Molecular Pathology Flashcards
Molecular Techniques have led to:
- Better understanding of mechanisms of disease e.g. carcinogenesis
- New Diagnostic Methods
- New Prognostic markers - new molecular markers
- New treatments due to knowledge of cell receptors and pathways
- Predicting which patients will respond to Tx
- Early detection of residual or recurrent disease
- Screening for diseases
what is protein-based Immunohistochemistry
The identification of specific proteins in a histological specimen using antibodies that are tagged with an indicator (e.g., enzymes, radioactive molecules, or fluorescent dyes).
what is the nucleic acid probe?
A nucleic-acid fragment that is complementary to another nucleic-acid sequence and thus, when labeled in some manner, as with a radioisotope, can be used to identify complementary segments present in the nucleic-acid sequences of various microorganisms.
what are the Protein Detection and Molecular Techniques
• Tissue based
– Protein based Immunohistochemistry detects Protein using antibodies with a label directed against the protein
– Most commonly used to detect antibodies for diagnosis, prognosis, screening, prediction of response to therapy
– Nucleic acid based
– Extract DNA/RNA from tissue
– Identify areas of interest
– Use complementary nucleic acid probes for Translocations
– Use Sequencing for mutations etc.
what is the immunohistochemistry?
- An antibody is raised against the protein under investigation e.g. HER2 protein. The antibody is labelled with a brown dye which can be seen on a tissue section
- If the antibody recognises the antigen in the tissue it binds to it.
- The antibody does therefore not wash off when the tissue is rinsed
- The brown dye indicates that the protein is in the tissue.
Why identify the protein if we can identify the gene?
As protein is the end product of genes its detection is important
• Gene may be normal but protein function may be abnormal
• Detection is by immunohistochemistry
what are the techniques for DNA or RNA?
—ISH
• Fluorescence In Situ Hybridisation (FISH) fluorescence probe
• Chromogenic In Situ Hybridisation (CISH) chromogen probe
the probe is made up of?
Probe made up of DNA/RNA complementary to the sequence of interest
Labelled with a colour (Chromogen) or a fluorescence dye
FISH/CISH
(In Situ Hybridisation)
• BCL2 in lymphoma
• Her2 in Breast carcinoma
• C-myc in Burkitt’s lymphoma
To detect these mutations we use what molecular technique?
FISH
A molecular cytogenetic technique that uses fluorescent DNA probes to identify the presence or absence of specific DNA sequences on chromosomes. It is used for the specific staining of DNA sequences by a fluorescence-labeled DNA or RNA probe, e.g., to stain chromosomes in karyograms, in tumor diagnosis, or to map specific genes on chromosomes in metaphase.
FISH is not limited to chromosome visualization. The method can also be used to provide evidence of specific nucleic acid sequences in tissue samples or fixed cells, e.g., in embryology or pathology.
what is the HER2 ISH
• To determine if HER2 gene is amplified • Compare number of chromosome 17 signals with number of HER2 signals • Positive – Ratio >2 – HER2 copy number >6
Chromosomal rearrangements are detected by
FISH
• Has the patient follicular lymphoma? Follicular lymphoma is caused by t (14;18) which activates an anti-apoptotic gene BCL2.
• We use FISH to look for this t(14;18).
• If it is present follicular lymphoma is diagnosed.
what is the sequencing of DNA/RNA?
determination of the exact sequence of base pairs of a gene; e.g., to demonstrate an unknown mutation on a disease-causing gene
This method is used to detect the BRCA1 gene in familial breast cancer, for example.
It is the gold standard in characterizing a mutation.
It is now automated.
what is the gold standard for characterizing mutation?
sequencing
what are the techniques for mutation detection
– PCR
– Sequencing
gene abnormality is detected by…
- PCR
- Sequencing (automated NGS)
- FISH
- CISH
Detect protein expression by
immunohistochemistry
Alterations of 4 types of genes → cancer
what are these genes?
– Oncogenes
– Tumor suppressor genes,
– Mismatch repair genes
– Anti-apoptotic genes
what are the Receptor Tyrosine Kinases (RTK)s?
- Receptor tyrosine kinases (RTK)s are the high-affinity cell surface receptors for many polypeptide growth factors, cytokines, and hormones.
- Kinase enzymes that specifically phosphorylate tyrosine on amino acids are termed tyrosine kinases.
- This phosphorylation leads to activation of cell signalling and may cause a constant “on” activation.
- Tyrosine kinase inhibitors (TKI’s) can inhibit this
describe The 4 important Genes altered in tumors:
- Tumour suppressor genes
- -Recessive
- -Inactivation due to either loss of both alleles eg LOH or methylation
- -Double hit required - Oncogenes
- -Dominant
- -Activation by mutation, translocation, amplification etc
- -Mutation detection by techniques such as
- - Protein level – IHC
- - mRNA – RT-PCR
- -DNA – PCR and sequencing - Mismatch Repair genes
- Anti apoptotic genes
give examples of activated proto-oncogenes
c-ras -50% of cancers
c-myc - Burkitt’s Lymphoma
c-abl Chronic myeloid leukemia
c-ret -Thyroid carcinoma
describe tumor suppressor genes
common tumour mutations
- -These accumulate over time
- Found in all types of neoplasia
- Can be caused by e.g. LOH (Loss of heterozygosity) or methylation
Important TSGs
• Retinoblastoma
• APC
• P53
Epidemiological studies of both retinoblastoma and Wilm’s tumor led Knudson to propose the two-hit model of tumourigenesis e.g. LOH and methylation.
–Retinoblastoma
–APC
–P53
are proto-oncogenes True/False
False
They are tumor suppressor genes
FAP is caused by loss of function of tumor suppressor genes or proto-oncogenes?
Cause of APC Loss of APC TSG (inherited)
This accounts for approximately 1% of cases of colon cancer inherited as autosomal dominant.
Patients have thousands of adenomatous (neoplastic) polyps, some of which undergo malignant change
Li-Fraumeni syndrome is caused by mutations in what tumor suppressor gene?
Li Fraumeni syndrome is a rare autosomal dominant disorder that predisposes to a range of tumors including sarcoma, leukemia, breast and brain tumors. Affected family members have one mutant p53 gene in their germline. An acquired abnormality of their second p53 allele causes a loss of p53 activity and malignant change.
what is the microsatellite instability?
repetitive, 2-6 nucleotide sequence of DNA. Defects in mismatch repair genes can cause these DNA regions to expand (microsatellite instability).
what are the mismatch repair genes?
A collection of genes that encode proteins that recognize and excise mismatched nucleotides in newly synthesized DNA strands. Essential for the maintenance of genomic stability. Examples include MLH1 and MSH2.
The events which are required for DNA to replicate itself are so complex that it is amazing that the system works with so few errors. Damage to the DNA occurs via the environment in the form of ultraviolet and ionizing radiation and exposure to chemicals.
Mismatch match repair genes “proofread” the DNA and correct it before cells can divide
i.e. the damage is corrected by mismatch repair genes (MMR).
what are the ways too look for defective MMR?
– IHC – looking for a loss of the normally expressed enzyme (lack of staining in section)
– Mutation analysis
what is the Lynch syndrome
• Due to abnormality in DNA mismatch repair genes
» MLH1, PMS2, MSH2, MSH6
» Leads to microsatellite instability
• Associated with Colorectal carcinoma, Endometrial carcinoma, others
what cancers are associated with Lynch syndrome?
Colorectal carcinoma, Endometrial carcinoma, Gastric cancer
