Molecular Genetics - Lung Cancer (III)

Question 1

4 major histological subtypes of Primary lung carcinoma classification
Prevalence?

Answer 1

Adenocarcinoma 45-55%
Squamous cell 20-30%
Large cell carcinoma 5-10%
Small cell carcinoma 5-10%

Question 2

Which major histological subtypes of Primary lung carcinoma is most important for genetic testing?
Why?

Answer 2

Adenocarcinoma

Only subtype with existing targeted drug treatment
Other subtypes lack clinically validated targeted drug treatment

Question 3

2 key oncogenic targets in Lung cancer

Answer 3

EGFR mutation *
ALK Translocation*

common downstream MAPK and PI3K/AKT pathways&raquo_space; cancer function, proliferation, metastasis …etc

Question 4

Compare response of EGFR wild-type and mutated lung cancer to targeted Gefitinib therapy

Answer 4

Wild type: treat with chemotherapy = better response than gefitinib

EGFR mutation: treat with gefitinib = better response than chemotherapy

Question 5

2 reasons why EGFR and ALK mutation status may change during lung cancer progression?

Answer 5

1) Clonal evolution: selection and expansion of cells with EGFR bypass mutations
2) Direct resistant mutations

Question 6

Lung cancer management.

When to conduct mutation testing?

Answer 6

1) Before systemic treatment +/- metastasis

2) After first line tyrosine kinase inhibitor

Question 7

3 types of sequencing for lung cancer mutation?

Answer 7

1) Direct Sanger sequencing
2) Mutation-specific quantitative PCR (qPCR)
3) Targeted next generation sequencing

Question 8

4 types of samples for EGFR mutation detection in Lung CA

Answer 8

1) Tumour biopsy > isolate tumour cells for DNA enrichment (microdissection)
2) Peripheral blood cell-free DNA
3) Pleural fluid (malignant pleural effusion): dissolved DNA and tumour cells
4) Surgically resected tumor

Question 9

3 criteria for high quality sample for EGFR testing

Answer 9

1) Adequate tumor amount
2) High tumor cell proportion in sample
3) Good DNA quality

Question 10

Define the 4 mutation hotspots in EGFR mutation in lung CA

Which hotspots have highest activity

Answer 10

EGFR mutation: Kinase domain mutation

Exon 18, 19, 20, 21

Exon 19,21 have highest freq. of mutation

Question 11

Define the exact mutations in the 4 exon hotspots in EGFR

Define TKI resistance in each exon

Answer 11

Not all genotypes respond equally to TKI

Exon 18: G719A - sensitive
Exon 19: In-frame deletion - sensitive, D761Y - resistant
Exon 20: In-frame insertion, T970M - resistant
Exon 21: L858R - sensitive, L854A - resistant

Question 12

Which EGFR mutations are covered by Mutation-specific quantitative PCR (qPCR)?
Mechanism of mutation detection?

Answer 12

Common mutations only

Exon 18 mutation
Exon 19 in-frame deletion
Exon 20 T970M
Exon 21 L858R

Fluorophore-labeled primers bind to specific sequence&raquo_space; give fluorescence signal after binding

Question 13

Compare direct sanger sequencing and qPCR for EGFR mutation

• Turnaround time
• Types of EGFR mutation
• Sample quality

Answer 13

• Turnaround time
  RT-qPCR = 2-3 days
  Sanger = 3-4 days
• Types of EGFR mutation
  RT-qPCR = Common mutations, primer dependent
  Sanger = All mutations
• Sample quality
  Sanger needs higher quality DNA

Question 14

Compare direct sanger sequencing and qPCR for EGFR mutation

• Sensitivity
• Specificity

Answer 14

• Sensitivity
  Sanger = affected by sample quality
  RT-qPCR = Higher
• Specificity
  Both high. Sanger = higher

Question 15

Describe ALK fusion gene in lung carcinoma

Answer 15

EML4 or other genes dimerize with ALK via coiled-coil domain&raquo_space; ALK activation

EML4 constitutively expressed for intracellular transport

Question 16

2 methods to detect ALK translocation

Answer 16

IHC - ALK protein overexpression

break-apart FISH - ALK gene translocation

Question 17

Mechanism of break-apart FISH for ALK translocation

Answer 17

ALK kinase domain and remaining sequence tagged with 2 different fluorescent probes

Any gene fusion (EML4) with ALK kinase domain (to activate ALK) breaks the kinase domain from remaining sequence

No fusion = 2 probes stick together
Fusion EML4-ALK= 2 probes break apart = +ve for ALK activation