Immunohistochemistry
sarcomas
SARCOMAS
The protein expression levels of CDK4, cyclin E, and E2F1 were assessed by immunohistochemistry (IHC) in formalin-fixed paraffin-embedded (FFPE) samples to determine their prognostic/predictive value. Four-μm sections were cut from the FFPE and stained with hematoxylin and eosin and with immunohistochemistry staining. Immunohistochemistry used an anti-CDK4 mouse monoclonal antibody (sc-56277; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), an anti-cyclin E mouse monoclonal antibody (sc-247; Santa Cruz Biotechnology, Inc.) or an anti-E2F1 rabbit polyclonal antibody (#3742; Cell Signaling Technology, Danvers, MA, USA). The extension of protein expression was evaluated in 4 levels (0—negative; 1—extension between 5% and 24%; 2—extension between 25% and 49%; and 3—extension between 50% and 100%) and the strength of IHC was evaluated in 3 levels (0—negative; 1—weak; and 2—strong). An immunohistochemical score was determined for CDK4 (CDK4 score) expression levels, by multiplying the extension of protein expression (level 0–3) by the strength of IHC (level 0–2). The CDK4 score was grouped as low expression (score of 2–3) and high expression (score of 4–6) for statistical analysis. A pathologist with great expertize in sarcomas (RR) was responsible for reviewing protein expression, blinded to clinical data.