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.
abstract: OBJECTIVE
  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.
  
  
  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 4 log(10) 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 pO(2). 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.
authors.affiliation: Translational Radiobiology Group, Institute of Cancer Sciences, Manchester Academic Health Science Centre, Christie Hospital, Wilmslow Road, Manchester M20 4BX, United Kingdom.
authors.forename: Guy N J
authors.initials: GN
authors.lastname: Betts
authors.forename: Amanda
authors.initials: A
authors.lastname: Eustace
authors.forename: Shalini
authors.initials: S
authors.lastname: Patiar
authors.forename: Helen R
authors.initials: HR
authors.lastname: Valentine
authors.forename: Joely
authors.initials: J
authors.lastname: Irlam
authors.forename: Anassuya
authors.initials: A
authors.lastname: Ramachandran
authors.forename: Ashirwad
authors.initials: A
authors.lastname: Merve
authors.forename: Jarrod J
authors.initials: JJ
authors.lastname: Homer
authors.forename: Carla
authors.initials: C
authors.lastname: Möller-Levet
authors.forename: Francesca M
authors.initials: FM
authors.lastname: Buffa
authors.forename: Gillian
authors.initials: G
authors.lastname: Hall
authors.forename: Crispin J
authors.initials: CJ
authors.lastname: Miller
authors.forename: Adrian L
authors.initials: AL
authors.lastname: Harris
authors.forename: Catharine M L
authors.initials: CM
authors.lastname: West
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002294:Carcinoma, Squamous Cell / Q000235:genetics* / Q000473:pathology; D015687:Cell Hypoxia / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D006258:Head and Neck Neoplasms / Q000235:genetics* / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D059467:Transcriptome