BIO3CBH – Haematological

Malignancies

Lecturer
Helen Irving
Intended learning outcomes

By the end of this topic, you should be able to:

• Describe the differences between leukaemias, lymphomas and myelomas
in general terms highlighting which cells the malignancies occur
• Outline the aetiology of haematological malignancies and the underlying
genetic changes that occur in general terms.
• Use examples to explain how flow cytometry (FACS), cytogenetic (e.g. FISH)
and molecular biology tests can be used to support the diagnosis of
suspected malignancies (start with one or two and refer to the workshop)
• Interpret blood test results and explain your reasoning on various blood
disorders such as haematological malignancies
• Identify tests that will distinguish different haematological malignancies
from white cell disorders
Haematological Malignancies
- aetiology and detection
Helen Irving
Reference text: Hoffbrand & Moss (2016) Hoffbrand’s Essential Haematology
(pages 123-9, 216)
Haematological malignancies
Ref: Hoffbrand & Moss (2016) Hoffbrand’s Essential Haematology (pages 123-9, 216)

• Haematological malignancies account for ~8% of all cancers

• The major types are:
• Non-Hodgkin’s lymphoma, Leukaemia, Myeloma, Hodgkin’s lymphoma

Haematological malignancies ~9% in males and 7 % in females

 Aetiology of haematological malignancies
Ref: Hoffbrand & Moss (2016) Hoffbrand’s Essential Haematology (pages 123-9, 216)

• Inherited factors can predispose

patients to some haematological Leukemia cells containing EBV
malignancies.
• Viral infections can cause
malignancies (contributing to ~18%
of cases) and this especially so with
lymphomas
• Epstein Barr Virus (EBV)
• human T-lymphotrophic virus type 1 (HTLV-1)
• human herpes virus 8 (HHV-8)
• human immunodeficiency virus (HIV-1)
• Hepatitis C (HCV)

Image source: Centers for Disease Control and Prevention Dr Paul M Feorino
 Basic genetics of haematopoietic malignancy
Gene class Oncogenes Tumor Suppressor
Normal role Positive regulator Negative regulator
Cancer role Gain of function of Loss of function of a
protein that drives protein involved in
cell division mortality (or
positioning)
Viral cancer Carried or activated Bound and
by virus inactivated by viral
oncoproteins
Non-viral cancer Dominant mutation Recessive mutation
Source of Somatic tissues Either germ
mutation (not inherited) (inherited) or
somatic cells

Source: Figure 11.4, Hoffbrand & Moss (2016) Hoffbrand’s essential Haematology 7th ed
Cytogenetics in cancer diagnosis
Ref: Hoffbrand & Moss (2016) Hoffbrand’s Essential Haematology
(pages 129-34)

• Schematic of a chromosome is shown

Image source: Hoffbrand & Moss figure 11.8

• Chromosome abnormalities can range from point FISH analysis

mutations in genes, to deletions and
rearrangements and can also include epigenetic
changes

• Cytogenetics – microscopic examination of

chromosomes for abnormalities and various
rearrangements that are associated with different
leukaemias
• Fluorescence in situ hybridization (FISH) analysis
Al-Achkar et al (2010) Oncology Letters 1:445-7
To find out more about the FISH technique see:
O'Connor, C. (2008) Fluorescence in situ
hybridization (FISH). Nature Education 1(1):171
 Human chromosome translocations can lead to cancer
• Some chromosomal translocations are restricted to few types of leukaemia (e.g.
Philadelphia chromosome) while others can be found in a wide variety of leukaemia

Nambiar et al. (2008) Biochim Biophys Acta 1786: 139-152

O'Connor, C. (2008) Human chromosome translocations and cancer. Nature Education 1(1):56
Molecular Analysis of Genes in Haematology Labs
• Microarray-based gene expression. Heat map of RNA gene expression where red
shows increased and green decreased expression.
• Changes in expression have been monitored in control (untreated) versus treatments and
clusters of genes with similar behaviours (up or down expression) have been grouped.
• Molecular amplification of specific genes and/or
sequencing can reveal mutations at the
nucleotide level or at the epigenetic level
(e.g. gene methylation)
• Quantitative polymerase chain reaction (PCR) to detect
residual cancer cells
Quantitative PCR

Image source: Hoffbrand & Moss figure 11.12 Karen S. Hathcock et al. Blood 2015;126:2291-2301
Flow Cytometry in Blood Cancers
• Antibodies to specific cell surface markers (often CD proteins) are used to detect
different cell lineages and determine malignancy type
• The antibodies are labelled with different fluorochromes and thus multiple
surface markers per cell can be detected.

