1

Recalling gene expression

Why we study the molecular genetics of hematologic
malignancies ?
Objective
• Molecular mechanism is complex process.
• To understand pathogenesis of hematological diseases/ to identify
the underlying molecular deregulation in haematopoietic
malignancies
– Hyperproliferation
– block in differentiation
– sensitivity to growth factors
• To Develop Diagnostic/Predictive And Prognostic Biomarkers For
Different Type Of Hematologic Malignancies
• To identify potential or specific therapeutic target
• Risk stratification—into low, intermediate, or high risk group

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 Recalling gene expression
• All the genetic information that makes up an organism is
encoded in the DNA.
• This information is transcribed into mRNA, is translated into
protein.
• Changes that affect the DNA or RNA sequence or its
expression, either in the germ line or acquired after birth, can
cause many hematologic disorders.

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Compartmentalization of the cell

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 Compartmentalization of the cell
• The cell has membrane, mitochondria & Golgi complex
and rough endoplasmic reticulum -post‐translational
modification protein .
• Nucleus, contain DNA, is tightly packaged by proteins
and the DNA/protein complex is known as chromatin.
• Heterochromatin-when DNA is tightly packaged, the
genes more likely to be not expressed
• Euchromatin- DNA is less tightly packaged and is
lighter in appearance.

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Nucleus
• The DNA in the nucleus is
distributed among 22 pairs of
autosomal chromosomes
(numbered 1–22, in order of
size) and two sex chromosomes
• Chromosomes are divided into
two arms: a short arm (P) &
longer arm (q).
• chromosomes joining region is
centromere.
• Chromosomes are further
subdivided into light and dark
bands (depending on how they
stain with the Giemsa dye)

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• Only 1-2% of human genome are genes.
• Genes are divided into:
• Protein-coding genes- provide instructions for making
proteins.
• RNA-coding genes- provide instructions for making
functional RNA molecules (e.g., rRNA, tRNA, miRNAs)
• Up of 98% DNA are non-coding sequences
– Important roles in regulating genes, maintaining
chromosome structure, and more.
Example: regulatory sequences
• These control how genes are expressed (turned on/off, or how
much protein/RNA is made).
• Promoters- DNA sequences near genes that help initiate
transcription.
• Silencers- DNA sequences that reduce or silence gene
expression. 7
 Conti…
• Transcription begins- at the Promoters sequences of DNA
located upstream (near the 5' end) of the gene
• Binding of Transcription Factors-
• Unwinding of DNA
• Initiation of RNA Synthesis

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 Conti…
• A copy of the DNA of genes
is transcribed into RNA by
transcription in the nucleus.
• RNA is processed and
transported into the
cytoplasm.
• RNA corresponding to protein
genes is then translated in the
cytoplasm.
• Not surprisingly, these
processes are very complex,
affording opportunities for the
cell to exquisitely regulate the
complement of proteins made
but also vulnerable to errors
that lead to disease.

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 Methods of control of gene expression
• Gene, expression is regulated to ensure genes are expressed at
the right time and in the right cells.
Transcriptional Control
Cis-Regulatory Elements/ DNA sequences
• Promoters: DNA sequences near the transcription start site
(TSS) where RNA polymerase binds
• The promoter defines the location of transcription start site(s)
and the directionality of transcription
• Enhancers/Silencers: Distal DNA regions that
increase/decrease transcription via transcription factor (TF)
binding.
• Insulators: Block enhancer-promoter interactions to prevent
aberrant activation

