Application of a BRAF pyrosequencing assay for mutation detection and copy number analysis in malignant melanoma - PubMed (original) (raw)

Application of a BRAF pyrosequencing assay for mutation detection and copy number analysis in malignant melanoma

Cynthia Spittle et al. J Mol Diagn. 2007 Sep.

Abstract

Mutations in the BRAF gene are found in the majority of cutaneous malignant melanomas and subsets of other tumors. These mutations lead to constitutive activation of BRAF with increased downstream ERK (extracellular signal-regulated kinase) signaling; therefore, the development of RAF kinase inhibitors for targeted therapy is being actively pursued. A methodology that allows sensitive, cost-effective, high-throughput analysis of BRAF mutations will be needed to triage patients for specific molecular-based therapies. Pyrosequencing is a high-throughput, sequencing-by-synthesis method that is particularly useful for analysis of single nucleotide polymorphisms or hotspot mutations. Mutational analysis of BRAF is highly amenable to pyrosequencing because the majority of mutations in this gene localize to codons 600 and 601 and consist of single or dinucleotide substitutions. In this study, DNAs from a panel of melanocyte cell lines, melanoma cell lines, and melanoma tumors were used to validate a pyrosequencing assay to detect BRAF mutations. The assay demonstrates high accuracy and precision for detecting common and variant exon 15 BRAF mutations. Further, comparison of pyrosequencing data with 100K single nucleotide polymorphism microarray data allows characterization of BRAF amplification events that may accompany BRAF mutation. Pyro-sequencing serves as an excellent platform for BRAF genotyping of tumors from patients entering clinical trial.

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Figures

Figure 1

Figure 1

Detection of V600E in melanoma cell lines. Pyrograms generated for BRAF wild-type (WM3208) (A), heterozygous mutant (WM 902B) (B), and homozygous mutant (WM39) (C) cell lines distinguish T versus A peaks (yellow shading). Nucleotide dispensation order is given below each pyrogram. Numerical position for each nucleotide is indicated at the top. Dispensations of C at positions 1 and 6 are included as controls for misincorporation. Dideoxy cycle sequence tracings for each cell line are shown for comparison.

Figure 2

Figure 2

Detection of BRAF variant mutations. Pyrograms for two variant mutations, V600R (B) and V600K (C), show distinct patterns compared with V600E (A). Changes in relative A/G peak heights at pyrogram positions 2, 3, 5, 7, and/or 8 reflect dinucleotide substitutions given at right. The specific variants are confirmed by dideoxy cycle sequencing.

Figure 3

Figure 3

Applicability of pyrosequencing assay to FFPE tissue. Pyrograms generated from a V600E mutant tumor (A) and a V600K mutant tumor (B) are identical for paired frozen and FFPE tumors.

Figure 4

Figure 4

Cell line DNA mixing study. Theoretical peak heights, calculated from initial percent mutant A peak in undiluted heterozygous V600E mutant tumor, are correlated with actual peak heights generated for each dilution (Pearson’s correlation = 0.96), indicating a linear relationship.

Figure 5

Figure 5

Analytic sensitivity for V600E detection in a heterozygous tumor. Pyrograms generated from dilution series for two separate experiments (day 1 and day 2) are shown with corresponding dideoxy sequence tracings for day 1 data. The percent tumor corresponding to each data set is indicated at left.

Figure 6

Figure 6

Comparison of pyrosequencing data with SNP microarrays. A: SNP _Xba_I 50K microarray data for chromosome 7 displayed with CNAT 3.0 software. Melanoma cell lines analyzed and location of BRAF gene are indicated. For each cell line, genome smoothed chromosome copy number data are shown at the top and the LOH score is shown at the bottom. Chromosome location in megabases is given at the bottom of the data set for each cell line. Scale for copy number and LOH score indicated at right. B: Corresponding BRAF pyrosequencing data including percent T and A peaks are indicated for each cell line.

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