Predictive value of CD73 expression for the efficacy of immune checkpoint inhibitors in NSCLC

Background CD73 induces the dephosphorylation of adenosine monophosphate converting it to adenosine, enabling malignancies to escape from immune surveillance. Although CD73 overexpression has been reported to be a poor prognostic factor in several malignancies including non‐small cell lung cancer (NSCLC), its predictive relevance in NSCLC patients receiving immune checkpoint inhibitors is unknown. The present research was conducted to investigate the prognostic significance of CD73 expression in NSCLC patients receiving immune checkpoint inhibitors (ICIs). Methods We screened 91 patients with advanced or recurrent NSCLC who received immune checkpoint inhibitors. CD73 expression was evaluated immunohistochemically using tissue specimens obtained just before treatment with ICIs. Results Analysis of progression‐free survival (PFS) and overall survival (OS) in relation to several levels of CD73 expression (1%, 10%, 30%, and 50%) showed that both tended to be more favorable as expression of CD73 increased. PFS and OS were longer for patients in whom at least 50% of the tumor cells expressed CD73 than for those in whom <50% of the tumor cells did so. In patients who were positive for EGFR mutation, immune checkpoint inhibitors were significantly more effective in those with high CD73 expression, whereas CD73 expression did not significantly affect the efficacy in patients with EGFR mutation‐negative NSCLC. Furthermore, CD73 expression was predictive factor for the PFS independent of PD‐L1 expression in patients with EGFR mutation. Conclusions High CD73 expression may predict a favorable response to ICIs in NSCLC patients, especially those harboring EGFR mutations. Key points Significant findings of the study: In patients who were positive for EGFR mutation, immune checkpoint inhibitors (ICIs) were significantly more effective in those with high CD73 expression, whereas CD73 expression did not significantly affect the efficacy in patients with EGFR mutation‐negative NSCLC. What this study adds: High CD73 expression may predict a favorable response to immune checkpoint inhibitors in NSCLC patients, especially those harboring EGFR mutations.


Introduction
Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide. 1 Immune checkpoint inhibitors (ICIs) have been recently used as a new strategy for treatment in various malignancies. 2,3 Several clinical trials demonstrated that treatment with antiprogrammed cell death 1 (PD-1)/PDligand 1 (PD-L1) antibodies showed a promising efficacy. [4][5][6][7] ecto-5 0 -nucleotidase, catalyzes the dephosphorylation of extracellular adenosine monophosphate (AMP) to adenosine and inorganic phosphate. The released extracellular adenosine inhibits the antitumor function of T cells and induces T cell apoptosis, thus helping tumors to escape immune surveillance. 8,9 CD73 is frequently expressed in a large number of malignancies and is correlated with poor patient survival. [10][11][12][13] Streicher et al. have reported that epidermal growth factor receptor (EGFR) mutation-positive cell lines show significantly high CD73 expression, and that expression of CD73 is decreased by EGFR inhibition in NSCLC cell lines. 14 However, the prognostic value of CD73 expression in NSCLC patients, including those with EGFR mutation-positive NSCLC, receiving ICIs is unclear. This retrospective study aimed to evaluate the prognostic significance of CD73 expression in NSCLC patients receiving ICIs.

Patients
We retrospectively examined consecutive NSCLC patients who had been treated with immune checkpoint inhibitors (ICIs) at Kurume University Hospital between February 2016 and September 2018. Of these, 91 patients who had adequate biopsy tissues with enough tumor cells were enrolled into the present study. Among these patients, we screened 25 with advanced or recurrent EGFR mutationpositive NSCLC and 66 with EGFR mutation-negative NSCLC who had received ICIs. All EGFR mutationpositive patients were treated with ICIs after developing resistance to EGFR-TKI therapy.
We followed the provisions of the Declaration of Helsinki and obtained study approval from the Institutional Review Board of Kurume University Hospital.

Tumor samples
Formalin-fixed paraffin-embedded tumor slices obtained by biopsy before treatment with immune checkpoint inhibitors (ICIs) were used for analysis. EGFR mutations were detected using the Cobas EGFR Mutation Test (Roche Diagnostics Deutschland GmbH, Mannheim, Germany).

Immunohistochemical analysis for CD73 and PD-L1 expression
The sections were mounted onto slides and incubated with antirabbit monoclonal antibodies against CD73 and PD-L1 (Cell Signaling Technology, Danvers, MA, USA) for immunohistochemical (IHC) analysis using a Bench-Mark XT slide staining system (Ventana Automated Systems, Inc., Tucson, AZ, USA). For both PD-L1 and CD73 IHC analysis, each specimen had to contain more than 100 viable malignant cells and the percentage of stained malignant cells in the entire area of the tumor (tumor proportion score; TPS) was determined.

Statistical analysis
The association between CD73 expression level and the efficacy of immune checkpoint inhibitors (ICIs) was evaluated regarding the overall response rate (ORR), progression-free survival (PFS), and overall survival (OS). ORR was defined as the proportion of patients who achieved a complete response or partial response according to the Response Evaluation Criteria in Solid Tumors (ver. 1.1). PFS was estimated as the period from the start of treatment with ICIs to the date of disease progression or death due to any cause. OS was defined as from the start of treatment until the death or the date of last follow-up. Survival curves were analyzed by the Kaplan-Meier method and a log-rank test was performed to analyze the significance of differences between two groups. All statistical analyses were conducted using JMP version 12 software (SAS Institute Inc., Cary, NC, USA).

Patient characteristics
The characteristics of the enrolled patients are shown in Table 1. We observed positive expression of PD-L1 in 56 of the patients (67.0%). Additionally, 25 patients (27.5%) had EGFR mutations. All patients positive for EGFR mutation received immune checkpoint inhibitors (ICIs) subsequently developed resistance to EGFR-TKI treatment. A total of 55 patients received nivolumab, 33 patients received pembrolizumab, and three patients received atezolizumab as an ICI.