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 Control of gene expression
Transcription Factors (TFs)
• Sequence-specific DNA-binding proteins (e.g., SP1, NF-κB, p53) that
recruit RNA polymerase.
• E.g.
– RUNX1 (hematopoiesis), MYC (cell proliferation), OCT4 (stem cell
pluripotency).
Epigenetic/Chromatin Structure/- regulation of gene expression
through control of packing and unpacking of DNA
• E.g. Histone Modifications & TADs
• Topologically Associating Domains (TADs)boundaries :
• TAD boundaries are marked by proteins called cohesins & CTCF
(CCCTC‐binding factor).
• Cohesins: Protein complexes that loop DNA and stabilize TAD
structures.
• Thus, abnormalities in have the potential to disrupt TAD
boundaries and alter gene expression. They associate with AML
and myelodysplastic syndromes.
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 Control of gene expression
Post-Transcriptional Control
• Regulation of RNA processing, stability, and translation.
E.g. RNA Splicing
• Nuclear Export
• NXF1/NXT1 mediate mRNA export; dysregulated in cancer
(e.g., NUP214 fusions in T-ALL).
Translational Control
• Regulation of protein synthesis from mRNA.
• E.g. Regulatory RNAs
• miRNAs: Fine-tune translation (e.g., miR-15/16 target BCL2 in
CLL). miR-15/16 deletion → BCL2 overexpression → CLL
progression.
Environmental & External Signals
 E.g. Hormones, Stress Responses, Nutrient Availability…

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• Gene expression is controlled by transcription factors that bind cis‐elements.
DNA‐binding TFs binding to cis‐elements then recruit cofactors and other
transcriptional regulators. It is also likely that DNA‐binding TFs and
non‐DNA‐binding TFs/cofactors may form preformed complexes that bind directly
to DNA. Binding of RNA polymerase II, the preinitiation complex, and DNA
looping between different cis‐elements triggers transcription and elongation of
polymerase II along the gene
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Genes are in yellow boxes. Unwrapped genes (1) are in an open chromatin,
euchromatic state. These genes have unmethylated CpG DNA residues (shown as pink
lollipops) and are transcribed (green arrow). Genes that are not transcribed (2 and 3)
often have methylated CpG residues (blue lollipops). Closed chromatin is shown as
closely packaged nucleosomes. In cancer, gene expression is often inappropriate, and
this is reflected in the changed chromatin structure. Thus genes such as p16, VHL, and
E‐cadherin (E‐cad) are not transcribed. CpG residues are methylated and the chromatin
is in a closed conformation.
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15
 Introduction to Hematopoiesis
• During life blood cells need to be replaced frequently,
– they undergo many divisions during growth.

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 Introduction Genetic Basis of Hematopoiesis

• Hematopoiesis requires the constant production of large

numbers of peripheral blood cells.
• This process is under-tight control of :
 transcription factor networks
 Cytokines
 growth factors
 hormones
 Epigenetics
 Microenvironment (Niche) Regulation

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 Transcription factors (TFs) networks
• Are proteins that regulate gene expression by binding to specific
DNA sequences in the promoter, enhancer, or silencer regions of
genes.
– Their primary role is to activate or repress transcription of target genes
• The function of TFs helps regulate the cell‘s potential to make
blood cells of different lineages, proliferate, undergo apoptosis, and
self renew.
• E.g.
– GATA1: essential for erythroid and megakaryocyte development.
– PU.1: critical for myeloid (Granulocytes, monocytes) and lymphoid (B cells,
T cells) differentiation.
– RUNX1: required for the development of all blood cell lineages.
– CEBPΑ: promotes granulocyte differentiation.
– NOTCH1: regulates t-cell development.
• Acquired mutations TFs encoding genes cause lymphoma and
leukemia. 18
Cytokines- signaling proteins that regulate the proliferation,
differentiation, and survival of hematopoietic cells

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 Epigenetic
• Epigenetic mechanisms control gene expression without
altering the DNA sequence.
• They ensure that genes are turned on or off at the right
time during blood cell development.
E.g.
• DNA methylation: Silences genes that are not needed in
specific blood cell lineages.
• Histone modifications: Alter chromatin structure to make
genes accessible or inaccessible for transcription.
• Non-coding RNAs (e.g., microRNAs): Regulate gene
expression post-transcriptionally.

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Introduction Genetic Basis of Hematologic Hematopoiesis

• These factor have the pivotal role in programing:

 Proliferation
 Differentiate
 Maturation
 Apoptosis… through signaling
• Cell signaling Cell signaling is the intracellular part of cell-to-
cell communication, the conversion of the extracellular
Messenger into an intracellular response.

