Trophoblast Cell-surface Antigen 2 Expression in Advanced Lung Cancer Patients and the Effects of Anti-cancer Treatments

Background: Trophoblast cell-surface antigen 2 (TROP2) is expressed on the surface of trophoblast cells and many malignant tumor cells. However, data on TROP2 expression in advanced lung cancer is insucient, and its changes have not been fully evaluated. Methods: We assessed the prevalence and changes in TROP2 expression in lung cancer patients receiving anti-cancer treatments using immunohistochemical (IHC) analysis with an anti-TROP2 (clone: SP295). IHC scores were graded from 0–3; grade ≥ 2 was considered positive for TROP2 expression. We dened a difference in IHC score, before and after anti-cancer treatments, as the change in TROP2 expression. Results: Before anti-cancer treatment, TROP2 expression was observed in 89% (143/160) of patients and was signicantly more common in adenocarcinoma and squamous cell carcinoma than in neuroendocrine carcinoma (P < 0.001). After anti-cancer treatment, TROP2 expression was observed in 87% (139/160) of patients. The distribution of TROP2 expression in post-treatment samples was analogous to that in pre-treatment samples when compared using the Wilcoxon signed-rank test (P = 0.509). However, an increase in TROP2 expression was seen in 19 (12%), and a decrease in 20 (13%) patients. Patients treated with targeted therapy showed signicantly higher changes in TROP2 expression (P = 0.019) and thoracic radiotherapy was more likely to increase TROP2 expression than chemotherapy alone. Conclusion: TROP2 was expressed in most lung cancer specimens before and after anti-cancer treatments. Additionally, some anti-cancer treatments might alter the TROP2 expression. These results may provide a strong rationale for TROP2-directed therapy against advanced lung cancer.


Introduction
Trophoblast cell-surface antigen 2 (TROP2), also known as tumor-associated calcium signal transducer 2 (TACSTD2) or epithelial glycoprotein-1 (EGP-1), is a single-pass transmembrane glycoprotein that is expressed on the surface of trophoblast cell and many malignant tumor cells (Cubas et  Although its function has not been fully clari ed, previous reports suggest that overexpression of TROP2 has an anti-apoptotic effect and promotes tumor survival by regulating the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway or phosphoinositide 3-kinase/protein double-stranded DNA breaks, eventually leading to apoptosis. Previous studies have shown that sacituzumab govitecan has been effective for triple-negative breast cancer, metastatic small cell lung cancer, and metastatic non-small cell lung cancer (NSCLC) that are resistant to other cytotoxic chemotherapy (Bardia et al. 2017; Bardia et al. 2019; Gray et al. 2017; Heist et al. 2017). DS-1062 is also a TROP2-directed ADC that contains a derivative of exatecan (DX-8951), which is a novel type I topoisomerase inhibitor, and has demonstrated clinical activity in advanced NSCLC (Sands et al. 2019).
Currently, clinical trials are ongoing to evaluate the safety and e cacy of DS-1062 in advanced NSCLC patients.
According to previous reports, TROP2 expression has been associated with various clinicopathological features and prognosis, in lung cancer patients (Inamura et al. 2017;Jiang et al. 2013;Kobayashi et al. 2010;Pak et al. 2012). However, information on TROP2 expression in advanced lung cancer is insu cient because previous publications have mainly targeted surgical cases, such as early stage lung cancer. Therefore, it is necessary to investigate the prevalence of TROP2 expression in lung cancer tumor cells in a cohort comprising advanced lung cancer patients. In addition, it is still unclear whether TROP2 expression is altered after therapeutic intervention. In this study, we aimed to assess the prevalence of TROP2 expression in lung cancer specimens, before and after administering various anti-cancer treatments, in an advanced lung cancer population.

