Molecular Diagnostics in Cancer

Question 1

What are the types of molecular diagnostics in cancer?

Answer 1

Genetic screening and biomarkers.

Question 2

What is genetic screening?

Answer 2

Genetic analysis can identify the specific changes to the genome which have resulted in cancer.
They can indicate whether a particular drug is suitable or not.
May identify mutations which may have one day result in cancer.

Question 3

What are biomarkers?

Answer 3

Substances, whose production is increased in cancer cells, or by cells in response to cancer.
May be secreted and found in blood or other clinical samples – blood, urine, stool sample, biopsy.
Cam be used to diagnose, monitor and manage treatment – concentration often related to prognosis, reduction may indicate efficacy of treatment.

Question 4

What is hybridisation?

Answer 4

Most genetic analysis techniques rely on the hybridisation – joining of two complementary strands of nucleic acids.
Complementary probe can bind to DNA/RNA to detect the presence of a specific sequence.
Complementary primer can bind to DNA/RNA to amplify a specific sequence.
Occurs below the melting temperature of the nucleic acid, DNA and RNA sequences can hybridise together.

Question 5

What is fluorescence in situ hybridisation?

Answer 5

Modern method for looking at gross changes and chromosomes.
Probe hybridised to a specific target which must be of sufficient size but not too large.
The diagnostic tool – gene and chromosome duplications or loss.
There are numerous types of sample which can be used.
Many cancers have significant alterations to the structure of genes or chromosomes, which fluorescence in situ hybridisation can detect. These changes often affect the activity of the tumour suppressor gene or turn on a signaling pathway resulting in aberrant cell growth, the type of genetic changes can dictate treatment.

Question 6

What do the changes in chromosome structure or genes mean in cancer?

Answer 6

Many cancers have significant alterations to the structure of genes or chromosomes, which fluorescence in situ hybridisation can detect. These changes often affect the activity of the tumour suppressor gene or turn on a signaling pathway resulting in aberrant cell growth, the type of genetic changes can dictate treatment.

Question 7

What happens in chromosome translocate in leukemia?

Answer 7

Chromosome translocation results from the fusion of genes ABL and BCR. The result is the Philadelphia chromosome which leads to the BCR-ABL fusion protein – an always on tyrosine kinase.

Question 8

What happened in chromosome inversion/fusion?

Answer 8

A number of changes in the ALK gene have been implicated in a variety of cancers.
ALK is a receptor tyrosine kinase – its activity can be altered by mutation of its gene, gene amplification or chromosomal rearrangement.
A well known fusion/inversion of ALK is with the EML4 gene, implicated in 2-5% of NSCLC.
Altered ALK activity can be targeted with an ALK inhibitor.
The EML4-ALK fusion can be screened for using a FISH assay.

Question 9

What is ALK chromosome inversion/fusion detected by?

Answer 9

A positive test can lead to targeted treatment with ALK inhibitors eg Crizotinib, Ceritinib, Alectinib.
Can also be detected by PCR.

Question 10

What is PCR?

Answer 10

A method of detecting and amplifying DNA.
PCR allows the DNA from a selected region of genome to be amplified by more than a million fold, provided that at least parts of its nucleotide sequence is already known.

Question 11

What are the ingredients in polymerase chain reaction?

Answer 11

Template – DNA of any origin or cDNA.
Primers – two synthetic oligonucleotides usually 18 to 22 nucleotides, complementary to the 3’ end region of each strand of template amplified.
DNA polymerase – replicates the template DNA from the 3’ end of each primer.

Question 12

What are the steps in polymerase chain reaction?

Answer 12

Denaturation of the template; takes it above the melting point of the double stranded DNA until there is a single stranded DNA.
Primers bind to complementary region they have been primed to bind to which spans the region wanted to be amplified. Both the primed pair need a similar melting point so they bind at the same rate.
Synthesis of new DNA complementary to the target sequence, addition of dNTP is catalysed by DNA polymerase. Binds to the 3’ end of the primer, reads the 3 nucleotide on the template strand and adds complementary bases and moves on until the conditions are changed.
The enzyme is thermostable, bacterial enzyme need to be used.
The cycle is repeated and there is 30 second denaturation, shorter DNA strands so there is less time needed to denature, primers anneal and hybridise and the temperature is raised for DNA polymerase.
By the end of the 3rd cycle there are 2 strands of the region wanted to amplify, for every 1 strand used in cycle 1.

