Development of a real-time RT-PCR assay for detecting EGFRvIII in glioblastoma samples

Koji Yoshimoto, Julie Dang, Shaojun Zhu, David Nathanson, Tiffany Huang, Rebecca Dumont, David B. Seligson, William H. Yong, Zhenggang Xiong, Nagesh Rao, Henrik Winther, Arnab Chakravarti, Darell D. Bigner, Ingo K. Mellinghoff, Steve Horvath, Webster K. Cavenee, Timothy F. Cloughesy, Paul S. Mischel

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Abstract

Purpose: Epidermal growth factor receptor variant III (EGFRvIII) is an oncogenic, constitutively active mutant form of the EGFR that is commonly expressed in glioblastoma and is also detected in a number of epithelial cancers. EGFRvIII presents a unique antigenic target for anti-EGFRvIII vaccines and it has been shown to modulate response to EGFR kinase inhibitor therapy. Thus, detection in clinical samples may bewarranted. Existing patents preclude the use of anti-EGFRvIII antibodies for clinical detection. Further, frozen tissue is not routinely available, particularly for patients treated in the community. Thus, detection of EGFRvIII in formalin-fixed paraffin-embedded (FFPE) clinical samples is a major challenge. Experimental Design: We developed a real-time reverse transcription-PCR (RT-PCR) assay for detecting EGFRvIII in FFPE samples and analyzed 59 FFPE glioblastoma clinical samples with paired frozen tissue from the same surgical resection. We assessed EGFRvIII protein expression by immunohistochemistry using two distinct specific anti-EGFRvIII antibodies and examined EGFR gene amplification by fluorescence in situ hybridization. Results: The FFPE RT-PCR assay detected EGFRvIII in 16 of 59 (27%) samples, exclusively in cases with EGFR amplification, consistent with the expected frequency of this alteration. The FFPE RT-PCR assay was more sensitive and specific for detecting EGFRvIII than either of the two antibodies alone, or in combination, with a sensitivity of 93% (95% confidence interval, 0.78-1.00) and a specificity of 98% (95% confidence interval, 0.93-1.00). Conclusion: This assay will facilitate accurate assessment of EGFRvIII in clinical samples and may aid in the development of strategies for stratifying patients for EGFRvIII-directed therapies.

Original languageEnglish
Pages (from-to)488-493
Number of pages6
JournalClinical Cancer Research
Volume14
Issue number2
DOIs
Publication statusPublished - Jan 15 2008

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Glioblastoma
Epidermal Growth Factor Receptor
Reverse Transcription
Polymerase Chain Reaction
Paraffin
Formaldehyde
Antibodies
Confidence Intervals
erbB-1 Genes
Patents
Gene Amplification
Fluorescence In Situ Hybridization
Research Design
Phosphotransferases
Vaccines
Immunohistochemistry

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research

Cite this

Yoshimoto, K., Dang, J., Zhu, S., Nathanson, D., Huang, T., Dumont, R., ... Mischel, P. S. (2008). Development of a real-time RT-PCR assay for detecting EGFRvIII in glioblastoma samples. Clinical Cancer Research, 14(2), 488-493. https://doi.org/10.1158/1078-0432.CCR-07-1966

Development of a real-time RT-PCR assay for detecting EGFRvIII in glioblastoma samples. / Yoshimoto, Koji; Dang, Julie; Zhu, Shaojun; Nathanson, David; Huang, Tiffany; Dumont, Rebecca; Seligson, David B.; Yong, William H.; Xiong, Zhenggang; Rao, Nagesh; Winther, Henrik; Chakravarti, Arnab; Bigner, Darell D.; Mellinghoff, Ingo K.; Horvath, Steve; Cavenee, Webster K.; Cloughesy, Timothy F.; Mischel, Paul S.

In: Clinical Cancer Research, Vol. 14, No. 2, 15.01.2008, p. 488-493.