Patients
Retrospectively, we screened 169 consecutive patients from the medical records to identify those who were histologically diagnosed with lung cancer and from whom tumor specimens were obtained after treatment, at the Shizuoka Cancer Center between July 2003 and June 2017. This study was approved by the Institutional Review Board of Shizuoka Cancer Center (Approval No. T29-17-29-1; July 27, 2017).
This study included patients with formalin-xed para n-embedded tumor tissue samples su cient to evaluate TROP2 expression. Pleural uid cell blocks were evaluated as well as biopsy or surgical tissue samples. If a patient had three or more samples, two samples were selected: one taken at diagnosis and the other obtained after anti-cancer treatment.
Patients who had received one or more anti-cancer treatments, including cytotoxic chemotherapy, epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI), anaplastic lymphoma kinase (ALK) inhibitor, and curative thoracic radiotherapy, were eligible. Patients who only received surgical resection or palliative radiotherapy were excluded from this study. We evaluated the changes in TROP2 expression before and after anti-cancer therapeutic intervention. Overall survival (OS) was de ned as the time from histological diagnosis of lung cancer to the date of death or last contact.

Immunohistochemical assessment
Patients' formalin-xed para n-embedded samples were sectioned at a thickness of 3 μm, mounted onto glass slides, and incubated with an anti-rabbit monoclonal antibody against TROP2 (clone: SP295, Daiichi Sankyo Co., Ltd., Tokyo, Japan; diluted 1:72 and clone: SP295, Spring Bioscience Co., California, USA; diluted 1:50). All of the slides were processed on the Autostainer Bond-III platform (Leica Biosystems) and visualized with a Leica Bond Polymer Re ne Detection kit (DS9800, Bond Polymer Re ne Detection Leica). The sections were incubated at a pH of 9 for 20 min at 100°C. After washing in a wash buffer, the slides were incubated for 30 min at room temperature with the primary antibody. The nuclei were lightly counter-stained with Mayer's hematoxylin. Positive and negative staining controls were provided by Daiichi Sankyo Co., Ltd., Tokyo, Japan.
A blinded histopathological evaluation of TROP2 expression on the membrane of tumor cells was interpreted by two pathologists (Takuya Kawata and Takashi Sugino), with no prior clinical information.
Tumor cells with stained cell membranes of the samples were evaluated, and the immunohistochemistry (IHC) scores were graded from 0 to 3 based on IHC human epidermal growth factor receptor 2 (HER2) testing for gastric cancer, as recommended in the American Society of Clinical Oncology/College of American Pathologists Clinical Practice guideline (Bartley et al. 2017). In this study, grade ≥ 2 was considered to be positive for TROP2 expression. In case of a discrepancy, the pathologists reviewed their assessments and established a consensus. We de ned a difference in the IHC score before and after anticancer treatment as the change in TROP2 expression. Additionally, the proportions of changes in IHC scores were evaluated for each anti-cancer treatment.

Statistical methods
To analyze the correlations between TROP2 expression and patient characteristics, we used Fisher's exact test for categorical variables. The distribution of TROP2 expression between pre-treatment samples and post-treatment samples was compared using the Wilcoxon signed-rank test. Furthermore, we evaluated OS using the Kaplan-Meier method and compared it among lung adenocarcinoma patients diagnosed as stage IV using the log-rank test. Because this study population included various histological types and stages, we selected and analyzed the most predominant population, that is, stage IV adenocarcinoma patients. P values of less than two-sided 0.05 were considered to be statistically signi cant. All analyses were implemented by JMP 10 for Windows statistical software (SAS Institute Japan Inc., Tokyo, Japan).

Patient characteristics
Among the 169 patients who were screened, 9 patients were excluded because the cancer cells in their specimen were not adequate for evaluation; thus, 160 patients were included in this study. The patient characteristics are listed in Table 1. The median patient age at diagnosis was 65 years (range, 39-89 years). Of the 160 patients, 92 (58%) were male, 49 (31%) had never smoked, 130 patients (81%) were diagnosed with adenocarcinoma, 19 (12%) with squamous cell carcinoma, and 8 (5%) with neuroendocrine carcinoma. Fifty patients (31%) were diagnosed as having stage I or II lung cancer, and stage III or IV lung cancer was observed in 110 (69%) patients. Among the 130 adenocarcinoma patients, 42 patients had tumors with deletions in EGFR exon 19, 30 carried the L858R mutation in EGFR exon 21, 3 carried the G719X mutation in EGFR exon 18, and 1 carried an insertion mutation in EGFR exon 20. Among the 45 patients with EGFR wild type adenocarcinoma, 7 had ALK-positive lung cancer. Genetic alterations were unknown in 9 patients.

