Detection of BRAF V600 Mutations in Metastatic Melanoma

Qu, Kevin; Pan, Qiulu; Zhang, Xi; Rodríguez, L. Raposo; Zhang, Ke; Li, Hairong; Ho, Albert K.; Sanders, Heather; Sferruzza, Anthony; Cheng, Shih Min; Nguyen, Diedre; Jones, Dan; Waldman, Frederic M.

doi:10.1016/j.jmoldx.2013.07.003

Cited by 49 publications

(17 citation statements)

References 21 publications

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“…Details about testing protocols are described elsewhere. [13–15] In selected cases mutational status was analyzed using a standardized real-time PCR (COBAS ® 4800) system provided by Roche. [15] The BRAF mutation was determined on the most recent available tumor tissue.…”

Section: Methodsmentioning

confidence: 99%

Which melanoma patient carries a BRAF-mutation? A comparison of predictive models

Eigentler¹,

Assi²,

Hassel

et al. 2016

Oncotarget

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BackgroundIn patients with advanced melanoma the detection of BRAF mutations is considered mandatory before the initiation of an expensive treatment with BRAF/MEK inhibitors. Sometimes it is difficult to perform such an analysis if archival tumor tissue is not available and fresh tissue has to be collected.Results514 of 1170 patients (44%) carried a BRAF mutation. All models revealed age and histological subtype of melanoma as the two major predictive variables. Accuracy ranged from 0.65–0.71, being best in the random forest model. Sensitivity ranged 0.76–0.84, again best in the random forest model. Specificity was low in all models ranging 0.51–0.55.MethodsWe collected the clinical data and mutational status of 1170 patients with advanced melanoma and established three different predictive models (binary logistic regression, classification and regression trees, and random forest) to forecast the BRAF status.ConclusionsUp to date statistical models are not able to predict BRAF mutations in an acceptable accuracy. The analysis of the mutational status by sequencing or immunohistochemistry must still be considered as standard of care.

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Section: Methodsmentioning

confidence: 99%

Which melanoma patient carries a BRAF-mutation? A comparison of predictive models

Eigentler¹,

Assi²,

Hassel

et al. 2016

Oncotarget

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“…In this study, pathologist-generated estimates of tumor cellularity served as a QA measure for a clinical BRAF pyrosequencing assay. Detection of a lower-than-expected p.V600E signal in specimens with a high tumor cellularity prompted an investigation, which revealed that our original pyrosequencing assay, similar to the Cobas 4800 BRAF V600 mutation test [22–24, 35, 36], may show a weak p.V600E signal in p.V600K positive specimens. Our revised pyrosequencing assay could distinguish p.V600E mutation from other non-p.V600E mutations and confirmed a higher incidence of p.V600K mutation than previously reported in melanomas [4, 6, 9–11].…”

Section: Discussionmentioning

confidence: 99%

“…However, the probes have been shown to also cross-hybridize with the p.V600K allele and other non-p.V600E mutant alleles such as p.V600D [22–24, 35, 36]. In addition, Cobas 4800 and other PCR-based assays detect non-p.V600E mutations with a lower analytic sensitivity.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, Cobas 4800 and other PCR-based assays detect non-p.V600E mutations with a lower analytic sensitivity. This leads to a low false-positive p.V600E signal in non-p.V600E positive specimens with high tumor cellularity, or possibly a false-negative result when the tumor cellularity is low [22–24, 35]. The 3′ nucleotide of the sequencing primer of our initial pyrosequencing assay ended at the first nucleotide of codon 600.…”

Section: Discussionmentioning

confidence: 99%

“…The Cobas 4800 assay was designed to detect the p.V600E mutation, however it also cross-reacts with non-p.V600E mutations, including 70 % of p.V600K mutations [22, 23]. The analytic sensitivity (or limit of detection) of Cobas 4800 for the p.V600E mutation may be different for non-p.V600E mutations; some patients with non-p.V600E mutations who might otherwise benefit from vemurafenib therapy could therefore be missed [11, 13, 16, 24]. A variety of other assays to detect BRAF mutations have been developed [22, 23, 25–28], including a clinical pyrosequencing assay used in our laboratory.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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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.

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BRAF-V600E-Testung beim metastasierten kolorektalen Karzinom und neue, chemotherapiefreie Therapieoptionen

et al. 2021

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ZusammenfassungDie Therapie des metastasierten kolorektalen Karzinoms (mKRK) hat in den letzten 25 Jahren tief greifende Veränderungen erfahren. Auf die Zulassung neuerer Chemotherapeutika folgten ab 2005 die ersten zielgerichteten Therapien, die sich gegen den epidermalen Wachstumsfaktorrezeptor (EGFR) bzw. gegen Rezeptoren vaskulärer endothelialer Wachstumsfaktoren (VEGFR) richteten. Mit der fortschreitenden molekularen Charakterisierung des mKRK in den letzten 10 Jahren und der Einteilung der Erkrankung in 4 Konsensus-Subtypen zeichnet sich weiterer Wandel ab, unter anderem durch Einführung speziell entwickelter Proteinkinaseinhibitoren wie auch Immuncheckpoint-Inhibitoren in den Therapiealgorithmus.Eine angepasste molekularpathologische Testung ist heute für eine leitliniengerechte Behandlung von mKRK-Patienten unabdingbar. Neben der RAS-Testung als Voraussetzung für die Therapieentscheidung bezüglich Cetuximab und Panitumumab ist die BRAF-Testung äußerst relevant, um – im Falle des Nachweises einer BRAF-V600E-Mutation – eine Therapieentscheidung zugunsten der neu zugelassenen, chemotherapiefreien Kombination aus dem BRAF-Inhibitor Encorafenib und Cetuximab treffen zu können. Eine erweiterte Diagnostik sollte auch die Genominstabilität (Mikrosatelliten-Instabilität) einbeziehen. Insgesamt müssen immer mehr molekulare Alterationen simultan untersucht werden, sodass sich zunehmend die Verwendung des fokussierten Next Generation Sequencing empfiehlt.Diese Übersichtsarbeit beschreibt die prognostische Relevanz der BRAF-Testung im Rahmen der molekularpathologischen Diagnostik des mKRK, stellt neue Therapieoptionen zur Behandlung BRAF-mutierter mKRK-Patienten vor und erläutert, welche modernen DNA-analytischen und immunohistochemischen Verfahren zur BRAF-Diagnostik von mKRK-Patienten zur Verfügung stehen.

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Detection of BRAF V600 Mutations in Metastatic Melanoma

Cited by 49 publications

References 21 publications

Which melanoma patient carries a BRAF-mutation? A comparison of predictive models

Which melanoma patient carries a BRAF-mutation? A comparison of predictive models

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

BRAF-V600E-Testung beim metastasierten kolorektalen Karzinom und neue, chemotherapiefreie Therapieoptionen

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