Research output: Contribution to journalArticle

Yoshimoto, K, Dang, J, Zhu, S, Nathanson, D, Huang, T, Dumont, R, Seligson, DB, Yong, WH, Xiong, Z, Rao, N, Winther, H, Chakravarti, A, Bigner, DD, Mellinghoff, IK, Horvath, S, Cavenee, WK, Cloughesy, TF & Mischel, PS 2008, 'Development of a real-time RT-PCR assay for detecting EGFRvIII in glioblastoma samples', Clinical Cancer Research, vol. 14, no. 2, pp. 488-493. https://doi.org/10.1158/1078-0432.CCR-07-1966
Yoshimoto, Koji ; Dang, Julie ; Zhu, Shaojun ; Nathanson, David ; Huang, Tiffany ; Dumont, Rebecca ; Seligson, David B. ; Yong, William H. ; Xiong, Zhenggang ; Rao, Nagesh ; Winther, Henrik ; Chakravarti, Arnab ; Bigner, Darell D. ; Mellinghoff, Ingo K. ; Horvath, Steve ; Cavenee, Webster K. ; Cloughesy, Timothy F. ; Mischel, Paul S. / Development of a real-time RT-PCR assay for detecting EGFRvIII in glioblastoma samples. In: Clinical Cancer Research. 2008 ; Vol. 14, No. 2. pp. 488-493.
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abstract = "Purpose: Epidermal growth factor receptor variant III (EGFRvIII) is an oncogenic, constitutively active mutant form of the EGFR that is commonly expressed in glioblastoma and is also detected in a number of epithelial cancers. EGFRvIII presents a unique antigenic target for anti-EGFRvIII vaccines and it has been shown to modulate response to EGFR kinase inhibitor therapy. Thus, detection in clinical samples may bewarranted. Existing patents preclude the use of anti-EGFRvIII antibodies for clinical detection. Further, frozen tissue is not routinely available, particularly for patients treated in the community. Thus, detection of EGFRvIII in formalin-fixed paraffin-embedded (FFPE) clinical samples is a major challenge. Experimental Design: We developed a real-time reverse transcription-PCR (RT-PCR) assay for detecting EGFRvIII in FFPE samples and analyzed 59 FFPE glioblastoma clinical samples with paired frozen tissue from the same surgical resection. We assessed EGFRvIII protein expression by immunohistochemistry using two distinct specific anti-EGFRvIII antibodies and examined EGFR gene amplification by fluorescence in situ hybridization. Results: The FFPE RT-PCR assay detected EGFRvIII in 16 of 59 (27{\%}) samples, exclusively in cases with EGFR amplification, consistent with the expected frequency of this alteration. The FFPE RT-PCR assay was more sensitive and specific for detecting EGFRvIII than either of the two antibodies alone, or in combination, with a sensitivity of 93{\%} (95{\%} confidence interval, 0.78-1.00) and a specificity of 98{\%} (95{\%} confidence interval, 0.93-1.00). Conclusion: This assay will facilitate accurate assessment of EGFRvIII in clinical samples and may aid in the development of strategies for stratifying patients for EGFRvIII-directed therapies.",
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AU - Yoshimoto, Koji

AU - Dang, Julie

AU - Zhu, Shaojun

AU - Nathanson, David

AU - Huang, Tiffany

AU - Dumont, Rebecca

AU - Seligson, David B.

AU - Yong, William H.

AU - Xiong, Zhenggang

AU - Rao, Nagesh

AU - Winther, Henrik

AU - Chakravarti, Arnab

AU - Bigner, Darell D.

AU - Mellinghoff, Ingo K.

AU - Horvath, Steve

AU - Cavenee, Webster K.

AU - Cloughesy, Timothy F.

AU - Mischel, Paul S.

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Y1 - 2008/1/15

N2 - Purpose: Epidermal growth factor receptor variant III (EGFRvIII) is an oncogenic, constitutively active mutant form of the EGFR that is commonly expressed in glioblastoma and is also detected in a number of epithelial cancers. EGFRvIII presents a unique antigenic target for anti-EGFRvIII vaccines and it has been shown to modulate response to EGFR kinase inhibitor therapy. Thus, detection in clinical samples may bewarranted. Existing patents preclude the use of anti-EGFRvIII antibodies for clinical detection. Further, frozen tissue is not routinely available, particularly for patients treated in the community. Thus, detection of EGFRvIII in formalin-fixed paraffin-embedded (FFPE) clinical samples is a major challenge. Experimental Design: We developed a real-time reverse transcription-PCR (RT-PCR) assay for detecting EGFRvIII in FFPE samples and analyzed 59 FFPE glioblastoma clinical samples with paired frozen tissue from the same surgical resection. We assessed EGFRvIII protein expression by immunohistochemistry using two distinct specific anti-EGFRvIII antibodies and examined EGFR gene amplification by fluorescence in situ hybridization. Results: The FFPE RT-PCR assay detected EGFRvIII in 16 of 59 (27%) samples, exclusively in cases with EGFR amplification, consistent with the expected frequency of this alteration. The FFPE RT-PCR assay was more sensitive and specific for detecting EGFRvIII than either of the two antibodies alone, or in combination, with a sensitivity of 93% (95% confidence interval, 0.78-1.00) and a specificity of 98% (95% confidence interval, 0.93-1.00). Conclusion: This assay will facilitate accurate assessment of EGFRvIII in clinical samples and may aid in the development of strategies for stratifying patients for EGFRvIII-directed therapies.

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