Survival analysis according to CD73 expression level
For several cutoff levels of CD73 expression, we divided the patients into two groups and compared the PFS and OS between them for each level. Both PFS (Fig 1a) and OS (Fig 1b) tended to become more favorable as the expression of CD73 increased. Patients with a TPS of ≥50% for CD73 expression had longer PFS and OS than those with a CD73 TPS of <50% (Fig 2a: median PFS in the high and low CD73 groups, 2.8 months and 1.6 months, respectively; P = 0.225. Figure 2b: corresponding median OS 11.5 months and 7.1 months, respectively; P = 0.099).

Efficacy of immune checkpoint inhibitors in EGFR mutation-positive patients
Among patients positive for EGFR mutation, the ORR in those receiving immune checkpoint inhibitors (ICIs) was significantly higher in patients with high CD73 expression than in those with low CD73 expression (60.0% vs. 6.7%, respectively, P = 0.007; Table 2). PFS was significantly longer in patients with high CD73 expression than in those with low expression after treatment with ICIs (median PFS, 7.6 vs. 1.0 months, respectively, P = 0.043 ; Fig 3a), and the OS also tended to be longer in those with high CD73 expression (median OS not reached vs. 10.4 months, respectively, P = 0.077; Fig 3b).    Fig 3d), unlike the situation in patients with EGFR mutation.

Association between CD73 expression and patient characteristics
The association between patient characteristics and CD73 expression are shown in Table 3. High CD73 expression and PD-L1 expression were significantly correlated in all patients. In EGFR mutation-positive patients, high expression of CD73 was also associated with PD-L1 expression (Table SS1). CD73 expression was not correlated with age, gender, smoking status, or performance status.

Discussion
As immune checkpoint inhibitors (ICIs) have been recently used as a new strategy for treatment in NSCLC, it is necessary to identify biomarkers that are able to predict the efficacy to anti-PD-1/PD-L1 treatment. Although the results   of preliminary phase III clinical trials of anti-PD-1/PD-L1 treatment for NSCLC have suggested that expression of PD-L1 can predict the response to this type of treatment, expression of PD-L1 is not considered to be a perfect biomarker. In this study, high expression of CD73 in NSCLC patients receiving ICIs tended to be more prognostically favorable (CD73 TPS ≥50%) than low expression (TPS <50%), particularly in patients with EGFR mutationpositive NSCLC. CD73 has been reported to be an important predictor of poor prognosis in several cancers. [10][11][12][13] To investigate the prognostic relevance of CD73 expression in EGFR mutation-positive NSCLC patients untreated with ICIs, we further screened 67 patients with stage I-III EGFR mutation-positive NSCLC who had undergone complete resection. Patients with high CD73 expression had a relatively shorter disease-free survival and OS than those with low CD73 expression ( Figure SS1). As far as we are aware, no correlation between CD73 expression and efficacy of treatment with ICIs has been previously investigated for individuals with NSCLC. The present study demonstrated that high expression of CD73 was a factor predictive of a positive response to ICIs in EGFR mutation-positive NSCLC patients. Furthermore, in patients with EGFR mutation-negative NSCLC, expression of CD73 was not associated with the outcome of treatment with ICIs, unlike the situation in patients who were EGFR mutation-positive. Our findings appear to be consistent with those of previous preclinical studies, which have shown that the mechanism of CD73-induced immune tolerance involves the EGFR signaling pathway. 15 The present study demonstrated that high CD73 expression was significantly correlated with PD-L1 expression in also patients with EGFR mutations. The results of preliminary phase III clinical trials of anti-PD-1/PD-L1 therapy against NSCLC have suggested that PD-L1 expression is predictive of the response to this type of treatment. 4,6 However, it has been reported that PD-L1 expression is an imperfect biomarker for predicting the efficacy of ICIs in EGFR mutation-positive NSCLC. 16 We analyzed the predictive value of PD-L1 expression in enrolled EGFR mutation-positive patients. PD-L1 expression was not associated with the PFS of ICIs (median PFS was 1.5 months in the PD-L1-negative group and 3.0 months in the PD-L1-positive group, P = 0.714) and OS (median OS was 15.6 months in the PD-L1-negative group and 10.4 months in the PD-L1-positive group, P = 0.592) as previously reported. 16 Furthermore, multivariate analysis showed that CD73 expression was a predictive factor for the PFS independent of PD-L1 expression (Table 4).
Recently, a new therapeutic product designed to target CD73 has been developed. MEDI9447, a human monoclonal antibody, selectively binds to CD73 and inhibits its ectonucleotidase activity. 17 On the basis of the addictive activity of MED9447 with ICIs revealed in preclinical studies, a phase I trial is currently ongoing to investigate the safety and efficacy of MEDI9447 as monotherapy, or in combination with durvalumab for advanced solid tumors. In addition, a clinical trial of MEDI9447 plus osimertinib is currently being planned for patients with previously treated EGFR mutation-positive NSCLC. The results obtained from those studies might help to clarify the clinical significance of CD73 in immune target treatment for patients with EGFR mutation-positive NSCLC.
This study had some limitations. A major point was that the number of patients analyzed was relatively small. Next, the data was collected retrospectively. Further prospective clinical researches are therefore warranted to clarify the value of CD73 expression, and the therapeutic effect of anti-PD-1/PD-L1 treatment.
In conclusion, we have showed that high CD73 expression is correlated with favorable clinical efficacy of ICIs in patients with EGFR mutation-positive NSCLC who have developed resistance to EGFR-TKI treatment. These findings may have important implications for immunotherapy in such patients.