Question 13

What is reverse transcriptase PCR?

Answer 13

A technique for amplifying a specific sequence of RNA by initial conversion to its cDNA.
Starts with RNA template.
RNA has a polyA tail so the primer needs to be complimentary to tail.
Hybrid RNA, DNA double stranded molecule.
RNA destroyed with RNAse – leaves with a single strand of cDNA.

Question 14

What is Slab Gel electrophoresis?

Answer 14

A way to detect PCR products.

Question 15

How does Slab Gel electrophoresis work?

Answer 15

Ethidium bromide is used to stain nucleic acid PCR products in agarose gel followed by visualisation under UV light.
Primers with fluorescent label or conjugated enzyme can be used.
Multiple products can be detected on a single gel if their sizes are sufficiently different, more than one set of primers can be used per tube.
The laser source detects the DNA passing through it and there are lots of reaction products which can be separated out based on their size.
PCR products are separated by size within a capillary and can be combined with the used of primers tagged with different fluorophores.

Question 16

What is real time PCR?

Answer 16

Enables quantification of the product in real time.
Several types used eg TaqMan.
There is a fluorescent reporter and an oligonucleotide sequence which is complementary to a region of the target between the forward and reverse primers.
The forward and reverse primers, DNA pol and TaqMan probe are added to the DNA sample.
Following denaturing and annealing of priers and probe, DNA polymerase synthesises new DNA.
Reverse strand synthesised normally.
On forward strand, DNA polymerase encounters the TaqMan probe.
5’ nuclease activity degrades the probe, releasing the fluorescent reporter.

Question 17

What is a cycle threshold?

Answer 17

Cycle number where fluorescence is detected. Gives an idea of how much template is in the cycle.

Question 18

How can real time PCR detect cancer?

Answer 18

In breast cancer, TOP2A gene is often co-amplified with HER2.
Amplification of both genes can be detected with PCR.

Question 19

How is PCR used to detect mutations in the genome?

Answer 19

PCR can detect different types of mutations eg point mutations, deletions and insertions.
They rely on reaction products being different sizes in the absence or presence of a mutation or the reaction not working when a mutated template is used.

Question 20

What is a point mutation?

Answer 20

Alteration in a single nucleotide pair in the DNA molecule and usually leads to a change in only one biochemical function.

Question 21

What is a single nucleotide polymorphism?

Answer 21

Loci with alleles that differ at a single base, with rarer allele having a frequency of at least 1% in a random set of individuals in a population.

Question 22

What are epidermal growth factor receptor mutations and how are they treated?

Answer 22

NSCLC account for 80% of all lung cancers.
Mutations which increase sensitivity to 1st , 2nd and 3rd generation EGFR TKIs and can inform treatment options.
Drugs including gefitinib and erlotinib inhibit EGDR tyrosine kinase and are used to treat NSCLC.
PCR can be used to detect these changes by designing primers to give different sized PCR products in the absence and presence of the mutation.

Question 23

What is allele specific PCR?

Answer 23

Detects point mutations without relying on changes in restriction sites.
Relies on the fact that the correct base at the 3’ end of a primer is crucial for DNA synthesis.
Carefully designed primer sets differentiate between wild type and mutant alleles.

Question 24

What is the q-PCR detection of mutations?

Answer 24

Unlabelled forward and reverse primers. Two fluorscent taqman probes; one is specific for wild type sequence, the other for mutation.

Question 25

What are microarray technologies?

Answer 25

Microchips or flow cells which contain many microscopic spots of different DNA and RNA probes which are used to assess expression of large number of genes and determine the genotype or sequence of nucleic acids.
The probes are specific sequences of DNA or RNA which is used to capture complementary sequences from the test sample.

Question 26

How does our genetic makeup affect drug response?

Answer 26

Drug absorption – transporters
Drug metabolism – cytochrome P450
Adverse drug reactions
Drug efficacy – subtle differences in protein structure

Question 27

What is the purpose of DNA sequencing?

Answer 27

To result in huge amounts of information which can inform clinical decisions of therapy even before the development of cancer.

Question 28

what is a benefit of population-based screening?

