Tumor cellularity as a quality assurance measure for accurate clinical detection of braf mutations in melanoma

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

Research output: Contribution to journalArticle

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 themolecular assays.

Original languageEnglish (US)
Pages (from-to)409-418
Number of pages10
JournalMolecular Diagnosis and Therapy
Volume18
Issue number4
DOIs
StatePublished - 2014

Fingerprint

Melanoma
Mutation
Neoplasms
Molecular Pathology
Alleles
Standard of Care
Paraffin
Formaldehyde

ASJC Scopus subject areas

  • Genetics
  • Molecular Medicine
  • Medicine(all)
  • Pharmacology

Cite this

Tumor cellularity as a quality assurance measure for accurate clinical detection of braf mutations in melanoma. / Dudley, Jonathan C.; Gurda, Grzegorz T.; Tseng, Li Hui; Anderson, Derek A.; Chen, Guoli; Taube, Janis M; Gocke, Christopher; Eshleman, James; Lin, Ming-Tseh.

In: Molecular Diagnosis and Therapy, Vol. 18, No. 4, 2014, p. 409-418.

Research output: Contribution to journalArticle

@article{63e1fd2927c747b8a25e48f613923d0c,
title = "Tumor cellularity as a quality assurance measure for accurate clinical detection of braf mutations in melanoma",
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 themolecular assays.",
author = "Dudley, {Jonathan C.} and Gurda, {Grzegorz T.} and Tseng, {Li Hui} and Anderson, {Derek A.} and Guoli Chen and Taube, {Janis M} and Christopher Gocke and James Eshleman and Ming-Tseh Lin",
year = "2014",
doi = "10.1007/s40291-014-0091-6",
language = "English (US)",
volume = "18",
pages = "409--418",
journal = "Molecular Diagnosis and Therapy",
issn = "1177-1062",
publisher = "Adis International Ltd",
number = "4",

}

TY - JOUR

T1 - Tumor cellularity as a quality assurance measure for accurate clinical detection of braf mutations in melanoma

AU - Dudley, Jonathan C.

AU - Gurda, Grzegorz T.

AU - Tseng, Li Hui

AU - Anderson, Derek A.

AU - Chen, Guoli

AU - Taube, Janis M

AU - Gocke, Christopher

AU - Eshleman, James

AU - Lin, Ming-Tseh

PY - 2014

Y1 - 2014

N2 - 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 themolecular assays.

AB - 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 themolecular assays.

UR - http://www.scopus.com/inward/record.url?scp=84905686054&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84905686054&partnerID=8YFLogxK

U2 - 10.1007/s40291-014-0091-6

DO - 10.1007/s40291-014-0091-6

M3 - Article

C2 - 24604154

AN - SCOPUS:84905686054

VL - 18

SP - 409

EP - 418

JO - Molecular Diagnosis and Therapy

JF - Molecular Diagnosis and Therapy

SN - 1177-1062

IS - 4

ER -