Za 0508

Paraffin sections were deparaffinized, dehydrated, and antigen retrieved. The sections were blocked by 5% goat serum for 1 hour to reduce nonspecific background staining and incubated with primary antibody against CCL23 (abcam #ab171751) or CD8 (ZSGB‐BIO #ZA‐0508) overnight at 4°C. Subsequently, the sections were rinsed with TBS, treated with 3% H₂O₂ to block endogenous peroxidase activity, incubated with horseradish peroxidase–conjugated secondary antibody, and visualized by using a DAB Kit (ZSGB‐BIO #ZLI‐9018). Immunostaining degree of each sample was scored based on staining intensity (intensity score: 0, no staining; 1, weak staining; 2, intermediate staining; 3, strong staining) and proportion of positive cells (extent score: 0, < 5%; 1, 5%‐25%; 2, 26%‐75%; 3, 75%‐100%). The final immunoreactivity score of CCL23 expression for each case is the product of the intensity score and the extent score. The counts for CD8⁺ T cells represent an average number of positively stained cells within three random intratumor areas under a 20X objective.

PD-L1, CD8, and p16 (as a surrogate of HPV infection)20 (link),21 (link) were stained using a standard protocol as previously described.22 (link) Briefly, freshly cut formalin-fixed paraffin-embedded specimens were dewaxed in xylene, hydrated in graded alcohol, and washed in phosphate-buffered saline; after neutralizing endogenous peroxidase (0.3% H₂O₂ for 10 min), the microwave antigen retrieval method was applied using Tris–EDTA (pH 9.0). Subsequently, the slides were preincubated with blocking serum and then were incubated overnight at 4°C with each primary antibody: p16 (1:100, ZM0205; ZSGB-BIO, Beijing, People’s Republic of China), CD8 (1:100, ZA0508, ZSGB-BIO), and PD-L1 (1:100, #13684; Cell Signaling Technology, Shanghai, People’s Republic of China). Once the above procedure was completed, the sections were serially rinsed, incubated with secondary antibodies, visualized using diaminobenzidine, and counterstained with hematoxylin. Tonsil tissues were used as positive controls for PD-L1 and CD8, and cervical squamous cell carcinoma specimens were used as positive controls for p16 (Figure S1). Specimens of identical tissues stained without primary antibody were used as negative controls.

Pretreatment biopsy samples were obtained before the initiation of nCRT. TILs were assessed by immunohistochemical staining of slides from formalin-fixed, paraffin-embedded tumor blocks using mouse monoclonal antibodies against CD8 (1:100, ZA0508; ZSGB-BIO, Beijing, China). For each slide, three random fields were chosen for the enumeration of CD8+ TIL within the area where tumor cells were considered to be most strongly stained. Then, an average was reached and considered as the number of CD8+ TIL per field. The density of TILs was defined as the number of positive CD8 lymphocytes per square millimeter and was then graded as either “high” or “low” (cutoff=80/mm²). This cutoff value yielded a minimal “P-value” in the analysis of correlation with pCR and was thus applied.