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 Signaling
• Blood cells, like other cells, receive signals that influence how
they proliferate, differentiate and apoptoses
• Signaling inputs can be transmitted in a number of different
ways
• Cytokines or growth factors are released systemically into the
bloodstream by organs bind to their receptors with very tightly
and specifically-
• w/h activates downstream signaling pathways, which
transmit signals into the cell ultimately lead to changes in gene
expression, to regulate processes like cell growth, survival,
and differentiation.

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 Signaling
 These pathways are often depicted as linear, but in reality, they
are highly interconnected and can interact with other
pathways, especially when multiple ligands/receptors are
involved.
Activation of downstream signaling pathways
When proteins interact with themselves or other proteins to
regulate biological processes, including gene expression, signal
transduction, and enzymatic activity

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 signaling pathways interactions in hematopoietic cells.
1. The Wnt pathway is critical for the self-renewal of HSCs
2. Cytokine Signaling Pathways
• 2.1. PI3K Pathway:
– promotes cell survival, growth, and metabolism, and
interacts with the RAF/MAPK pathway.
• 2.2. RAS/MAPK Pathway-cell proliferation/gene expression
– Differentiation
– Survival
– Apoptosis resistance
• 2.3. JAK-STAT Pathway:
• Hematopoiesis
• Immune responses
• Cell proliferation/survival
• Inflammation
• Dysregulation of these pathways is often associated with diseases
like leukemia.

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• HEMATOLOGIC MALIGNANCIES

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Objectives

At the end of this chapter, the student shall be able

to:
 Define and differentiate the terms neoplasm and malignant and
identify hematopoietic disorders that can be included in each
category.
 understanding of the genetic alterations that occur in the
leukemia genome,

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 Objectives
• Explain how proto-oncogenes are activated and the role that
oncogenes and tumor suppressor genes and their protein
products play in the etiology of hematopoietic neoplasms.
• Name the leukemogenic factors of leukemia and propose how
each contributes to the development of leukemia.
• Describe the World Health Organization (WHO) classification
system used for MDSs, MPNs, the leukemias, and lymphoid
neoplasms.

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 Hematopoietic neoplasms…
• Neoplasm (tumor) means ―new growth.‖ Neoplasms arise as a
consequence of dysregulated proliferation of a single
transformed cell
• Neoplasms are either benign or malignant
• A malignant neoplasm is a clone of abnormal, anaplastic,
proliferating cells, which often have the potential to
metastasize
• Only malignant tumors are correctly referred to as cancer.
• Hematological malignancies are cancers of the blood, bone
marrow, and lymphatic system
• Benign myeloid and lymphoid proliferation is usually a
reactive process (reactive leukocytosis; leukemoid reaction).
• A benign neoplasm can be premalignant and progress with
further genetic mutations to a malignant neoplasm. 28
 Hematopoietic neoplasms…

• The spectrum of hematopoietic proliferation ranges from benign to

malignant outcomes-derived from a mutated precursor cell that
divides incessantly

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 Clonal…
Clonality of Leukemic cell
• There are evidences serve to establish the fact that
haematological malignancies are clonally derived from a
single ancestral cell.
• Hematopoietic neoplasms are believed to occur as the result of
a somatic mutation(s) of a single hematopoietic stem or
progenitor cell.
• Evidence for the clonal evolution of neoplastic cells comes
from cytogenetic studies.
• More than 50% of individuals with leukemia show an acquired
abnormal karyotype in hematopoietic cells whereas other
somatic cells are normal.

