Tumor samples from a total of 275 patients with ESCC were included in this analysis; 55 patients had received no neoadjuvant therapy (treatment-naïve cohort), and 200 and 20 had received neoadjuvant chemotherapy or CRT, respectively. Patient demographics were comparable across cohorts (Table 1). Pathologic T status was 1A and 1B in 2 (3.6%) and 23 (41.8%) patients in the treatment-naïve cohort, 5 (2.5%) and 50 (25.0%) patients in the chemotherapy cohort, and no patients in the CRT cohort; pathologic N status was 0 in 35 (63.6%), 21 (10.5%), and 5 (25.0%) patients, respectively; and pathologic stage was IA and IB in 23 (41.8%) and 1 (1.8%) patients, respectively, in the treatment-naïve cohort, no patients in the chemotherapy cohort, and 3 (15.0%) and no patients in the CRT cohort. The most commonly used chemotherapy regimens across the chemotherapy and CRT cohorts were cisplatin plus 5-fluorouracil (5-FU; n = 173/220; 78.6%) and docetaxel plus cisplatin plus 5-FU (n = 46/220; 20.9%).

Image analysis of IHC stained tumor samples for CD73, CD39, PD-L1, and CD8/FoxP3/PanCK in the TME showed that median CD73 expression in the TME overall was higher in the chemotherapy (7.2% CD73-positive tissue) and CRT (6.4%) cohorts compared with the treatment-naïve cohort (2.8%; p < 0.0001 and p < 0.01, respectively) (Fig. 1A). No significant differences in PD-L1 expression were observed between cohorts (Fig. 1B). Median CD39 expression was lower in the CRT cohort (4.9% CD39-positive tissue) compared with either the chemotherapy (14.0%, p < 0.0001) or treatment-naïve (11.7%, p < 0.001) cohorts (Fig. 1C).

Expression of A CD73, B PD-L1, and C CD39 in the TME of resected ESCC tumor samples from patients who received no treatment, chemotherapy, or CRT prior to surgery. A CD73 expression was increased in the chemotherapy and CRT cohorts versus the treatment-naïve cohort. B There were no significant differences in PD-L1 expression across cohorts. C CD39 expression was lower in the CRT cohort versus the treatment-naïve and chemotherapy cohorts. Solid red lines represent the medians; dashed red lines represent the quartiles. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. CD cluster of differentiation, CRT chemoradiotherapy, ESCC esophageal squamous cell carcinoma, PD-L1 programmed death-ligand 1, TME tumor microenvironment

Using HALO® AI digital image analysis software to classify and annotate the tumor epithelium, stroma, necrotic areas, and immune infiltrates and to assess the expression of CD73 across different compartments (Supplementary Fig. 1) demonstrated that CD73 expression was primarily observed in stromal and immune cells compared with in epithelial tumor cells (Fig. 2). Tumor samples from patients in the chemotherapy and CRT cohorts showed higher CD73 expression in the stroma (median CD73-positive area: 9.3% and 8.1% vs 4.1%; p < 0.0001 and p < 0.01, respectively) (Fig. 2B) and tumor epithelium (1.5% and 3.2% vs 0.6%; p < 0.05 and p < 0.01, respectively) (Fig. 2A), and CD73 expression was also higher in the immune infiltrate for the chemotherapy versus treatment-naïve cohort (median 8.0% vs 3.2%, p < 0.0001) (Fig. 2C). The difference in CD73 expression between cohorts appeared to be most profound in the stroma.

Expression of CD73 in A tumor epithelium, B stroma, and C immune infiltrate compartments of resected ESCC tumor samples from patients who received no treatment, chemotherapy, or CRT prior to surgery. A, B CD73 expression was increased in the tumor epithelium and stroma in the chemotherapy and CRT cohorts versus the treatment-naïve cohort. C CD73 expression was increased in the immune infiltrate in the chemotherapy cohort versus the treatment-naïve cohort. Solid red lines represent the medians; dashed red lines represents quartiles. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. CD cluster of differentiation, CRT chemoradiotherapy, ESCC esophageal squamous cell carcinoma

In the treatment-naïve cohort, median CD8-positive cell density was higher in the stroma (745.6 cells/mm2) than in the tumor epithelium (200.7 cells/mm2) (p < 0.0001; Supplementary Fig. 2a). In contrast, median Foxp3-positive cell densities were similar (529.8 vs 579.2 cells/mm2) between compartments (Supplementary Fig. 2b). CD8-positive cell density was significantly lower in the tumor epithelium in the chemotherapy and CRT cohorts compared with the treatment naïve cohort (median 93.9 and 30.5 cells/mm2 vs 200.7 cells/mm2, p < 0.0001 and p < 0.01, respectively) (Fig. 3A), whereas there were no significant differences in stromal CD8-positive cell density between the different cohorts (Fig. 3D). In contrast, FoxP3-positive cell densities were significantly lower in both the epithelial (median 118.3 and 36.6 vs 579.2 cells/mm2, p < 0.0001 and p < 0.001, respectively) (Fig. 3B) and stromal (median 324.3 and 267.9 vs 529.8 cells/mm2, p < 0.0001 and p < 0.001, respectively) (Fig. 3E) compartments in the chemotherapy and CRT cohorts compared with the treatment-naïve cohort. As CD8-positive cells were abundant in the stromal compartment compared with the epithelium, the differential effect of chemotherapy and CRT on CD8-positive cells compared with FoxP3-positive cells resulted in significantly increased CD8/FoxP3 ratios in the tumor epithelium in the chemotherapy versus treatment-naïve cohort (median ratio 0.8 vs 0.4, p < 0.001) (Fig. 3C) and in the stroma in both the chemotherapy and CRT versus tumor-naïve cohorts (2.1 and 4.5 vs 1.5, p < 0.05 and p < 0.0001, respectively) and in the CRT versus chemotherapy cohort (p < 0.0001) (Fig. 3F).

