[99mTc]Tc-Galacto-RGD2 integrin αvβ3-targeted imaging as a surrogate for molecular phenotyping in lung cancer: real-world data

Background Epidermal growth factor receptor tyrosine kinase inhibitors (TKIs) are beneficial in patients with lung cancer. We explored the clinical value of [99mTc]Tc-Galacto-RGD2 single-photon emission computed tomography (SPECT/CT) in patients with lung cancer, integrin αvβ3 expression, and neovascularization in lung cancer subtypes was also addressed. Methods A total of 185 patients with lung cancer and 25 patients with benign lung diseases were enrolled in this prospective study from January 2013 to December 2016. All patients underwent [99mTc]Tc-Galacto-RGD2 imaging. The region of interest was drawn around each primary lesion, and tumour uptake of [99mTc]Tc-Galacto-RGD2 was expressed as the tumour/normal tissue ratio(T/N). The diagnostic efficacy was evaluated by receiver operating characteristic curve analysis. Tumour specimens were obtained from 66 patients with malignant diseases and 7 with benign disease. Tumour expression levels of αvβ3, CD31, Ki-67, and CXCR4 were further analysed for the evaluation of biological behaviours. Results The lung cancer patients included 22 cases of small cell lung cancer (SCLC), 48 squamous cell carcinoma (LSC), 97 adenocarcinoma (LAC), and 18 other types of lung cancer. The sensitivity, specificity, and accuracy of [99mTc]Tc-Galacto-RGD2 SPECT/CT using a cut-off value of T/N ratio at 2.5 were 91.89%, 48.0%, and 86.67%, respectively. Integrin αvβ3 expression was higher in non-SCLC compared with SCLC, while LSC showed denser neovascularization and higher integrin αvβ3 expression. Integrin αvβ3 expression levels were significantly higher in advanced (III, IV) than early stages (I, II). However, there was no significant correlation between tumour uptake and αvβ3 expression. Conclusions [99mTc]Tc-Galacto-RGD2 SPECT/CT has high sensitivity but limited specificity for detecting primary lung cancer, integrin expression in the tumour vessel and tumour cell membrane contributes to the tumour uptake.


Background
Lung cancer is the leading cause of cancer mortality worldwide [1,2]. The incidence and mortality of lung cancer in China have increased rapidly in the last three decades, associated with increases in air pollution and tobacco consumption [3,4]. However, new clinical treatment strategies, such as antiangiogenic epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) and immunotherapy, have significantly improved the outcomes of patients with lung cancer in the last decade [5]. TKIs have a cytostatic effect on tumour cells by slowing their growth and preventing the development of distant metastases [6,7]. Multiplex genetic sequencing has been used to select appropriate treatment, based on the recommendation of the American Society of Clinical Oncology (ASCO); however, this requires obtaining enough tumour tissue by biopsy or surgery. Unfortunately, suitable tumour specimens are unavailable for some patients due to the tumour heterogeneity or undetermined primary lesion.
Radiolabelled RGD peptide as a target ligand for angiogenesis imaging has been well documented in preclinical and clinical studies [12,21,22]. In a previous multicentre study, we showed that [ 99m Tc]Tc labelled RGD dimers, such as [ 99m Tc]Tc-3PRGD 2 , had high sensitivity for the detection of lung cancer, including primary and metastatic tumours [21,23,24]. [ 99m Tc] Tc-Galacto-RGD 2 , with higher affinity to α v β 3 and a favourable biodistribution, has been synthesized and utilized for the quantitative evaluation of α v β 3 expression and of tumour angiogenesis [25].
Clinically, multiple lymphadenopathy and remote metastasis were developed rapidly in higher aggressive lung cancer even with radical resection and comprehensive treatment, we suppose some key molecules medicate the tumour development and metastasis. Therefore, we conducted a longitudinal study to evaluate the clinical role of [ 99m Tc]Tc-Galacto-RGD 2 SPECT/CT in a large population of patients with lung neoplasms. We also explored the expression of integrin α v β 3 protein in tumour cells and in the neovasculature, and determined the capability of the technique to detect lymphadenopathy and bone metastasis in patients with advanced lung cancer. Herein, we investigated the value of RGD-based imaging as a surrogate for molecular phenotyping in lung cancer.

Patients
This prospective, single-centre study enrolled patients referred to our centre with suspected lung neoplasms from January 2013 to December 2016. [ 99m Tc]Tc-Galacto-RGD 2 SPECT/CT was performed in all patients; the final diagnosis was confirmed by histopathology based on acupuncture biopsy or surgery. A total of 210 consecutive patients (147 male, 63 female; mean age 63.80 ± 10.51 years, range 21-85 years) were enrolled and analysed. Of the 210 patients, 185 were confirmed with lung cancer and the other 25 patients had benign pulmonary diseases and served as the control. Patients who had undergone perioperative chemotherapy or radiotherapy were excluded from this study; the schema of study is shown in Fig. 1.

