Targeted therapy has significantly improved the outcome for patients with lung cancer in the last decade . For example, EGFR is a major driver of NSCLC tumorigenesis , and tumour growth can be inhibited by treating lung tumours expressing somatic mutations of the EGFR gene with TKIs. This strategy revealed the potential for precise biomarker-directed and personalized treatments for lung cancer [34, 35]. However, EGFR status is determined by histological tumour biopsy, and it is not always possible to obtain a representative biopsy suitable for precise histopathology because of tumour localization and heterogeneity, and small tumour specimens. Selecting TKI-sensitive patients thus remains a challenge, highlighting the need for alternative (preferably non-invasive) means of patient selection. We previously validated the ability of 99mTc-Galacto-RGD2 to identify iodine-refractory status in patients with thyroid cancer . In a rare case with a solitary fibrous tumour located in the main pulmonary artery, 99mTc-Galacto-RGD2 imaging played an important role in detecting the primary tumour and predicting the metastatic potential . In the current study, we evaluated the use of 99mTc-Galacto-RGD2 SPECT/CT for the detection of lung cancer. We also explored the expression of integrin αvβ3 and CXCR4 in different lung cancer subtypes, and compared the neovasculature among these subtypes. We also examined the correlations between tumour uptake of 99mTc-Galacto-RGD2 and integrin αvβ3 expression and neovascularization. Finally, we validated the use of integrin molecular imaging as a surrogate for phenotyping.
High-contrast images acquired 1 h after injection of 99mTc-Galacto-RGD2 showed a significantly higher T/NT ratio in malignant compared with benign lung lesions. Malignant primary tumours and metastatic lymph nodes showed higher focal uptake, while benign lesion showed significantly lower uptake. 99mTc-Galacto-RGD2 SPECT/CT showed high sensitivity for detecting primary tumours and remote metastases. ROC analysis showed a sensitivity and accuracy of 91.89% and 86.67%, respectively, for 99mTc-Galacto-RGD2 SPECT/CT, using a cut-off value of 2.5. However, the specificity for differentiating between malignant and benign disease was limited, possibly because of the involvement of integrin αvβ3 in various benign diseases. Overlap usually occurs between tuberculosis and inflammatory pseudo-tumours, which usually show higher uptake of 99mTc-Galacto-RGD2 than other types of benign diseases, such as pneumonia .
In the current study, IHC showed that αvβ3 levels were higher in advanced lung cancer, and proliferation index, represented by Ki-67, was significantly increased in advanced stages of SCLC, associated with metastatic potential [18, 24, 37]. Patients with lung cancer, even in the early stages, may develop multiple metastases several months after thorough tumour resection, possibly related to specific tumour types with higher metastatic potential. In the current study, CXCR4 expression levels were higher in lung cancer compared with benign disease, though the differences were not significant. Its expression was correlated with both integrin αvβ3 and CD31 expression in primary lung tumours, while integrin αvβ3 was also correlated with CD31. These findings validate our hypothesis that lymphadenopathy and remote metastasis are mediated by specific biological molecules. Integrin αvβ3 and CXCR4 may mediate angiogenesis, which may further promote lymph node and remote metastases. Imaging targeting integrin αvβ3 may thus improve our understanding of the interactions between cancer cells and their microenvironment, which is a necessary prerequisite for the development of treatment strategies specifically targeting cancer-induced invasion and metastases. This information is significant in light of the correlations of integrin αvβ3 overexpression with recurrence and poor prognosis, and in relation to early diagnosis and treatment-response monitoring. These findings demonstrated that expression levels of integrin αvβ3 were strongly correlated with tumorigenic and aggressive behaviours in lung cancer cells. CXCR4 has been implicated in the chemotactic migration of cancer cells . CXCR4 and integrin might synergistically promote lymphatic metastasis in lung cancer, and might act as clinical predictors of lymph node metastasis in NSCLC [38–40]. High expression levels of chemokines are related to a poor prognosis and chemotherapy tolerance in cancer patients [41–44]. CXCR4 is a chemokine receptor that plays a critical role in the process of lymphocyte homing to lymphatic vessels and secondary lymphoid organs, including the lymph nodes .
Integrin αvβ3 was expressed not only in the tumour cells, but also in the endothelium, though there was a lack of a correlation between tumour uptake of 99mTc-Galacto-RGD2 and integrin αvβ3 expression because of the heterogenicity of lung cancer, however, both 99mTc-Galacto-RGD2 imaging and integrin αvβ3 expression behaved well in distinguishing lung cancer and benign lung disease. We supposed that tumour uptake of 99mTc-Galacto-RGD2 was related to integrin αvβ3 expression, neovascularization, and tumour stage. Integrin αvβ3 expression in tumour cells promoted lymphatic and distant metastases, as observed (Fig. 1). However, benign diseases showed variable degrees of angiogenesis, also associated with higher expression of integrin αvβ3, as shown in one patient with thymus adenoma and in another with pulmonary sequestration (Figs. 3, 4). We hypothesized that tumour uptake of 99mTc-Galacto-RGD2 depended on the neovasculature and integrin αvβ3 expression in the tumour cell, and focal uptake in RGD-targeted imaging would thus be higher in primary tumours with more neovasculature and higher integrin αvβ3 expression. Regarding the different subtypes of lung cancer, LSC usually had more neovascularization and higher integrin αvβ3 expression, followed by LAC, while SCLC usually showed less neovascularization and a higher proliferation index. The highest T/NT ratio was therefore found in LSC (8.53), and was significantly higher than that in LAC and SCLCs (6.84 and 4.73, respectively) (Fig. 2). RGD-targeted imaging may thus serve as a useful tool for the phenotyping of lung cancer, which will in turn be important for helping to select suitable treatment strategies.
In conclusion, this was the first extensive longitudinal study to investigate the expression of integrin αvβ3 in lung cancer. 99mTc-Galacto-RGD2 imaging showed high sensitivity for the detection of primary lung cancer, but limited specificity. 99mTc-Galacto-RGD2 uptake in the primary tumour was attributed to integrin αvβ3 expression in the endothelial cells and tumour cells, and greater focal uptake occurred in primary lung cancers with more neovascularization and high levels of integrin αvβ3 in the tumour cells. LSC had a higher density of neo-vessels and higher integrin αvβ3 expression, followed by LAC and then SCLC. Furthermore, advanced lung cancer showed higher expression levels of integrin αvβ3 compared with early stages, and higher integrin αvβ3 and CXCR4 expression in the tumour cells may mediate lymphatic and distant metastases. These two molecules might thus serve as independent predictors of patient prognosis. These findings suggest that RGD based imaging might be a useful tool for lung cancer phenotyping and for evaluating tumour biological behaviours, such as aggressiveness. integrin αvβ3 targeted imaging might thus be a valuable tool to aid the selection of molecular targeted treatment strategies. However, further studies are needed to validate the current findings and to address the issue that tumour specimens suitable for IHC are not obtained from all patients.