Tumor epithelium and stromal A, D density of CD8-positive cells, B, D density of FoxP3-positive cells, and C, F CD8/FoxP3 ratio in resected ESCC tumor samples from patients who received no treatment, chemotherapy, or CRT prior to surgery. A CD8-positive cell density was lower in the tumor epithelium in the chemotherapy and CRT cohorts versus the treatment-naïve cohort, but D there were no significant differences in stromal CD8-positive cell density across cohorts. B, E FoxP3-positive cell density was lower in the tumor epithelium and stroma in the chemotherapy and CRT cohorts versus the treatment-naïve cohort. The CD8/FoxP3-positive cells ratio c in the tumor epithelium was higher in the chemotherapy cohort versus the treatment-naïve cohort and F in the stroma was higher in the chemotherapy and CRT cohorts versus the treatment-naïve cohort. Solid red lines represent the medians; dashed red lines represent the quartiles. *p < 0.05; **p < 0.01, ***p < 0.001, ****p < 0.0001. CD cluster of differentiation, CRT chemoradiotherapy, ESCC esophageal squamous cell carcinoma

Median RFS was not reached (NR), 95.9 months, and 6.3 months in the treatment-naïve, chemotherapy, and CRT cohorts, respectively, and 3-year RFS rates (95% CIs) were 73.0% (61.8–86.2), 58.0% (51.5–65.4), and 30.0% (15.4–58.6), respectively (Supplementary Fig. 3A, C, E). Median OS was 99.6, 141.2, and 25.6 months in the treatment-naïve, chemotherapy, and CRT cohorts, respectively, and 3-year OS rates (95% CIs) were 78.2% (67.5–90.5), 71.4% (65.2–78.3), and 33.5% (17.5–64.3), respectively (Supplementary Fig. 3b, d, f).

Analysis of RFS and OS in the treatment-naïve and chemotherapy cohorts according to marker levels, dichotomized around the median, showed that the percentage tumor epithelium in a sample was a negative biomarker and that the percentage of CD73-positive tissue in the TME was a prognostic biomarker for both RFS and OS in both cohorts. CD8-positive cell density, FoxP3-positive cell density, percentage CD39-DAB-positive tissue, and percentage PD-L1-positive tissue did not show any prognostic association in these cohorts (Supplementary Fig. 4).

RFS was significantly longer in the CD73-high versus CD73-low groups, defined by overall TME CD73 expression, in both the treatment-naïve (median RFS NR vs 55.9 months, HR 0.16, 95% CI 0.05–0.58, p = 0.0014) (Fig. 4A) and chemotherapy (median RFS 129.3 vs 13.3 months, HR 0.53, 95% CI 0.35–0.79, p = 0.0018) (Fig. 4B) cohorts. Assessment of RFS by CD73 expression in the epithelial, stromal, and immune compartments demonstrated differences in the contributions by compartment to the RFS benefit. Analysis of RFS by CD73 expression in the stromal compartment, in which CD73 expression was higher than in the other compartments (Fig. 2), showed that there was a pronounced RFS benefit in the CD73-high versus CD73-low group in both the treatment-naïve (median RFS NR vs NR, HR 0.25, 95% CI 0.08–0.80, p = 0.012) (Fig. 4C) and chemotherapy (median RFS 129.3 vs 31.1 months, HR 0.61, 95% CI 0.40–0.91, p = 0.014) (Fig. 4D) cohorts. Similarly, benefit was seen in the CD73-high versus CD73-low group on analysis of RFS by CD73 expression in the immune infiltrate compartment in both the treatment-naïve (median RFS NR vs NR, HR 0.41, 95% CI 0.14–1.19, p = 0.09) (Fig. 4E) and chemotherapy (median 129.3 vs 31.2 months, HR 0.54, 95% CI 0.36–0.82, p = 0.0029) (Fig. 4F) cohorts. In contrast, analysis of RFS by CD73 expression in the epithelial compartment, in which CD73 expression was limited (Fig. 2), demonstrated no difference in RFS between the CD73-high and CD73-low groups in the treatment-naïve cohort (median NR vs NR, HR 0.96, 95% CI 0.35–2.65, p = 0.94) (Fig. 4G) and a numerically shorter RFS in the CD73-high versus CD73-low group in the chemotherapy cohort (median 54.1 vs 141.0 months, HR 1.26, 95% CI 0.84–1.88, p = 0.26) (Fig. 4H). Interestingly, the group with high tumor epithelial CD73 expression had lower CD8-positive and FoxP3-positive cell densities compared with the corresponding CD73-low group, whereas there were no significant differences in these cell densities between the CD73-high and CD73-low groups defined by stromal CD73 expression (Supplementary Table 1).

RFS in patients with ESCC who received no treatment or chemotherapy prior to surgery according to high versus low CD73 expression (dichotomized by the respective medians) in A, B the overall TME, or the C, D stromal, E, F immune infiltrate, or G, H epithelial compartments. In the treatment-naïve cohort, RFS was significantly longer in patients with high versus low CD73 expression in the A overall TME and C stromal compartment but not the E immune infiltrate or G epithelial compartments. In the chemotherapy cohort, RFS was significantly longer in patients with high versus low CD73 expression in the B overall TME, and D stromal and F immune infiltrate compartments but not the H epithelial compartment. Red lines represent the groups with high CD73 expression above the median cut; blue lines represent the groups with low CD73 expression below the median cut. CD cluster of differentiation, CI confidence interval, ESCC esophageal squamous cell carcinoma, HR hazard ratio, NC not calculable, RFS recurrence-free survival, TME tumor microenvironment