Image sources: Keeney et al. 2017 International Journal of Laboratory Hematology 39S:86-92
 Haematological Malignancies -
Leukaemias
Helen Irving
Reference text: Hoffbrand & Moss (2016) Hoffbrand’s Essential Haematology
Leukaemia classification
• Leukaemias are heterogeneous group of haematological malignancy. Leukaemias are caused by
unregulated proliferation of a clone of immature blood cells derived from mutant haematopoietic stem
cells. Multiple genetic and epigenetic changes are acquired leading to the cancers.
• Leukaemias basically classified as acute or chronic and consideration is paid to their cell or origin
• Presence of granulocytes
• Myeloid or non-lymphocytic leukaemia
• Lymphocytes present
• Lymphocytic leukaemia

• Major types:
• Acute myeloid leukaemia (AML)
• Chronic myeloid leukaemia (CML)
• Acute lymphoblastic leukaemia (ALL)
• Chronic lymphoblastic leukaemia (CML)
• Rare type – hairy cell leukaemia
• Classification based on cell differentiation, types of CD markers expressed and genetic mutations
Image sources: : Hoffbrand & Moss figure 11.8 and 11.12
Keeney et al. 2017 International Journal of Laboratory Hematology 39S:86-92
Leukaemia classification - Immunophenotyping (CD markers)
• CD markers via flow cytometry (FACS) are used to identify the exact lineage
of a leukaemia.
• It is important to know if the cells are of myeloid or lymphoid lineage as that
will affect treatment options
Cell line Antigens
Myeloid CD13, CD33, CD117
Monocytic CD14, CD11c, CD64
Erythroid Glycophorin (CD235a)
Megakaryoblastic (platelet) Platelet antigens (CD41, CD42, CD61)
Undifferentiated Myeloperoxidase
Lymphoid TdT
B cell lineage CD19, cCD22, cCD79a, CD10 (+/-) clg (+ - preB), slg (-)
T cell lineage CD7, cCD3, CD2
Comparison between acute and chronic leukaemias

Presentation Acute leukaemia Chronic leukaemia

Onset Rapid Insidious
Death Months Years
Age All Adults
WBC number Raised/normal/low Raised
Appearance of cells Blast (immature) usually >20% Mature
Neutropenia Present Absent
Anaemia Present Present
Platelets Low Normal/increased
Organomegaly Mild Severe
Acute myeloid leukaemia (AML)
Ref: Hoffbrand & Moss (2016) Hoffbrand’s Essential Haematology (Chapter 13)

• AML is the most common acute leukaemia and incidence increases with age
• Gene mutations are important in classifications and these are indicated by
mutation details and chromosomal location.
• Variability in symptomology of presenting patients
• Some asymptomatic while others very ill with
symptoms of anaemia, neutropenia and
infection due to bone marrow infiltration into blood
• Acute promyelocytic leukaemia is associated with Auer rod
disseminated intravascular coagulation (DIC)
• Diagnosis
• Blood count and film, bone marrow aspirate and Acute myelogenous leukaemia
trephine, immunophenotyping, cytogenetics and (AML) – note the Auer rods
molecular biology
Blood smear image source: https://library.med.utah.edu/WebPath/HEMEHTML/HEMEIDX.html
Acute lymphoblastic leukaemia (ALL)
Ref: Hoffbrand & Moss (2016) Hoffbrand’s Essential Haematology (Chapter 17)

• ALL is a clonal malignancy of lymphoid precursor cells. ALL is most common in

children.
• For treatment options, need to classify - generally as early pre-B, pre-B, B-cell
and T-cell subtypes
• Variability in symptomology of presenting patients
• Accumulation of malignant lymphoblasts leads to
decreased normal cells in blood and symptoms of
anaemia, infection and haemorrhage.
• Diagnosis
• Blood count and film, bone marrow aspirate and
trephine, cytochemistry (Sudan black and
myeloperoxidase are negative in ALL; while Acute lymphoblastic leukaemia (ALL) –
T-ALL is reactive with acid phosphatase stain) note the immature lymphocytes (blasts)
, immunophenotyping, cytogenetics and molecular biology
Blood smear image source: https://library.med.utah.edu/WebPath/HEMEHTML/HEMEIDX.html
Chronic myelogenous leukaemia (CML)
Ref: Hoffbrand & Moss (2016) Hoffbrand’s Essential Haematology (Chapter 14)
• Chronic leukaemias have a slower progression than acute leukaemias and are broadly classified into
myeloid or lymphoid
• CML accounts for ~15% of leukaemias and arises in bone marrow stem cells precursors to
granulocytes & megakaroyocytes
• Hallmark identification of CML is the Philadelphia chromosome due to a reciprocal translocation
resulting in different ends to chromosomes 9 and 22 and expression of in frame fusion gene –
chimeric BCR-ABL that can be detected by FISH etc.