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 Clonality

Evidence: of clonal
• lymphoproliferative disorders- In nearly all
lymphoproliferative disorders, the malignant cells carry a
unique rearrangement in their immunoglobulin or T cell
receptor (TCR) genes.
– In contrast, proliferating normal lymphocytes show polyclonal and
therefore diverse patterns of antigen receptor rearrangements, each with
a different antigenic specificity
• In CML - Philadelphia (Ph) chromosome- are usually found in
all cells of the clone

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X chromosome inactivation patterns (XCIPs) in female

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 Clonality…
• The standard model of leukaemogenesis postulates the
stepwise acquisition cell and of genetic mutations in a
susceptible its progeny, leading to autonomous the
development of an clone that expands enough to cause a
clinical syndrome.
• The phenotype of the resulting malignancy is dependent on the
host of the specific mutations and the cells
• The core challenge of this model :
• Discovery of ontogeny of the identical mutations in
phenotypically diverse malignancies
• the flexibility of the leukemic stem cell compartment have
added new layers of complexity

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 Cancer Stem Cells
• The cell w/h acquires a cancer-initiating mutation is termed the
cell of origin or cancer-initiating cell.
• The cancer-initiating cell can be
• HSC or more differentiated progenitor cells.
• Restricted progenitor cells CLP & CMP) may acquire
mutation(s) that reactivate the self-renewal program(s) and that
allow them to become cancer-initiating cells
• Identification of the hematopoietic form of a cancer stem cell,
or leukemic stem cell (LSC), for each type of leukemia has
become a major focus of research
• Both populate the marrow simultaneously. In untreated
leukemias and during relapse, the leukemic cells dominate

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• Clonal expansion of neoplastic cells
in the bone marrow over a period of
time leads to a decrease in the
concentration of normal cells in both
the bone marrow and peripheral blood

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 Terms: neoplasm precursor lesions/ Myeloid
• Clonal haematopoiesis (CH) - the presence of a population of
cells derived from a mutated multipotent stem/ progenitor cell
harbouring a selective growth advantage in the absence of
• unexplained cytopenias,
• haematological cancers, or
• other clonal disorders.
– The incidence of CH increases with age
• Clonal haematopoiesis of indeterminate potential (CHIP)
referring specifically to CH harbouring somatic mutations of
myeloid malignancy-associated genes detected in the blood or
bone marrow at a variant allele fraction (VAF) of ≥ 2% (≥4%
for X-linked gene mutations in males)
• in individuals without a diagnosed haematologic
disorder or unexplained cytopenia
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 Myeloid neoplasm precursor lesions
• Clonal cytopenia of undetermined significance (CCUS)- is
defined as CHIP detected in the presence of one or more persistent
cytopenias that are otherwise unexplained by haematologic or non-
haematologic conditions and that do not meet diagnostic criteria for
defined myeloid neoplasms.
• Cytopenia definitions are harmonized for CCUS, MDS, and
MDS/MPN; they include
• Hb Hb <13g/dL in males and <12g/dL in females for anaemia,
absolute
• neutrophil count <1.8 ×109/L for leukopenia, and
• platelets <150 ×109/L for thrombocytopenia
Tumour-like lesions with B-cell predominance
• Reactive B-cell-rich lymphoid proliferations that can mimic
lymphoma
• IgG4-relateddisease
• Castlemandisease
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Precursor lesions and their evolution to myeloid neoplasm

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 Mutations leading to malignant transformation
• The mutations leading to malignant transformation of the
cancer stem cell or LSC often are associated with:
 1.large scale/ Genomic mutation- chromosomal Mutation
like translocations, inversions and numerical aberrations)-
That is observed as an abnormal karyotype when
studying cells in mitosis
 2. Aberrations in DNA at the molecular level (e.g. point
mutations, microdeletions and epigenetic changes)
• The KIT (c - KIT ) gene- leading to activation of its kinase
activity.
• Mutation of fms like tyrosine kinase 3 gene (flt3)

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 Large scale
• Chromosomal translocations
• Chromosomal translocations are probably the most
extensively studied genetic abnormalities in haematological
malignancies
• Balanced translocations: Involve a reciprocal exchange of
genetic material between two chromosomes
• may result in aberrant function of genes adjacent to the
breakpoint

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 Conti…
• Fusion( chimeric) genes:
• encode a fusion protein with oncogenic properties
• This mechanism is seen in in many myeloid malignancies and
some ALL
• Fusion genes can also result from interstitial deletions
– E.g. deletion of chromosome 4 giving rise to the FIP1L1 PDGFRA
fusion gene in chronic eosinophilic leukaemia)
• Intrachromosomal inversions E.g. inversion 16 in AML

