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Optimisation of an immunohistochemistry method for the determination of androgen receptor expression levels in circulating tumour cells

BACKGROUND: AZD3514 inhibits and down regulates the androgen receptor (AR) and has undergone clinical trials in prostate cancer. To provide proof-of-mechanism (POM) in patients, an immunohistochemistry (IHC) method for determination of AR in circulating tumour cells (CTC) was developed and validated...

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Autores principales: Cummings, Jeffrey, Sloane, Robert, Morris, Karen, Zhou, Cong, Lancashire, Matt, Moore, David, Elliot, Tony, Clarke, Noel, Dive, Caroline
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977890/
https://www.ncbi.nlm.nih.gov/pubmed/24674711
http://dx.doi.org/10.1186/1471-2407-14-226
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author Cummings, Jeffrey
Sloane, Robert
Morris, Karen
Zhou, Cong
Lancashire, Matt
Moore, David
Elliot, Tony
Clarke, Noel
Dive, Caroline
author_facet Cummings, Jeffrey
Sloane, Robert
Morris, Karen
Zhou, Cong
Lancashire, Matt
Moore, David
Elliot, Tony
Clarke, Noel
Dive, Caroline
author_sort Cummings, Jeffrey
collection PubMed
description BACKGROUND: AZD3514 inhibits and down regulates the androgen receptor (AR) and has undergone clinical trials in prostate cancer. To provide proof-of-mechanism (POM) in patients, an immunohistochemistry (IHC) method for determination of AR in circulating tumour cells (CTC) was developed and validated. METHODS: After an assessment of specificity validation focused on intra- and inter-operator reproducibility utilising a novel modification of incurred sample reanalysis (ISR). β-Content γ-confidence tolerance intervals (BCTI) and Cohen’s Kappa (κ) were employed in statistical analysis of results. RESULTS: In a first set of IHC reproducibility experiments, almost perfect agreement was recorded (κ=0.94) when two different operators scored CTC as overall positive or negative for AR. However, BCTI analysis identified a specific bias in scoring staining intensity, where one operator favoured moderate over strong assignments, whereas the reverse was the case with the second operator. After a period of additional training involving deployment of a panel of standardised images, a second set of validation experiments were conducted. These showed correction of the inter-operator bias by BCTI with κ for scoring intensity increasing from 0.59 to 0.81, indicative of almost perfect agreement. CONCLUSIONS: By application of BCTI to the validation of IHC, operator bias and therefore poor reproducibility can be identified, characterised and corrected to achieve a level of error normally associated with a quantitative biomarker assay, such as an ELISA. The methodological approach described herein can be applied to any generic IHC technique.
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spelling pubmed-39778902014-04-21 Optimisation of an immunohistochemistry method for the determination of androgen receptor expression levels in circulating tumour cells Cummings, Jeffrey Sloane, Robert Morris, Karen Zhou, Cong Lancashire, Matt Moore, David Elliot, Tony Clarke, Noel Dive, Caroline BMC Cancer Technical Advance BACKGROUND: AZD3514 inhibits and down regulates the androgen receptor (AR) and has undergone clinical trials in prostate cancer. To provide proof-of-mechanism (POM) in patients, an immunohistochemistry (IHC) method for determination of AR in circulating tumour cells (CTC) was developed and validated. METHODS: After an assessment of specificity validation focused on intra- and inter-operator reproducibility utilising a novel modification of incurred sample reanalysis (ISR). β-Content γ-confidence tolerance intervals (BCTI) and Cohen’s Kappa (κ) were employed in statistical analysis of results. RESULTS: In a first set of IHC reproducibility experiments, almost perfect agreement was recorded (κ=0.94) when two different operators scored CTC as overall positive or negative for AR. However, BCTI analysis identified a specific bias in scoring staining intensity, where one operator favoured moderate over strong assignments, whereas the reverse was the case with the second operator. After a period of additional training involving deployment of a panel of standardised images, a second set of validation experiments were conducted. These showed correction of the inter-operator bias by BCTI with κ for scoring intensity increasing from 0.59 to 0.81, indicative of almost perfect agreement. CONCLUSIONS: By application of BCTI to the validation of IHC, operator bias and therefore poor reproducibility can be identified, characterised and corrected to achieve a level of error normally associated with a quantitative biomarker assay, such as an ELISA. The methodological approach described herein can be applied to any generic IHC technique. BioMed Central 2014-03-28 /pmc/articles/PMC3977890/ /pubmed/24674711 http://dx.doi.org/10.1186/1471-2407-14-226 Text en Copyright © 2014 Cummings et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Technical Advance
Cummings, Jeffrey
Sloane, Robert
Morris, Karen
Zhou, Cong
Lancashire, Matt
Moore, David
Elliot, Tony
Clarke, Noel
Dive, Caroline
Optimisation of an immunohistochemistry method for the determination of androgen receptor expression levels in circulating tumour cells
title Optimisation of an immunohistochemistry method for the determination of androgen receptor expression levels in circulating tumour cells
title_full Optimisation of an immunohistochemistry method for the determination of androgen receptor expression levels in circulating tumour cells
title_fullStr Optimisation of an immunohistochemistry method for the determination of androgen receptor expression levels in circulating tumour cells
title_full_unstemmed Optimisation of an immunohistochemistry method for the determination of androgen receptor expression levels in circulating tumour cells
title_short Optimisation of an immunohistochemistry method for the determination of androgen receptor expression levels in circulating tumour cells
title_sort optimisation of an immunohistochemistry method for the determination of androgen receptor expression levels in circulating tumour cells
topic Technical Advance
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3977890/
https://www.ncbi.nlm.nih.gov/pubmed/24674711
http://dx.doi.org/10.1186/1471-2407-14-226
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