Samples and anti-cancer treatments
The breakdown of pre-treatment samples was as follows: 95 (59%) biopsy samples, 58 (36%) surgically resected specimens, and 7 (5%) pleural uid cell blocks. Post-treatment samples included 104 (65%) biopsy samples, 30 (19%) pleural uid cell blocks, 21 (13%) surgically resected specimens, and 5 (3%) autopsy samples. Pleural uid cell blocks were more frequently seen in post-treatment samples compared to pre-treatment samples. Table 2 shows the anti-cancer treatments that patients received before their post-treatment samples were obtained. In their clinical course, 123 (77%) patients received cytotoxic chemotherapy, including postoperative adjuvant chemotherapy, and 67 (42%) received targeted therapy, including EGFR-TKI and ALK-TKI. Thirty-one (19%) patients received radiation therapy for the primary lesion. However, only 5 (3%) patients were treated with immune checkpoint inhibitors as most patients in this cohort were treated before the approval of immune checkpoint inhibitors in Japan.
TROP2 expression in pre-treatment samples The relationship between patient demographics and TROP2 expression in pre-treatment samples is listed in Table 1. TROP2 was signi cantly more expressed in adenocarcinoma and squamous cell carcinoma samples than in neuroendocrine carcinoma (P < 0.001). While, there was no signi cant correlation between TROP2 expression and age, sex, smoking status, stage at diagnosis, or the status of EGFR mutation. Similarly, among 110 patients that were diagnosed with stage III or IV lung cancer, positive TROP2 expression was observed in 97 (88%) patients. However, no signi cant correlation was observed between TROP2 expression and clinical characteristics except for histology (Table 3).

Changes in TROP2 expression
Positive TROP2 staining of tumor cells (IHC score 2 or 3) in post-treatment samples was observed in 139 (87%) patients. Among 110 patients with stage III or IV lung cancer, positive TROP2 expression was observed in 94 (85%) patients. The distribution of TROP2 expression of the post-treatment samples was analogous to those in pre-treatment samples when compared using the Wilcoxon signed-rank test (P = 0.509). However, an increase in TROP2 expression was seen in 19 (12%) patients, while a decrease was observed in 20 (13%) other patients (Table 4). In adenocarcinoma patients, IHC scores of TROP2 expression were changed by approximately 30%. Among them, a positive or negative conversion of TROP2 expression was observed in 11 (8%) patients, respectively.
Regarding the evaluation of anti-cancer treatment intervention, 12 (10%) of 123 patients treated with cytotoxic chemotherapy showed an increase in TROP2 expression, while a decrease was seen in 15 (12%) patients ( Figure 2). Six (19%) of 31 patients treated with thoracic radiotherapy showed an increase in TROP2 expression, while a decrease in the same was not observed. Among 67 patients treated with targeted therapy, consisting of EGFR-TKI or ALK-TKI, 10 (15%) patients showed an increase in TROP2 expression, and 14 (21%) showed decreased TROP2 expression. Of these patients that were treated with targeted therapy, positive or negative conversion in TROP2 expression occurred in 6 (9%) patients. Of 63 NSCLC patients with EGFR mutations who were treated with EGFR-TKI, an increase in TROP2 expression was observed 9 (14%) patients, while a decrease was observed in 14 (22%). While comparing each anticancer treatment, patients treated with targeted therapy alone showed a signi cant change in TROP2 expression compared to those treated with chemotherapy alone (P = 0.019). Similarly, patients treated with thoracic radiotherapy were more likely to have an increased TROP2 expression than those undergoing chemotherapy alone (P = 0. 063).

