Vascular endothelial growth factor receptor 2 expression and immunotherapy efficacy in non–small cell lung cancer

Abstract It is unclear whether tumor vascular endothelial growth factor receptor 2 expression affects the therapeutic efficacy of immune‐checkpoint inhibitors and antiangiogenic agents. This retrospective, multicenter study included patients with advanced non–small cell lung cancer who were treated with immune‐checkpoint inhibitors. We constructed tissue microarrays and performed immunohistochemistry with an anti‐vascular endothelial growth factor receptor 2 antibody. We analyzed immune and tumor cell staining separately in order to determine their correlation with the objective response rate, progression‐free survival, and overall survival in patients receiving immune‐checkpoint inhibitors. Of 364 patients, 37 (10%) expressed vascular endothelial growth factor receptor 2 in immune cells and 165 (45%) in tumor cells. The objective response rate, progression‐free survival, and overall survival were significantly worse in patients treated with immune checkpoint inhibitor monotherapy who expressed vascular endothelial growth factor receptor 2 in immune cells than those who did not (10% vs 30%, p = 0.028; median = 2.2 vs 3.6 months, p = 0.012; median = 7.9 vs 17.0 months, p = 0.049, respectively), while there was no significant difference based on tumor cell expression (24% vs 30%, p = 0.33; median = 3.1 vs 3.5 months, p = 0.55; median = 13.6 vs 16.8 months, p = 0.31). There was no significant difference in overall survival between patients treated with and without antiangiogenic agents in any treatment period based on vascular endothelial growth factor receptor 2 expression. Immune checkpoint inhibitor efficacy was limited in patients expressing vascular endothelial growth factor receptor 2 in immune cells.


| INTRODUC TI ON
Lung cancer remains the leading cause of cancer-related death worldwide. 1 Although the prognosis of lung cancer patients is gradually improving with the emergence of various therapeutic agent classes, such as molecular-targeted drugs and immune checkpoint inhibitors (ICIs), 2-6 achieving long-term survival remains a challenge.
Thus, improved therapeutic strategies need to be developed.
Increased vascularization and the expression of proangiogenic factors in tumors are associated with poor prognosis in various cancer types. [7][8][9] In particular, the vascular endothelial growth factor/vascular endothelial growth factor receptor (VEGF/ VEGFR) signaling pathway plays a key role in angiogenesis, which promotes cancer proliferation and metastasis. 10,11 The resultant aberrant vessels exhibit vascular permeability and induce hypoxia, leading to an immunosuppressive tumor microenvironment (TME). 12,13 Therapy based on the VEGF/VEGFR axis blockade has been effective in various types of cancer. [14][15][16][17] For example, bevacizumab, a humanized monoclonal antibody against VEGF-A, in combination with platinum doublet chemotherapy, was effective against advanced non-squamous non-small cell lung cancer (NSCLC). 18,19 In a phase III trial of ramucirumab, a recombinant human IgG1 monoclonal antibody against VEGFR2, combination with docetaxel achieved longer overall survival (OS) than docetaxel alone as second-line treatment for stage IV NSCLC patients experiencing disease progression following platinum-based chemotherapy. 20 Ramucirumab together with erlotinib also exhibited significant efficacy in nonsquamous NSCLC patients harboring epidermal growth factor receptor (EGFR) mutations. 21 The VEGF/VEGFR pathway negatively regulates the immune system through various mechanisms, such as the recruitment of regulatory T cells, 22,23 inhibition of dendritic cell maturation, 24 conversion of tumor-associated macrophages from the M1 to the M2 phenotype, 25 and the induction of T cell exhaustion. 26 Therefore, blocking the VEGF/VEGFR pathway represents a potential strategy for reprogramming the immunosuppressive TME and improving the efficacy of ICIs. In fact, combination treatment of anti-programmed death-ligand 1 (PD-L1) monoclonal antibody atezolizumab together with carboplatin, paclitaxel, and bevacizumab resulted in longer progression-free survival (PFS) as well as OS than without atezolizumab in advanced non-squamous NSCLC patients. 3 Thus, drugs targeting the VEGF/VEGFR pathway are expected to have a synergistic effect with ICIs. However, it is unclear which patients are most likely to experience these synergistic effects and whether the expression of VEGF/VEGFR in tumor tissues is associated with the efficacy of ICIs and antiangiogenic agents. Hence, we conducted a multicenter, retrospective study to investigate the relationship between expression of VEGFR2, the main signaling tyrosine kinase receptor of the VEGF/VEGFR pathway, and the therapeutic effects of ICIs as well as antiangiogenic agents in patients with advanced NSCLC.