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 Conti…
Large deletions and aneuploidy
• Chromosome deletions and disorders of chromosome
number(aneuploidy) are frequently seen in haematological
malignancies.
• Hyperdiploidy is the most frequent cytogenetic abnormality in
childhood all and can involve any chromosome
• Trisomy 8: AML, myelodysplasia and myeloproliferative
disorders
 In either case, genomic changes in the cancer cells lead to a
survival and/or proliferation advantage over normal cells
 In acute leukemia, this unregulated proliferation is accompanied by an
arrest in maturation at the blast cell stage

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43
 Etiology
o several factors have been suggested as playing roles in
leukemogenesis:
o (1) genetic susceptibility
o (2) somatic mutation
o (3) viral infection, and
o (4) immunologic dysfunction

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 Genetic Susceptibility
• Strong evidence suggests that hereditary factors and abnormal
genetic material have important leukemogenic effects.
• A number of individuals who have congenital abnormalities
associated with karyotypic abnormalities have a markedly
increased risk of developing acute leukemia
• Down syndrome in which the extra chromosome 21 may
participate
• Various other congenital disorders also are associated with an
increased risk for leukemia

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 Environmental factors

Somatic Mutation
• an acquired change in the genetic material of cells other than
those involved in reproduction.
• Mutations in the chromosome near proto-oncogenes likely
play a role in neoplasm development.
• More than 50% of patients with leukemia have acquired
abnormal karyotypes and cytogenetic studies have revealed
specific, consistent mutations in certain subgroups of
hematopoietic neoplasms
• Radiation, some chemicals (benzene), and drugs

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 Viral Infection
Retroviruses
• human T-cell leukemia/lymphoma virus (HTLV-I, II, V) and
• HIV-1 from cell lines of patients with mature T-cell
malignancies.
• Exactly how viruses induce leukemia is unclear, but the
incorporation of the viral genome into host DNA is suspected
to lead to activa tion of proto-oncogenes.
• IMMUNOLOGIC DYSFUNCTION
• MISCELLANEOUS FACTORS

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 Disease phenotype
• The cancer cell genotype is generally maintained (stably
inherited) during cell division.
– This implies that the tumor cell DNA determines the
disease phenotype.
• Chromosomal alterations
• Micro-mutation
• Change in Epigenetic
• contribute to tumorigenesis by altering the
expression levels of key:
A. Oncogenes or
B. Tumour-suppressor genes.

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 A. Oncogene activation/Changes proto-oncogenes to
oncogenes
• Proto-oncogenes are genes that regulate the initiation of DNA
replication, cell division, the commitment to cellular
differentiation, and/or apoptosis
• One of the defining features of cancer cells is their ability to
proliferate under conditions in which normal cells do not.
• The proteins encoded by proto-oncogenes function in the
signaling pathways by which cells receive and execute growth
instructions.
• The mutations that convert proto-oncogenes to oncogenes are
often either resulting in the continuous (constitutive) activity
of a protein without an incoming signal or production of a
protein at the wrong place or time.

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 Proto-oncogenes
• Growth factors These molecules provide the signals to grow
and when activated to an ―oncogene‖ result in an autocrine
growth stimulation.
• Growth factor receptors When activated to an oncogene, the
mutated receptors are capable of triggering growth-promoting
signals, even in the absence of ligand (cytokine) binding.

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 Proto-oncogenes
• Signal transducers
• The normal function of proto-oncogenes) is to pass receptor
signals to downstream targets.
• Many of these proto-oncogenes encode protein-tyrosine
kinases found on the inner surface of the membrane.
• Often the oncogenic form of these genes produces signaling
molecules that exist in a constantly activated state in the
absence of growth factor/receptor interaction and signaling.
• Transcription factors These proteins bind DNA and function
to control the expression of cellular genes required for
proliferation.

