nach oben

Erschienen in:

01.08.2014 | Original Research Article

Tumor Cellularity as a Quality Assurance Measure for Accurate Clinical Detection of BRAF Mutations in Melanoma

verfasst von: Jonathan C. Dudley, Grzegorz T. Gurda, Li-Hui Tseng, Derek A. Anderson, Guoli Chen, Janis M. Taube, Christopher D. Gocke, James R. Eshleman, Ming-Tseh Lin

Erschienen in: Molecular Diagnosis & Therapy | Ausgabe 4/2014

Einloggen, um Zugang zu erhalten

Abstract

Background

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.

Objective

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.

Methods

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.

Results

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.

Conclusions

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.

Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, et al. Mutations of the BRAF gene in human cancer. Nature. 2002;417(6892):949–54.PubMedCrossRef

Kong Y, Kumar SM, Xu X. Molecular pathogenesis of sporadic melanoma and melanoma-initiating cells. Arch Pathol Lab Med. 2010;134(12):1740–9.PubMedCentralPubMed

Roring M, Brummer T. Aberrant B-Raf signaling in human cancer: 10 years from bench to bedside. Crit Rev Oncog. 2012;17(1):97–121.PubMedCrossRef

Amanuel B, Grieu F, Kular J, Millward M, Iacopetta B. Incidence of BRAF p.Val600Glu and p.Val600Lys mutations in a consecutive series of 183 metastatic melanoma patients from a high incidence region. Pathology. 2012;44(4):357–9.PubMedCrossRef

Curtin JA, Fridlyand J, Kageshita T, Patel HN, Busam KJ, Kutzner H, et al. Distinct sets of genetic alterations in melanoma. N Engl J Med. 2005;353(20):2135–47.PubMedCrossRef

Long GV, Menzies AM, Nagrial AM, Haydu LE, Hamilton AL, Mann GJ, et al. Prognostic and clinicopathologic associations of oncogenic BRAF in metastatic melanoma. J Clin Oncol. 2011;29(10):1239–46.PubMedCrossRef

Ellerhorst JA, Greene VR, Ekmekcioglu S, Warneke CL, Johnson MM, Cooke CP, et al. Clinical correlates of NRAS and BRAF mutations in primary human melanoma. Clin Cancer Res. 2011;17(2):229–35.PubMedCentralPubMedCrossRef

Ugurel S, Thirumaran RK, Bloethner S, Gast A, Sucker A, Mueller-Berghaus J, et al. B-RAF and N-RAS mutations are preserved during short time in vitro propagation and differentially impact prognosis. PLoS One. 2007;2(2):e236.PubMedCentralPubMedCrossRef

Greaves WO, Verma S, Patel KP, Davies MA, Barkoh BA, Galbincea JM, et al. Frequency and spectrum of BRAF mutations in a retrospective, single-institution study of 1112 cases of melanoma. J Mol Diagn. 2013;15(2):220–6.PubMedCrossRef

10.

Menzies AM, Haydu LE, Visintin L, Carlino MS, Howle JR, Thompson JF, et al. Distinguishing clinicopathologic features of patients with V600E and V600K BRAF-mutant metastatic melanoma. Clin Cancer Res. 2012;18(12):3242–9.PubMedCrossRef

11.

Rubinstein JC, Sznol M, Pavlick AC, Ariyan S, Cheng E, Bacchiocchi A, et al. Incidence of the V600K mutation among melanoma patients with BRAF mutations, and potential therapeutic response to the specific BRAF inhibitor PLX4032. J Transl Med. 2010;8:67.PubMedCentralPubMedCrossRef

12.

Bollag G, Hirth P, Tsai J, Zhang J, Ibrahim PN, Cho H, et al. Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature. 2010;467(7315):596–9.PubMedCentralPubMedCrossRef

13.

Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med. 2011;364(26):2507–16.PubMedCentralPubMedCrossRef

14.

Klein O, Clements A, Menzies AM, O’Toole S, Kefford RF, Long GV. BRAF inhibitor activity in V600R metastatic melanoma. Eur J Cancer. 2013;49(5):1073–9.PubMedCrossRef

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.

