Molecular Diagnostics in Neoplasia

Question 1

PCR

Answer 1

Denaturation
Annealing
Elongation

Question 2

Sanger sequencing

Answer 2

• Labeled ddNTPs
• Capillary electrophoresis
• Can detect subs, insertions, deletions
• Won’t detect large rearrangements

Question 3

Pyrosequencing

Answer 3

• Sequencing by synthesis
• Luciferase to interact with ATP released when dNTP is added
• Detect base pair substitutions

Question 4

Reverse transcription PCR

Answer 4

Use of RNA made into cDNA and then amplified

Question 5

Size discrimination by capillary electrophoresis

Answer 5

• Amplify target regions

- Run through capillary electrophoresis with control fragments

Question 6

Next generation sequencing: massively parallel sequencing

Answer 6

DNA fragment
DNA ligated to coded sequence
DNA hybridized to substrate
Sequenced
Bioinformatics

Question 7

Lynch syndrome

Answer 7

• Hereditary cancer caused by mutation in mismatch repair proteins
• Deletion in 3’ exons of EPCAM gene causes methylation of MSH2
• BRAF mutations almost never found in Lynch, but occasionally see mutations in RAS
• HNPCC

Question 8

Endometrial cancer

Answer 8

• 2-3% associated with Lynch
• Increased risk for women who have Lynch
• Sentinel cancer in 50% of women with Lynch

Question 9

What is Lynch Syndrome

Answer 9

• Microsatellite instability
• 15-20% of sporadic are also microsatellite unstable
• MSI in sporadic tumors caused by hyper-met of MLH1, not germline mutations in MMR

Question 10

Testing for Lynch

Answer 10

MSI

• Reflex MLH1 hypermet
• Reflex BRAF testing

MMR protein expressed by IHC
-MLH1 hypermet

Gene sequencing

Question 11

EGFR mutations in non small cell lung cancer

Answer 11

• EGFR mutations in Exon 18,19,20,21
• Rarely found with other mutations
• Primarily in adenocarcinoma histology never smokers, females, asians
• FDA approved targeted small molecular inhibitors