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
what other cancers are caused by microsatellite instability?
Many other cancers associated with MMR
» Skin cancers
» Brain tumors
Why do we want to identify patients with MMR mutations?
» 1 patient identified, other family members may also be at risk
what are the newer molecular technologies?
– Arrays DNA, RNA, Protein
– Genomics
•Gene Information Technology e.g. gene arrays
– Proteomics
– Bioinformaticswhat are the 3 types of DNA microarrays?
- DNA Microarrays
- RNA Microarrays
- Protein Microarrays
what is the array?
Arrays are the arrangement of DNA, RNA or a protein on a large platform to allow us examine large numbers of cases in one study
DNA microarrays allow as to…
We can examine DNA between disease tissue and normal tissue
• This allows the identification of genes which are over expressed or under expressed in diseased tissue
• From the genetic code the protein can be determined
• The function of this protein can be ascertained
• Drugs can be manufactured to interfere with the action of a drug that causes over activity of a protein e.g. growth factor receptor
what is bioinformatics?
Bioinformatics is the method of analysing data on tissues from gene or protein microarray studies
list viruses that can cause cancer?
• HPV and cervical carcinoma • HPV and oropharyngeal carcinoma • EB virus –Burkitt’s lymphoma (c-myc translocation) –Nasopharyngeal carcinoma –Hodgkin’s disease • Hepatitis B virus • Hepatitis C virus
how HPV cause cancer?
High risk or oncogenic viruses are characterised by their ability to integrate into the host genome compared to the more benign papilloma viruses which remain non-integrated.
E6 and E7 proteins of HPV combine to inactivate the gene products P53 and Retinoblastoma thereby promoting further growth advantage of the infected cells and the development of cancer.
What is the significance of identifying HPV as the cause of cervical carcinoma?
- Protection eg condoms
- Vaccine
- HPV also common in oropharyngeal carcinomas
- Clinical point: If HPV is detected in a cervical nodal metastatic carcinoma probably an oropharyngeal primary present
give examples when molecular techniques have led to discovery of cancer mutations?
- BCL2 activation in follicular lymphoma for t(14;18)
- Philadelphia chromosome in chronic myeloid leukemia t(9;22) → BCR Abl gene
- c-kit mutation in Gastrointestinal stromal tumor
- Translocation in Burkitt’s Lymphoma t(8;14)
- Certain translocations in sarcomas
examples when gene abnormalities can also be used as prognostic tools
- Translocations in some lymphomas identify worse prognostic groups e.g. small cell lymphocytic lymphoma.
- Certain molecular changes in multiple myeloma have a worse prognosis e.g. deletion 17p13.
examples when gene alterations can also be used to assess a tumor’s likely response to therapy
- The finding of amplified Her2 receptor in breast cancer cells identifies tumors that will respond to Herceptin
- The finding of c-kit mutation in a gastrointestinal stromal tumor will identify tumors that will respond to Imatinib (Glivec)
- The finding of BCR-Abl gene mutation in CML (chronic myeloid leukemia) (Glivec)
what is the targeted therapy?
Molecular techniques and new cancer drugs
• New technologies have allowed us to compare the molecular profile of cancer cells compared to normal cells
• This has given us an understanding of how neoplasms develop and grow
• This information has provided drugs which target specific pathways of cell division which stops tumor growth
• Targeted drugs- “Right drug for the right patient”
• Block membrane receptor (Monoclonal antibodies name ends in “ab”)
• Block signaling pathways (Small molecule inhibitors name ends in “ib”)
examples of targeted therapy?
- Right drug for the right patient
- Maximum benefit with minimal side effects
- Breast (Oes receptor-Tamoxifen and Her2 receptor-Herceptin)
- Imatinib in CML
- BRAF inhibitors- Vemurafenib Melanoma
- Cetuximab in Lung carcinoma
- Rituximab in B cell lymphomas
what is the role of Pathologist in targeted Tx
- Selection of patients based on the presence of the therapeutic target is essential
- Clinician’s question for pathologist:Does the tumour have receptors for the targeted therapy e.g. EGFR receptor in adenocarcinoma of lung?
- If yes, treat
- If not, don’t treat, tumour will not respond
what is the vemurafenib?
A small molecule inhibitor used in the treatment of malignant melanoma and other cancers with a V600E mutation of the BRAF gene (i.e., valine (V) replaced with glutamic acid (E) at amino acid position 600).
what is the Herceptin?
Trastuzumab
A monoclonal antibody against the HER2 tyrosine kinase receptor that inhibits cellular signaling and causes cytotoxicity. Used in the treatment of HER2-positive breast and gastric cancer. Causes cardiotoxicity.
what is the cetuximab?
A monoclonal antibody against the epidermal growth factor receptor (EGFR) often used in the treatment of colorectal cancer and head and neck cancer. Adverse effects include elevated liver function tests, diarrhea, and an acneiform rash on the face, upper chest, and upper back.
what is the imatinib (Glivec)?
A biologic anti-neoplastic agent used in the treatment of Philadelphia chromosome (t(9:22) translocation) positive neoplasms, chronic myelogenous leukemia (CML), and acute lymphocytic leukemia (ALL). It functions by competitively inhibiting the tyrosine kinase that results from the abnormal BCR-ABL gene.
what is the treatment of cancers 2 decades ago and now?
Treatment of cancer two decades ago • Surgery • Radiotherapy • Chemotherapy • Immunotherapy
NOW
• Hormonal Therapy
• Immunotherapy
• Personalised/Targeted Therapy
A very commonly used example of a monoclonal antibody
• Trastuzumab (Herceptin)
• This is a monoclonal antibody that binds to the extracellular domain of the Her2 receptor
• Her2 is a member of a tyrosine kinase family along with EGFR (Her 1). It is a surface receptor with epidermal growth factor activity.
