1. KRAS testing in colorectal cancer
Philippe Taniere
Birmingham

2. Targeted therapy in colorectal cancer (CRC)
Two anti EGFR monoclonal antibodies are licensed for CRC
Cetuximab (Erbitux, Merck Serono)
first line treatment in combination with irinotecan-based chemotherapy or FOLFOX4
2nd or 3rd line treatment as a single agent
Panitumumab (Vectibix, Amgen)
monotherapy after failure of fluoropyrimidine, oxaliplatin and irinotecan-containing chemotherapy regimens.

4. Mandatory molecular testing
KRAS mutation testing is mandatory prior to prescription since trials have clearly shown that KRAS mutated tumours will never respond to the drug
Negative predictive marker

5. KRAS mutation Licensing for wild type codons 12 and 13
KRAS gene mutated in 40 to 45% of CRC
2 main hotspots in exon 2 at codons 12 and 13
1 rarely mutated site in exon 3 at codon 61
1 even more rarely mutated site at codon 146
Licensing for wild type codons 12 and 13

6. Sources of material Sections from paraffin blocks Stained sections
Cytology specimens

7. DNA extraction and PCR Scraping from slides
Tissue in DNA extraction buffer

8. Direct sequencing (Sanger)
Substitution

9. Pyrosequencing
G to T codon 12
GGT
Codon 12
GGC
Codon 13

10. Real time PCR View: All Wells View: Control (3A) and 12 Val mix (3G)
Exogenous Control and Single Mutation: 12 Val Positive
all 7 K-RAS Mutation Mixes
Exogenous Control. Well 3A
12 Val: Positive. Well 3G

11. Others, …
HRM
Snapshot
Etc,..

12. KRAS and CRC
In practice, tests to be performed within 3 to 5 working days

13. In practice Who is paying for testing? How much does it cost?
NHS
Merck Serono
How much does it cost?
Very cheap!
When to start on the testing?
Who can do the testing?

14. Perspective
Only a proportion of patients with wild-type KRAS respond to anti EGFR monoclonal antibody
A more advanced assessment of tumour cells may become justified in the near future
?More codons of KRAS (61 and 146)
?BRAF, PIK3CA, pTEN, etc,..
Need for platforms, kits, etc,..for cheap and quick multiple screening