OVERVIEW OF

GENOMICS IN
HAEMATOLOGICAL
MALIGNANCIES
DR. OJETUNDE B.A
 OUTLINE
INTRODUCTION
OVERVIEW OF GENOMICS
GENOMICS IN HEALTHCARE
THE HUMAN GENOME PROJECT
CLINICAL APPLICATION OF GENOMICS
GENOMICS OF HAEMATOLOGICAL MALIGNANCES
CONCLUSION
REFERENCES
 WORLD CANCER DAY Feb 2014
By 2025, there will be more than 20 million
new cancer cases per year, compared with
14.1 million in 2012, according to the World
Cancer Report 2014, released on 3 February
by the World Health Organizations
International Agency for Research on Cancer.
IARC World Cancer Report 2014
 INTRODUCTION
All diseases have a genetic component, whether
inherited or resulting from the body's response to
environmental stresses like viruses or toxins
Inside the nucleus of every cell in the body, a
complex set of genetic instructions, known as the
human genome, contained on pairs of
chromosomes.
Errors in genes--the smallest units of heredity--
may cause or contribute to disease
 CONCEPTS

A gene
Chromosomal region that is capable of making a functional
transcript.
Genes are both units of inheritance and encoded messages for
the creation of a functional unit in a cell.
Genetics Focuses on individual genes in isolation.

A genome
All the DNA in the cell, including its genes.
The precise ordering of As, Ts, Cs and Gs in organisms genomes
is the foundation of lifes diversity.
Genomics examines all the genes together, all their
interactions and effects
 Genetics Genomics
Rare disease oriented Common Disease
Single gene oriented
Absolute risk Multiple Genes
Direct results Relative Risk
Currently indirect
inference
 1953

The molecular characteristics of DNA

were first described in 1953
(Watson and Crick, 1953).
Francis Crick This molecule serves as the blueprint
for determination of structure and
function of all living organisms.

James Watson
BASICS
 Basic Concepts
Humans & Other organisms comprise of
individual cells
Cells communicate, interacts and work together
to form
Tissues (Collection of cells doing similar process)
Organs
Entire organism
The Cell
Nucleus within Where all the DNA is contained
 Basics
Somatic cell is any biological cell forming the body of
an organism
Germ cells are cells that give rise to gametes
Gametes : is a cell that fuses with another cell
during fertilization (conception) in organisms that
sexually reproduce, witch carry half the genetic
information of an individual.
Stem cells are cells that can divide through mitosis
and differentiate into diverse specialized cell types.
 BASICS
Somatic cells contain DNA arranged
in chromosomes.
If a somatic cell contains chromosomes arranged in
pairs, it is called diploid and the organism is called a
diploid organism.
The gametes of diploid organisms contain only single
unpaired chromosomes and are called haploid.
Each pair of chromosomes comprises one
chromosome inherited from the father and one
inherited from the mother
 BASICS
In humans, somatic cells contain 46 chromosomes
organized into 23 pairs.
By contrast, gametes of diploid organisms contain
only half as many chromosomes.
In humans, this is 23 unpaired chromosomes.
When two gametes (i.e. a spermatozoon and an
ovum) meet during conception, they fuse together,
creating a zygote.
Due to the fusion of the two gametes, a human
zygote contains 46 chromosomes (i.e. 23 pairs)
BASICS
 THE DNA
DNA is composed of 3 basic components:
Base : Pyrimidine or purine
Sugar : (2-deoxyribose)
Phosphate
The Nucleic Acid alphabet consists of 4
bases:
1. purines adenine (A)
2. purines guanine (G)
3. pyrimidines thymine (T)
4. pyrimidines cytosine (C).
5. There is a fifth base that can be
found in DNA known as 5-
methylcytosine (5-mC).
Uracil (U) is substituted for thymine
in the case of RNA.
The combination of a base and a
sugar (deoxyribose) is referred to as
a nucleoside
THE DNA
 DNA
Hydrogen bonding occurs specifically between
the purine adenine (A) and the pyrimidine
thymine (T) and between the purine guanine
(G) and the pyrimidine cytosine (C)
The connection between repeating
phosphates and sugars creates a Helical
Chain.
In the RNA molecule, adenine base pairs with
uracil (U).
THE DNA
The combination of a sugar phosphate
group and a base constitutes a
nucleotide.
The double helix is made from two
polynucleotide chains, each of which
consists of a series of 5 to 3sugar
phosphate links that form a backbone
from which the bases protrude.
The double helix maintains a constant
width because purines always face
pyrimidines in complementary A-T and G-
C base pairs, respectively.
 GENE EXPRESSION
Transcription is the first step in converting DNA into protein

During the process of transcription, linear DNA is converted to linear messenger RNA
(mRNA)

The process of translation consists of the conversion of linear mRNA to a linear set of
amino acids that will eventually form a functional protein

Single strand of RNA is copied from one of the strands of DNA.

