Molecular Methods in Haematology

Match these to their type of test: Diagnostic, Prognostic, Monitoring, Predictive

• NPM1, FLT3 in AML

• MRD testing

• Tazemetostat for EZH2 mutation

• BCR-ABL in CML

•Diagnostic: Identify the precise genetic abnormality (e.g., BCR-ABL in CML)

•Prognostic: Predict disease behaviour (e.g., NPM1, FLT3 in AML)

•Monitoring: Track residual disease (MRD testing)

•Predictive: Guide targeted therapy (Tazemetostat for EZH2 mutation)

__________ are cancers of the white blood cells, which begin in the bone marrow

Leukaemia

Leukaemias are grouped in two ways by the type of white blood cell affected - what 2 cells

lymphoid or myeloid

Lymphoma is what

Lymphoma is a cancer of the lymphatic system, which includes the lymph nodes, spleen, thymus gland and bone marrow

What is myeloma

Plasma cell disorder

Affecting marrow, bones, kidneys, and other organs

Between Leukaemia, Lymphoma, & Myeloma, which are these terms used for:

• Acute v Chronic

• High v Low grade

Leukaemia - Acute v Chronic

Lymphoma - High v Low grade

Genomics

Genomics is an all-encompassing term that takes into account all the DNA in a person or organism’s genome – both the genes that code for proteins and the non-coding regions. It looks at how genes are expressed and the interplay between different genes

What was the the Human Genome Project (HGP) and what was its significance

•The Human Genome Project (HGP) was an international research effort aimed at mapping and sequencing the entire human genome. It was initiated in 1990 and completed in 2003, costing millions

•The primary goal of the Human Genome Project was to determine the complete nucleotide sequence of the human genome and to identify and map all the genes present within it to understand their structure and regulation, determine the extent of variation in these genes in different populations, and uncover how genetic variation contributes to disease.

•This has provided a comprehensive understanding of human genetics and has catalyzed significant advancements in technology, particularly in DNA sequencing methods.

•The entire human genome can now be sequenced in 24 hours

Differentiate between genomics & genetics

Proto-oncogenes may be converted to oncogenes by a number of mechanisms - name 3

Point Mutation

Gene Amplification

Translocation

Match the condition to the type of mechanism of Converting Proto-Oncogenes to Oncogenes

Point Mutation

Gene Amplification

Translocation

•  Sickle Cell Disease

• BCR-ABL in Chronic Myeloid Leukaemia

• HER2 amplification in breast cancer

Point Mutation -Point mutation in a proto-oncogene gene or gene regulatory element (e.g. Sickle Cell Disease).

Gene Amplification -Copy number variation leading to gene amplification (e.g. HER2 amplification in breast cancer).

Translocation - Translocation causing disruption of regulatory elements, or by production of a constitutively activated fusion protein product (e.g. BCR-ABL in Chronic Myeloid Leukaemia).

Which nucleobases bond to which

Role of Histone proteins

Special proteins that DNA wraps around to stay compact and organised

Role of Non-histone proteins

Additional structural and regulatory proteins that help control gene expression and chromosome structure

Each chromosome contains how many genes

Each one has many hundreds or thousands of genes.

Describe the numbers of pairs of chromosomes in human diploid cells

Human diploid cells contain 23 pairs of chromosomes,including 22 autosomes, named in order of decreasing size from 1 to 22, and one pair of sex chromosomes

what is a gene

Genes are sequences of DNA which encode for specific molecules, usually proteins. Humans have approximately 20,000 genes, the function of ~7000 of which is known

Coding DNA accounts for only what % of all of our genomic information. What does the other % do

Coding DNA accounts for only 2% of all of our genomic information. The other 98% of human nuclear DNA is non-coding, but some of this non coding DNA is functional, by virtue of regulatory activity, control of gene expression, and structural maintenance

Human genomes are about what % identical – approximates to 3 million ‘differences’ between individuals

These ‘differences’ are called _______

Human genomes are about 99.9% identical – approximates to 3 million ‘differences’ between individuals

These ‘differences’ are called genetic variants

What haem test is used for Molecular assessment at a protein level (protein expression)

IHC/Flow

What haem test is used for Molecular assessment at a chromosome level

Cytogenetics, FISH

What haem test is used for Molecular assessment at a nucleotide level

PCR, Sanger, NGS, WES

What does cytomorphology detect

Cytomorphology examines the size, shape and appearance of cells under a microscope

Explain the process of flow cytometry

Name a test used to perform immunophenotyping

Flow cytometry

What is Flow cytometry - method & use

Flow cytometry is a laboratory technique that allows for the analysis of cells based on their physical and chemical properties. E.g. clonality

It involves passing cells in a fluid suspension, one at a time, through a laser beam.

