Solid Tumors BRAF Mutation Analysis

Melanoma - Mutations in the BRAF gene are the most common oncogenic alterations in malignant melanoma, present in approximately 45% of tumors arising from cutaneous sites and also occurring in melanomas at other locations. The V600E mutation is the most common BRAF alteration, and this mutation is the target for BRAF kinase inhibitors such as vemurafenib.^1,2
Colorectal adenocarcinoma - BRAF gene mutations are present in approximately 12% of cases and correlate with resistance to treatment with cetuximab.³ In addition, BRAF mutations are a significant negative prognostic factor in patients with stage II and III colorectal carcinoma.^4-7

1. Microscopic examination of the specimen and macrodissection of tumor-rich areas.
2. DNA extraction and purification.
3. PCR amplification of BRAF exon 15.
4. Screening for mutations by one of two methods, depending on tumor cellularity.
   1. Real-time PCR with high resolution melting curve analysis (HRM). DNA sequencing is used to confirm any potential mutations identified by this approach.
   2. Bidirectional Sanger sequencing using an LNA-modified oligonucleotide to suppress wild-type allelic amplification (LNA-sequencing).
5. Estimated sensitivity:
6. 20% mutant allele for HRM.
7. 1% mutant allele for LNA-sequencing (used for samples with low tumor cellularity)
8. Estimated specificity: 99.5% of BRAF mutations reported in melanoma and colorectal carcinoma.

Paraffin blocks or unstained slides:

• A paraffin block or
• 10 unstained sections of tumor (4-5 microns)(15 sections for small biopsies). 

• Due to DNA damage, decalcified specimens are not recommended.

Blood or bone marrow:

• 2-10 mL Yellow (ACD) or purple (EDTA) tube (unspun)
• Bone marrow aspirates or cores are acceptable if received in the lab within 24 hours (room temperature) or 4 days (refrigerated; never frozen).

• Frozen cell pellets of bone marrow-derived leukocytes (without red blood cells) are also acceptable.

Deliver to lab at shipping address above within 24 hours of collection;  if sample cannot arrive within 24 hours, refrigerate until sample can be transported. 

A REQUISITION FORM MUST ACCOMPANY ALL SAMPLES.  Please include detailed clinical information.

Mon - Fri

7-10 days

• Package and ship specimen to remain cool during transit, but not frozen, unless shipping frozen pellets.
• Please use the cold pack provided in the KDL shipping kit.
• Ship the specimen via overnight express, using the FedEx priority overnight label provided. 
• Contact Client Services for shipping materials and procedures at (855) 535-1522.

1. K. Flaherty, I. Puzanov, J. Sosman, K. Kim, A. Ribas, G. McArthur, J.A. Sosman, P.J. O’Dwyer, R.J. Lee, J. F. Grippo, K. Nolop, P.B. Chapman. Inhibition of Mutated, Activated BRAF in Metastatic Melanoma. N Engl J Med 2010;363:809-19.
2. Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, Dummer R, Garbe C, Testori A, Maio M, Hogg D, Lorigan P, Lebbe C, Jouary T, Schadendorf D, Ribas A, O'Day SJ, Sosman JA, Kirkwood JM, Eggermont AM, Dreno B, Nolop K, Li J, Nelson B, Hou J, Lee RJ, Flaherty KT, McArthur AG; BRIM-3 Study Group. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 364(26):2507-16, 2011
3. Loupakis F, Ruzzo A, Cremolini C, Vincenzi B, Salvatore L, Santini D, Masi G, Stasi I, Canestrari E, Rulli E, Floriani I, Bencardino K, Galluccio N, Catalano V, Tonini G, Magnani M, Fontanini G, Basolo F, Falcone A, Graziano F. KRAS codon 61, 146 and BRAF mutations predict resistance to cetuximab plus irinotecan in KRAS codon 12 and 13 wild-type metastatic colorectal cancer. Br J Cancer. 2009 Aug 18;101(4):715-21.
4. Price TJ, Hardingham JE, Lee CK, Weickhardt A, Townsend AR, Wrin JW, Chua A, Shivasami A, Cummins MM, Murone C, Tebbutt NC. Impact of KRAS and BRAF Gene Mutation Status on Outcomes From the Phase III AGITG MAX Trial of Capecitabine Alone or in Combination With Bevacizumab and Mitomycin in Advanced Colorectal Cancer. J Clin Oncol. 2011 Jul 1;29(19):2675-82.
5. Yokota T, Ura T, Shibata N, Takahari D, Shitara K, Nomura M, Kondo C, Mizota A, Utsunomiya S, Muro K, Yatabe Y. BRAF mutation is a powerful prognostic factor in advanced and recurrent colorectal cancer. Br J Cancer. 2011 Mar 1;104(5):856-62.
6. Fariña-Sarasqueta A, van Lijnschoten G, Moerland E, Creemers GJ, Lemmens VE, Rutten HJ, van den Brule AJ. The BRAF V600E mutation is an independent prognostic factor for survival in stage II and stage III colon cancer patients. Ann Oncol. 2010 Dec;21(12):2396-402.
7. Roth AD, Tejpar S, Delorenzi M, Yan P, Fiocca R, Klingbiel D, Dietrich D, Biesmans B, Bodoky G, Barone C, Aranda E, Nordlinger B, Cisar L, Labianca R, Cunningham D, Van Cutsem E, Bosman F. Prognostic role of KRAS and BRAF in stage II and III resected colon cancer: results of the translational study on the PETACC-3, EORTC 40993, SAKK 60-00 trial. J Clin Oncol. 2010 Jan 20;28(3):466-74.