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52
 B. Tumor Suppressor Genes
• Specific tumor suppressor genes function to inhibit cell growth
in normal cells.
• Block cell cycle progression in the event of damaged DNA or
to trigger apoptosis if the damaged DNA cannot be repaired.
• Tumor suppressor mutations, are recessive, loss-of-function
mutations.
• tumor suppressor gene that normally functions to arrest
excessive growth of cells.
• Thus, in addition to oncogene activation that results in growth-
promoting activity, tumor cells often have inactivating
mutations of growth-suppressing genes that can also contribute
to tumor development.

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 Tumor Suppressor Genes…
• Tumor suppressor mutations run in families in which affected
family members appear to inherit susceptibility to and develop
certain kinds of tumors at rates much higher than the normal
population.
• The first of these familial cancers to be explained at the
molecular level was the inherited susceptibility to
retinoblastoma (a tumor of the eye) in certain families
• mutations that inactivate both of the RB loci on each of
the chromosomes 13
• Acquired mutations of RB (i.e., nonfamilial) are found in
about 25% of sporadic cancers and have been observed in
various hematopoietic neoplasms.

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 Tumor Suppressor Genes
• Inactivation of the p53 gene/tumor suppressor gene, is the
most common genetic defect detectable in human tumors
• damaged p53 gene can be inherited (like familial
retinoblastoma) resulting an inherited susceptibility to a
variety of cancers including hematopoietic neoplasms.
• The p53 protein is a major component of the body‘s anti tumor
army, serving as a ―molecular policeman‖ monitoring the
integrity of the genome

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 Inactivating point mutations
• E.g. inactivating point mutations of the CEBPA gene
encoding the transcription factor C/EBP α
• are found in 20% of cases of M2 AML.
• The gene is critical for differentiation of myelomonocytic cells
• The mutation leads to truncation of the normal protein, but
allows the translation of a smaller protein initiated downstream
of the mutation.
• leading to a differentiation block in myelomonocytic cells.

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 Classification of hematological malignancy
• Multiple classification schemes have been employed for these
diseases over the years.
• The earliest classification systems were based upon tissue
architecture and the cytologic appearances of the neoplastic
cells
Revised European-American classification (REAL)
• In 1994 developed the diagnosis of lymphoid malignancies by
integrating morphology, immunophenotype, genetic
features, and clinical behavior for the first time.
• It served as the blueprint for the modern WHO classification
of hematological cancers.

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 French-American-British system(FAB)
 neoplastic disorders of hematopoietic cells typically were
grouped into three main categories:
 myeloproliferative disorders (MPDs),
 myelodysplastic states or syndromes (MDSs), and
 acute leukemias (ALs), including both myeloid and lymphoid
 This classification system used
 blast count
 lineage commitment
 cell morphology,
 level of differentiation of the neoplastic cells,
 cytochemistry, and
 immunophenotyping
• Blast Threshold for Acute Leukemia
– Defined ≥30% blasts in marrow as acute leukemia
•

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 The FAB classification system
M0 AML with minimal differentiation
M1 Myelogenous Blasts & Promyelocyte without further maturation
M2 Myelogenous Myelogenous cells with maturation beyond blast
and promyelocyte stage
M3 Promyelocytic Promyelocytes predominate in the bone marrow
M4 Myelomonocyitic Both myelogenous and monoctic cells are present (at least 20% of the total
leukocytes)
M5 Monocytic Most cells monocytic
M6 Erythroleukemia Known as Di Guglielmo syndrome; abnomal proliferation of both erythroid
and granulocytic precursors; may include abnormal megakaryocytic and
monocytic proliferations
M7 Megakaryocytic Large and small megakaryoblast with a high nuclear-cytoplasm ratio; pale
agranular cytoplasm
L1 Homogenous One population of cells
Small cells predominant;
Nuclear shape is regular with an occasional cleft; Chromatin pattern is
homogenous &
Nucleoli are rarely visible; Cytoplasm moderately basophilic
L2 Heterogenous Large cells
Nuclear shape irregular; cleft in the nucleus common
1 large Nucleoli, Cytoplasm varies in colour
L3 Burkitt‘s lymphoma Cells are large and homogenous in size
type Nuclear shape round or oval;
1-3 prominent nucleoli 59
Cytoplasm is deeply basophilic; often with prominent vacuoles
 Classification of hematological malignancy…
• The WHO Classification (2001, updated in 2008, 2016,
2022) expanded on REAL & FAB by:
• Adding molecular genetics.
• Defining new entities
• Integrating disease-specific prognostic markers
• The WHO & International Consensus Classification (ICC)
systems are the most widely used
• WHO Classification (2022)
– Integrates morphology, immunophenotype, genetics, and
clinical features.
– Example: Defines AML by ≥20% blasts or AML-defining
mutations (e.g., RUNX1::RUNX1T1).
• ICC (International Consensus Classification, 2022)
– Emphasizes molecular drivers (e.g., TP53-mutant AML as
a distinct entity).