More Her2 receptors expose cell to more growth factor leading to more cell growth
Why is the blockage of Her 2 receptor so important?
- One in 10 to 12 women develop breast cancer
- The Her2 gene is overexpressed in 20-30% of breast cancers. We can block this by Herceptin
- Amplification of Her2 is associated with large tumour size, high tumour grade, aneuploidy and negativity for oestrogen receptor
how does Herceptin help in Her2 positive breast cancers?
causes dimerization and downregulation of growth receptors
what is the small molecule inhibitor?
– A small molecule inhibitor inhibits at a site different from the cell membrane binding site
– It inhibits a protein eg tyrosine kinase involved in a particular signalling pathway
– Overactive Abl kinase proteins in chronic myeloid leukaemia
– Overactive Kit protein in GIST’s
In GISTs, there are mutations of
the kit gene that produce a constitutively active (always turned on) receptor protein
• Therefore a monoclonal antibody that binds to and that blocks this ligand is of no value
• Therefore a small molecule inhibitor is more useful
example of a small molecule inhibitor?
Imatinib mesylate (Gleevec) is a small molecule inhibitor of tyrosine kinase
• GISTS (Gastrointestinal stromal tumors) have a response rate of 7% to chemotherapy
• But 50% response to Imatinib
• Imatinib is highly successful in the treatment of chronic Myeloid Leukaemia 91% 2y survival
what is the bevacizumab
A humanized monoclonal antibody against vascular endothelial growth factor (VEGF) used in the treatment of neovascular age-related macular degeneration and malignancies (e.g., colorectal cancer, renal cell carcinoma).
what is the erlotinib?
A tyrosine kinase epidermal growth factor receptor inhibitor (EGFRi). Used to treat non-small cell lung and pancreatic cancer. Adverse effects include xerosis and an acneiform rash on the face, upper chest, and upper back.
examples of predictive biomarkers for cancers that are commonly tested?
1)HER2 Testing – IHC & FISH – Breast & Gastric cancer – National FISH testing center since 2004 - >300 cases pa – Reference laboratory for UK NEQAS 2)KRAS testing – Colorectal cancer 3)EGFR testing – NSCLC 4)BRAF testing – Metastatic melanoma 5)Oncotype
what is the BRAF gene?
A proto-oncogene that encodes B-Raf, a serine/threonine kinase. Activating mutations in the BRAF gene are associated with melanoma, non-Hodgkin lymphoma, papillary thyroid carcinoma
what is the KRAS gene?
A proto-oncogene that encodes a GTPase (a ras protein) that regulates cellular growth. Mutation is associated with colon, lung, and pancreatic cancers.
what is the EGFR gene?
A gene located on chromosome 7 that encodes the epidermal growth factor receptor protein. Activating mutations are associated with lung adenocarcinoma.
Newer therapies do not “cure” cancer
True/False
True
• They cause disease stabilization and decrease further growth rather than tumor regression
• Newer agents inhibit the growth of cancer cells rather than killing cancer cells
• The tumor does become resistant
what is the role of histopathology in these new therapies
. The tumour has to be tested to determine if it has the receptor to be blocked
– Why: Must give the drug only if it will work
– Avoid side effects in non responsive patients and cost (Herceptin 50,000 euro for a year’s Tx)
The tumor has to be tested to determine if it has the receptor
Performed by
• Immunohistochemistry and or genetic analysis e.g. FISH or mutation analysis
post commencement of therapy it is helpful to know at an early stage:
- Lack of response to treatment
- Residual disease post-treatment
- Early recurrence of disease after initially successful treatment
what is the importance of early detection of response to therapy or of recurrence
- E.g. Patient with lymphoma may be prescribed 6 doses of chemotherapy at monthly intervals
- Why continue to give 6 cycles if after 2 cycles the tumor burden is not shrinking
- Knowledge of lack of early response (after 2 chemotherapy cycles) will allow us to change Chemotherapy agent
- How do we detect a lack of response?
How do we detect a lack of response?
- PET scan
- Evaluation of tumour markers
- Molecular analysis of blood e.g. molecular marker eg translocation still present
what is the PET scan?
A diagnostic imaging technique that uses a radiotracer to provide information about blood flow and/or metabolic processes in the body. The most common radiotracer used is fluorodeoxyglucose (FDG). Uptake of FDG by tissues indicates the metabolism of glucose. Thus, FDG-avid lesions are metabolically active, while FDG-nonavid lesions are not.
what is the immunotherapy?
- Cancers may have genetic and epigenetic alterations → Neo antigens that can be attached by the immune system of the patient
- Neo antigens are recognizable by the immune system that can be attacked by the immune system of the patient which prevents tumor growth
Tumor cells evade immune detection by:
– Tolerance
– Local immune suppression
– Dysfunction in T cell signalling
– Activating endogenous immune check points (that terminate immune responses)
examples of cancer immunotherapy
- Stimulate immune response e.g. Vaccination (BCG for in situ bladder ca)
- Immune checkpoint pathway inhibitors e.g. Anti CTLA-4 antibody (Ipilimumab)
- These prevent “switching off”/inactivation of the immune response by proteins such as PD 1 and CLT4 and thereby enhance the immune response of the patient against the tumour
what is the ipilimumab?
A monoclonal antibody that is used in combination with nivolumab for the treatment of metastatic colorectal cancer with deficient mismatch repair or high microsatellite instability. It is also used to treat melanoma and renal cell carcinoma. It activates the immune system by targeting CTLA-4 which downregulates T-cell activation pathways.
