lab 6

what type of epithelium is the cervix

stratified squamous

what area of the cervix is most susceptible to HPV infection

squamous columnar junction (SCJ) or transition zone

because columnar up inside and squamous on the outside - squamous cells taken in smear

how does the cervix change over a womans life 

SCJ becomes more exposed in puberty → so need to make sure vaccinated before then

is HPV alone enough for cancer 

probably not - need a second hit which we dont really understand yet 

cervical intraepithelial neoplasia (CIN)

potentially premalignant transformation and dysplasia of squamous cells on the surface of the epithelium 

pleomorphic 

when the cells are dysplastic and are different sizes 

CIN 2 and 3 cytology

reduction in size of some cells

increase in nuclear/cytiplasmic ratio

hyperchromatic nuclei

pleomorphism

halos around nuclei - koilocytes

neutrophils or bacteria 

CIN 2 and 3 carcinoma in situ - histology

loss of maturity of basal cells that extends to various levels of epithelial thickness

loss of cellular orientation

hyperchromatic nuclei

increase in nuclear/cytoplasmic ratio

pleomorphism

koilocytes

what causes halo effects

less protein being produced as the virus does not care about trying to preserve cell function

immunocytochemistry

use of antibodies to label specific antigens in cells

antibody-antigen reaction binding is identified by a number of labelling methods and enzyme substrate reaction produced colourful product

important tumour suppressor genes 

p53, RB, BRCA1, PTEN

p53 mutations involved in breast and brain but not cervical cancer 

BRCA1 in breast and ovarian cancer 

why test for p53 

can label cells for p53 using immunocytochemistry

tightly negatively regulated in cells - so mutations mean there are high levels of p53 in cells as mutant is more stable (labels both wild type and mutated p53)

imunocytochemistry - how

treat cells with DNA damaging agent → should see an increase in p53 to fix damage and then go back down as it is negatively regulated 

DNA damage → primary antibody to p53 (mouse) → excess washed off → secondary antibody conjugated to a chromagen 

FISH is used 

to label specific DNA or mRNA (transcript sequences) using a labelled single strand probe targeting the sequence 

often there are gene or chromosome abnormalities (amplification) in cancer cells 

what is the most commonly amplified gene in cervical cancers 

PIK3CA

what looking for in FISH

one colour highlights one gene so should see two of each colour in a nucleus

if see more than 2 of each colour in nucleus know that the gene has been amplified