Prospective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma
Background and purpose: Tumour hypoxia is associated with a poor prognosis in head and neck squamous cell carcinoma (HNSCC), however there is no accepted method for assessing hypoxia clinically. We aimed to conduct a technical validation of a hypoxia gene expression signature using the TaqMan Low De...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Journal article |
| Language: | English |
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2013
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| _version_ | 1826293004849119232 |
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| author | Betts, G Eustace, A Patiar, S Valentine, H Irlam, J Ramachandran, A Merve, A Homer, J Möller-Levet, C Buffa, F Hall, G Miller, C Harris, A West, C |
| author_facet | Betts, G Eustace, A Patiar, S Valentine, H Irlam, J Ramachandran, A Merve, A Homer, J Möller-Levet, C Buffa, F Hall, G Miller, C Harris, A West, C |
| author_sort | Betts, G |
| collection | OXFORD |
| description | Background and purpose: Tumour hypoxia is associated with a poor prognosis in head and neck squamous cell carcinoma (HNSCC), however there is no accepted method for assessing hypoxia clinically. We aimed to conduct a technical validation of a hypoxia gene expression signature using the TaqMan Low Density Array (TLDA) platform to investigate if this approach reliably identified hypoxic tumours. Materials and methods: Tumour samples (n = 201) from 80 HNSCC patients were collected prospectively from two centres. Fifty-three patients received pimonidazole prior to surgery. TaqMan Low Density Array-Hypoxia Scores (TLDA-HS) were obtained by quantitative real-time PCR (qPCR) using a 25-gene signature and customised TLDA cards. Assay performance was assessed as coefficient of variation (CoV). Results: The assay was sensitive with linear reaction efficiencies across a 4log10 range of inputted cDNA (0.001-10 ng/μl). Intra- (CoV = 6.9%) and inter- (CoV = 2.0%) assay reproducibility were excellent. Intra-tumour heterogeneity was lower for TLDA-HS (23.2%) than for pimonidazole (67.2%) or single gene measurements of CA9 (62.2%), VEGFA (45.0%) or HIG2 (39.4%). TLDA-HS in HNSCC cell lines increased with decreasing pO2. TLDA-HS correlated with Affymetrix U133 Plus 2.0 microarray HS (p < 0.01) and positive pimonidazole scores (p = 0.005). Conclusions: Gene expression measurements of hypoxia using a 25-gene signature and TLDA cards are sensitive, reproducible and associated with lower intra-tumour heterogeneity than assaying individual genes or pimonidazole binding. The approach is suitable for further assessment of prognostic and predictive capability in clinical trial material. © 2012 Elsevier Ltd. All rights reserved. |
| first_indexed | 2024-03-07T03:23:25Z |
| format | Journal article |
| id | oxford-uuid:b837bd70-324f-4c0b-93a3-e1cd4d2dda1d |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T03:23:25Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | oxford-uuid:b837bd70-324f-4c0b-93a3-e1cd4d2dda1d2022-03-27T04:54:22ZProspective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinomaJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:b837bd70-324f-4c0b-93a3-e1cd4d2dda1dEnglishSymplectic Elements at Oxford2013Betts, GEustace, APatiar, SValentine, HIrlam, JRamachandran, AMerve, AHomer, JMöller-Levet, CBuffa, FHall, GMiller, CHarris, AWest, CBackground and purpose: Tumour hypoxia is associated with a poor prognosis in head and neck squamous cell carcinoma (HNSCC), however there is no accepted method for assessing hypoxia clinically. We aimed to conduct a technical validation of a hypoxia gene expression signature using the TaqMan Low Density Array (TLDA) platform to investigate if this approach reliably identified hypoxic tumours. Materials and methods: Tumour samples (n = 201) from 80 HNSCC patients were collected prospectively from two centres. Fifty-three patients received pimonidazole prior to surgery. TaqMan Low Density Array-Hypoxia Scores (TLDA-HS) were obtained by quantitative real-time PCR (qPCR) using a 25-gene signature and customised TLDA cards. Assay performance was assessed as coefficient of variation (CoV). Results: The assay was sensitive with linear reaction efficiencies across a 4log10 range of inputted cDNA (0.001-10 ng/μl). Intra- (CoV = 6.9%) and inter- (CoV = 2.0%) assay reproducibility were excellent. Intra-tumour heterogeneity was lower for TLDA-HS (23.2%) than for pimonidazole (67.2%) or single gene measurements of CA9 (62.2%), VEGFA (45.0%) or HIG2 (39.4%). TLDA-HS in HNSCC cell lines increased with decreasing pO2. TLDA-HS correlated with Affymetrix U133 Plus 2.0 microarray HS (p < 0.01) and positive pimonidazole scores (p = 0.005). Conclusions: Gene expression measurements of hypoxia using a 25-gene signature and TLDA cards are sensitive, reproducible and associated with lower intra-tumour heterogeneity than assaying individual genes or pimonidazole binding. The approach is suitable for further assessment of prognostic and predictive capability in clinical trial material. © 2012 Elsevier Ltd. All rights reserved. |
| spellingShingle | Betts, G Eustace, A Patiar, S Valentine, H Irlam, J Ramachandran, A Merve, A Homer, J Möller-Levet, C Buffa, F Hall, G Miller, C Harris, A West, C Prospective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma |
| title | Prospective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma |
| title_full | Prospective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma |
| title_fullStr | Prospective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma |
| title_full_unstemmed | Prospective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma |
| title_short | Prospective technical validation and assessment of intra-tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma |
| title_sort | prospective technical validation and assessment of intra tumour heterogeneity of a low density array hypoxia gene profile in head and neck squamous cell carcinoma |
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