[ 99m Tc]Tc-Galacto-RGD 2 imaging and interpretation
The radiochemical purity was 95.1% ± 2.9%. [ 99m Tc] Tc-Galacto-RGD 2 was administered at 555-740 MBq (15-20 mCi) and whole-body images were acquired at 1-h post-injection. The chest images, including the upper abdomen and adrenal glands, were performed using a combined transmission and emission device with x-ray tube and detector. All-purpose collimator centred on the140-keV energy peak with a 20% symmetrical energy window. Thirty projection images were acquired over a 180° arc at 6° intervals for each SPECT head. The acquisition time was 30 s at each projection. The transaxial data were reconstructed using Ordered Subset Expectation Maximization, 2 iterations, 8 subsets (Symbia T6 SPECT/ CT; Siemens AG, Germany). Anatomic CT images were performed for attenuation correction and tumour localization. If unexpected lesions were detected by wholebody imaging, additional abdomen or pelvis images were also acquired.
All images were interpreted independently on the computer monitor in three orthogonal planes by nuclear medicine physicians and a radiologist who were unaware of the clinical information and other imaging examinations. Significantly greater local uptake of [ 99m Tc] Tc-Galacto-RGD 2 compared with the adjacent surrounding lung was interpreted as demonstrating a malignant lesion, and uptake less than or equal to the adjacent or surrounding lung was interpreted as a benign lesion. Focal activity in the hilum and mediastinum greater than the surrounding mediastinal activity was interpreted as lymphadenopathy. Regions of interest (ROI) were drawn around the primary lesion and contralateral lung tissue, respectively, and [ 99m Tc]Tc-Galacto-RGD 2 uptake was measured and expressed as the tumour/normal tissue ratio (T/N).

Composite reference standard
All available cytologic, histologic, follow-up, and imaging findings were used as a composite reference standard for the presence of tumour lesions. This is considered the optimal gold standard because cytologic or histologic verification of every lesion was not feasible or justifiable in these patients. Whenever possible, new findings on [ 99m Tc]Tc-Galacto-RGD 2 SPECT-CT were verified by additional investigations.

Statistical analysis
All statistical analyses were carried out using R software (version 3.6.1) and graphs were constructed using GraphPad Prism software (version 7). Continuous variables with a non-normal distribution were expressed as median (interquartile range). Differences in T/NT and protein expression levels among groups were compared using Wilcoxon's rank-sum or Kruskal-Wallis tests. The sensitivity, specificity, area under the curve (AUC), and cut-off value of T/NT were evaluated by receiver operating characteristic curve (ROC) analysis. Correlations between continuous variables with non-normal distributions were evaluated by Spearman's rank correlation analysis. Bonferroni's correction was applied for multiple comparisons. Statistical significance was established at p < 0.05.

Patient characteristics
The clinical characteristics of the patients are shown in Table 1

[ 99m Tc]Tc-Galacto-RGD 2 imaging and interpretation
High-contrast images acquired 1 h after injection of [ 99m Tc]Tc-Galacto-RGD 2 showed higher focal uptake in malignant primary tumours and metastatic lymph nodes (Fig. 2), compared with significantly lower uptake in benign lesions, the ratio (median (interquartile range)) of T/N in malignant disease was 6.84 (4.62, 9.86), whereas that of benign diseases was 2.53 (1.24, 3.91), p < 0.01. We also compared the uptake in different lung cancer subtypes (Fig. 3).   also compared uptake by the primary tumour between locoregional and advanced stages. T/N was significantly lower in stage I-II (5.78 (3.62, 7.95)) compared with advanced stages (III -IV; 7.28 (5.43, 10.34)), p < 0.01. However, there was overlap with inflammatory pseudotumours or tuberculosis. RGD avidity was found in in two cases of pulmonary sequestration and thymoma, respectively, due to higher density of micro-vessels (Figs. 4, 5). ROC analysis indicated that the sensitivity, specificity, and accuracy of [ 99m Tc]Tc-Galacto-RGD 2 were 91.89%, 48.0%, and 86.67%, respectively, using a cut-off value of 2.5. With a T/N cut-off value of 3.94, the AUC was 0.83 and the sensitivity and the specificity were 82.7% and 76.0%, respectively.

