into to molecular pathology Flashcards
surgical pathology role in cancer includes:
diagnosis
prognosis
prediction
- The understanding of the way DNA, RNA and protein synthesis function and interact has changed the landscape of medicine.
- “an emerging discipline within pathology which is focused in the study and diagnosis of disease through the examination of molecules within organs, tissues or bodily fluids” are both definition of
we are trying to asses — , – , –
molecular pathology
an exon , a methyl group , and micro dna
techniquees used can be — based or — based and examples of each are:
- Tissue based
– Protein using immunohistochemistry
– Nucleic acid probes - Nucleic acid based
– Extract DNA/RNA from tissue
– Identify areas of interest
– Size on gel/Flourescence
– Sequencing
1- imunohistochemistry refers is – based by which it identifies —
* As protein is the end product of genes its – is important
* Gene may be – but protein function may be –
* Detection is by immunohistochemistry
* An — is raised against the
protein under investigation e.g.
HER2 protein. The antibody is
labelled with a – which
can be seen on a – section
* If the antibody recognises the
antigen in the tissue it — to it.
* The antibody does therefore not
— when the tissue is rinsed
* The brown dye indicates that the
protein is in the – .
tissue
protein
detection
normal
abnormal
antibody
brown dye
tissue section
binds
wash off
tissue
( example: Brown dye means Her 2 is present in the tissue and the patient will respond to Trastuzumab)
- ISH
- Fluorescence In Situ Hybridisation (FISH)
- Chromogenic In Situ Hybridisation (CISH)
- Probe made up of DNA/RNA complementary to the
sequence of interest - Attached fluorophore/chromogen
are all under – - these are examined under —- microscopy
tissue based nucleic acid probes
fluorescence/bright field
HER2 ISH :
* Determine if HER2 gene is –
* Compare number of chromosome – signals with number of HER2 signals
* –
– Ratio > -
– HER2 copy number > –
amplified
17
positive
2
6
- fish to determine chromosomal rearrangement – the probes apart and uses – probes
- other uses of ISH includes the diagnostics of — , — , —
- clinical example:
- 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.
break
fusion
lymphoma sarcoma and viral infection
difference between IHC ( Immunohistochemistry) and FISH :
* — to detect the protein in tissue
– Uses –
* — to detect the altered gene on the chromosome or to detect the chromosome
– Uses a —
Immunohistochemistry
antibodies
FISH
nucleic acid probe
we can detect mutation by using – based probes and these include:
1- —
* Traditional
* Real time
2- –
* Traditional
* Next generation
nucleic acid
PCR
sequencing
PCR technique can be — PCR or — PCR and the advantages are — and its relatively — for small numbers of assays , the disdavantages is that full mutation info is not always –
tradition PCR or real time PCR (rtPCR)
sensitive and cheap
not always available
- Sanger
- Pyrosequencing
are for –
advantages:
1- —-
2- relatively — for small numbers of assay
3- wealth of —
disadaavtegs :
1- — for large number of targets
2- max read –
traditional sequencing
gold standard
cheap
experience
inefficient
Lengths
the next generation sequencing has massively — sequencing by which multiple — relatively – reads
—- and — approached used to generate sequence
parallel
overlapping
short
compared and bio informatics
understanding carcinogenesis:
* “Cancer is a – disease”
* — vs — mutations
– BRCA
– FAP
* Alterations of cancer genes, 2 broad categories:
– —
– —
genetic
somatic vs gremlin
oncogenes
Tumour suppressor genes
1- Oncogenes
is —
Activation by – , – , — etc
Mutation detection by techniques such as:
— level using —
—- using RT-PCR
– using PCR and sequencing
2- Tumour suppressor genes
is —
Inactivation due to either loss of – alleles eg LOH or —
– hit required
- examples of oncogenes:
Ras commonly mutated in –
Myc for —
Abl for —
Ret for — carcinoma
dominant
mutation translocation and amplification
protein level by IHC
mRNA
DNA
recessive
both
methylation
double hit
cancer
burkitts lymphoma
chronic myeloid leukoma
thyroid
Changes in – cause changes in — that control the pathways
of growth, pathways in signalling
in cells, cell cycle control and
differentiation etc
genes
proteins
receptor tyrosine kinase (RTKs):
* Receptor tyrosine kinases (RTK)s are the – affinity — receptors for many polypeptide – factors, – , and — .
