Clinical Approach to the Diagnosis and Classification of Leukaemia

Question 1

Leukaemia

Answer 1

Clonal diseases resulting from neoplastic proliferation of immature haemopoietic cells. This results in a loss of normal bone marrow function

Question 2

Acute Leukaemia

Answer 2

Imbalance between proliferation and maturation
Immature cells dominate
Variable degree of proliferation drive
Defect in maturation at the precursor level

Question 3

Chronic leukaemia

Answer 3

Mature cell dominate
Expanded pool of proliferating cells
Retain capacity to differentiate to mature cells
Can lose differentiation capacity -> acute disease

Question 4

Consequences of leukaemia

Answer 4

Marrow failure - anaemia, neutropenia
Infiltration of other tissues - lymph nodes, spleen, liver
Metabolic consequences of ⬆️ cell turnover - electrolyte disturbance, gout
Hyperleucocytosis

Question 5

WHO classification of tumours is based on:

Answer 5

Clinical features
Morphology
Immunophenotype
Cytogenetic
Molecular genetics

Question 6

WHO classification of leukaemias according to:

Answer 6

Lineage - myeloid, lymphoid, histiocytic
Precursor v mature

Question 7

Investigation of haematopoietic malignancies:

Answer 7

Clincal - history and exam
Routine blood test
Routine Biochem
Blood film
Bone marrow aspirate and trephine
Other tissue biopsies
Immunophenotype
Cytogenetic and molecular genetic techniques
Radiology, X rays, CT/MRI scans

Question 8

Symptoms of ⬆️ Hb, WBC and Platelets

Answer 8

Visual disturbance
Headaches

Question 9

Symptoms of pancytopenia:

Answer 9

Fatigue
Infection
Haemorrhage

Question 10

B symptoms

Answer 10

Fever
Sweats
Weight loss

Question 11

Clinical assessment of leukaemia (symptoms)

Answer 11

Symptoms of ⬆️ WBC, Hb and platelets
Pancytopenia
Bony problems - pain, fracture, spinal cord compression
B symptoms
Itch and hyper viscosity
Petechiae, bruising, gum hypertrophy, lymphadenopathy, splenomegaly

Question 12

Basics of clonality

Answer 12

Environmental influence
Epigenetic changes
Random acquired mutations
Genetic predisposition

Question 13

How to test for immunophenotype

Answer 13

Flow cytometry - identified by physical characteristic or antigen expression

Question 14

Flow cytometry can determine:

Answer 14

Number of cells +ve for given antigen
Intensity of antigen expression on individual cell

Question 15

Antibody panels designed to assess:

Answer 15

Cell lineage - myeloid or lymphoid
If lymphoid - B/T/NK

Stage of differentiation

Question 16

Principles of flow cytometry

Answer 16

Cells in suspension pass in single file through laser beam @ appropriate wavelength
Cell breaks beam and scatters light

Rapid, specific and sensitive

Question 17

Flow cytometry can establish:
(Leukaemia)

Answer 17

Haempoietic or not
Clonality
Lineage
Stage of maturation
Characterisation of B cell neoplasms

Question 18

Cytogenetics

Answer 18

Culture cells and stimulate mitosis
Arrest cell division in metaphase stage - colcemid
Giemsa staining
Examine under high power microscope

Question 19

Advantages of FISH

Answer 19

Can visual abnormalities in non dividing cells
Performed directed on prepared cells -> more rapid -> more cells analysed
Cryptic rearrangements detected
Characterise complex rearrangements
Can be preformed on tissue sections

Question 20

FISH probes

Answer 20

Chromosome paints - hybridise to metaphases

Locus Specific Probes - target genes of interest to identify rearrangements, deletions and gains in metaphase and interphase cells eg MLL locus on 11q23

Question 21

NGS

Answer 21

1️⃣DNA attaches to flow cell via complementary sequences
2️⃣DNA folds over to form bridge
3️⃣Primers added and polymerase synthesises reverse strand
4️⃣2 strands release, straighten and form new bridge
5️⃣Cluster of DNA clones
6️⃣Sequencing by synthesis - each cycles allows 1 base w/ fluorescently labelled tag to be added
7️⃣After each base, sequencer can detect fluorescent label and record what base was added for each DNA cluster

Question 22

Clinical applications for PCR

Answer 22

Diagnosis
- CML: detection of BCR::ABL1
- MPN: detection of MPL mutation

Prognosis
- FLT3 and NPM mutations give info about prognosis

Molecular monitoring by RQ-PCR
- MRD: response to therapy
- Follow up for evidence of molecular relapse

Question 23

PCR

Answer 23

Denaturation - @95ºc to separate dsDNA
Annealing - add primers to provide initiation site for elongation 45-60ºc
Extension - taq polymerase extends primers by adding dNTPs 72ºc

Question 24

Reverse Transcription PCR (RT-PCR)

Answer 24

Convert mRNA to complementary DNA which is then the substrate for PCR amplification

Question 25

Allele-specific PCR (AS-PCR)

Answer 25

The primers include a known mutation eg. JAK2 V617F

Question 26

Real-time quantitative PCR (RQ-PCR)

Answer 26

Used to measure absolute or relative amount of a cDNA or DNA molecule in a sample
Molecular monitoring

Question 27

NGS

Answer 27

DNA attaches to flow cell via complementary sequences
DNA folds over to form bridge
Primers added and polymerase synthesises reverse strand
2 strands release, straighten and form new bridge
Clusters of DNA clones
Sequencing by synthesis - each cycle allows 1 base w/ fluorescently labelled tag to be added
After each base, sequencer can detect fluorescent label and record what base was added for each cluster