Molecular Pathology in Cancer Diagnostics Flashcards
T/F- the first molecular biomarker was t(9:22) BCR-ABL1 and was discovered in 1972
true
What was the first molecular targeted therapy?
imatinib (2001)
Name 3 types of tissue specimins that can be collected for solid tumor molecular testing
- fresh or frozen tissue
- formalin fixed paraffin embedded (FFPE) tissue
- fine needle aspiration smear
If a patient has leukemia what type of tissue collection will be used for molecular testing?
Blood or bone marrow aspirate (use EDTA anticoagulant purple tube)
What elements are required in a PCR tube mixture?
- oligonucleotide primers complementary to the target sequence
- the four deoxynucleotides (dNTPs)
- DNA polymerase (Taq)
- buffer
- Mg++, water
- patient’s DNA specimen.
What are the steps for PCR
- mixture heated to denature DNA strands
- temp decreased to let primers anneal to template DNA
- heat stable DNA pol extends primers and synthesizes complementary DNA strands
- cycle is repeated
Name the steps for single nucleotide primer extension (SNPE)
- PCR amplify the target sequence
- incubate amplicons with allele- specific primer and fluorescent tagged dideoxynucleotides (blocks addition after 1st base)
- separate blocks by capillary electrophoresis to size and visualize the fluorescent signal
In SNPE, if only a wild type sequence is observed how many peaks will you see? How about it a mutation is present?
1 peak if wt only
2nd peak if mutation is present in sequence
Describe the method of Sanger Dideoxynucleotide chain termination of DNA sequencing
Sequencing using mix of deoxy- and dideoxyribonucleic (fluorescently labeled) acids to generate random lengths of DNA covering the span of sequence then separated by capillary electrophoresis
What are the pros and cons of sanger sequencing?
- Comprehensive method for mutation testing
- Used to discover new mutations
- Limit of detection is less sensitive so the mutation must be fairly prevalent among all the cells in the tissue sample in order to be detected
T/F- Next generation sequencing (NGS) simultaneously seqeunces billions of individual target sequences
true
What is the utility of next gen sequencing (NGS)?
- Identify cancer mutations in low frequency or with limited amount of specimen DNA
- Identify mutations in known genes when individuals have atypical features
- Mutation discovery
Next gen is _______based and “next next gen” is _______ based
- fluorescent based
2. hydrogen ion based
How does Ion Torrent PGM semiconductor screening work?
- PCR amplification of patient DNA
- Library preparation of amplicons for microdroplet or emulsion PCR
- Prepare DNA ends for binding one sequence per bead - Clonal PCR amplification of individual sequence on each bead
- Load semiconductor chip with sequence covered beads (one bead/well)
- Simultaneous semiconductor sequencing by flowing each base one at a time and detection of H+ release by pH change.
- Conversion of data into sequence, alignment to a reference and identification of mutations
What mutations would you use FISH to detect? How is it’s limit of detection?
- aneuploidy, large deletion, defined RAR’s (retinoid acid receptors)
- limit of detection: 0.1-1% (good)
What 2 types of probes can be used in FISH?
Fusion probes
Break apart probes
What is Sanger chain termination DNA sequencing used for? How is its limit of detection?
- defined and new (discovery) point mut, small in/dels, translocations
- 10-20% (okay, but lowest of all discussed)
What is single base primer extension used for? How is it’s limit of detection?
- point (single substitution) mutations
- 1-5% (good)
What is next ben sequencing used for? limit of detection?
- whole exome or genome assessment; mutation discovery
- 0.1-10% (good)
Qualitative PCR fragment analysis? limit of detection?
- point mut, small ins/dels, defined rearrangements (RAR)
- 0.1-5% (good)
RT-PCR? limit of detection?
- defined translocation fusion transcripts, gene expression
- 0.001-0.1% (excellent)
Real time fluorescent PCR? limit of detection?
- point mutations
- 0.01-5% (excellent)
Overall, what is the primary method for mutation detection?
PCR (The complementary oligonucleotide primers and probes confer the specificity of the reaction)
___% of colorectal cancer is sporadic and ___% is inherited
- 70%
2. 10%
Name 4 inherited forms of colorectal cancer
- HNPCC (lynch syndrome, DNA mismatch repair gene)
- FAP (APC mutation)
- MYH-associated polyposis
- Peutz-Jeghers syndrome (STK11 mutation)
What are 4 gain of function mutations that can be tested for in colon cancer?
KRAS
HRAS
NRAS
BRAF
A normal colon acquires an APC mutation, what happens next?
forms an aberrant crypt focus
An aberrant crypt focus obtains a KRAS mutation and progresses to an early adenoma. An EGFR mutation is also gained. What’s next?
Late adenoma with increasing CIN
A late adenoma gains a p53, PIK3CA, loss of 18q mutation, what happens?
invasive cancer
EGFR is a ________ receptor and a _____oncogene
tyrosine kinase
proto-oncogene
A person with EGFR mutation is prescribed a monoclonal antibody to block the tyrosine kinase receptor but does not respond. Why?
Tumors resistant to anti-EGFR mAb therapy frequently have mutations in downstream signalling pathway genes. The most commonly mutated gene is KRAS.
___ and ____ mutations predict lack of response to EGFR mAb therapy
RAS (e.g. KRAS) and RAF (e.g. BRAF)
The ras oncogenes have homology to ___ proteins. Point mutations reduce ____ hydrolysis resulting in constituitively active growth stimulus
G proteins
GTP hydrolysis
What molecular methods would you use to detect RAS and BRAF mutations?