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 Classification…
• A deliberate attempt is made to prioritize classifying tumour
types based on defining genetic abnormalities where possible.
• reason
 To identify the underlying molecular deregulation
 To develop diagnostic/predictive and prognostic
biomarkers for different type of
 To identify potential or specific therapeutic target
 Risk stratification—into low, intermediate, or high risk
groups

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 WHO classification

• The most recent classification scheme is the 2022 WHO

classification of tumors of hematopoietic and lymphoid tissues
 refined and expanded upon the use of objective diagnostic
criteria for clearly defined entities
(cell lineage, degree of cell differentiation, Morphology,
Cytochemistry, immunophenotype, genetic
abnormalities, cytogenetics, DNA analysis, and clinical
features)

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 Hierarchical Classification Structure

• The classification structure follows a lineage-based

framework, flowing broadly from benign to malignant and
branching down to category, family, type (disease/tumour),
and subtype.
• Hierarchical Classification Structure
• The framework flows from broad categories to precise
molecular subtypes:
example:
• Lineage: Myeloid
• Category: Acute Myeloid Leukemia (AML)
• Family: AML with defining genetic abnormalities
• Subtype: AML with PML::RARA

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 WHO classification
• Core Diagnostic Triad: Integrating Key Attributes
• Each tumor is defined by three pillars:
• lineage + dominant clinical attribute + dominant biologic
attribute.
• Lineage attribution rests on immunophenotyping with flow
cytometry and/or immunohistochemistry.
• Dominant clinical attributes are general features of the
untreated disease and include descriptors such as acute,
chronic, cytopenia(s) (myelodysplasia) and cytosis(es)
(myeloproliferation).
• Most biologic attributes include
– gene fusions
– rearrangements, and
– mutations.
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 WHO classification/Handling Emerging Entities

• Emerging entities are listed as disease subtypes under a novel

rubric of other defined genetic alterations.
• This is envisioned as a landing spot in the classification to
incorporate new/rare entities whose recognition is increasing
as high-throughput molecular diagnostic tools become more
available
• However, diagnosis of such subtypes might not be feasible in
all practice settings.
• A set of decision support guide lines was adopted to aid in
determining what subtypes would qualify in this context;

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they include:
• (1) having distinct molecular or cytogenetic features driven by
established oncogenic mechanisms;
• (2) not meeting subtype criteria under other tumour types with
defining genetic abnormalities;
• (3) having distinct pathologic and clinical features, including- but
not limited to- response to therapeutic interventions; and,
• (4) at least two quality peer-review publications by distinct
investigator groups.

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 Practical Implementation
• Required Diagnostics: Integration of:
– Morphology (blasts, dysplasia).
– Immunophenotype (flow/IHC).
– Genetics (NGS, FISH, PCR).
• Resource Flexibility:
– High-resource settings: Full molecular profiling (e.g.,
NPM1 mutation testing).
– Low-resource settings: Fall back to family-level
classification (e.g., "AML with recurrent genetic
abnormalities" without subtype specification).

