Exploring the Expression of CD73 in Lung Adenocarcinoma with EGFR Genomic Alterations

Abstract

Background/objectives: Immune checkpoint inhibitors (ICIs) benefit some lung cancer patients, but their efficacy is limited in advanced lung adenocarcinoma (LUAD) with EGFR mutations (EGFRm), largely due to a non-immunogenic tumour microenvironment (TME). Furthermore, EGFRm LUAD patients often experience increased toxicity with ICIs. CD73, an ectonucleotidase involved in adenosine production, promotes tumour immune evasion and could represent a novel therapeutic target. This study investigates CD73 expression in LUAD with EGFR alterations and its clinico-pathological correlations.

Methods: CD73 expression in tumour (CD73_TC) and stromal (CD73_SC) cells was assessed in 76 treatment-naive LUAD patients using immunohistochemistry (IHC) (D7F9A clone) alongside IHC PD-L1 (22C3 clone). EGFR alterations were identified by molecular sequencing and FISH. Event-free survival (EFS) was analysed based on CD73_TC expression.

Results: CD73_TC expression was observed in 66% of cases, with high expression (Tumour Proportion Score > 50%) correlating with improved EFS (p = 0.045). CD73_TC and PD-L1 expression were not significantly correlated (p = 0.44), although a weak inverse trend was observed. CD73_SC expression was detected in 18% of cases, predominantly in early-stage (p = 0.037), PD-L1-negative (p = 0.030), and non-EGFR-amplified (p = 0.0018) tumours. No significant associations were found with disease stage, histological subtype, EGFR mutation type, and amplification.

Conclusions: CD73 expression in EGFRm LUAD is heterogeneous and associated with diverse TME profiles. These findings support the potential of CD73 as a predictive biomarker and therapeutic target, highlighting its clinical relevance in EGFRm LUAD.

Keywords: CD73; EGFR; PD-L1; immunohistochemistry; immunotherapy; lung adenocarcinoma; non-small cell lung cancer.

Figure 1

Expression of CD73. (a) Representative image of expression of CD73 in adjacent normal lung tissue and tumour cells. Analysis of adjacent normal lung tissue showed consistent negative staining for CD73 in pneumocytes (black arrows and lower right insert), macrophages (black asterisk and upper left insert), and smooth muscle (black arrowheads) (A,B, ×40). Normal bronchial epithelium was mostly negative (red arrows) (A) but occasionally showed positive staining in apical ciliated cells (green arrow) (B). Blood vessels (Vx) were positive and served as internal control (A,B, ×40). In LUAD, CD73 was expressed on cancer cell membranes with luminal (C,D, ×40), apico-lateral (E, ×40), or complete membrane staining (F,G, ×40) with infrequent cytoplasmic reinforcement (G, ×40). Intracytoplasmic dot staining in tumour cells (H, ×40) and positive tumour-infiltrating lymphocytes ** (C, ×40) were also observed. Intensity of CD73 staining varied among LUAD, and within same adenocarcinoma (I, ×4), with weak (1+) (J, ×40), medium (2+) (K, ×40), and strong (3+) (L, ×40) intensity staining, highlighting complexity of calculated H-score. (b) Representative image of expression of CD73 in stromal cells. Cancer-associated fibroblasts (black arrow) exhibited CD73 overexpression in 3% of cases, even when LUAD cells (black arrowheads) were negative (M, ×40). Stromal fibroblasts can be difficult to differentiate from small vessels (VX) in neovascularised tumours (M, ×40). In addition, CD73 expression was observed in macrophages (*) and tumour-infiltrating lymphocytes (**) in subset of cases (N,O,P, ×40) regardless of expression of CD73 in cancer cells. (c) Bar graph illustrating different patterns of expression of CD73 in tumour cells. Patterns observed include complete membrane staining, apical–lateral staining, luminal staining, and intracytoplasmic dot staining. These expression patterns often coexisted within same tumour sample.

Figure 2

Comparison of PD-L1 expression and CD73 expression in tumour cells (CD73_TC) using Tumour Proportion Score (TPS). Dot Tumour plot illustrating relationship between expression of PD-L1 and CD73 in tumour cells. High expression of both PD-L1 and CD73_TC was mutually exclusive. Plot suggests trend of inverse correlation, where high levels of PD-L1 are generally associated with low levels of expression of CD73. However, this correlation is not statistically significant.

Figure 3

Kaplan–Meier curves illustrating event-free survival in patients categorised by high and low levels of CD73 expression in tumour cells (CD73_TC) using the Tumour Proportion Score (TPS). Patients with a high level of expression of CD73_TC (red line) had a significantly extended event-free survival in contrast to patients with a low level of expression of CD73_TC (blue line). The median event-free survival for patients with a low level of expression of CD73_TC was 24 months, while for those with a high level of expression of CD73, it was yet to be reached (log-rank test, p = 0.045 with TPS). The number of patients at risk at each time point is specified below the x-axis.