Download BRAF V600E Mutation Detection

LAB TEST CONNECT
BRAF V600E Mutation Detection
by Sequence Analysis
Danbin Xu, M.D., Ph.D., Co-Director, Molecular Diagnostic Laboratory
Marcy Bauman, Ph.D., Director, Molecular Diagnostic Laboratory
CLINICAL APPLICATION
ORDER CODE:
Currently, the BRAF (V600E) mutation has been used as a screening biomarker in three
major areas:
BRAFSQ
1.
2.
3.
In Metastatic Melanoma, patients with previously untreated melanoma, positive for the
BRAF V600E mutation, show improved rates of overall and progression-free survival
when treated with Vemurafenib, a potent inhibitor of mutated BRAF recently approved
by the FDA. The package insert requires confirmation of BRAF V600E mutation
positivity prior to treatment with Vemurafenib. The BRAF V600E mutation can be
detected in 40-60% of cutaneous melanomas.
In Metastatic Colorectal Cancer (mCRC), the occurrence of BRAF V600E mutation
is predictive of resistance and poor survival in patients treated by anti-epidermal
growth factor receptor (EGFR) antibody. Therefore, the National Comprehensive
Cancer Network (NCCN) recommends analysis of BRAF V600E mutation status on
tumors negative for KRAS mutations prior to initiating anti-EGFR therapy. BRAF V600E
mutation can be detected in approximately 8-12% of CRC patients.
The presence of the BRAF V600E mutation in tumors can rule out Hereditary
Nonpolyposis Colon Cancer (HNPCC, also called Lynch Syndrome), by excluding
germline mutations in mismatch repair genes that cause microsatellite instability (MSI)
or loss of MLH1 protein expression in tumors. Therefore, the BRAF V600E mutation
can be used as a screening marker to quickly rule out the hereditary form of CRC.
CLINICAL BACKGROUND
EGFR, along with the signaling pathways downstream, plays a critical role in regulation of
cell growth and cell cycle progression. Over activation of EGFR, due to enhanced ligand
response or complete ligand independence, is associated with unregulated growth and
proliferation of tumor cells.
KRAS and BRAF are key downstream genes in the EGFR pathway. Certain gain-offunction mutations in these genes could lead to stimulation of the EGFR signaling pathway
independent of EGFR. Therefore, CRC in patients with KRAS or BRAF mutations (mutually
exclusive to each other) will not respond to anti-EGFR treatment (Cetuximab and
Panitumumab).
The highest incidence of BRAF mutations occurs in malignant melanoma (40% to 60%).
Oncogenic mutations in BRAF appear at a very early stage and result in a gain-of-kinasefunction in tumorigenesis.Vemurafenib is a potent inhibitor of BRAF, which has been used
in patients with late-stage melanoma, and only works in patients whose cancer has a BRAF
V600E mutation.
BRAF V600E mutation has been detected in other tumor types, such as thyroid papillary
cancer, low-grade ovarian serous carcinoma, and non-small cell lung cancer. The clinical
utility of BRAF V600E in these tumor types is not fully confirmed.
(BRAF only)
KRASRF
(KRAS testing +BRAF Reflex
if KRAS negative)
Quick Facts
• BRAF kinase inhibitors increase
survival of patients with BRAF
V600E-positive metastatic
melanoma
• Patients with the BRAF V600E
mutation should not be treated
with EGFR inhibitors
• Metastatic colorectal carcinoma
patients who have tested
negative for a KRAS mutation
should be tested for BRAF gene
mutations
• BRAF V600E detection is a
reliable, efficient, and costeffective alternative strategy for
HNPCC screening
• This assay is able to detect
all mutations in codons
600 and 601
• Order Codes: BRAFSQ
(BRAF only) & KRASRF
(KRAS testing +BRAF Reflex
if KRAS negative)
Mutations other than V600E have been detected at codon 600, and within other codons of
the BRAF gene. The clinical significance of these mutations remains unclear to date.V600
substitutions account for 91% of BRAF mutations in cancer. This sequencing-based assay will
allow the detection of all mutations at codons 600 and 601.
www.paml.com
BRAF V600E Mutation Detection_PAML_BRAF_0001
032813
BRAF V600E Mutation Detection
by Sequence Analysis
TECHNICAL NOTES
Tumors with lymph node or distant metastases can be used for KRAS
and BRAF mutation detection.
Tissue sections are reviewed by a pathologist and manually dissected to
improve tumor yield.
DNA is extracted from the enriched tumor section and amplified
by PCR. PCR products are sequenced and analyzed by capillary
electrophoresis and fluorescence detection.
V600E1 and other mutations previously reported to occur in codons
600 such as V600E2,V600K,V600G,V600D,V600R,V600M, and 601 such
as K601E may be detected by this assay, provided that the tissue sections
submitted to the laboratory have sufficient tumor burden.
The analytical sensitivity of this test is approximately 10%
BRAF mutation-positive tumor cells within a background of BRAF
mutation-negative cells following tumor enrichment.
TEST INFORMATION
BRAF V600E MUTATION DETECTION
DESCRIPTION BRAF V600E Mutation Detection by Sequencing Analysis (Exon15, Codons 600 & 601)
METHOD PCR and Sequence Analysis
ORDER CODE BRAFSQ
CPT CODE 81210
SPECIMEN Formalin-fixed paraffin-embedded tissue block or 6 unstained 7-micron slides with an additional H&E stained slide containing at least
REQUIREMENTS 50% tumor cells.
COMMENTS Unacceptable Conditions: No tumor in tissue. (Specimens that contain less than 20% tumor will be tested and reported with a disclaimer.)
Specimens fixed/processed in alternative fixatives (alcohol, Prefer®). Frozen specimens.
Stability: Room Temp: Indefinitely; Refrigerated: Indefinitely; Frozen: Unacceptable.
RANGES Positive, negative and indeterminate for BRAF mutation.
REFLEX TESTING Test Code: KRASRF
KRAS WITH BRAF
IF KRAS IS NEGATIVE Condition: KRAS Negative
KRAS ASSAY CPT CODE: 81275. If reflex from KRAS is needed, add CPT code: 81210.
NOTES Panel of Mutations:V600E1,V600E2,V600K,V600G,V600D,V600R,V600M, K601E.
SELECTED REFERENCES
1.
2.
3.
4.
Garnett MJ, Marais R. Cancer Cell (2004). Guilty as charged: B-RAF is a human oncogene.6(4):313-9
www.nccn.org/professionals/physician_gls/f_guidelines.asp
Domingo E, et al. (2004). BRAF screening as a low-cost effective strategy for simplifying HNPCC genetic testing. J Med Genet;41(9):664-8
Chapman PB. et. al. (2011) Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med.;364(26):2507-16
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