Tissue microarrays were de-waxed and hydrated, boiled in 0.01 M citrate buffer, and treated with 3% hydrogen peroxide after natural cooling. The primary polyclonal rabbit anti CALM1(catalog number: #10,541–1-AP; dilution at 1:400; Proteintech, Wuhan, China), EGFR (catalog number: # 18,986–1-AP;1:600; Proteintech), cleaved caspase-3(Catalog number: 19677–1-AP, dilution at 1:200; Proteintech, Wuhan, China) and Ki-67(Catalog number: 27309–1-AP, dilution at 1:10,000; Proteintech, Wuhan, China) were incubated overnight in 4 °C by adding drop of glass slide, followed by treatment with biotinylated anti-rabbit secondary antibody (Catalog number:# ZLI‐9032, Zhongshan Jinqiao Biotechnology) for 60 min at 37 °C overnight in 4 °C. The immunostainings results were evaluated by two pathologists (Qing Liu and Xiaomei Lu) under optical microscopy and cellular sub-localization of immunostaining was assessed in each section. The intensity of staining was divided into four grades (0, none; 1, weak; 2, moderate; and 3, strong) and percentage of positive cells (0, < 10%; 1, 10%-25%; 2, 25%–50%; and 3, > 50%). According to the immunoscoring (staining intensity plus positive cell score), ESCC patients were divided into two groups, specifically "low expression "(total score,0–3) and" high expression "(total score,4–6), which were used to analyze the prognostic significance of EGFR and CALM1 in ESCC.

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