Histology and immunohistochemistry
Hematoxylin and eosin (H&E) stained slides were carefully analysed and validated by pathology specialists and appropriate tumor samples were selected for DNA isolation with a tumor percentage >20% (in samples where lower tumor ratio was detected, microdissection was performed). IHC of neuron specific enolase (NSE, BBS/NC/VI-H14 clone, 1:800 dilution, Agilent Technologies, Santa Clara, CA, USA) and synaptophysin (27G12 clone, 1:100 dilution, Leica Biosystems, Wetzlar, Germany) was performed to confirm Rb diagnosis. Anti-RB monoclonal antibody (1F8 (RB1) clone, 1:200 dilution, Invitrogen, Carlsbad, CA, USA) was used on all samples of the patient and on a control colorectal adenocarcinoma sample.