MRNA expression and BRAF mutation in circulating melanoma cells isolated from peripheral blood with High molecular weight melanoma-associated antigen-specific monoclonal antibody beads

Minoru Kitago, Kazuo Koyanagi, Takeshi Nakamura, Yasufumi Goto, Mark Faries, Steven J. O'day, Donald L. Morton, Soldano Ferrone, Dave S B Hoon

Research output: Contribution to journalArticle

51 Citations (Scopus)

Abstract

The detection of circulating tumor cells (CTCs) in the peripheral blood of melanoma patients by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis correlates with a poor prognosis. The assessment of CTCs from blood has been difficult because of lack of a good monoclonal antibody (mAb) directed against surface cell antigens to capture melanoma cells. methods: Blood was collected prospectively from 57 melanoma patients (43 test and 14 test-development cases) and 5 healthy donors. High molecular weight melanoma-associated antigen (HMW-MAA)-specific mAbs bound to immunomagnetic beads were used to isolate CTCs. mRNA and/or DNA were extracted from CTCs. Testing for the expression of a melanomaassociated gene panel (MLANA, MAGEA3, and MITF) with qRT-PCR and for the presence of BRAFmt (a BRAF gene variant encoding the V600E mutant protein) verified the beads-isolated CTCs to be melanoma cells. A peptide nucleic acid-clamping PCR assay was used for BRAFmt analysis. results: Spiking of peripheral blood cells (PBCs) with melanoma cells showed that the beads-based detection assay can detect approximately 1 melanoma cell in 5 X 10 6 PBCs. qRT-PCR analysis detected MLANA, MAGEA3, and MITF expression in 19 (44%), 29 (67%), and 19 (44%) of the patients, respectively. At least one biomarker of the panel was positive in 40 (93%) of the 43 melanoma patients. BRAFmt was detected in 17 (81%) of the 21 assessed stage IV melanoma patients. CONCLUSION: The assay of bead capture coupled with the PCR has utility for assessing CTCs in melanoma patients, which can then be characterized for both genomic and transcriptome expression.

Original languageEnglish
Pages (from-to)757-764
Number of pages8
JournalClinical Chemistry
Volume55
Issue number4
DOIs
Publication statusPublished - 2009 Apr 1
Externally publishedYes

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Circulating Neoplastic Cells
Tumors
Melanoma
Blood
Monoclonal Antibodies
Mutation
Transcription
Assays
Polymerase Chain Reaction
Reverse Transcription
Genes
Cells
Peptide Nucleic Acids
Blood Cells
Biomarkers
Mutant Proteins
Surface Antigens
HMW-MAA
Transcriptome
Constriction

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry, medical

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MRNA expression and BRAF mutation in circulating melanoma cells isolated from peripheral blood with High molecular weight melanoma-associated antigen-specific monoclonal antibody beads. / Kitago, Minoru; Koyanagi, Kazuo; Nakamura, Takeshi; Goto, Yasufumi; Faries, Mark; O'day, Steven J.; Morton, Donald L.; Ferrone, Soldano; Hoon, Dave S B.

In: Clinical Chemistry, Vol. 55, No. 4, 01.04.2009, p. 757-764.

Research output: Contribution to journalArticle

Kitago, Minoru ; Koyanagi, Kazuo ; Nakamura, Takeshi ; Goto, Yasufumi ; Faries, Mark ; O'day, Steven J. ; Morton, Donald L. ; Ferrone, Soldano ; Hoon, Dave S B. / MRNA expression and BRAF mutation in circulating melanoma cells isolated from peripheral blood with High molecular weight melanoma-associated antigen-specific monoclonal antibody beads. In: Clinical Chemistry. 2009 ; Vol. 55, No. 4. pp. 757-764.
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title = "MRNA expression and BRAF mutation in circulating melanoma cells isolated from peripheral blood with High molecular weight melanoma-associated antigen-specific monoclonal antibody beads",
abstract = "The detection of circulating tumor cells (CTCs) in the peripheral blood of melanoma patients by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis correlates with a poor prognosis. The assessment of CTCs from blood has been difficult because of lack of a good monoclonal antibody (mAb) directed against surface cell antigens to capture melanoma cells. methods: Blood was collected prospectively from 57 melanoma patients (43 test and 14 test-development cases) and 5 healthy donors. High molecular weight melanoma-associated antigen (HMW-MAA)-specific mAbs bound to immunomagnetic beads were used to isolate CTCs. mRNA and/or DNA were extracted from CTCs. Testing for the expression of a melanomaassociated gene panel (MLANA, MAGEA3, and MITF) with qRT-PCR and for the presence of BRAFmt (a BRAF gene variant encoding the V600E mutant protein) verified the beads-isolated CTCs to be melanoma cells. A peptide nucleic acid-clamping PCR assay was used for BRAFmt analysis. results: Spiking of peripheral blood cells (PBCs) with melanoma cells showed that the beads-based detection assay can detect approximately 1 melanoma cell in 5 X 10 6 PBCs. qRT-PCR analysis detected MLANA, MAGEA3, and MITF expression in 19 (44{\%}), 29 (67{\%}), and 19 (44{\%}) of the patients, respectively. At least one biomarker of the panel was positive in 40 (93{\%}) of the 43 melanoma patients. BRAFmt was detected in 17 (81{\%}) of the 21 assessed stage IV melanoma patients. CONCLUSION: The assay of bead capture coupled with the PCR has utility for assessing CTCs in melanoma patients, which can then be characterized for both genomic and transcriptome expression.",
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AU - Koyanagi, Kazuo

AU - Nakamura, Takeshi

AU - Goto, Yasufumi

AU - Faries, Mark

AU - O'day, Steven J.

AU - Morton, Donald L.

AU - Ferrone, Soldano

AU - Hoon, Dave S B

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N2 - The detection of circulating tumor cells (CTCs) in the peripheral blood of melanoma patients by quantitative real-time reverse-transcription PCR (qRT-PCR) analysis correlates with a poor prognosis. The assessment of CTCs from blood has been difficult because of lack of a good monoclonal antibody (mAb) directed against surface cell antigens to capture melanoma cells. methods: Blood was collected prospectively from 57 melanoma patients (43 test and 14 test-development cases) and 5 healthy donors. High molecular weight melanoma-associated antigen (HMW-MAA)-specific mAbs bound to immunomagnetic beads were used to isolate CTCs. mRNA and/or DNA were extracted from CTCs. Testing for the expression of a melanomaassociated gene panel (MLANA, MAGEA3, and MITF) with qRT-PCR and for the presence of BRAFmt (a BRAF gene variant encoding the V600E mutant protein) verified the beads-isolated CTCs to be melanoma cells. A peptide nucleic acid-clamping PCR assay was used for BRAFmt analysis. results: Spiking of peripheral blood cells (PBCs) with melanoma cells showed that the beads-based detection assay can detect approximately 1 melanoma cell in 5 X 10 6 PBCs. qRT-PCR analysis detected MLANA, MAGEA3, and MITF expression in 19 (44%), 29 (67%), and 19 (44%) of the patients, respectively. At least one biomarker of the panel was positive in 40 (93%) of the 43 melanoma patients. BRAFmt was detected in 17 (81%) of the 21 assessed stage IV melanoma patients. CONCLUSION: The assay of bead capture coupled with the PCR has utility for assessing CTCs in melanoma patients, which can then be characterized for both genomic and transcriptome expression.

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