Cancer Flashcards
genetic tetsing of cancer is done for one (or more) of for reseaons:
diagnosis
prognosis
therapeutic choice
identification of inherited predispositions
how do we test for cancers?
amplification-based assays
probe-based assays
sequencing-based assays
types of PCR
- end point/ regular
- allele-specific (ARMS)
- reverse transcription (RT)
- real-time/quantitative
endpoint pcr
evaluated at endpoint or plateau phase
binary detection = amplified or non-amplified
pros of endpoint pcr
efficient; yes/no answers
quick, cheap
easy detection of insertions/deletions via size analysis
cons of endpoint pcr
not useful for quantitation
does not detect point mutations
nonspeicifc amplification possible
allele specific PCR
one prime set for mutant allele
one primer set for ‘normal’ allele
amplification of mutant allele indicates patient harbours the pt mutation
pros of allele-specific PCR
simple detection of pt mutations
heterozygosity testing possible
cons of allele-specific PCR
only amplifies specific SNP targeted
poor priming
non-specific amplification due to GC ‘clamping’
RT PCR
RNA to cDNA before PCR (PCR only recognizes DNA)
one-step = RT/PCR in same tube
two-step = RT in on tube, PCR in second tube
pros of RT PCR
allows detection of fusion genes
can be used to detect splice variants
detects gene expression
can be quantitated (qRT-PCR)
cons of RT PCR
if RT doesn’t work, assay doesn’t work
highly depndent on RNA sability
highly dependent on pretest knowledge of breakpts
pros of RT-PCR
direct measurement of gene activity
extremely sensitive
allows monitoring overtime
easier to test for fusion genes (qRT-PCR)
cons of RT-PCR
requires extensive pretest knowledge
standard ctls required
extremely sensitive to contam by inhibitors
canonnical probe-based assay
FISH
types of FISH
Enumeration - HER2? breast cancer gene
break-apart
fusion
pros of FISH for cancer
can do copy number analysis
high sensitivity and specificity
require less specific knowledge of affected region
direct visualization of neoplastic vs non-neoplastic cells
cons of FISH for cancer
detects fairly large scale changes
low sensitivity if number of neoplastic cells is low
technically demanding
intrachromosomal variants can be difficult to assess
pros of sanger
well estbalished technique; relatively cheap
can determine zygosity from tracing
insensitive to poylmerase introduced errors
cons of sanger sequencing
unable to multiplex (one rxn/one sequence)
insensitive = 10-20% allele fraction required
slow
need to have positional knowledge of region of interest (Gene,exon)
how to detect translocations?
karyotype
FISH
RT PCR
qPCR
RT-qPCR
oldest available way of detecting chromosome-level changes in cancer
karyotype
what does karyotype rely on?
ability to grow cancer cells in culture and induce mitosis
benefits of RT-qPCR
highly sensitive
- capable of detecting fewer than 1 abn cell in 10 000
- god for minimal residual disease detection
highly specific
quick
quantitative
