Mib 1

All specimen slides were evaluated by two pathologists. The background liver was evaluated to confirm the presence of NAFLD or HBV. The presence and type of cirrhosis were evaluated microscopically. The tumor was then evaluated using gross and microscopic sections. Characteristics, including microvascular invasion, serosal invasion, and bile duct invasion were evaluated. The ES grade was noted and the margin status was evaluated. Immunochemistry was performed using 4-µm-thick paraffin-embedded sections. Sections were stained with specific antibodies for p53 (DO-7, 1:1,000, Dako), Ki-67 (MIB-1, 1:200, Dako), and COX2 (COX229, 1:300, Invitrogen) using an automated immunostainer (BenchMark XT, Ventana Medical Systems, Tucson, AZ, USA) in accordance with the manufacturer’s protocol. For Ki-67, cells with any intensity of nuclear staining were considered positive whereas only strong nuclear staining was considered positive for p53 (10 (link)). For COX2, cytoplasmic staining was considered positive. After counting 500–1,000 cells per slide, the percentages of hepatocytes positive for each antibody were calculated.

The status of the hormone receptor was analyzed by core needle biopsy at the time of initial diagnosis. Hormone receptor expression including ER, PR, and AR was defined by the Allred score [11 (link)]. Scores from 0 to +2 were negative, and scores from +3 to +8 were regarded as positive (Fig. 1).
Immunohistochemistry (IHC) was performed using the following primary antibodies: prediluted anti-ER rabbit monoclonal antibody (SP1, Ventana-Diapath, Tucson, AZ); prediluted anti-PgR rabbit monoclonal antibody (1E2, Ventana-Diapath); anti-AR rabbit monoclonal antibody (SP107, diluted 1:50, Cell Marque, Rocklin, CA); anti-Ki67 monoclonal antibody (MIB-1, diluted 1:100, Dako, Glostrup, Denmark) and c-erbB2 (4B5, prediluted, Ventana Medical, Tucson, AZ). Sliver in situ hybridization (SISH) assays for assessing HER2 gene amplification were performed for IHC equivocal (score 2+) cases. A positive HER2 SISH result was a HER2/chromosome enumeration probe 17 (CEP17) ratio ≥ 2.0 regardless of the average HER2 copy number or a HER2/CEP17 ratio < 2.0 with an average HER2 copy number ≥ 6.0 signal/cell [12 (link)]. IHC results were reviewed by one pathologist. pCR was defined as ypT0N0 or ypTisN0.

To evaluate p16 and K_i-67 expression levels, immunohistochemistry was performed using monoclonal antibody MIB 1 (Dako) for the K_i-67 in the dilution of 1:100, and G175–405 (Zeta) in the dilution of 1:100 for the p16. Both antigens were detected using HiDef Detection System, HRP Polymer System (Cell Marque, Rocklin, USA). All immunohistochemical studies were performed in the laboratory of Instituto Moacyr Junqueira, in Belo Horizonte, according to standard protocols.
The readings were done by two independent examiners who classified the slides according to the percentage of positive cells, as described by Zhong et al5 (link) (Table 1).

Ten micrometre sections of formalin‐fixed paraffin‐embedded tissue from the tumour TMA blocks were immunostained in a single batch. Immunohistochemistry was performed using the appropriate antigen retrieval methods for each primary antibody. Biotinylated secondary antibodies (rabbit anti‐mouse, swine anti‐rabbit and rabbit anti‐goat) were from Dako, normal serum and avidin‐biotin complex were from Vector Laboratories. Bound antibody was visualized using the avidin‐biotin‐peroxidase complex method (Vectastain Elite ABC) with 3′3 diaminobenzidine as chromogen and 0.05% hydrogen peroxide as substrate to obtain a brown precipitate. All sections were dehydrated before to be mounted in DePeX (BDH Laboratory Supplies).
The primary antibodies used were: microglia: Iba1 (all microglia‐Wako Laboratories), CD68 (phagocytic activity – clone PG‐M1, Dako); stem cell: CD133 (orb18124, Biorbyt), nestin (ab22035, Abcam), SOX2 (clone Y‐17, Santa Cruz Biotechnology); GFAP (glial acid fibrillary protein – Z0334, Dako), and Ki67 (cell proliferation – clone MIB1, Dako).