BRAF Mutation Testing (including V600E)

Performing Lab:

UCSF Clinical Cancer Genomics Lab

CPT Code:

81210, 88381

Specimen Type:
Formalin-fixed, paraffin-embedded tissue. Tissue block or unstained slides. If sending unstained slides, we require five (5) 10-micron tissue sections on uncharged glass slides. One adjacent H&E stained slide should be included. Cytology smears may also be used for testing if there is sufficient tumor present. Contact the laboratory at 415.502.3252 or [email protected] if testing on a cytology smear is desired.
Methodology:
The BRAF Mutation Testing assay utilizes capture-based next generation sequencing of exon 15 of the BRAF gene. A histologic section of formalin-fixed, paraffin-embedded tissue is examined by a pathologist to identify an area of tissue with sufficient tumor for detection (>=25% tumor if possible). DNA is extracted from tumor areas on adjacent unstained slides. Target enrichment is performed by hybrid capture using custom oligonucleotides. Sequencing of captured libraries was performed on an Illumina HiSeq 2500 by the UCSF Genomic Sequencing Services Lab at Institute for Human Genetics CLIA laboratory (San Francisco, CA). Sequence reads are de-duplicated to allow for accurate allele frequency determination. The analysis uses open source or licensed software for alignment to the human reference sequence UCSC build hg19 (NCBI build 37) and variant calling.

This assay is designed to detect single nucleotide variants (SNVs) and small to medium insertion/deletions (indels). Sequencing of target intervals is performed to high depth, with greater than 250x mean target coverage.

Mutations are reported based on NCBI Reference Sequence: NM_004333.

In clinical validation studies, for SNVs, this assay has >95% sensitivity at >=15% mutant allele frequency, 65-80% sensitivity at 10-15% mutant allele frequency. For indels <40 bp, there is >98% sensitivity at >=7% mutant allele frequency. Testing of 19 pathogenic BRAF mutations in 18 different specimens showed the mutations were detected 100% of the time. Testing of 35 samples without pathogenic BRAF mutation showed that no mutation was detected in any of the specimens.

Turnaround Time: 7-14 days

TEST INDICATIONS:

This test detects the V600E mutation in the BRAF gene from formalin-fixed paraffin-embedded tumor tissue. BRAF V600E mutation has been shown to be present in a number of tumors, including thyroid and colorectal carcinomas, melanomas, histiocytic tumors, and certain brain tumors.

In the thyroid, presence of the mutation is associated with more aggressive clinical behavior. BRAF mutation in Lynch syndrome-associated colorectal cancers is extremely rare, and the presence of BRAF mutation is a strong indication of a sporadic (i.e., non-Lynch) colorectal cancer. However, note that not all MSI-H colorectal cancers that lack BRAF mutation are due to Lynch syndrome. BRAF mutation in colon cancer is associated with a lack of response to EGFR therapy. In melanoma, BRAF V600E mutation predicts response to vemurafenib. BRAF mutation testing may also be useful in evaluation for EGFR therapy in lung cancer.

HOW THE TEST WORKS:

A histologic section of formalin-fixed, paraffin-embedded tissue is examined by a pathologist to identify areas of tumor. DNA is extracted from these areas and the region of BRAF exon 15 that includes codon 600 is captured and sequenced by next generation sequencing.

LIMITATIONS OF THE TEST:

Results of this test should be correlated with the patient’s other clinical and laboratory information. This assay is designed to detect BRAF mutations in exon 15, including the common V600E mutation and less-frequent V600K, V600R, c.1794_1796dupTAC (insT), and K601E mutations.

The test was validated by the UCSF Clinical Cancer Genomics Laboratory to confirm performance characteristics, in compliance with current guidelines for clinical implementation.

SPECIMEN REQUIREMENTS FOR A SUCCESSFUL TEST:

In general, the tumor should be a minimum of 0.4 cm in size. Blocks selected for BRAF Mutation Testing must contain tumor tissue. Areas of tumor must contain at least 50% tumor cells. The lab can remove adjacent non-tumor tissue, so the entire slide does not need 50% tumor cells, just the area with tumor. Contact the laboratory at 415.502.3252 or [email protected] if the specimen suitability is uncertain. Label slides with pathology case number and block identification.

Specimen rejection criteria include: All required slides not included. Insufficient tumor tissue present on slide as determined by pathologist. Outside slides not labeled or not accompanied by printed copy of test order.

HOW TO ORDER THE TEST:

UCSF Clinicians — this test can be ordered through APeX
Outside Physicians — this test can be ordered using the CCGL Requisition Form

For all specimens, an interpretation of this test by a laboratory physician will automatically be performed and billed for separately.