As each cell passes through the laser, it scatters light and emits fluorescence, providing information about its size, granularity, and specific markers or molecules expressed on its surface or within the cell

What is Clonality

A single cell with an advantageous mutation (a "clone") begins to divide and outcompete its neighbors. This leads to the expansion of this clone, forming a tumor that is genetically similar, as all cells originate from the same ancestral cell

What is an Immunophenotype

“Signature” eg. CLL – CD5/CD19/CD23/CD43/CD200 Positive, CD20, CD79b and surface Immunoglobulin (sIg) DIM, CD10 negative,FMC7 negative

Recurrent cytogenetic abnormalities are a hallmark of & can be diagnostic for what

Recurrent cytogenetic abnormalities are a hallmark of many, and can be diagnostic of some haematological malignancies

What stage of cell cycle are cells in for cytogenetic examination

Metaphase

Process of conventional cytogenetics

How common are Chromosomal Translocations

Translocations do not occur or occur rarely in normal cells

Translocations are specific for various types of leukaemia and lymphoma

Name 2 Chromosomal Translocations & their resulting protein & condition

BCR-ABL t(9;22) in CML

PML-RARA (t15;17) in APML

How does FISH (Flourescent in situ Hybridisation) work & what is it used for

Read this FISH report: nuc ish(ABL1,BCR)x3(ABL1 con BCRx2)[138/200]

Rearrangement was observed in 138/200 nuclei

What is the lowest % of cells that must have a translocation for a lab to report it

1%

Acute Promyelocytic Leukemia (APML) is a subtype of acute myeloid leukemia (AML) characterized by what specific chromosomal translocation resulting in what fusion gene

Retinoic acid receptor alpha (RARA) gene on chromosome 17 & the promyelocytic leukemia (PML) gene on chromosome 15 → PML-RARA fusion gene.

How does APML (/APL) present

APL is associated with a high risk of disseminated intravascular coagulation (DIC) → bleeding complications → easy bruising, petechiae, and mucosal bleeding

Prognosis for APML

Up to 30% mortality in first two weeks – but with long term survival of 90%

What molecular test is used to monitor APML response to tx

RT-PCR is used to assess the clearance of PML-RARA transcripts, indicating successful treatment

PCR works at what level

Nucleotides

What does PCR do

Polymerase Chain Reaction (PCR) amplifies a specific DNA region exponentially

(described as photocopying a single page millions of times so it can be easily read)

Describe the steps of PCR

Denaturation → Annealing → Extension (repeated ~30 cycles).

Name 3 uses of PCR

Detect BCR-ABL in CML, PML-RARA in APL, MRD monitoring

What’s the difference between Conventional PCR & qPCR/RT-PCR

Conventional PCR has end-point detection – is the target DNA present or absent?

qPCR / RT-PCR has quantitative measurement – how much target DNA is present?

Is PCR sensitive/specific

Highly sensitive

Highly specific

PCR products can be used for a variety of downstream molecular assays such as…

- Chain termination sequencing (Sanger or capillary sequencing)

- NGS

- Gel electrophoresis

- Single nucleotide variants

- Not suitable for large genome analysis

Once PCR has amplified the DNA sequence - what reads it

Sanger

How does Sanger sequencing work

Reads DNA base by base using chain-terminating nucleotides

Strengths of sanger sequencing

Accurate, simple, good for confirming known variants.

Limitations of sanger sequencing

Slow, not suitable for large regions or mixed populations

May not detect low VAF (VAF = Variant Allele Frequency – the percentage of DNA copies that contain a specific mutation)

Give some clinical uses of sanger sequencing

Confirm NPM1 mutation in AML or validate NGS findings

What does Next Generation Sequencing do

Sequences millions of DNA fragments in parallel

How does NGS work

Extract DNA fragment & add adapters

Sequence thousands of fragments simultaneously

Analyse & interpret results computationally

Clinical uses of NGS

Myeloid gene panels (e.g. FLT3, NPM1, DNMT3A in AML)

True/False NGS can detect multiple mutations in one test

True

Whole Exome Sequencing does what

Reads only coding regions (~2% genome)

Use of Whole Exome Sequencing

Identify inherited or rare variants

Limitation of Whole Exome Sequencing

Misses non-coding or structural variants

What does Single Cell Sequencing do

Analyses the genetic profile of individual cells.

What does Single Cell Sequencing detect

Detects subclones, resistance mechanisms, tumour evolution

What test is done on Patient with high WCC, basophilia, splenomegaly

PCR for BCR-ABL

What test is done on Patient with new AML

NGS myeloid panel to guide prognosis

What test is done on Patient with Chronic cytopenia, negative marrow

WES for inherited marrow failure