Nucleic acids were extracted from frozen tumor tissue (pre-NAT samples) using AllPrep DNA/RNA mini kit (Qiagen France SAS, Courtaboeuf, France) according to the manufacturer’s protocol.
The extracted material was sent to Helixio (Saint-Beauzire, France), where it was hybridized with gene arrays (Human SurePrint, Agilent Technologies France, Les Ullis, France)
Limma package was applied to process Agilent microarray data and identify differentially expressed genes between progressive tumors and tumors with pathological complete response. Background correction was performed on expression intensities before normalization by the parameter of “expnorm”. Median value was taken when a gene has multiple probes. Then, “cyclicloess” method, which applies loess normalization to all possible pairs of arrays, was used to normalize corrected intensities. The probes have been filtered for those close to background and without gene name.