Table 8. BRAF Pyrosequencing analysis of cases with discordant results between ASLNAqPCR and Sanger sequencing.

Case number	BRAF-SSEQ	BRAF-ASLNAa	BRAF mutated/wild type (%)b	Pyrosequencing	Sample	Tumor cells/Non neoplastic cells (%)c
1	WT	V600E	1.25	V600E	metCRC, LN biopsy	5
2	WT	V600E	1.25	V600E	CRC, resection,	75
3	WT	V600E	1.5	V600E	CRC, resection	55
4	WT	V600E	3.0	NP	PTC, resection	80
5	WT	V600E	3.0	NP	CRC, resection	10

SSEQ, Sanger sequencing; ASLNA, allele specific quantitative PCR using 3′-locked nucleic acid modified primers (ASLNAqPCR); WT, wild type; NP, not performed due to lack of additional DNA; CRC, colonic adenocarcinoma; met, metastatic; LN, lymph node; PTC papillary thyroid carcinoma. ^aASLNAqPCR primers designed to identify only the BRAF V600E mutation. ^bReal time ASLNAqPCR quantitative data. ^cPercentage of the tumor/non neoplastic cells ratio estimated in the area dissected for DNA extraction.