16.

Sosman JA, Kim KB, Schuchter L, Gonzalez R, Pavlick AC, Weber JS, et al. Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib. N Engl J Med. 2012;366(8):707–14.PubMedCentralPubMedCrossRef

17.

Ascierto PA, Minor D, Ribas A, Lebbe C, O’Hagan A, Arya N, et al. Phase II trial (BREAK-2) of the BRAF inhibitor dabrafenib (GSK2118436) in patients with metastatic melanoma. J Clin Oncol. 2013;31(26):3205–11.PubMedCrossRef

18.

Falchook GS, Lewis KD, Infante JR, Gordon MS, Vogelzang NJ, DeMarini DJ, et al. Activity of the oral MEK inhibitor trametinib in patients with advanced melanoma: a phase 1 dose-escalation trial. Lancet Oncol. 2012;13(8):782–9.PubMedCrossRef

19.

Falchook GS, Long GV, Kurzrock R, Kim KB, Arkenau TH, Brown MP, et al. Dabrafenib in patients with melanoma, untreated brain metastases, and other solid tumours: a phase 1 dose-escalation trial. Lancet. 2012;379(9829):1893–901.PubMedCrossRef

20.

Flaherty KT, Robert C, Hersey P, Nathan P, Garbe C, Milhem M, et al. Improved survival with MEK inhibition in BRAF-mutated melanoma. N Engl J Med. 2012;367(2):107–14.PubMedCrossRef

21.

Hauschild A, Grob JJ, Demidov LV, Jouary T, Gutzmer R, Millward M, et al. Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial. Lancet. 2012;380(9839):358–65.PubMedCrossRef

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.PubMedCrossRef

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.PubMedCrossRef

24.

Qu K, Pan Q, Zhang X, Rodriguez L, Zhang K, Li H, et al. Detection of BRAF V600 mutations in metastatic melanoma: comparison of the Cobas 4800 and Sanger sequencing assays. J Mol Diagn. 2013;15(6):790–5.PubMedCrossRef

25.

Fadhil W, Ibrahem S, Seth R, Ilyas M. Quick-multiplex-consensus (QMC)-PCR followed by high-resolution melting: a simple and robust method for mutation detection in formalin-fixed paraffin-embedded tissue. J Clin Pathol. 2010;63(2):134–40.PubMedCrossRef

26.

Flaherty KT, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N Engl J Med. 2010;363(9):809–19.PubMedCentralPubMedCrossRef

27.

Kotoula V, Charalambous E, Biesmans B, Malousi A, Vrettou E, Fountzilas G, et al. Targeted KRAS mutation assessment on patient tumor histologic material in real time diagnostics. PLoS One. 2009;4(11):e7746.PubMedCentralPubMedCrossRef

28.

Tan YH, Liu Y, Eu KW, Ang PW, Li WQ, Salto-Tellez M, et al. Detection of BRAF V600E mutation by pyrosequencing. Pathology. 2008;40(3):295–8.PubMedCrossRef

29.

Lin MT, Tseng LH, Rich RG, Hafez MJ, Harada S, Murphy KM, et al. Delta-PCR, a simple method to detect translocations and insertion/deletion mutations. J Mol Diagn. 2011;13(1):85–92.PubMedCentralPubMedCrossRef

30.

Chen G, Olson MT, O’Neill A, Norris A, Beierl K, Harada S, et al. A virtual pyrogram generator to resolve complex pyrosequencing results. J Mol Diagn. 2012;14(2):149–59.PubMedCentralPubMedCrossRef

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.PubMedCentralPubMedCrossRef

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)

33.

Lin MT, Tseng LH, Beierl K, Hsieh A, Thiess M, Chase N, et al. Tandem duplication PCR: an ultrasensitive assay for the detection of internal tandem duplications of the FLT3 gene. Diagn Mol Pathol. 2013;22(3):149–55.PubMedCrossRef

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.PubMedCentralPubMedCrossRef

35.