-Mutation/allele-specific methods
(Single nucleotide primer extension)
-DNA sequencing
RAS and RAF are gain or loss of function mutations?
gain
What two mechanisms cause DNA mismatch repair defects?
- Germline mutations, most commonly in MLH1 or MSH2, result in hereditary non-polyposis CRC or Lynch syndrome
- MLH1 promoter hypermethylation silences gene expression and are associated with sporadic colorectal adenocarcinoma (~15% of cases)
Whats a microsatellite area?
small areas of repetitive dna
Lynch syndrome displays an autosomal ______ pattern of inheritance
dominant
Where can you develop tumors with lynch syndrome?
colon, rectum, endometrium, stomach, ovary, ureters, small bowel, hepatobiliary tract, brain, and skin
Patients with lynch syndrome present with R or L sided tumors?
right sided tumors affecting the cecum and proximal colon
If you have the lynch gene, what type of surveillance should you get?
- colonoscopy every 1-2 years at age 20 (or 10 years before earliest dx in familY)
- endometrial and ovarian carcinoma screening at age 30 (or 10 year rule as above)
- annual skin and urinalysis
- periodic upper endoscopy
- discussion of hysterectomy or salpingo-oophorectomy at 35
Microsatellite unstable tumors in HNPCC and sporadic CRC are: (more/less) likely to be lower stage, (longer/shorter survival), and have a (poor/good response) to 5-FU
more likely lower stage
longer survival
poor response to 5-fu
T/F- IHC for loss of MLH1, MSH2, MSH6, or PMS2 (all DNA MMR defects) can identify MMR gene affected by either germline mutation or hypermethylation silencing of the gene
true
What method would you use to test DNA for microsatalite instability?
MSI PCR Assay (minimum panel of 5 markers)
How do you interpret a MSI PCR Assay?
- MSI High (MSH) if >20% (2 or more) markers are altered
- MSI Low (MSL) if 20% (1 of 5) markers are mutated showing loss or addition of the repeats
- Microsatellite stable (MSS) if all of the markers are stable
T/F- MLH1 promoter methylation is consistent with sporadic colorectal adenocarcinoma
true
T/F- BRAF p.VAL600GLU mutation if present is not consistent with sporadic colorectal adenocarcinoma
False, it is
T/F- DNA sequencing of MLH1, MSH2, MSH6, PMS2, or EPCAM for DNA MMR gene mutations, if present in the germline confirm LYNCH SYNDROME
TRUE
Tumors arising in patients with HNPCC have a germline mutation of one allele of a DNA MMR protein (e.g. MLH1) and ____mutation in the other allele.
somatic
A patient is at risk for HNPCC, what mutation would you test for to rule this out?
BRAF (will not respond to EGFR mAb therapy)
Micro satellite instability (MSI) results from defects in ______ ezymes
DNA mismatch repair
Name 2 ways MSI can be screened for
- IHC
2. PCR
The MSH phenotype of Microsatellite instability indicates a patient is at risk for_____
HNPCC
Regarding testing for non-small cell lung carcinoma, ROS1 and EML4/ALK are both ____ of function mutations and can be detected by ____ and ____
gain
FISH and RT PCR
EGFR is a _____ of function mutation and can be tested for using____ and _____
gain
DNA sequencing and mutation specific methods
T/F- EGFR mutation in lung cancer confers a favorable prognosis
true
What are the most common exon mutations in EGFR/tyrosine kinase receptor mutations?
Exon 19 in-frame deletions and exon 21 L858R account for almost 90% of the tyrosine kinase inhibitor sensitizing mutations
EGFR mutations are present in 10% of NSCLC adenocarcinoma in the US, who is this most common in?
nonsmokers, younger age, female sex and Asian descent
T/F- EGFR testing is indicated for any patient with adenocarcinoma of the lung
true
EML4-ALK rearrangement in NSCLC predicts for sensitivity to what?
ALK tyrosine kinase inibitor (crizotinib)
What is the gold standard of dx for ALK mutations?
FISH
ROS1 mutations are a member of the _______ receptor family
tyrosine kinase insulin (sensitive to crizotinib)
Most common way of detecting ROS1 translocations?
- FISH
- RT-PCR
T/F- ALL patients with NSCLC should have tumor tissue assessed for the presence of EGFR mutations and ALK and ROS1 translocations if EGFR is negative
TRUE
_____ is associated with longer survival than either MSI-L or MSS in both HNPCC and sporadic CRC
MSI-H
Epigenetic changes due to _______can be identified using molecular methods that incorporate bisulfite modification
methylation
Chromosomal changes such as translocations or inversion require use of _____ or _____
FISH or RT-PCR
Substitutions and small insertions and deletions can be detected effectively and efficiently with ______ based methods
PCR
MLH1 promoter hypermethylation or BRAF V600E mutation identify sporadic ____
colorectal carcinoma
Germline mutations in the DNA MMR genes (eg. MLH1, MSH2) identify patients with _____
lynch syndrome
KRAS mutations in colorectal adenocarcinoma (indicated/contraindicate) anti-EGFR therapy
contraindicate
EGFR or ALK mutations in non-small cell lung carcinoma indicated what therapy?
EGFR TKI (tyrosine kinase inhibitor) ALK TKI
