leukaemia (most common, AML + ALL: >60%) (high risk at first remission / at relapse)
2. ***MDS (high risk)
3. ***CML (T315I mutation (resistant to most TKI), accelerated phase / blastic crisis)
4. ***Transformed MPN (leukaemic transformation, fibrotic phase of MPN)
5. Aplastic anaemia
6. Lymphoma (mainly Autologous HSCT)
7. Myeloma (mainly Autologous HSCT)

Paediatric indication for HSCT (Immunodeficiency / Haemoglobinopathy):
1. **Thalassaemia major
2. **Sickle cell anaemia
3. Fanconi anaemia
4. Blackfan-Diamond
5. **SCID
6. **Wiskott-Aldrich
7. ***Inborn errors of metabolism
8. Other solid tumours
9. Neuroblastoma
10. Ewing sarcoma

Question 14

Different types of Allo-HSCT donors, Different types of HSC donor grafts

Answer 14

1. HLA matched family donors
2. HLA matched unrelated donors
3. HLA mismatched family donors (i.e. Haploidentical)

3 main types of grafts:
1. BM
2. **PBSC (peripheral blood stem cell) (*main type ∵ ease + ↓ morbidity for donor)
3. Cord blood cells (seldom used in adults due to dosage problems)

Question 15

How to get HSC grafts from donor?

Answer 15

Both are referred to HSCT
1. BM
- directly get cells from BM (i.e. BMT)

2. PBSC
   - medications to mobilise stem cells in BM into circulation —> collect using apheresis machine (i.e. PBSCT)
   - HSC mobilisation by growth factors (G-CSF)
   —> promote myeloid cell proliferation + cut off adhesion ties between stem cells and BM stroma

Comparing BM vs PBSC source for HSCT:
BM
Advantages:
- ***single collection already have enough cells
- no need for growth factor injection
- no need for double lumen catheter for collection (apheresis)

Disadvantages:
- **require GA, performed in OT
- **higher rates of morbidity in donors (e.g. wound pain, infection (rare))
- ***slower count recovery in recipients

PBSC
Advantages:
- **not require GA, can be performed out-patient
- **lower rates of morbidity in donors
- ***faster count recovery in recipients

Disadvantages:
- collection may take **several days
- donor may require **double lumen catheter for collection
- citrate toxicity risk (anti-coagulant during apheresis)

Question 16

***How is Allo-HSCT performed?

Answer 16

Steps:
1. ***Conditioning chemotherapy / TBI (total body irradiation)
- eradicate residual leukaemia
- suppress patient’s immune system —> not to reject donor graft

2. ***Donor graft infusion (BM / PBSC)
   - just like blood transfusion (done at bedside)
3. Cytopenic phase (2-3 weeks)
4. Engraftment (recovery of neutrophils and platelets) (10-14 days (CL Lai))
   (- can give G-CSF to speed up)
5. **Immunosuppression for **GVHD prophylaxis after infusion

Question 17

Pre-transplant conditioning

Answer 17

Myeloablative (清髓性) vs Non-myeloablative (非清髓性)

• High doses of **chemotherapy +/- **radiation
  —> **eradicate residual malignancy
  —> induce **immunosuppression to allow engraftment
• Require healthy stem cells to restore marrow function

Agents used:
1. **Cyclophosphamide
2. Busulfan
3. **Fludarabine

Question 18

Reduced intensity conditioning / Non-myeloablative (NMA) transplants (非清髓性)

Answer 18

Less intensive chemotherapy
- less organ toxicity
- **shorter cytopenic phase
- achieve adequate immunosuppression to allow engraftment
- allow **older patients to benefit from curative potential of HSCT

Question 19

How does HSCT cure Leukaemia?

Answer 19

1. ***Eradicate leukaemia cells with high dose chemotherapy / radiotherapy
2. ***Replace the disease involved BM with healthy BM from normal donor
3. **Graft versus Leukaemia effect (GVL)
   - chronic GVHD associated with ↓ relapse
   - donor immune system-mediated reaction against host / patient organ / tissue —> donor immune system will also seek **residual tumour and eradicate them
   - but too much GVHD not good

GVHD:
- important complication after allogeneic HSCT

Question 20

***Complications related to Allo-HSCT

Answer 20

Conditioning phase:
1. Toxicities related to conditioning regimen (Chemotherapy / TBI)
- N+V, hair loss, mucositis, infections, bleeding
- **Cardiotoxicity due to Cyclophosphamide (e.g. Haemorrhagic myocarditis)
- **Haemorrhagic cystitis due to Cyclophosphamide (Acrolein, liver metabolite excreted in urine cause inflammation of bladder, use IV **Mesna to neutralise)
- **Veno-occlusive disease (VOD) of liver (i.e. chemotherapy toxicity on liver cells)