The sugar element in the RNA molecule is ribose and the pyrimidine uracil substitutes for
thymine

RNA polymerase II is the enzyme that synthesizes the first copy of RNA
This primary strand of RNA is called heterogeneous nuclear RNA (hnRNA)

hnRNA contains coding sequences (exons) of DNA and noncoding sequences (introns).
RNA Ribonucleic Acid
GENE EXPRESSION

During transcription, a section of

one DNA strand, or the other, is
used as a template for the
synthesis of mRNA.
This synthesis always occurs in a
5 to 3 direction.
 Protein Translation
Translation of mRNA into protein occurs in the
cytoplasm where ribosomes are located.
Two other forms of RNA are important for
protein translation: transfer RNA (tRNA) and
ribosomal RNA (rRNA)
The mRNA message is translated in segments
of three adjacent nucleotides called a codon
Each codon is translated into one of 20 amino
acids
The information contained in DNA is
transcribed into RNA and then translated into
protein.
Transcription of RNA is tightly regulated and is
tissue specific.
A single gene can encode for multiple unique
proteins by including or excluding certain exons
in the mRNA transcript by alternative splicing.
Post-transcriptional gene regulation can occur
by a mechanism involving the expression of
noncoding RNAs that have the capability of
binding to and degrading mRNAs
Epigenetics
THE HUMAN GENOME PROJECT
TARGETS IN GENOMICS

Whole Genome Sequencing (WGS)

E.g. Rearrangements outside genes
Whole Exome Sequencing (WES)
E.g. Gene Discovery (Rare/unknown tumors)
Custom target
MSK-IMPACT (Integrated Mutation Profiling
of Actionable Cancer Targets)
410 genes related to cancer
>15K patents profiled at MSKCC
https://cbioportal.mskcc.org/study?id=mskimpact
NGS PRINCIPLES
NGS Principles and Coverage
NGS Principle -Coverage

Not all reads retrieved are correct

Many errors when sequencing
DNA Library prep protocol
Sequencing error rate

Sequencing groups of cells

Certain genetic changes only in small fraction
of cells
Need to sequence the same part multiple
times to get confidence
Amount depends on analysis & expectation
Identify the Genetic Changes
Identify The Genetic Changes

Compare against reference human

genome
Gives both germline and somatic mutations
How to differentiate?
Databases with common germline variants misses
many

Somatic mutations
Take DNA from normal cells and tumor cells
Filter mutations in normal
Identify Somatic Mutatons
 ONCOGENESIS
Cellular growth and differentiation
carefully controlled processes
regulated by several interconnected pathways.
This facilitates
development,
responses to normal and abnormal stimuli
the replacement of dying cells.
In this context, the growth of individual cells is
restrained in the interest of their cognate tissue
and by extension of the organism as a whole.
 ONCOGENEIS
The progressive corruption of this order.
The stepwise escape of an individual cell and
its progeny from checks on their growth.
This corruption occurs in the form of the serial
acquisition of genetic mutations, which
disrupt the genome of the fateful cell and
morph it into a cancer genome
 MALIGNANCIES GENOME DISEASE
Loss of DNA
Gain of DNA
Changes in nucleotides
Epigenetic effects
 Causes of Malignancies
Cancer is caused by alterations or mutations in the
genetic code
Can be induced in somatic cells by:
Carcinogenic
chemicals
Radiation
Some viruses
Heredity - 5%
 In turn, the ontogeny of the cell of origin and
the nature of the mutations are the primary
determinants of the cancer phenotype
including
its histological type
biological behaviour,
clinical characteristics
responsiveness to therapies
 Molecular basis of neoplasia
Normal cellular growth and differentiation
depends precise control of gene expression
Alterations in the quantity or timing of gene
expression can affect the survival and
function of a cell.
When such alterations occur in certain types
of genes known as oncogenes or tumor suppressor
genes, the cell may gain abnormal growth or survival
properties, and accumulations of such mutations may
lead to cancer
 MOLECULAR BASIS OF CANCERS
Nonlethal genetic damage lies at the heart of carcinogenesis
A tumor is formed by the clonal expansion of a single precursor cell that
has incurred genetic damage (i.e., tumors are monoclonal).
most commonly used method to determine tumor
clonality involves the analysis of methylation patterns adjacent to the
highly polymorphic locus of the human androgen receptor gene, AR

Four classes of normal regulatory genesthe growth-promoting proto-

oncogenes, the growth-inhibiting tumor suppressor genes, genes that
regulate programmed cell death (apoptosis), and genes involved in DNA
repair-are the principal targets of genetic damage