Colomba E, Helias-Rodzewicz Z, Von Deimling A, Marin C, Terrones N, Pechaud D, 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. 2013;15(1):94–100.PubMedCrossRef

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.PubMedCentralPubMedCrossRef

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.PubMedCrossRef

38.

Schwartz KM, Pike-Buchanan LL, Muralidharan K, Redman JB, Wilson JA, Jarvis M, et al. Identification of cystic fibrosis variants by polymerase chain reaction/oligonucleotide ligation assay. J Mol Diagn. 2009;11(3):211–5.PubMedCentralPubMedCrossRef

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.PubMedCentralPubMedCrossRef

40.

Lade-Keller J, Romer KM, Guldberg P, Riber-Hansen R, Hansen LL, Steiniche T, et al. Evaluation of BRAF mutation testing methodologies in formalin-fixed, paraffin-embedded cutaneous melanomas. J Mol Diagn. 2013;15(1):70–80.PubMedCrossRef

41.

Magnin S, Viel E, Baraquin A, Valmary-Degano S, Kantelip B, Pretet JL, et al. A multiplex SNaPshot assay as a rapid method for detecting KRAS and BRAF mutations in advanced colorectal cancers. J Mol Diagn. 2011;13(5):485–92.PubMedCentralPubMedCrossRef

42.

McCourt CM, McArt DG, Mills K, Catherwood MA, Maxwell P, Waugh DJ, et al. Validation of next generation sequencing technologies in comparison to current diagnostic gold standards for BRAF, EGFR and KRAS mutational analysis. PLoS One. 2013;8(7):e69604.PubMedCentralPubMedCrossRef

43.

Rechsteiner M, von Teichman A, Ruschoff JH, Fankhauser N, Pestalozzi B, Schraml P, et al. KRAS, BRAF, and TP53 deep sequencing for colorectal carcinoma patient diagnostics. J Mol Diagn. 2013;15(3):299–311.PubMedCrossRef

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.PubMedCrossRef

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.PubMedCrossRef

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.PubMedCrossRef

47.

Long GV, Wilmott JS, Capper D, Preusser M, Zhang YE, Thompson JF, et al. Immunohistochemistry is highly sensitive and specific for the detection of V600E BRAF mutation in melanoma. Am J Surg Pathol. 2013;37(1):61–5.PubMedCrossRef

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.PubMedCrossRef

49.

Marin C, Beauchet A, Capper D, Zimmermann U, Julie C, Ilie M, et al. Detection of BRAF p. V600E mutations in melanoma by immunohistochemistry has a good interobserver reproducibility. Arch Pathol Lab Med. 2014;138(1):71–5.PubMedCrossRef

50.

Hoorens A, Jouret-Mourin A, Sempoux C, Demetter P, De Hertogh G, Teugels E. Accurate KRAS mutation testing for EGFR-targeted therapy in colorectal cancer: emphasis on the key role and responsibility of pathologists. Acta Gastroenterol Belg. 2010;73(4):497–503.PubMed

51.

van Krieken JH, Jung A, Kirchner T, Carneiro F, Seruca R, Bosman FT, et al. KRAS mutation testing for predicting response to anti-EGFR therapy for colorectal carcinoma: proposal for an European quality assurance program. Virchows Arch. 2008;453(5):417–31.PubMedCrossRef

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.PubMedCentralPubMedCrossRef

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.

54.

Viray H, Li K, Long TA, Vasalos P, Bridge JA, Jennings LJ, et al. A prospective, multi-institutional diagnostic trial to determine pathologist accuracy in estimation of percentage of malignant cells. Arch Pathol Lab Med. 2013;137(11):1545–9.PubMedCrossRef

55.

Boissiere-Michot F, Lopez-Crapez E, Frugier H, Berthe ML, Ho-Pun-Cheung A, Assenat E, et al. KRAS genotyping in rectal adenocarcinoma specimens with low tumor cellularity after neoadjuvant treatment. Mod Pathol. 2012;25(5):731–9.PubMedCrossRef

56.

