27. Introduction to molecular pathology Flashcards
Molecular techniques
Tissue based:
- protein-based
- immunohistochemistry
- detects protein using antibodies with a label directed against the protein - nucleic acid based
- extract DNA/RNA from tissue
- identify area of interest
- use complementary nucleic acid probes for translocation
- use sequencing for mutation
Technique for DNA or RNA
FISH (Flourescence In Situ Hybridisation)
-fluorescence probe
CISH (Chromogenic In Situ Hybridisation)
-chromogen probe
When is FISH/CISH used
good for translocations
- Bcl2 in lymphoma
- Her2 in breast carcinoma
- c-Myc in Burkitt’s lymphoma
Genetic changes in follicular lymphoma
-caused by t(14; 18) which activates an anti-apoptotic gene Bcl2
What is sequencing of DNA/RNA for
process of determining the identity and order of nucleotides in a segment of DNA/RNA
How is mutation detected
- PCR
2. Sequencing
Gene abnormality is detected by
- PCR
- sequencing (automated next generation sequencing)
- FISH
- CISH
Protein expression is detected by
immunohistochemistry
Alteration of which 4 types of gene will lead to cancer
- oncogenes
- tumour suppressor gene
- mismatch repair gene
- anti-apoptotic gene
What are receptor tyrosine kinase
high affinity cell surface receptors for many polypeptide growth factors, cytokines and hormones
What are tyrosine kinase
kinase enzymes that specifically phosphorylate tyrosine on amino acid
Effects of phosphorylation of tyrosine on amino acid
activation of cell signalling and may cause a constant “on” activation
How does oncogene works
- dominant
- activation by mutation, translocation, amplification
- mutation detected by:
a) protein level - immunohistochemistry
b) mRNA - RT-PCR
c) DNA - PCR and sequencing
How does tumour suppressor gene works
- recessive
- inactivation due to either loss of both alleles or methylation
- double hit required
- found in all types of neoplasia
- most common
Example of activated proto-oncogenes
- c-ras
- 50% of cancers - c-Myc
- Burkitt’s lymphoma - c-abl
- chronic granulocytic leukemia - c-ret
- thyroid carcinoma
Example of tumour suppressor genes - inhibit cell prolfieration
- retinoblastoma
- adenomatous polyposis coli (APC)
- p53
What is Li-Fraumeni syndrome
rare autosomal dominant disorder which predisposes to a range of tumours including sarcoma, leukarmia, breast and brain tumours
- affected family members have one mutant p53 gene in their germ line
- an acquired abnormality of thier second p53 allele causes a loss of p53 activity and malignant change
What does mismatch repair genes do
maintains integrity of genome
-proofread the DNA and correct it before cells can divide
What is the result of abnormal function of mismatch repair genes
microsatellite instability
How to test for abnormal function of mismatch repair genes
- immunohistochemistry
- look for loss of normal expressed enzyme (lack of staining in section) - mutation analysis
What is Lynch syndrome
- due to abnormality in DNA mismatch repair genes
- MLH1, PMS2, MSH2, MSH6
- leads to microsatellite instability
- associated with colorectal carcinoma, endometrial carcinoma
What is microsatellite instability
- condition of genetic hypermutability that results from impaired DNA mismatch repair
- many other cancers associated with mismatch repair:
a) skin cancer
b) brain tumour
Newer technologies
- DNA, RNA, protein microarrays
- genomics
- Gene Information Technology
- e.g. gene arrays - proteomics
- bioinformatics
What are arrays
arrangement of DNA, RNA or a protein on a large platform to allow us to examine large number of cases in one study
What is bioinformatics
method of analysing data on tissues from gene or protein microarray studies