Carcinogenesis is a multistep process at both the phenotypic and the

genetic levels, resulting from the accumulation of multiple mutations
 CARCINOGENESIS
Oncogenesis
Oncogenes (the gas)
Cell growth
Activation causes cancer
Tumor Suppressor Genes (the breaks)
DNA repair, slow down cell division
Loss of function causes cancer
Two Hit Hypothesis (Knudson 1971)
 Characteristics of Tumors
1. Genetic instability
2. Autonomous growth
3. Insensitivity to internal and external antiproliferative
signals
4. Resistance to apoptosis and other forms of induced cell
suicide
5. Unlimited cell division potential
6. The ability to induce new blood vessel formation , a
process termed angiogenesis.
7. Locally invasive behavior, which uniquely distinguishes
malignant from benign neoplasms.
8. Evasion of the immune system
 Categorize Mutations
Silent/Nonsilent
Does the mutation alter phenotype?
In exonic region
Synonymous: Amino Acid Code stay the same
Nonsynonymous: Changes Amino Acid Code of protein
 Mutational Signatures
Find activated mutational processes
Use the identified SNVs (single nucleotide variants)
to determine
Use 1 base context on both 5 and 3 side
.C. > .T.
6 base transition classes
C>A, C>G, C>T, T>A, T>C, T>G
4 possible bases on both sides
Total: 6 * 4 * 4 = 96 possible transitions
 GENOMICS OF HAEMATOLOGICAL
MALIGNANCIES
LEUKEMIAS
MYELOMAS
LYMPHOMAS
MPDS
MPNS
 MDS
Neoplastic disorders with bone
marrow dysplasia and insufficiency leading to
one or more cytopenia in
the peripheral blood
MYELOPROLIFERATIVE NEOPLASMS
A group of clonal stem-cell malignancies
characterized by the overproduction of mature
blood cells.
The predominant cell type in excess defines the
specific disease entity and is closely correlated
with clinical complications
Share a variable tendency to phenotypic
transformation, which may manifest as
uncontrolled cellular proliferation, bone marrow
failure, bone marrow fibrosis or development
of acute leukaemia.
 MPN Genetic Pathogenesis
Mutations in cytokine signalling pathways
mutations in pathways controlling transcriptional
regulation
Events associated with transformation to advanced-
phase disease (including acute leukaemia).
Constitutional alleles that are associated with an
increased risk of developing a clonal MPN.
Inherited conditions characterized
by polyclonal blood cell overproduction that
phenocopy the clonal MPN.
 Acquired mutations in cytokine
signalling pathways
Signalling pathway mutations target either
cytokine receptors or downstream signalling
intermediaries.
Result in increased signalling that is limited to
a specific cytokine pathway and is therefore
usually associated with overproduction of a
single blood lineage
 Case Scenario
3yo male
Acute lymphoblastic leukemia
Being treating with mercaptopurine
 Heterogeneity in patient response to
chemotherapy is consistently observed across
patient populations, including both efficacy
and toxicity. Much of this variance is likely
due to genetic differences that affect drug
metabolism.
 One of the most common of these situations
involves the use of Purinethol
(mercaptopurine) to treat leukemia in
children. One in 300 children (0.3%) has a
gene defect for the enzyme thiopurine
methyltransferase (TPMT) which can result in
mercaptopurine toxicity and associated bone
marrow failure. If this defect is identified, the
drug can still be safely used at a lower dose.
 New labeling now includes information about
the risk of severe bone marrow suppression in
TPMT activity-deficient genotypes and TMPT
testing has now become standard practice for
children who may receive this medication.
 Pharmacogenomic Alert
Pharmacogenomic Alert
This patient has a TMPT gene defect which
indicates a high sensitivity to standard doses
of mercaptopurine.
 In the ideal scenario, a patient with a TPMT deficiency
has that genetic information stored in their electronic
medical record. So when mercaptopurine is ordered,
the physician will get an alert explaining that they need
to lower the dose of the medication or consider a
separate treatment.

Ideally, the alert would actually allow them to modify

the order at the same time. If they ignore this alert,
they will need to explain their reasoning.
 CONCLUSIONS
The ability to obtain full genomic data on a
given tumor will allow us to make rational
choices for therapy
Functional genomics may provide help in
choosing combination therapy Combinations
will not be easy due to enhanced toxicities
Cancer as a chronic disease is not a bad thing
as long as we recognize rapid development of
resistance and clonal evolution
 But very important to differentiate genomic
testing from traditional genetic testing for
highly penetrate, Mendellian disorders that
have specific therapeutic interventions
available whether for cancer syndromes,
cardiac disorders or any number of other
disorders
 The Promise Imagine when
Hematologists can:
Predict Disease - pre-symptomatically with simple testing
Prevent Disease - by identifying risks, early interventions
Diagnose Conditions - less invasively, more accurately
Select Drugs - that maximize benefits and minimize risks
Calibrate Treatments - to heighten efficacy and recovery
Treat/Cure Disease - using our own genes