| Study design and patients
We conducted a retrospective, multicenter study of advanced NSCLC patients treated with ICIs at Osaka Metropolitan University Hospital, Ishikiriseiki Hospital, and Bell Land General Hospital between December 2015 and July 2020. The protocol was approved by the institutional review boards and ethics committees of all participating institutions. We obtained written informed consent from most patients. Because the application of the opt-out method in this research is permitted under Japan's most preferential law governing clinical research, additional informed consent was obtained in the form of an opt-out on the website.
Patients who had previously received anticytotoxic T lymphocyte-associated antigen 4 therapy or durvalumab as consolidation therapy after chemoradiation, as well as those with insufficient residual tissue for creating tissue microarrays or evaluating immunostaining results due to too few tumor cells were excluded.

| Microarray construction
To prepare the tissue microarrays, formalin-fixed paraffin-embedded tumor blocks from surgically resected and biopsy specimens were collected in a routine clinical setting. The most representative tumor areas were carefully selected based on the matched H&E-stained slides and marked directly on the donor block. We then removed a tissue sample 2.0 mm in diameter from the selected region in each donor block using a manual tissue microarrayer and embedded it directly into the premade recipient block. The manual microarrayer and premade recipient block were purchased from Funakoshi Co., Ltd. A total of ten tissue microarray blocks were constructed.

| Statistical analyses
We analyzed the association of IC-VEGFR2 and TC-VEGFR2 positivity with ORR, PFS, and OS. ORR was assessed in patients who received ICI monotherapy, while PFS and OS were assessed in patients who received ICI monotherapy as well as in those who received ICI concurrently with chemotherapy. We also analyzed OS in all patients based on the administration of antiangiogenic agents in any treatment line. We compared the categorical variables using Fisher's exact test. Survival curves were estimated via the Kaplan-Meier method, and the differences between groups were compared using the log-rank test. Univariate and multi-

| Patient characteristics
We reviewed the medical records of 496 NSCLC patients who received ICI therapy at participating institutions during the study period. Eventually, immunohistochemically evaluable specimens on microarray blocks were available for 364 patients (Table 1) TC-VEGFR2 (+) group had a lower rate of EGFR mutant, had more frequently received ICI concurrent with chemotherapy, were less frequently treated with antiangiogenic agents in any treatment period, and were more frequently treated with ICI as first-line treatment than the TC-VEGFR2 (−) group (2% vs 8%, p = 0.020, 35% vs 22%, p = 0.007, 18% vs 32%, p = 0.004, 67% vs 49%, p = 0.001, respectively) ( Table 1).

| Overall survival based on the use of antiangiogenic agents
We analyzed whether there was a significant difference in the me- 55 (17) 17 (10) 42 (21) VEGFR2 in immune cells Positive Angiogenesis has major influence on immune activity in tumors, 36 and VEGF/VEGFR modulates tumor immunity both directly and indirectly. [22][23][24][25][26] Therefore, the combination of ICIs and angiogenesis inhibitors is expected to have a synergistic effect. 37 In an analysis of 20 patients with advanced gastric cancer, VEGFR2 was highly expressed in regulatory T cells, with PD-L1 expression and CD8 + T cell infiltration increasing after the administration of ramucirumab, which inhibits VEGFR2. 38 Moreover, blocking VEGFR2 in a breast cancer mouse model increased immune cell infiltration and improved the efficacy of anti-PD-1 therapy, especially at low doses of anti-VEGFR2. 39 In a meta-analysis assessing the impact of timing and the sequence of treatment with ICIs and antiangiogenic agents for NSCLC, combination therapy for both improved the ORR, PFS, and OS. However, when ICIs were administered immediately after antiangiogenic agents, there were no benefits compared with ICI monotherapy. 40 Regarding the association between VEGFR2 and PD-L1 expression, a significant positive correlation was reported in the immunohistochemical analysis of 96 patients with renal cell carcinoma 41 and 93 patients with osteosarcoma. 42 In contrast, a significant negative correlation was obtained for 92 patients with large cell neuroendocrine

ACK N OWLED G M ENTS
We would like to thank all the participating patients. Watanabe, and K. Asai have nothing to disclose.