Tumor Cellularity as a Quality Assurance Measure for Accurate Clinical Detection of BRAF Mutations in Melanoma
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Detection of BRAF mutations is an established standard of care to predict small-molecule inhibitor (vemurafenib) response in metastatic melanoma. Molecular assays should be designed to detect not only the most common p.V600E mutation, but also p.V600K and other non-p.V600E mutations.
The purpose of this study was to assess if tumor cellularity can function as a quality assurance (QA) measure in molecular diagnostics. Potential causes of discrepancy between the observed and predicted mutant allele percentage were also explored.
We correlated pathologist-generated estimates of tumor cellularity versus mutant allele percentage via pyrosequencing as a QA measure for BRAF mutation detection in formalin-fixed, paraffin-embedded melanoma specimens.
BRAF mutations were seen in 27/62 (44 %) specimens, with 93 % p.V600E and 7 % non-p.V600E. Correlation between p.V600E mutant percentage and tumor cellularity was poor–moderate (r = −0.02; p = 0.8), primarily because six samples showed a low p.V600E signal despite high tumor cellularity. A QA investigation revealed that our initial pyrosequencing assay showed a false positive, weak p.V600E signal in specimens with a p.V600K mutation. A redesigned assay detected BRAF mutations in 50/131 (38 %) specimens, including 30 % non-p.V600E. This revised assay showed strong correlation between p.V600E BRAF mutant percentage and tumor cellularity (r = 0.76; p ≤ 0.01). Re-evaluation of the previously discordant samples by the revised assay confirmed a high level of p.V600K mutation in five specimens.
Pathologists play important roles in molecular diagnostics, beyond identification of correct cells for testing. Accurate evaluation of tumor cellularity not only ensures sufficient material for required analytic sensitivity, but also provides an independent QA measure of the molecular assays.
KeywordsMutant Allele BRAF Mutation Vemurafenib Tumor Cellularity BRAF Gene
Acknowledgement and Disclosure
The authors have no conflicts of interest pertaining to this work. This work will be presented, in part, at the 103rd Annual Meeting of the United States and Canadian Academy of Pathology (USCAP) in San Diego, CA, USA, in March 2014. We would like to thank Molly Van Appledorn for statistical analysis consultation, and Norman J. Barker for assistance in generating figures.
- 15.McArthur G, Hauschild A, Robert C, Larkin J, Haanen JB, Ribas A, et al. Efficacy of vemurafenib in BRAFV600K mutation-positive melanoma disease: results from phase 3 clinical study BRIM3. Pigment Cell Melanoma Res. 2012;25(6):871.Google Scholar
- 22.Anderson S, Bloom KJ, Vallera DU, Rueschoff J, Meldrum C, Schilling R, et al. Multisite analytic performance studies of a real-time polymerase chain reaction assay for the detection of BRAF V600E mutations in formalin-fixed, paraffin-embedded tissue specimens of malignant melanoma. Arch Pathol Lab Med. 2012;136(11):1385–91.PubMedCrossRefGoogle Scholar
- 23.Halait H, Demartin K, Shah S, Soviero S, Langland R, Cheng S, et al. Analytical performance of a real-time PCR-based assay for V600 mutations in the BRAF gene, used as the companion diagnostic test for the novel BRAF inhibitor vemurafenib in metastatic melanoma. Diagn Mol Pathol. 2012;21(1):1–8.PubMedCrossRefGoogle Scholar
- 31.Tsiatis AC, Norris-Kirby A, Rich RG, Hafez MJ, Gocke CD, Eshleman JR, et al. Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. J Mol Diagn. 2010;12(4):425–32.PubMedCentralPubMedCrossRefGoogle Scholar
