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BRAF V600E mutation

BRAFV600E mutation analysis

  • Clinical Implications
    • CNS Tumors
      • BRAF mutation is reported in 33% of midline tumors, and has the highest incidence (66%) in pleomorphic xanthoastrocytomas.
      • BRAF V600E mutation in pediatric Low Grade Gliomas decreases 10-year progression-free survival rate almost 40%, however, the use of BRAF inhibitors have shown a successful response.
    • Melanoma
      • BRAF mutation is the most common oncogenic driver in melanoma (~ 60%)
      • Many BRAF fusions contribute to different responses to MAPK inhibition
      • BRAF inhibitors
        • Small molecule that binds mutated BRAF and renders protein inactivation
        • FDA-approved inhibitors: Vemurafenib, dabrafenib, trametinib
    • Colorectal Carcinoma
      • BRAF mutations 6-15%
      • Sporadic CRC with microsatellite instability high (MSI-H) vs. hereditary nonpolyposis colorectal carcinoma (HNPCC)
        • Majority of sporadic CRCs with MSI-H have BRAF mutation
        • HNPCC almost never has BRAF mutation
        • Testing BRAF V600E status can avoid expense of full gene sequencing for mismatch repair genes
      • Resistance to EGFR-targeted therapy by cetuximab and panitumumab is common in CRC with BRAF mutations
      • BRAF mutation in non-MSI-H CRC is unfavorable prognostic marker
    • Papillary Thyroid Carcinoma
    • BRAF V600E mutation is most common genetic alteration

      • ~ 69% have V600E mutation
      • V600E occurs mainly in classic variant & rare in other variants
      • V600E is virtually absent in follicular, Hurthle cell, medullary thyroid carcinomas, and in benign thyroid tumors
      • Recurrence, lymph node metastases, extrathyroidal extension, and advanced stage is common in tumors with V600E
    • Non-Small Cell Lung Cancer
    • BRAF mutations are mutually exclusive with KRAS and EGFR mutations

    • Langerhans Cell Histiocytosis
      • BRAF V600E is found in ~ 60% of patients
      • Increased risk of initial treatment failure in patients with this mutation
      • Associated with 2x increased risk for recurrence or relapse

  • Test Description
  • Real-time PCR for quantitative detection of BRAF v600E mutation

  • Test approach
  • DNA was isolated and BRAF V600E mutation specific Competitive allele specific TaqMan assay along with its reference wildtype assay were performed. Results interpreted according to the manufacture user guide.

  • Reporting name
  • BRAF mutation

  • Test prerequisites (To ensure timely results)
    • Patient’s demographic data.
    • Clinicopathologic information
      • Pathology report (final or preliminary) including anatomic location.
      • History of any given therapy for cancer and its date and relation to sample sent for molecular study (i.e. pre & post therapy). Therapy includes chemo and radiotherapy, hormonal or targeted therapy.
      • Any other relevant clinical data or history.
    • Type of sample:
      • Preferred: Formalin-fixed, paraffin-embedded (FFPE) tumor tissue block.
      • Acceptable:
        • Section in Eppendorf: Up to 4 sections, each with a thickness of up to 10 μm and a surface area of up to 250 mm2 + good H&E slide for assessment.
        • Five unstained slides + one good H&E slide.
      • Specimen Minimum Volume: Two 10-micron sections of FFPE.

  • Quality Control measures
  • All samples are subject to stringent quality control measures that include:

    • From your side:
      • Double check you are fulfilling all required data before sending your sample.
      • Check that your pathologist has selected the best block in terms of tumor cellularity, with least presence of necrosis and inflammation.
      • Pretherapy sample is preferred (if underwent any cancer therapy).
    • In our lab:
      • Assessment of tissue for adequacy & tumor cellularity before any molecular analysis.
      • Matching block ID with the report ID and demographic data.
      • Matching the submitted block with the data reported in the pathology report.
    • N.B
      • If the sample sent in Eppendorf, it is your pathology lab’s responsibility to ensure the sample in Eppendorf is corresponding to the submitted H&E slide (we can’t prepare slide from Eppendorf).
      • This test does not include a pathology consultation.

  • Test Time
  • From 3 days to 5 working days.

  • Retention of the sample
  • Client provided paraffin blocks & unstained slides (if provided) will be returned after testing is complete.

  • Selected References
    1. Turner JA et al: BRAF fusions identified in melanomas have variable treatment responses and phenotypes. Oncogene. ePub, 2018
    2. Ross JS et al: The distribution of BRAF gene fusions in solid tumors and response to targeted therapy. Int J Cancer. 138(4):881-90, 2016
    3. Aramini JM et al: The RAS-binding domain of human BRAF protein serine/threonine kinase exhibits allosteric conformational changes upon binding HRAS. Structure. 23(8):1382-93, 2015
    4. Holderfield M et al: Targeting RAF kinases for cancer therapy: BRAF -mutated melanoma and beyond. Nat Rev Cancer. 14(7):455-67, 2014
    5. Colomba E et al: Detection of BRAF p.V600E mutations in melanomas: comparison of four methods argues for sequential use of immunohistochemistry and pyrosequencing. J Mol Diagn. 15(1):94-100, 2013
    6. Hamilton SR: BRAF mutation and microsatellite instability status in colonic and rectal carcinoma: context really does matter. J Natl Cancer Inst. 105(15):1075-7, 2013
    7. Bollag G et al: Vemurafenib: the first drug approved for BRAF -mutant cancer. Nat Rev Drug Discov. 11(11):873-86, 2012
    8. Chapman PB et al: Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 364(26):2507-16, 2011
    9. Wan PT et al: Mechanism of activation of the RAF-ERK signaling pathway by oncogenic mutations of B-RAF. Cell. 116(6):855-67, 2004
    10. Davies H et al: Mutations of the BRAF gene in human cancer. Nature. 417(6892):949-54, 2002
    11. García-Romero N, et al, BRAF V600E Detection in Liquid Biopsies from Pediatric Central Nervous System Tumors. Cancers. 2020.