The study was performed using 63 fresh pre-treatment primary tumor biopsies and five normal mucosa samples obtained from HNSCC patients. A sample aliquot was used for pathological diagnosis of the malignancy and another aliquot was immersed in RNAlater (Applied Biosystems Incorp, Foster City, CA), frozen in cold isopentane and kept in liquid nitrogen until RNA processing. Total RNA was extracted with Trizol (Invitrogen Ltd, Paisley, UK) and the phenol-chloroform method as previously described (Pavon, Parreno et al. 2008). Samples were, then, cleaned-up using RNeasy® Spin columns (Qiagen Incorp, Valencia, CA). Total RNA was quantified spectrophotometrically. The Agilent 2100 Bioanalyzer and the RNA Nano 6000 kit (Agilent Technologies, Santa Clara, USA) were used to determine the integrity of total RNA samples. Only samples with an RNA integrity number (RIN) higher than seven were included.
Label
biotin
Label protocol
cDNA and cRNA synthesis were performed using 2μg of total RNA from each biopsy as starting material and the GeneChip One-Cycle Target Labelling and Control Reagents Kit, following the instructions described in the Affymetrix user’s manual (Affymetrix, Santa Clara, CA, USA). After “in vitro” transcription, we obtained biotinylated cRNA. cRNA quality was checked using the Agilent 2100 Bioanalyzer.
Hybridization protocol
Ten micrograms of biotinylated cRNA, previously fragmented, were hybridized to the Affymetrix HG-U133A 2.0 microarray at 45ºC for 16 hours in the Affymetrix hybridization oven.
Scan protocol
Microarrays were scanned using a Gene Chip Scanner 3000 (Affymetrix, Santa Clara, CA, USA).The acquisition of microarray images was performed using GCOS software
Description
Gene expression data from a pre-treatment tumor biopsy Patients included in our microarray study (n=63) were treated from 1999 to 2007 at Hospital de la Santa Creu i Sant Pau (HSCSP). All had a pathologically confirmed, untreated, locally advanced (stage III, IVA & IVB) HNSCC. Patients defined as not operable at diagnosis were treated with concomitant CRT (stage IVB), whereas operable patients (stage III or IVA) were treated with IC followed by CRT/RT or surgery. Induction Chemotherapy consisted on the administration of cisplatin at a dose of 100 mg/m2 on day 1, and 5-FU at a dose of 1,000 mg/m2/day by continuous intravenous infusion on days 2 to 6 every 3 weeks, per three courses. After IC, patients whose tumour showed a complete response or a partial response above 50% received radiotherapy (IC+RT) or chemo radiotherapy (IC+CRT). Patients with stable or progressive disease after IC were treated with surgery (IC+SURGERY). Radiotherapy, at a total dose of 70 Gy, was administered to the primary tumor and clinically positive nodes, in 35 fractions of 2 Gy each, over a 7-week period. Nodal areas not clinically involved by tumor received a total dose of 50 Gy. Concomitant chemo radiotherapy (cCRT) consisted in RT at the same doses plus 3 cycles of cisplatin at a dose of 100mg/m2 on day 1 every 3 weeks. Probe microarray washing and staining was performed in a Fluidics Station 450 applying the midi_euk2v3_450 protocol.
Data processing
All the microarrays “.CEL” files included in the study were pre-processed applying the Robust Multi-array Average (RMA) algorithm (Irizarry, Hobbs et al. 2003) implemented in the Bioconductor statistical affy package (http://www.bioconductor.org).
