KRAS testing in colorectal cancer

Philippe Taniere

Birmingham

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.

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

KRAS mutation

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

Sources of material

Sections from paraffin blocks Stained sections Cytology specimens

Scraping from slides Tissue in DNA extraction buffer

DNA extraction and PCR

Direct sequencing (Sanger)

Substitution

G to T codon 12

Pyrosequencing

110

120

130

140

E S T A C G A C T C A G A T C G T A G

G:90.8%A:0.0%C:0.0%T:9.2%

G:100.0%A:0.0%

5 10 15

GGTCodon 12

GGCCodon 13

Real time PCR

Exogenous Control. Well 3A

12 Val: Positive. Well 3G

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

Others, …

HRM Snapshot Etc,..

KRAS and CRC

In practice, tests to be performed within 3 to 5 working days

In practice

Who is paying for testing? NHS Merck Serono

How much does it cost? Very cheap!

When to start on the testing? Who can do the testing?

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