* In biochemistry, a kinase is a type of – that transfers — groups from – energy donor molecules, such as — to specific target molecules ( — ); the process is termed — . The opposite, an enzyme that removes phosphate groups from targets, is known as a – .
* Kinase enzymes that specifically phosphorylate tyrosine
amino acids are termed —
* This phosphorylation leads to activation of — etc
high
cell surface receptor
growth factors cytokines and hormones
enzyme
phosphate
high energy
atp
substrate
phosphorylation
phosphatase
tyrosine kinases
cell signalling
- RAS family proteins are expressed in – cells.
- The RAS proteins are small – involved in —
- The RAS genes are the most — oncogenes in human cancer.
- Activation of RAS leads to activation of other – involved in cell – and –
pathways. - Activation is found in 20-25% of tumours and up to 90% in certain tumour types e.g. – cancer.
ALL?
gtpases
signal transduction
most common
protein
cell growth differentiation and survival
pancreatic
( check slide 57)
—- are the most common tumour mutation and these – over time and found in all types of — , can be caused by – or –
these include:
* APC
* TP53
* Retinoblastoma (Rb)
* — Genes:
– MMR genes
– BRCA
( also P53 which is very important TSG which is normally a molecular policeman and safeguards the integrity of genome)
tumour suppressor genes
accumulate
neoplasm
eg. LOH or methylation
dna repair genes
Familial Adenomatous Polyposis/Adenomatous
Polyposis Coli APC:
-This accounts for approximately 1% of cases of – cancer inherited as —
-Patients have thousands of adenomatous ( — ) polyps, some of which undergo – change
-APC discovered as the gene causing – , but APC mutated in the majority of —
colon
autosomal dominant
neoplastic
malignant
FAP
CRC
p53 and cancer evidence :
Transgenic mice which have both P53 genes inactivated by gene “—” studies are normal at birth but by – to –
months 100% develop a range of cancers. This exemplifies the importance of normal p53 function in preventing cancer.
knock off
6 to 9
1- Li Fraumeni syndrome is a rare — disorder which predisposes to a
range of tumours including – , — , – and – tumours.
Affected family members have – mutant - gene in their –
-An – abnormality of their second p53 allele causes a loss of p53 – and – change.
autosomal dominat
sarcoma leukaemia breast n brain
one mutated
germline
acquired
activity and malignant
1- dna repair genes as — genes and — :
-The events which are required for DNAto 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 — and — and exposure to – .
2- mismatch pair genes causes — instability and foud in ~15% of —
- these are associated w —- differentiated morphology , – and – locations , includes:
» — mutations
» – promoter methylation
» – syndrome (HNPCC)
mistamatch repair genes and BRACA
ultraviolet , ionising radiation and chemicals
microsatelite
CRC
mucinous/poorly differentiated
morphology,
TILs,
proximal location.
BRaf
MLH1
Lynch
Lynch syndrome:
* Due to abnormality in—- genes:
» MLH1, PMS2, MSH2, MSH6
» Leads to — instability
* – , — carcinoma, others
* Clinical criteria relatively —
- — can be used to screen
- — testing can also be done
- however majority of CRC w MMR gene abnormalities are —
- due to — phenotype which leads to – of — promoter
- confirmed by — testing through —
DNA mismatch repair
microstaltie
CRC and endometrial
insensitive
( chrck slide 67)
IHC
MSI
sporadic
hypermethylator
methylation
MLH1
genetic testing through sequencing