Histopathology and IHC
Of the 210 patients with suspected lung cancer, immunohistochemistry (IHC) was performed in 66 patients with lung cancer and seven patients with benign diseases. Expression levels (median (interquartile range)) of integrin α v β 3 were significantly higher in lung cancer (OD: 15,020.5 (4482.6, 44,455.2)) compared with benign diseases (OD: 1797.8 (794.0, 2943.6); p < 0.01) ( Table 2). CD31 levels were also elevated in lung cancer (OD: 21.9 (13.75, 34.35) vs 9.00 (8.90, 11.50); p < 0.01). Higher levels  , p < 0.05. Integrin α v β 3 was highly expressed not only in endothelial cells in the neovasculature, reflected by CD31 expression, but also in tumour cells (Fig. 6), with a higher density of neovasculature and integrin α v β 3 expression in the primary tumour. Integrin α v β 3 was also significantly correlated with CD31 expression in lung cancer (r = 0.30, p = 0.016). However, there was no correlation between tumour uptake of [ 99m Tc]Tc-Galacto-RGD 2 and integrin α v β 3 expression in the primary tumour in this study (Fig. 7). Squamous lung cancer usually showed higher level of α v β 3 in the tumour cell and the higher density of microvessel, which was consistent with RGD imaging as shown in Fig. 8. Aggressive LAC tends to higher express integrin α v β 3 in the tumour cell and has denser microvessels, which showed focal uptake in the RGD image, as shown in Fig. 9. Neo-vascularization varied in benign respiratory diseases, associated with higher integrin α v β 3 expression. In the current study, integrin α v β 3 correlated with CD31 expression in the neo-vessel, indicating that integrin α v β 3 mediated angiogenesis, leading to tumour development and metastasis. We also examined CXCR4 expression. CXCR4 was highly expressed in lung cancer, as demonstrated by IHC. Furthermore, expression levels of CXCR4 tended to be positively correlated with integrin α v β 3 levels in lung cancer specimens (r = 0.22, p > 0.05). In addition, the proliferation index (Ki-67, median (interquartile range)) in LSC and SCLC (27.45 (11.88, 42.00) and 70.00 (55.13, 73.48), respectively) were both significantly higher than in LAC (10.15 (2.98, 27.89)) ( Table 3).

Lymphadenopathy and distant metastasis
Of the 185 patients with lung cancer, 116 patients had lymphadenopathy, 87 had remote metastasis, 17 had multiple lung tumours including pleural invasion, and 70 patients had bone metastasis. The metastatic lymph However, although lymphadenopathy was evaluated by imaging follow-up, the final diagnosis was not confirmed, and we were therefore unable to evaluate the diagnostic value of [ 99m Tc]Tc-Galacto-RGD 2 imaging for lymphadenopathy and remote metastasis in this study.