Ondrejka SL, Schaeffer DF, Jakubowski MA, Owen DA, Bronner MP. Does neoadjuvant therapy alter KRAS and/or MSI results in rectal adenocarcinoma testing? Am J Surg Pathol. 2011;35(9):1327–30.PubMedCrossRef

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).

Titel: Tumor Cellularity as a Quality Assurance Measure for Accurate Clinical Detection of BRAF Mutations in Melanoma
verfasst von: Jonathan C. Dudley
Grzegorz T. Gurda
Li-Hui Tseng
Derek A. Anderson
Guoli Chen
Janis M. Taube
Christopher D. Gocke
James R. Eshleman
Ming-Tseh Lin
Publikationsdatum: 01.08.2014
Verlag: Springer International Publishing
Erschienen in: Molecular Diagnosis & Therapy / Ausgabe 4/2014
Print ISSN: 1177-1062
Elektronische ISSN: 1179-2000
DOI: https://doi.org/10.1007/s40291-014-0091-6

Leitlinien kompakt für die Innere Medizin

Mit medbee Pocketcards sicher entscheiden.

^{Seit 2022 gehört die medbee GmbH zum Springer Medizin Verlag}

Kostenlos registrieren

Neu im Fachgebiet Innere Medizin

Alphablocker schützt vor Miktionsproblemen nach der Biopsie

16.05.2024 alpha-1-Rezeptorantagonisten Nachrichten

Nach einer Prostatabiopsie treten häufig Probleme beim Wasserlassen auf. Ob sich das durch den periinterventionellen Einsatz von Alphablockern verhindern lässt, haben australische Mediziner im Zuge einer Metaanalyse untersucht.

Eingreifen von Umstehenden rettet vor Erstickungstod!

15.05.2024 Fremdkörperaspiration Nachrichten

Wer sich an einem Essensrest verschluckt und um Luft ringt, benötigt vor allem rasche Hilfe. Dass Umstehende nur in jedem zweiten Erstickungsnotfall bereit waren, diese zu leisten, ist das ernüchternde Ergebnis einer Beobachtungsstudie aus Japan. Doch es gibt auch eine gute Nachricht.

Neue S3-Leitlinie zur unkomplizierten Zystitis: Auf Antibiotika verzichten?

15.05.2024 Harnwegsinfektionen Nachrichten

Welche Antibiotika darf man bei unkomplizierter Zystitis verwenden und wovon sollte man die Finger lassen? Welche pflanzlichen Präparate können helfen? Was taugt der zugelassene Impfstoff? Antworten vom Koordinator der frisch überarbeiteten S3-Leitlinie, Prof. Florian Wagenlehner.

Schadet Ärger den Gefäßen?

14.05.2024 Arteriosklerose Nachrichten

In einer Studie aus New York wirkte sich Ärger kurzfristig deutlich negativ auf die Endothelfunktion gesunder Probanden aus. Möglicherweise hat dies Einfluss auf die kardiovaskuläre Gesundheit.

Update Innere Medizin

Bestellen Sie unseren Fach-Newsletter und bleiben Sie gut informiert.

Newsletter bestellen

Springer Medizin

Abstract

Background

Objective

Methods

Results

Conclusions

Bitte loggen Sie sich ein, um Zugang zu diesem Inhalt zu erhalten

Weitere Artikel der Ausgabe 4/2014

The Economic Considerations and Implications of the Stratification of Future Oncology Therapeutics

PAX2 Polymorphisms and Congenital Abnormalities of the Kidney and Urinary Tract in a Brazilian Pediatric Population: Evidence for a Role in Vesicoureteral Reflux

Interleukin-18 −607C/A Gene Polymorphism in Egyptian Asthmatic Children

Circulating Tumor Cells in Prostate Cancer Diagnosis and Monitoring: An Appraisal of Clinical Potential

Highly Sensitive KRAS Mutation Detection from Formalin-Fixed Paraffin-Embedded Biopsies and Circulating Tumour Cells Using Wild-Type Blocking Polymerase Chain Reaction and Sanger Sequencing