Al-Achkar et al (2010) Oncology Letters 1:445-7

BCR-ABL is a hybrid protein kinase
• The Philadelphia chromosome contains a CHIMERIC gene and translates a
functional protein BCR-ABL
• The BCR-ABL fusion protein is a constitutively active tyrosine protein kinase
• The tyrosine kinase inhibitor Imatinib mesylate (Gleevec) is used to reduce
BCR-ABL activity and is now a cornerstone first line therapy for CML

BCR-ABL gene
CML cont’d
• Symptomology of patients who usually present in chronic phase
• Anaemia, anorexia, and weight loss. Splenomegaly is common with associated pain and bloating.
Some patients present with gout and hyperviscosity due to very high white blood cell count.
• Diagnosis
• Blood count and film – very high white cell count of apparently normal myeloid cells (mainly
myelocytes and neutrophils but basophils regularly seen). Bone marrow aspirate and trephine
less informative, cytogenetics shows Ph chromosome and
molecular biology
• Monitor Ph chromosome by quantitative PCR throughout
treatment
• Management
• Tyrosine kinase inhibitors mainstay now
• Stem cell transplantation also considered
Chronic myelogenous leukaemia (CML) – note the
granulocytic cells including myeloid cells and bands
Blood smear image source: https://library.med.utah.edu/WebPath/HEMEHTML/HEMEIDX.html
 Chronic lymphoblastic leukaemia (CLL)
Ref: Hoffbrand & Moss (2016) Hoffbrand’s Essential Haematology (Chapter 18)
• CLL is a clonal proliferation of mature B lymphocytes and is a disease of the elderly
• Variability in symptomology of presenting patients
• Asymptomatic or minimal symptoms, to bulky lymphoadeonpathy and hepatosplenomegaly. Diagnosis
often by chance following routine blood count
• Diagnosis
• Blood count and film with some smear cells (cells burst during preparation), immunophenotyping to
show clonal nature of B lymphocytes with a single kappa band on cell surface (i.e. a monoclonal
population). Bone marrow aspirate and trephine to see cell numbers, molecular biology – PCR or
antigen testing

Blood smear image source: https://library.med.utah.edu/WebPath/HEMEHTML/HEMEIDX.html

Hairy Cell Leukaemia

• Relatively rare chronic leukaemia

• Cytoplasm of lymphocytes is often
ragged or “hairy”, giving rise to the
name
• Monocytopenia is a distinctive
feature of presenting patients
• Immunotyping shows CD11c, CD19,
CD25, CD103
• Due to a mutation in BRAF protein From Chris Bradley
kinase gene
• Several effective treatments
 Haematological Malignancies -
Lymphomas and Myelomas
Helen Irving
Reference text: Hoffbrand & Moss (2016) Hoffbrand’s Essential Haematology
Lymphomas

From Chris Bradley

 Thomas Hodgkin
Hodgkin’s lymphoma
Hoffbrand & Moss (2016)
Hoffbrand’s Essential Haematology (Chapter 19)
Dorothy Reed
• Symptoms
• Lymphoadenopathy and constitutional symptoms
• Diagnosis
• CBC ,chest x-rays, computed tomography (CT) scans, lymph node
biopsies, bone marrow aspirations (sometimes), additional
biochemical tests, etc.
• Arises from B cells in lymph nodes – presence of
Reed-Sternberg cell (binucleate) is a diagnostic feature in
https://askhematologist.com/hodgkins-disease/
lymph node biopsies
• Reed-Sternberg cells associated with mutations in immunoglobulin gene including
loss of HLA (human leukocyte antigen or major histocompatibility (MHC)) class I
expression
• Association with Epstein-Barr virus (EBV) is seen in >50% of cases
 Non-Hodgkin’s lymphoma (NHL)
Hoffbrand & Moss (2016) Chapter 20

• Malignant solid tumours in lymph nodes

• More common than Hodgkin’s and ~85%
due to B cell and the remainder T or NK
cells origin
• Irregular pattern of spread and disease
often developed outside lymph nodes
although presentation invariably includes
swollen lymph nodes
• Separation between lymphoma and
leukaemia blurred
• Some associated with virus infections

MM = multiple myeloma; WM = Waldenström macroglobulinemia; NHL = non-

Hodgkin lymphoma; and CLL = chronic lymphocytic leukemia Merlini and Stone (2006) Blood 108: 2520-2530
Multiple Myeloma
Hoffbrand & Moss (2016) Hoffbrand’s Essential Haematology (Chapter 21)

• Clonal proliferation of plasma cells in bone marrow

• Myeloma cells secrete monoclonal immunoglobulin fragment (M protein)
• Single heavy and single light chain class (κ or λ)
• Most produce IgG or IgA
• Symptoms
• Chronic bone pain, mainly elderly,
• Diagnosis
• Back pain and acute phase response (high ESR or C-peptide)
• indicative, bone marrow biopsies
• electrophoresis of serum (or urine) proteins

Image source: https://bpac.org.nz/bt/2011/july/serum-protein.aspx

The end