Answer 28

It is cost effective when compared to clinical criteria and family history BRCA1/2 screening.

Question 29

What is the mechanism of Sanger sequencing?

Answer 29

Dideoxy NTPs are chain terminators each labelled with a different fluorophore.
The rest is end labelled sequences of DNA with different lengths which is readily detected by GCE, enabling the sequence to be determined.

Question 30

What is microarray sequencing technology?

Answer 30

Second generation sequencing technique which allows more rapid sequencing. Millions of clusters can be sequences in a matter of hours and the systems are flow based.

Question 31

What is illumina sequencing technology?

Answer 31

Genomic DNA, fragmented DNA, adaptor ligation, amplification

Question 32

What is immunodetection?

Answer 32

Detection of a specific antigen using antibody binding. Used in:
Immunohistochemistry
ELISA
Flow cytometry
The antigen being detected can be in situ, in solution, on a membrane/ well plate, on a microparticle or in a gel

Question 33

When would ELISA be used?

Answer 33

To detect a specific antigen or antibody in a sample.
Useful to detect infection or disease markers.
Involves adsorption of antigen or antibody to multiwell plates

Question 34

How does ELISA work?

Answer 34

Secondary antibody has an enzyme conjugated to it
Substate converted by enzyme to chromogenic or fluorogenic product which can be detected using a plate reader.
Alkaline phosphate and horseradish peroxidase commonly used.

Question 35

What is sandwich ELISA?

Answer 35

ELISA to detect low concentrations of protein.

Analogous approaches are now used for high throughput screening of patient samples using microbeads instead of plates.

Question 36

What is the applications of ELISA?

Answer 36

In cancers, high levels of proteases can result in degradation of fibrin and these breakdown products can be detected by ELISA

Question 37

What is immunosorbent Assay technology?

Answer 37

Prostate specific antigen is an enzyme secreted by prostate epithelial cells
Levels more than 4ng/mL in blood may be a sign of prostate cancer.

Question 38

What is chromogenic in situ hydbridisation?

Answer 38

Combination of FISH and IHC.
Dual stains a sample using probes with different antigens and secondary antibodies with different enzymes.
The enzyme reaction can be used to cause certain ions to precipitate.

Question 39

What is flow cytometry?

Answer 39

A method for detecting specific molecules on and within cells.
Can be used for sorting and isolating specific sub-populations of cells and a powerful analytical technique for cell biology research.
It is important in medicine – immunophenotyping, diagnosis and cell sorting.

Question 40

How does flow cytometry work?

Answer 40

Multiple antigens can be quantified simultaneously, they are extra and intracellular.
The modern flow cytometers have up to 6 lasers and can measure 18 colours independently.
They are mainly used for proteins, but DNA and RNA can also be detected.

Question 41

What are the applications for flow cytometry?

Answer 41

Diagnosis 
Immunophenotyping 
Cell enumeration 
Basic research 
Cell sorting – purification of cell populations for therapeutic applications

Question 42

How is flow cytometry used in lymphocytic leukemia?

Answer 42

B-cell lymphocytic leukemia characterised by gradual accumulation of CD5, CD19+, malignant B cells.
Analysis of peripheral blood mononuclear cells, labelled with antibodies against CD5, CD19, and the receptor tyrosine kinases ROR1 and ROR2.

Question 43

What is high throughout screening?

Answer 43

High throughput screening screens hundreds of samples per hour
Multiplex – analytes quantified per tube
Low volume
Sensitive and accurate

Question 44

What is lumex technology?

Answer 44

A multiplex immunoassay system with many beads which are tagged with a different coloured fluorescent marker and captures a specific protein.
Secondary antibody has a PE tag. The beads are read in a flow-based system or in a layer at the bottom of the well if it is magnetic.
One laser classifies the type of bead, the second quantifies the PE signals.

Question 45

What is the cancerSEEK blood test?

Answer 45

Used on samples from patients with stage I to stage III cancers.
Supervised machine learning was used to predict the underlying cancer type in patients with positive cancerSEEK results – examining the combination of ctDNA and proteins.
There is cancer source localisation largely due to protein biomarkers.

Question 46

What are the limitations of cancerSEEK blood test?

Answer 46

Patient had known disease – should ideally be able to detect in general population at earlier stage
Controls were healthy individuals – some disease states may generate false positives.