An inexpensive, specific and highly sensitive protocol to detect the BrafV600E mutation in melanoma tumor biopsies and blood

Abstract

The Braf(V600E) mutation has been detected in patients with metastatic melanoma, colon, thyroid and other cancers. Recent studies suggested that tumors with this mutation are especially sensitive to Braf inhibitors, hence the need to reliably determine the Braf status of tumor specimens. The present technologies used to screen for this mutation fail to address the problems associated with infiltrating stromal and immune cells bearing wild-type Braf alleles and thus may fail to detect the presence of mutant Braf(V600E) tumors. We have developed a rapid, inexpensive method that reduces the contamination of wild-type Braf sequences from tumor biopsies. The protocol involves a series of PCR amplifications and restriction digestions that take advantage of unique features of both wild type and mutant Braf RNA at position 600. Using this protocol, mutant Braf can be detected in RNA from mixed populations with as few as 0.1% Braf(V600E) mutant cells.

Figure 1

(A) Schematic outline of the protocol determining the Braf V600 status, as described in results and discussion. (B) The rationale behind the final Xba 1 digestion. The final PCR uses a unique forward oligonucleotide with mismatches at positions -3 and -4 (bold letters). This creates an Xba 1 restriction site if the mutant Adenine (A) and not the wild type Thymidine (T) is present in the secondary PCR template. Digestion with Xba 1 produces products of 108 and 32 bp. Arrows indicate the potential Xba 1 cleavage site. (C) The sequence at Braf position 600 and the accompanying Xba 1 digestion for wild type (786-0) and mutant (A375) cell lines. The arrows in the sequence indicate the mutation site. The undigested (140 bp) and Xba 1 digested (108 and 32 bp) fragments are indicated. (D) Xba 1 digestion of Braf PCR from non-melanoma cell lines. U = PCR product prior to the addition of Xba 1; X = Xba 1 digested PCR product.

Figure 2

Accessing the sensitivity of the protocol in the presence (top) and absence (bottom) of TspR1. Varying ratios of wild type (786-0) to mutant (A375) RNA was subjected to the protocol. TspR1 was added according to the protocol or not added. The undigested and Xba 1 digested fragments are indicated.

Figure 3

Testing the protocol on melanoma biopsies. The sequence at Braf position 600 and the accompanying Xba 1 digestion for melanoma tumor biopsies. The arrows in the sequence indicate the mutation site. The undigested and Xba 1 digested fragment are indicated. The intensity of the 108 bp band correlates with the relative peak height of adenine (green) to thymidine (red) at postion 600.

Figure 4

Testing the protocol on peripheral blood lymphocytes. (A) Xba 1 digestion of the Braf PCR from a titration of mutant A375, A2058, SK MEL 5, WM 1976, WM 1862, WM 3163, and WM 3727 cells in 400,000 PBLs. Data is presented as the number of melanoma cells in 400,000 PBLs. (B) `Xba 1 digestion of the Braf PCR from PBLs from a normal donor (N) and a patient (B39) with a known V600E mutation. Either 1 or 10 ug of RNA was used in the assay.