- 32.Olson MT, Harrington C, Beierl K, Chen G, Thiess M, O’Neill A, Taube J, Zeiger MA, Lin MT, Eshleman JR. BRAF pyrosequencing analysis aided by a lookup table. Am J Clin Pathol. 2014 (in press)Google Scholar
- 34.Harada S, Henderson LB, Eshleman JR, Gocke CD, Burger P, Griffin CA, et al. Genomic changes in gliomas detected using single nucleotide polymorphism array in formalin-fixed, paraffin-embedded tissue: superior results compared with microsatellite analysis. J Mol Diagn. 2011;13(5):541–8.PubMedCentralPubMedCrossRefGoogle Scholar
- 36.Ihle MA, Fassunke J, Konig K, Grunewald I, Schlaak M, Kreuzberg N, et al. Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p. V600E and non-p.V600E BRAF mutations. BMC Cancer. 2014;14(1):13.PubMedCentralPubMedCrossRefGoogle Scholar
- 37.Mullins FM, Dietz L, Lay M, Zehnder JL, Ford J, Chun N, et al. Identification of an intronic single nucleotide polymorphism leading to allele dropout during validation of a CDH1 sequencing assay: implications for designing polymerase chain reaction-based assays. Genet Med. 2007;9(11):752–60.PubMedCrossRefGoogle Scholar
- 39.Ward KJ, Ellard S, Yajnik CS, Frayling TM, Hattersley AT, Venigalla PN, et al. Allelic drop-out may occur with a primer binding site polymorphism for the commonly used RFLP assay for the -1131T>C polymorphism of the Apolipoprotein AV gene. Lipids Health Dis. 2006;5:11.PubMedCentralPubMedCrossRefGoogle Scholar
- 44.Szankasi P, Reading NS, Vaughn CP, Prchal JT, Bahler DW, Kelley TW. A quantitative allele-specific PCR test for the BRAF V600E mutation using a single heterozygous control plasmid for quantitation: a model for qPCR testing without standard curves. J Mol Diagn. 2013;15(2):248–54.PubMedCrossRefGoogle Scholar
- 45.Tuononen K, Maki-Nevala S, Sarhadi VK, Wirtanen A, Ronty M, Salmenkivi K, et al. Comparison of targeted next-generation sequencing (NGS) and real-time PCR in the detection of EGFR, KRAS, and BRAF mutations on formalin-fixed, paraffin-embedded tumor material of non-small cell lung carcinoma-superiority of NGS. Genes Chromosomes Cancer. 2013;52(5):503–11.PubMedCrossRefGoogle Scholar
- 46.Busam KJ, Hedvat C, Pulitzer M, von Deimling A, Jungbluth AA. Immunohistochemical analysis of BRAF(V600E) expression of primary and metastatic melanoma and comparison with mutation status and melanocyte differentiation antigens of metastatic lesions. Am J Surg Pathol. 2013;37(3):413–20.PubMedCrossRefGoogle Scholar
- 48.Routhier CA, Mochel MC, Lynch K, Dias-Santagata D, Louis DN, Hoang MP. Comparison of 2 monoclonal antibodies for immunohistochemical detection of BRAF V600E mutation in malignant melanoma, pulmonary carcinoma, gastrointestinal carcinoma, thyroid carcinoma, and gliomas. Hum Pathol. 2013;44(11):2563–70.PubMedCrossRefGoogle Scholar
- 52.Bellon E, Ligtenberg MJ, Tejpar S, Cox K, de Hertogh G, de Stricker K, et al. External quality assessment for KRAS testing is needed: setup of a European program and report of the first joined regional quality assessment rounds. Oncologist. 2011;16(4):467–78.PubMedCentralPubMedCrossRefGoogle Scholar
- 53.Smits AJ, Kummer JA, de Bruin PC, Bol M, van den Tweel JG, Seldenrijk KA, et al. The estimation of tumor cell percentage for molecular testing by pathologists is not accurate. Mod Pathol. 2014;27(2):168–74.Google Scholar
- 57.Viray H, Coulter M, Li K, Lane K, Madan A, Mitchell K, et al. Automated objective determination of percentage of malignant nuclei for mutation testing. Appl Immunohistochem Mol Morphol (Epub 24 Oct 2013).Google Scholar