Discussion
We previously validated the ability of [ 99m Tc]Tc-Galacto-RGD 2 to identify iodine-refractory status in patients with thyroid cancer [27]. In a rare case with a solitary fibrous tumour located in the main pulmonary artery, [ 99m Tc]Tc-Galacto-RGD 2 imaging played an important role in detecting the primary tumour and predicting the metastatic potential [22]. In the current study, we evaluated the use of [ 99m Tc]Tc-Galacto-RGD 2 SPECT/CT for the detection of lung cancer. We also explored the expression of integrin α v β 3 and CXCR4 in different lung cancer subtypes, and compared the neovasculature among these subtypes. The correlations between tumour uptake of [ 99m Tc]Tc-Galacto-RGD 2 and integrin α v β 3 expression and neovascularization were also explored. High-contrast images of [ 99m Tc]Tc-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 lesions showed significantly lower uptake. [ 99m Tc]Tc-Galacto-RGD 2 SPECT/CT showed high sensitivity for primary tumours and remote metastases. ROC analysis showed a sensitivity and accuracy of 91.89% and 86.67%, respectively, for [ 99m Tc]Tc-Galacto-RGD 2 SPECT/CT, using a cut-off value of T/N ratio at 2.5. However, the specificity for differentiating between malignant and benign disease was limited, possibly because of the involvement of integrin α v β 3 in various benign diseases. Overlap usually occurs between tuberculosis and inflammatory pseudo-tumours, which usually show higher uptake of [ 99m Tc]Tc-Galacto-RGD 2 than other types of benign diseases, such as pneumonia [12].
In the current study, IHC showed that α v β 3 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 [12,19,28]. Patients with lung cancer, even in the early stages, may develop multiple metastases several months even after thorough tumour resection, possibly related to specific tumour types with higher metastatic potential [19]. 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 α v β 3 and CD31 expression in primary lung tumours, while integrin α v β 3 was also correlated with CD31. These findings validate our hypothesis that lymphadenopathy and remote metastasis are mediated by specific biological molecules. Integrin α v β 3 and CXCR4 may mediate angiogenesis, which may further promote lymph node and remote metastases. Integrin α v β 3 -targeted imaging thus improves our understanding of the interactions between cancer cells and their microenvironment, which is a necessary prerequisite for the development of treatment strategies. This study showed higher levels of integrin α v β 3 were expressed in advanced tumours, integrin α v β 3 was also highly expressed not only in endothelial cells in the neo-vasculature but also in tumour cells, higher uptake was found in the primary tumour with a higher density of neo-vasculature and higher α v β 3 expression, which associated with multiple lymphadenopathy and remote metastasis. This finding confirmed integrin α v β 3 overexpression as an important component of tumour microenvironment, which was related with tumourigenic and aggressive behaviour in lung cancer. CXCR4 has been implicated in the chemotactic migration of cancer cells [10]. CXCR4 and integrin might synergistically promote lymphatic metastasis in lung cancer, and act as clinical predictors of lymph node metastasis in non-SCLC [29,30]. High expression levels of chemokines are related to a poor prognosis and a poor   [31][32][33][34]. 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 [35].
Integrin α v β 3 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 [ 99m Tc] Tc-Galacto-RGD 2 and integrin α v β 3 expression related to the heterogenicity of lung cancer. We found that tumour uptake of [ 99m Tc]Tc-Galacto-RGD 2 was related to integrin α v β 3 expression, neovascularization, and tumour stage, and integrin α v β 3 expression in tumour cells may promote lymphatic and distant metastases (Fig. 2). However, benign diseases showed variable degrees of angiogenesis, also associated with higher expression of integrin α v β 3 , as shown in one patient with thymus adenoma and in another with pulmonary sequestration (Figs. 3, 4). We hypothesized that tumour uptake of [ 99m Tc]Tc-Galacto-RGD 2 depended on the neo-vasculature and integrin α v β 3 expression in the tumour cells, and focal uptake in RGD-targeted imaging would thus be higher in primary tumours with more neo-vasculature and higher integrin α v β 3 expression in the cell membrane. Regarding the different subtypes of lung cancer, LSC had more neovascularization and higher integrin α v β 3 expression, followed by LAC, while SCLC showed less neovascularization and a higher proliferation index. The highest T/N ratio was therefore found in LSC, and was significantly higher than that in LAC and SCLCs. RGD-targeted imaging may thus serve as a useful tool for the phenotyping of lung cancer.
[ 68 Ga]Ga and [ 18 F]F labelled RGD tracers have been used in the preclinical and clinical, [ 68 Ga]Ga-NODAGA-RGD provide a different spatial distribution than 2-[ 18 F] FDG. It is worth noting that [ 18 F]F-Galacto-RGD not only can be used for the assessment of α v β 3 expression in the tumour neovasculature, but also in human atherosclerotic carotid plaques, where it correlates with α v β 3 expression [36]. Compared with PET RGD tracer, [ 99m Tc] Tc-Galacto-RGD 2 SPECT imaging has a disadvantage in space resolution. However, if taking the expenditure into account, [ 99m Tc]Tc-Galacto-RGD 2 has a significant advantage, and the one-kit vial of [ 99m Tc]Tc-Galacto-RGD 2 makes the synthesis more convenient, both of them contribute to the clinical transformation and application of [ 99m Tc]Tc-Galacto-RGD 2 .
However, there are some limitations in this study should be taken into concern. First, the quantitation of integrin α v β 3 expression in the immunohistochemistry could be influenced by tumour specimen obtaining and vision field selection. Second, tumour specimens were achieved only in 66 patients, not in all suspected patients, which might influence the data analysis.

Conclusions
This was the first extensive longitudinal study to investigate the expression of integrin α v β 3 in lung cancer. [ 99m Tc]Tc-Galacto-RGD 2 imaging showed high sensitivity for the detection of primary lung cancer, but limited specificity. [ 99m Tc]Tc-Galacto-RGD 2 uptake in the primary tumour was attributed to integrin α v β 3 expression in the endothelial and tumour cells, and focal uptake occurred in primary lung cancers with more neovascularization and high levels of α v β 3 in the tumour cells. LSC had a higher density of neo-vessels and higher α v β 3 expression, followed by LAC and then SCLC, advanced lung cancer showed higher levels of integrin α v β 3 compared with early stage. These findings suggest that RGD-based imaging might be a useful tool for lung cancer phenotyping and tumour biological behaviour evaluation. Further studies are warranted to validate these findings.