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 Classification of hematological malignancy…
• Hematological malignancies are cancers of the blood, bone
marrow, and lymphatic system.
• Primarily, there are three basic types of blood cancer.
1. Leukemias- Leukaemia means ‗white blood‘
 are a group of cancers of the blood/bone marrow and are
characterized by an abnormal proliferation of blood cells,
usually WBCs.
 a myeloid or lymphoid neoplasm, characterized by circulating
neoplastic cells but also encompassing similar cases in which there
are neoplastic cells in the bone marrow but
not in the peripheral blood
 The name was given because the first cases of
leukaemia recognized had a marked increase in the
white cell count

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 Classification of hematological malignancy…
2. Lymphoma- a neoplasm of lymphocytes in contrast to lymphoid
leukaemias, lymphomas involve predominantly tissues, rather than
the bone marrow and blood.
 Two main categories of lymphomas are the non-Hodgkin
lymphoma (NHL) (90% of cases) and Hodgkin lymphoma
(HL) (10%); within these two broad categories, there are many
additional subtypes
 A diagnosis of lymphoma requires a tissue biopsy or a fine-needle
aspirate of a mass.
 Hodgkin's lymphoma Reed-Sternberg cell
 If the Reed-Sternberg cell is not present, the lymphoma is
classified as non-Hodgkin's
3. Myeloma- plasma cell neoplasm affecting predominantly the bone
marrow, most cases being characterized by synthesis of a monoclonal
immunoglobulin.
69
 Classification of hematological
malignancy (Can be classified by stem cell involved)

HEMATOLOGICAL
MALIGNANCY

MYELOID LYMPHOID HISTIOCYTIC/DENDR

NEOPLASMS NEOPLASMS ITIC CELL
NEOPLASMS

70
71
 Myeloid proliferations and neoplasms
include 9 sub-categories (families):
1. Myeloid precursor lesions (new),
2. Myeloproliferative neoplasms (MPNs),
3. Mastocytosis,
4. Myelodysplastic neoplasms (MDNs, previously known as
myelodysplastic syndrome, MDS),
5. MDN/MPNs,
6. Acute myeloid leukemia (AML),
7. Secondary myeloid neoplasms (new),
8. Myeloid/lymphoid neoplasms with eosinophilia and defining
gene rearrangement,
9. Acute leukemias of mixed or ambiguous lineage.
72
 Lymphoid neoplasm
• B-cell lymphoid proliferations and lymphomas consist of 5
sub-categories (families):
• Tumor-like lesions with B-cell predominance (new),
• Precursor B-cell neo plasms (12 sub-families/ entities: )
• Mature B-cell neoplasms (12 sub-families/ entities: )
• Hodgkin lymphoma, and
• Plasma cell neoplasms (PCNs) and other diseases with
paraproteins.
• T-cell and NK-cell lymphoid proliferations and neoplasms
include 3 sub-cate gories (families):
• Tumor-like lesions with T-cell predominance (new),
• Precursor T-cell neoplasms, and
• Mature T-cell and NK-cell neoplasms(9 sub-families/entities).
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 WHO 2022

MYELOID NEOPLASMS

ACUTE MYELOID
LEUKAEMIA.

MYELOPROLIFERATIVE
NEOPLASMS

MYELODYSPLASTIC
NEOPLASMS

MASTOCYTO
SIS
MYELODYSPLASTIC
/MYELOPROLIFERA
TIVE NEOPLASMS

74
 MYELOID NEOPLASMS
conti…
MYELOPROLIFERATIVE
NEOPLASMS

 MDNs, with defining genetic

MYELODYSPLASTIC
abnormalities;
NEOPLASMS
 MDNs, morphologically defined

 MDNs of childhood

75
 MASTOCYTOSIS

MYELODYSPLASTIC/MYELO
PROLIFERATIVE
NEOPLASMS

76
B CELL NEOPLASMS

77
B CELL NEOPLASMS

78
B CELL NEOPLASMS

79
B CELL NEOPLASMS

80
B CELL NEOPLASMS

81
T CELL NEOPLASMS

82
T CELL NEOPLASMS

83
• Thank you

84