Ata 27

TMAs were constructed as previously described^{14 (link)}. In brief, three 0.6 mm cores were taken from the tumorous areas and two 0.6 mm cores were taken from the surrounding tumor free liver tissue of each tissue block. The tumorous areas with vital tissue were marked by an experienced pathologist using archived H&E glass slides. The TMAs were made using an automated tissue-arrayer ATA-27 (Beecher Instruments, Silver Springs MD, USA).

Tumor cell analysis was conducted via large-core tissue microarray (TMA, OriGene, Rockville, MD, USA). TMA blocks were constructed by extracting 1.5-mm-diameter cylinders from the center of the tumor. A tissue array instrument (Model # ATA-27, Beecher Instruments, Sun Prairie, WI, USA) was used to extract tissue cores and arrange them into blank recipient paraffin blocks. The TMA blocks were cut into 4-µm sections and processed for IHC. Antibodies, including ER antibody (dilution 1:50), PR antibody (dilution 1:50), HER2 antibody (dilution 1:50), pCHK2-Thr68 and pCDC25C-Ser216 antibody (dilution 1:40) were all purchased from Cell Signaling Technology (CST, Boston, MA, USA). Immunostaining was performed using the Envision System with diaminobenzidine (Dako, Glostrup, Denmark).
The protein expression of pCHK2-Thr68 and pCDC25C-Ser216 was determined semi-quantitatively by two senior pathologists who were blinded to the clinicopathological status of the participants. The staining was quantified using visual scoring for the staining intensity (absent, 0; light brown, 1; brown, 2; dark brown, 3) and the extent of staining (percentage of positive tumor cells; ≤10%, 0; 11%–25%, 1; 26%–50%, 2; 51%–75%, 3; >75%, 4). The product of the two scores ranged from 0 to 12. High expression was classified with a score of ≥5 and low expression with a score of ≤4.