5. Neoplasia V- Molecular Diagnostics

Question 1

Define the function of tumor suppressor genes and oncogenes and how mutations in each lead to the development of malignancy (objective)

Answer 1

Answer later

Question 2

Name the basic steps of the polymerase chain reaction (PCR) and understand its role in current molecular diagnostics methodologies (objective)

Answer 2

Answer later

Question 3

Recognize the principles of some of the common basic molecular diagnostic techniques, including next generation sequencing, RT-PCR, Sanger sequencing, capillary electrophoresis, and pyrosequencing (objective)

Answer 3

Answer later

Question 4

Describe the role of molecular diagnostics in diagnosis, prognosis, treatment modality, and clinical trials eligibility in patients with solid and hematopoietic tumors (objective)

Answer 4

Answer later

Question 5

Describe the regulation of molecular diagnostic testing, including the difference between FDA-approved and laboratory developed tests (objective)

Answer 5

Answer later

Question 6

Need to know for summative

Answer 6

Oncogenes, tumor suppressor genes, Lynch syndrome, EGFR

Question 7

Tumor Suppressor Genes

Answer 7

1. Inhibit cell proliferation (Rb, TP53, P16)
2. Loss of 2 alleles
3. Accounts for many familial cancer syndromes (Lynch, BRA1/2)
4. Large variety of mutation types seen

Question 8

Tumor Suppressor HPV Induced Carcinogenesis

Answer 8

HPV leads to E6 and E7.

E6 shuts off P53/Bak, turns on telomerase and kinases leadings to cell immortalization

E7 does similar cell immortalization

Question 9

Oncogenes

Answer 9

1. Activation of cell cycle/cell proliferation (EGFR, growth and transcription factors)
2. Usually specific activating point mutations, gene amplification and chromosome translocation/rearrangement)-gain of function
3. Often therapeutic targets
4. Familial syndromes are rare

Question 10

2 Types of Tests in Molecular Lab

Answer 10

1. Laboratory developed tests

2. FDA approved tests

Question 11

Polymerase chain reaction- PCR

Answer 11

1. Denaturation (at 94-96 degrees C)
2. Annealing (at 68 degrees C)
3. Elongation (at 72 degrees C)

Question 12

Sanger Sequencing

Answer 12

1. Use of dye-labeled ddNTPs in elongation phase
2. Run through capillary electrophoresis
3. Can detect base pair substitutions and insertions/deletions
4. Sensitivity of 10-20%
5. Will NOT detect large rearrangements

Question 13

Pyrosequencing

Answer 13

1. Sequencing by synthesis (nucleotides added one at a time)
2. Use of Luciferase, which interacts with ATP released when dNTP is added to elongating chain
3. Sensitivity of 5%
4. Detect base pair substitutions

Question 14

Reverse Transcription PCR (RT-PCR)

Answer 14

1. RNA translated into cDNA, then DNA amplified by PCR
   (fusion transcripts/translocations, gene expression)
2. Don’t confuse with Real Time PCR (qPCR)
   - quantifies amount of DNA

Question 15

Size discrimination by capillary electrophoresis

Answer 15

• Amplification of Target Region of DNA

- Run through gel with control fragments of known size

Question 16

Next Generation Sequencing (Massively Parallel Sequencing)

Answer 16

1. DNA fragmented
2. DNA ligated to uniquely coded sequences (primer site, hybridization site)
3. DNA hybridized to substate (flow cell, bead)
4. Sequencing
5. Bioinformatics

Question 17

Lynch Syndrome

Answer 17

Hereditary cancer syndromes caused by mutation in 1 of 4 DNA mismatch repair proteins (form of tumor suppressor gene), MLH1, MSH2, MSH6, PMS2
-Deletion of 3’ exons of EPCAM gene, causing inactivation of MSH2
1. Hereditary non-polyposis colorectal cancer
2. Endometrial cancers
+ micro-satellite unstable (caused by hypermethylation of MLH1)

Question 18

Tests for Lynch Syndrome

Answer 18

• micro-satellite instability: reflex MLH1 hypermethylation, reflex BRAF mutation testing
• MMR protein expression by IHC: MLH1 hypermethylation
• Gene sequencing

Question 19

EGFR Mutations in Non-Small Cell Lung Cancer

Answer 19

EGFR mutations in Exons 18/19/20/21

Primarily in adenocarcinoma histology, never smokers, females and Asians