Immunohistochemistry was performed as previously described . Briefly, tissue sections (5 μm) underwent microwave antigen retrieval for 10 minutes at 100°C in a steam microwave (Sixth Sense, Whirlpool, VIC, Australia) in 10 mM Tris buffer, 1 mM EDTA (pH 9.0). Sections were incubated overnight with mouse monoclonal antibody which detects both K5 and K6 (1/50, clone D5/16 B4, Dako) or K5 rabbit monoclonal antibody (1/400, clone EPR1600Y, Abcam Ab75869), in blocking buffer (5% normal goat serum) at 4°C. Visualization of immunoreactivity was achieved using biotinylated anti-mouse or anti-rabbit immunoglobulins streptavidin-peroxidase conjugate, diaminobenzidine substrate as described previously . Human skin was used as a positive control and negative controls included tissues incubated with no primary antibody or with mouse or rabbit immunoglobulins. Slides were digitally scanned using the NanoZoomer Digital Pathology System (Hamamatsu Photonics, SZK, Japan) and images were collected using NDP view imaging software (NDP scan software v2.2, Hamamatsu Photonics). K5/6 or K5 expression was quantified using a visual grading system based on the extent of staining. Percentage of positive tumor cells was graded on a scale from 0–3; 0 = none, 1 = 1–9 %, 2 = 10–50%, 3 = > 50% by two independent assessors used previously for breast cancer [65, 66]. High K5/6 or K5 immunostaining was defined as ≥ 10% positivity and < 10% was defined as low K5/6 or K5 immunostaining.