Survival analysis in stage IV adenocarcinoma patients
The median follow-up time from diagnosis to censored case was 50.2 months in 71 patients with stage IV adenocarcinoma. There was no signi cant difference in OS between patients with high TROP2 expressing tumors (IHC score 3) and those without high TROP2 expressing tumors (IHC score 0, 1, 2) (median 38.1 months versus 46.2 months; P = 0.266; Fig. 3A). Similarly, no signi cant difference in OS was observed in patients with stage IV EGFR-mutated adenocarcinoma, who harbored high TROP2 expression and underwent EGFR-TKI treatment, when compared to those without high TROP2 expression (median 43.4 months versus 50.3 months, P = 0.329; Fig. 3B).

Discussion
In our study, of the 160 NSCLC patients, TROP2 expression was observed in 89% of pre-treatment samples. Furthermore, there was no signi cant change in the prevalence of TROP2 expression in tumor cells in pre-and post-treatment samples (P = 0.509). However, while assessing individual patients, changes were observed in 39 (24%) patients receiving anti-cancer treatments during their clinical course.
To the best of our knowledge, no reports that evaluated the changes in TROP2 expression among lung cancer patients, including advanced-stage lung cancer, have been published. Our analysis contributes novel data on TROP2 expression in patients with advanced lung cancer, adding value to the existing body of TROP2 research.
In previous reports, TROP2 expression in lung cancer has been described using archived surgically resected specimens. Inamura K, et al. reported the association of TROP2 expression in tumor cells by investigating the clinicopathological or molecular characteristics and prognosis in lung cancer patients.
Their results depicted a high TROP2 expression in 64% of adenocarcinoma, 75% of squamous cell carcinoma, and 18% of high grade neuroendocrine carcinoma patients using a rabbit monoclonal anti-TROP2 antibody (Clone 1E5-1E2) (Inamura et al. 2017). Pak MG et al. also reported that patients with lung squamous cell carcinoma showed signi cantly higher TROP2 expression in tumor cells, compared to patients with lung adenocarcinoma (64% vs 23%, P < 0.01) (Pak et al. 2012). Although the de nitions of positive TROP2 expression were different, these results are consistent with our ndings.
TROP2-directed ADCs have been reported to have potential as new therapeutic agents for lung and breast cancers (Gray et al. 2017;Heist et al. 2017). In a recent phase I study of a TROP2-directed ADC (DS-1062), drug clinical activity was observed irrespective of the levels of TROP2 expression (Lisberg et al. 2020). However, considering the mechanisms of action of ADC treatments, there is no doubt that the status of TROP2 expression in tumor cells plays an essential role in TROP2-directed ADC treatment. Our study demonstrated that TROP2 was expressed in most lung cancer specimens, before and after various anticancer treatments. This stable high expression rate of TROP2 in advanced lung cancer, during the clinical course, supports the rationale for TROP2-directed ADC treatments.
Although we observed that the frequency of TROP2 expression did not signi cantly differ after treatment, changes in IHC score were observed in approximately 20-30% of patients. Considering the results of our research, we hypothesize that anti-cancer treatments, including chemotherapy and radiotherapy, may change TROP2 expression in some lung cancer patients. Concerning the pathophysiology of changes in TROP2 expression, Zhao P et al. reported that tumor necrosis factor-alpha (TNF-α) can promote cancer cell migration and invasion by upregulating TROP2 expression (Zhao and Zhang 2018). Induction or reduction of TNF-α by therapeutic intervention may result in an altered tumor microenvironment, and consequently, change the TROP2 expression. In our study, patients that were only treated with targeted therapy showed a signi cant change in TROP2 expression than those who underwent chemotherapy alone (P = 0.019). Additionally, patients treated with radiotherapy were more likely to show an increase in TROP2 expression than those treated with chemotherapy alone (P = 0.063). Thus, targeted therapy and radiotherapy may enhance TNF-α production, as opposed to chemotherapy, and that may lead to increased TROP2 expression. However, in this retrospective study, we used archived tumor tissues, and therefore, we could not evaluate the changes in TNF-α. Since the pathophysiology of TROP2 expression is not fully interpreted, further research is needed to investigate whether TROP2 expression may change spontaneously, during the clinical course, regardless of intervention by anti-cancer treatments.
Regarding survival analysis, although there was no signi cant difference, we observed a potential for worse survival outcomes in patients with high TROP2-expressing lung adenocarcinoma, including EGFRmutated adenocarcinoma. This is consistent with previously reported ndings in lung cancer patients expression is a prognostic factor in adenocarcinoma patients without EGFR mutation as well as those with a high histological grade (Mito et al. 2020). They also reported an absence of correlation between TROP2 expression and prognosis for adenocarcinoma patients harboring EGFR mutation. Since more than 70% of the patients were stage 0 or 1, we assume that there was no correlation between TROP2 expression and survival in adenocarcinoma patients harboring EGFR mutation in their cohort (Kaplan-Meier OS curves had less than 20% of death events during the observation period) (Mito et al. 2020). Previous systematic reviews, involving other solid cancers, have also shown that TROP2 expression is associated with having a poor prognosis (Xu et al. 2017;Zeng et al. 2016). Conversely, Pak et al. suggested that TROP2 expression is associated with favorable OS in lung adenocarcinoma patients (Pak et al. 2012). The cause of such a discrepancy in lung cancer is still unknown and therefore, further studies are required.
Our study had several limitations. First, it was a single-institutional, retrospective study. Second, the patient characteristics were heterogeneous: we included patients in various cancer stages, undergoing various anti-cancer treatments. Third, it may not show an accurate prevalence of TROP2 expression, especially in small biopsy samples due to spatial heterogeneity in TROP2 expression within different regions of the same tumor tissue. To the best of our knowledge, present-day data showing intra-tumoral heterogeneity of TROP2 expression in lung cancer have not been reported. However, given the complexity of tumor microenvironment, it is likely that intra-tumoral heterogeneity of TROP2 expression may exist. Concerning the level of TROP2 expression based on the type of tumor specimens, there was no signi cant difference between surgically resected specimens and biopsy samples in our study. Therefore, even if intra-tumoral heterogeneity of TROP2 expression does exist, its assessment using biopsy samples would be acceptable. Fourth, as previously described, other molecules, such as TNF-α, were not evaluated, which may be involved in the pathophysiology of altered TROP2 expression.
In conclusion, TROP2 was expressed in most lung cancer specimens of both the early and advanced stages, particularly in those of adenocarcinoma and squamous cell carcinoma. Although there was no signi cant change in the prevalence of TROP2 expression in pre-and post-treatment samples, anti-cancer treatments, especially targeted therapy and radiotherapy, might alter TROP2 expression in advanced lung cancer patients. These results support the rationale for TROP2-directed therapy, which could yield a therapeutic effect against advanced lung cancer in different lines of treatment after various therapeutic interventions.

