Molecular Pathology

Question 1

How would you define histopathology and genomics? What is the difference between them?

Answer 1

Histo= use of molecular markers to supplement diagnostic, prognostic and predictive info provided by histopathology and IHC (usually to supplement diagnosis and prognosis in cancer)

Genomics= study of complete genetic material

Histopathology involves multiple different forms of DNA and RNA testing

Genomics only involves sequencing of DNA and RNA

Question 2

What are the different molecular markers in molecular pathology and how can they be used to influence patient care?

Answer 2

Diagnostic marker
-provide more precise diagnosis

Prognostic marker
-can be useful to grade the severity of the cancer and whether more aggressive or prompt treatment is required or if adjuvant chemo required

Predictive marker
-personalise patient treatment by targeting with drugs specific to their cancer
I.e. whether patient suitable for immunotherapy

Question 3

What is the difference between driver and passenger mutations in cancer?

Answer 3

Driver= directly responsible for driving carcinogenesis

Passenger= do not drive carcinogenesis

Question 4

What is the function of tyrosine receptor kinase inhibitors and what are the benefits of using them? How does this differ to immunotherapy?

Answer 4

Inhibit overactive cancer-associated tyrosine kinase receptors to prevent uncontrolled growth and proliferation

Benefits:
Highly effective treatment
Very low risk of serious side effects
Can be given to very frail patient

Immunotherapy requires patient to be relatively fit to receive

Question 5

What are the 2 main types of molecular alterations in cancers which be tested for?

Answer 5

Small-scale sequence changes
I.e. subsitutions/deletions/insertions (etc)

Large-scale chromosomal abnormalities
I.e. translocations/amplifications/inversions

Question 6

What tests are done to detect small-scale sequence changes? Give a brief outline of what these tests involve.

Answer 6

1. Target PCR
   Probes used which a complementary to pre-specified mutations
   Probe binds to sequence and causes fluorescence
2. Sequencing i.e. Next-generation sequencing
   Determining nucleic acid sequence of sample and comparing it to reference sequence

Question 7

How does next-generation sequencing work?

Answer 7

Software compares derived sequences with reference sequence to identify mutations and discard normal variants

Question 8

What are the pros and cons of PCR testing?

Answer 8

Pros
Fast= 1-2 hrs
Very little tissue required 
Works with low quality sample 
Little expertise required 

Cons
Misses mutations not targeted for by probes

Question 9

What are the pros and cons of NGS?

Answer 9

Pros
Can detect large and small scale alterations
Can detect all mutations in a sample

Cons
Slower 
More tissue required 
Not possible on low-grade samples 
Expensive equipment 
Expertise required

Question 10

When would you use sequencing over PCR?

Answer 10

When run out of other options for identifying cause of patients cancer

Need to test for many different targets

Question 11

What methods are used to detect large-scale chromosomal abnormalities? Give brief details of the process and use.

Answer 11

1.Fluorescence in situ hybridisation (FISH)
Probes used which are complementary to sequences associated with cancer
Used to detect amplification (number of signals with probe)
Used to detect translation (probes for 2 genes which should be next to each other appear far apart)

2. Sequencing (NGS)
   Will find part of sequence which is not expected when compared to reference sequence i.e. can then look up the unexpected sequence to identify which gene it is associated with to determine the nature of translocation

Question 12

What are the pros and cons of FISH?

Answer 12

Pros
Very fast
Works with low quality samples
Most reliable technique to detect amplification

Cons
Large number of tumour cells required for it to work
Quality of histological processing can affect results
Skill required for interpretation
Expensive
Can only look for one alteration at time
Can miss some alterations

Question 13

When would you use FISH and NGS?

Answer 13

FISH:
Need results quickly
Sample is poor quality

NGS:
Patient out of options
Need to test for multiple translocations
Need to test for translocations along side small-scale sequence changes

Question 14

What testing method is done to detect protein expression abnormalities? What does this involve?

Answer 14

Immunohistochemistry (IHC)

Primary antibodies which are complementary to protein of interest added to sample with secondary antibodies (has peroxidase enzyme attached)
Peroxidase enzyme converts a colourless substrate to colour product which indicates presence of protein of interest

Question 15

When can IHC be used?

Answer 15

Infering presence of small-scale sequence changes and large-scale sequence changes

Detect non genomic proteins expression changes

Question 16

What is the only way to confirm that a mutation is germ line?

Answer 16

Blood test or non-tumour sample

Question 17

What are examples of genetic cancer syndromes and their associated cancers?

Answer 17

Lynch syndrome (AD) = mutations of MMR genes 
Endometrial + colorectal 

BRCA1/2 mutation= loss of double-strand DNA breaks
Breast and ovarian

Li Fraumeni syndrome= inactivating mutation of p53
Childhood adenocarcinoma
Premenopausal breast cancer 
CNS tumours 
Osteosarcoma 
Soft tissue sarcoma 
Leukaemias
Colorectal cancer

Question 18

What is the main-stay in managing cancer syndromes?

Answer 18

Surveillance and prevention

Question 19

What are the 2 most common uses of genomic testing?

Answer 19

Cancer testing to personalise treatment

Inherited or rare disease testing