Graft infusion and Engraftment phase:
2. **Acute / Chronic GVHD
3. **Graft rejection (HvG) (i.e. Graft failure)

Cytopenia phase:
4. ***Infections
5. Haemorrhage

During immunosuppressants:
6. SE of immunosuppressants
- ***Cyclosporin: renal impairment, HT, PRES (Posterior reversible encephalopathy syndrome), TTP (Thrombotic thrombocytopenic purpura)

7. Late effects post-HSCT
   - Cataract (mainly due to TBI)
   - **Immunodeficiency (∵ immunosuppressants / GVHD)
   - Endocrine dysfunction and infertility (Metabolic syndrome, **Osteoporosis, **Hypogonadism)
   - **Second malignancy (e.g. PTLD (self notes))

Question 21

Graft vs Host disease (GVHD)

Answer 21

Acute GVHD:
- rash
- GI
- liver

Chronic GVHD:
- skin
- eye
- mouth
- GI
- liver
- lung
- MSS

Pathophysiology:
- **Alloreactivity
- **Autoimmunity
- Immunodeficiency
- Inflammatory activity —> Lesion —> Repair —> Fibrotic damage

Postulated pathologic mechanism of GVHD:
1. Conditioning chemotherapy cause tissue damage in patient
—> Cytokine release
—> ***Donor T cells activation
—> more inflammatory cytokine
—> Immune cascade + Recruitment of more effector cells
—> cause cell death in target organ tissue

2. Damage in GI mucosa
   —> **Release of bacterial LPS endotoxin
   —> **Immune effector cells (donor’s) activation
   —> Inflammation + Tissue damage

Question 22

Acute GVHD

Answer 22

Grade: Mild —> Intermediate —> Severe —> Life-threatening

Clinical features:
1. **Skin (grade by rule of 9)
- **maculopapular rash —> erythroderma with bullous formation / desquamation

2. ***Liver (grade by level of bilirubin)
   - impaired LFT (e.g. jaundice)
3. ***Lower GI (grade by volume of diarrhoea)
   - diarrhoea —> fresh blood / melena

Question 23

Chronic GVHD

Answer 23

• ***40-70% of patients receiving Allo-HSCT
• most common: first ***3-6 months post HCT, almost all within 1st year
• can affect ANY organs

Clinical features (very similar to **Autoimmune diseases):
1. **Skin changes
- Lichen-planus like
2. Dystrophic nails
3. Vitiligo
4. **Sjogren’s syndrome (dry eyes requiring frequent lubricant)
5. **Oral mucositis
6. **Bronchiolitis obliterans (airway obstruction) (GVHD of lungs)
7. **Impaired liver function
8. **GI (malabsorption)
9. **Sclerodermatous GVHD (fascia, joints affected)
10. Other: Genitalia, Muscles

Question 24

Infection Post-HSCT

Answer 24

Based on period after transplant:

Pre-engraftment phase (1st month) (**Neutropenia, **Barrier breakdown):
- Bacteria (Gram negative bacilli, Gram positive)
- Fungi (Aspergillus, Candida)

Post-engraftment phase (day 30 - 100)
- Fungi (Aspergillus)
- Virus (HSV, CMV)

Late phase (day 100 - >365) (Impaired B / T cell function from acute / chronic GVHD or immunosuppressants used to treat GVHD):
- Bacteria (Encapsulated)
- Fungi (Aspergillus, PCP)
- Virus (HSV, CMV, VZV, others)

Question 25

Late effect Post-HSCT

Answer 25

1. ***Accelerated metabolic syndrome (HL, DM, HT)
2. ***Atherosclerotic disease (CAD)
3. ***Cardiomyopathy secondary to chemotherapy
4. Fe overload
5. ***Renal dysfunction
6. ***Hypogonadism (∵ high TBI dose)
7. ***Osteoporosis / Osteonecrosis (∵ steroid use)
8. Thyroid disease
9. ***Cataract (∵ TBI)
10. ***Peripheral neuropathy (∵ vinca alkaloids)
11. Neurocognitive impairment (∵ cranial RT, high dose methotrexate / cytarabine)
12. ***Pulmonary dysfunction (∵ chemotherapy / cGVHD of lungs)
13. ***Second malignancy / Therapy-related myeloid diseases (e.g. MDS)