Table 2. Polyclonality of BRAF mutations in primary melanoma as revealed by the Mutector assay and subcloning.
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Subcloningc
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|---|---|---|---|---|---|---|---|---|---|
| Sample no. | Age (years) | Sex | Tumour thickness (mm) | Type of melanoma | Tissuea | Direct sequencing | Mutector (OD ratio)b | No. of colonies with mutant BRAF | No. of colonies with wild-type BRAF |
| PM-6 | 85 | M | 13 | CSD | FFPE | V600K | Positive (8.9) | 18 (V600K)+1 (V600E) | 21 |
| PM-7 | 57 | M | 4.8 | NCSD | FFPE | Wild type | Positive (9.04) | 3 (V600E) | 37 |
| PM-8 | 68 | F | 4.1 | NCSD | FFPE | Wild type | Negative (0.53) | 0 | 40 |
| PM-9 | 29 | F | 4 | NCSD | Frozen | V600E | Positive (32.51) | 20 (V600E) | 20 |
| PM-10 | 32 | F | 0.8 | NCSD | FFPE | Wild type | Positive (4.75) | 2 (V600E) | 38 |
| PM-11 | 50 | M | 4.1 | Acral | FFPE | Wild type | Positive (14.47) | 7 (V600E) | 33 |
| PM-12 | 69 | F | 3.5 | Acral | FFPE | V600E | Positive (27.34) | 18 (V600E)+1 (V600K) | 21 |
| PM-13 | 77 | F | 1.5 | Mucosal | Frozen | V600E | Positive (31.69) | 9 (V600E)+1 (K601E) | 30 |
| PM-14 | 74 | F | 30 | Mucosal | Frozen | Wild type | Positive (8.64) | 2 (V600E) | 38 |
| PM-15 | 79 | F | 6 | Mucosal | FFPE | Wild type | Negative (0.17) | 0 | 40 |
Abbreviations: Acral=acral melanoma; CSD=melanoma on chronic sun-damaged skin; FFPE=formalin-fixed paraffin-embedded tissues; MMG1=a cell line established from primary acral melanoma; Mucosal=mucosal melanoma; NCSD=melanoma on non-chronic sun-damaged skin; PM=primary melanoma.
a
All the FFPE sections were stained with gp100 protein, and pure tumour tissues were collected by laser microdissection.
b
OD ratio >2 were regarded as positive (Ichii-Nakato et al, 2006).
c
A total of 40 bacterial colonies were sequenced for each sample. V600E, T1799A; V600K, GT1798-99AA; K601E, A1801G.