Table 1 Specimen requirements and assay specifications of KRAS genotyping by laboratories

Specimen Requirements

Preferred sample type*:

Slides from FFPE block

1  H&E stained slide sections with tumor circled; 4 matching unstained slides,

10 microns each.

Preferred sample type: Archival FFPE or frozen surgical biopsies confirmed to contain >50% tumor by a surgical pathologist.

1 H&E slide;

5 unstained sections,

10 microns each.

Preferred sample type: FFPE tissue

6 unstained sections,

10 microns each.

Preferred sample type: Pre-cut slides from FFPE. Send all slides within 5–7 days of cutting. Air dry. Do not oven dry. Store specimen at room temperature (20–23.5°C).

5 unstained sections,

7 microns each

Preferred sample type: FFPE block, unstained slides, or fresh snap frozen biopsy

5 unstained sections,

7 microns each

Genotyping

Method: PCR amplification followed

by Direct Sanger sequencing

(Big Dye v. 1.1)

Detected mutations: KRAS codons 12 and 13

Method: PCR amplification followed by standard bidirectional sequencing on ABI 3100.

Detected mutations: KRAS codons 12 and 13

Method: PCR amplification followed by sequencing.

Detected mutations: KRAS codons 12,

13 and 61

Method: Single nucleotide primer extension with fragment analysis by capillary electrophoresis using a modified SNaPshot assay.

Detected mutations:

KRAS codons 12 and 13

Methods are propietary: qualitative real time PCR

Detected mutations: KRAS codons 12 and 13

Lower Limit of Detection

20% when ≥ 40% tumor cells present

20%

15-20%

10% when ≥ 2% tumor cells present

1-5%