Declarations
Funding: This study was supported by a contract research fund from Daiichi Sankyo Co., Ltd., Tokyo, Japan.

Con icts of interest/Competing interests:
The authors declare no con ict of interest directly relevant or directly related to the content of this article.

Availability of data and material:
The remaining data that support the ndings of this study are available from the corresponding author upon reasonable request. The data are not publicly available due to privacy or ethical restrictions.
Code availability:

Not applicable
Authors' contributions: Shota Omori was involved in study concept and design; acquisition, analysis, or interpretation of data; drafting of the manuscript; full access to all data in the study and responsible for the integrity of the data and accuracy of the data analysis. Koji Muramatsu, Takuya Kawata, and Takashi Sugino were involved in pathological support. Eriko Miyawaki, Taichi Miyawaki, Nobuaki Mamesaya, Takahisa Kawamura, Haruki Kobayashi, Kazuhisa Nakashima, Kazushige Wakuda, Akira Ono, Tateaki Naito, Haruyasu Murakami were involved in material support. Hirotsugu Kenmotsu was involved in administrative, technical or material support. Toshiaki Takahashi was involved in study supervision. All authors read and approved the nal manuscript.
Ethics approval: This study was approved by the Institutional Review Board of the Shizuoka Cancer Center, Japan (Approval No. T29-17-29-1; July 27, 2017).

Consent to participate:
An opt-out method was used on the basis of the Ethical Guidelines for Epidemiological Research in Japan.

Consent for publication:
Not applicable  Table 2 The anti-cancer treatments that patients received before posttreatment samples were obtained