99mTc-Galacto-RGD2 Integrin αvβ3 Targeted Imaging as a Surrogate for Molecular Phenotyping in Lung Cancer: Prospective Study in the Real World

Purpose: Epidermal growth factor receptor tyrosine kinase inhibitors (TKIs) are benecial in patients with lung cancer. We explored the clinical value of 99m Tc-Galacto-RGD 2 single-photon emission computed tomography (SPECT/CT) in patients with lung cancer, Integrin α v β 3 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 prostective study from January 2013 to December 2016. All patients underwent 99m Tc-Galacto-RGD 2 imaging. The region of interest was drawn around each primary lesion, and tumour uptake of 99m Tc-Galacto-RGD 2 was measured as the tumour/normal tissue ratio (cid:0) T/N (cid:0) . The diagnostic ecacy was evaluated by receiver operating characteristic curve analysis. Tumour tissues were obtained from 66 patients with malignant diseases and seven with benign disease. Tumour expression levels of α v β 3 , CD31, Ki67, and CXCR4 were analysed to determine their value for phenotyping and metastasis potential evaluation. 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, specicity, and accuracy of 99m Tc-Galacto-RGD 2 SPECT/CT using a cut-off value of 2.5 were 91.89 %, 48.0 %, and 86.67 %, respectively. Integrin α v β 3 expression was higher in non-SCLC compared with SCLC, while LSC showed denser neovascularization and higher integrin α v β 3 expression. Integrin α v β 3 expression levels were signicantly higher in advanced ( (cid:0) , (cid:0) ) than early stages ( (cid:0) , (cid:0) ). However, there was no signicant correlation between tumour uptake and α v β 3 expression. 99m 2 SPECT/CT has high sensitivity but limited specicity for detecting primary lung cancer. RGD imaging may help evaluate the biological behaviour and phenotyping, and thus aid management in lung cancer. correlation validated the use of integrin molecular imaging as a surrogate for phenotyping. High-contrast images acquired 1 h after injection of 99m Tc-Galacto-RGD2 showed a signicantly 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 signicantly lower uptake. 99m Tc-Galacto-RGD 2 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 99m Tc-Galacto-RGD 2 SPECT/CT, using a cut-off value of 2.5. However, the specicity 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


Introduction
Lung cancer is the leading cause of cancer mortality worldwide for both sexes combined [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 signi cantly 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 TKIs, 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, which limits the application of TKIs.
Nuclear medicine and molecular imaging are important for the quantitative evaluation of membrane receptors and biological tumour behaviour [8][9][10].
Positron emission tomography/computed tomography (PET/CT) can serve as a useful tool for identifying speci c biological behaviours. 18 Fuorodeoxyglucose (FDG)-PET has also been well-validated for the diagnosis, staging, and management of malignant tumours, and can guide the selection of suitable treatment strategies [11,12]. However, tumour uptake of FDG is in uenced by various factors, including blood glucose levels, tumour size,in ammation, and the acquisition protocol. Furthermore, FDG uptake by slow-growing and less metabolically active tumours, including highly differentiated lung cancers, was indistinguishable from that in normal tissues [13].
Overexpression of integrin α v β 3 was also correlated with tumour invasiveness in breast cancer, indicating a possible role in evaluating metastatic potential [24].
Radiolabelled RGD peptide as a target ligand for angiogenesis imaging has been well documented in preclinical and clinical studies [18,26,27]. In a previous multicentre study, we showed that Tc-labelled RGD dimers, such as 99m Tc-3PRGD 2 , had high sensitivity for the detection of lung cancer, including primary and metastatic tumours [26,28,29]. 99m Tc-Galacto-RGD 2 , with higher a nity 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, which may in turn serve as a prognostic hallmark and may aid treatment strategy selection [30].
In the clinical, multiple lymphadenopathy and remote metastasis was 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-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, and its potential use for selecting the appropriate treatment strategy, the schema of study was shown in (Fig. 1).

Patients
This prospective, single-centre study enrolled patients referred to our centre with suspected lung neoplasms from January 2013 to December 2016. 99m Tc-Galacto-RGD 2 SPECT-CT was performed in all patients. Written consent was obtained from all patients, and the study was approved by the local ethics committee of Nanjing Medical University. The nal diagnosis was con rmed 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 con rmed 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.
99m Tc-Galacto-RGD2 Imaging and Interpretation The radiochemical purity was 95.1% ± 2.9%. 99m 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 image, including the upper abdomen and adrenal glands, was performed using a combined transmission and emission device with x-ray tube and detector (Symbia T6 SPECT/CT; Siemens AG, Germany). Anatomic CT images were produced for attenuation correction and tumour localization. If unexpected lesions were detected by whole-body 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. Signi cantly greater local uptake of 99m 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-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 ndings were used as a composite reference standard for the presence of tumour lesions. This is considered the optimal gold standard because cytologic or histologic veri cation of every lesion was not feasible or justi able in these patients. Whenever possible, new ndings on 99m Tc-Galacto-RGD 2 SPECT-CT were veri ed by additional investigations.

Immunohistochemistry (IHC) Analysis
Tumour specimens were obtained from patients who underwent complete resection or biopsy. The sections were xed in formalin, embedded in para n, depara nised, and stained with hematoxylin and eosin (H&E). Integrin α v β 3 , Ki-67, CXCR4, and CD31 expression were analysed by IHC to evaluate the biological tumour behaviour. Sections were cut at 3-µm, dewaxed in xylene, and rehydrated in graded ethanols. Integrin α v β 3 and CXCR4 expression, microvascular density (CD31), and tumour cell proliferation (Ki-67) were detected by incubating the slides with monoclonal antibodies against human integrin Abcam), or CD31 (1:50, ab28364; Abcam), respectively, overnight, followed by horseradish peroxidase-conjugated anti-mouse IgG (1:1000, Earth Ox, Millbrae, California, US) with 3′3-diaminobenzidine as the chromogen. H&E staining was also performed. All images were obtained at 100× magni cation with the same exposure time. Brightness and contrast were adjusted similarly in all images. Integrin α v β 3 and CXCR4 expression levels were quanti ed by determining the optical density (OD) after immunostaining.

Statistical Analysis
All statistical analyses were carried out using R (version 3.6.1) and graphs were constructed using GraphPad Prism software. Continuous variables with a nonnormal 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, speci city, 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 signi cance was established at p < 0.05.

Histopathology and IHC
Of the 210 patients with suspected lung cancer, immunochemistry was performed in 66 patients with lung cancer and seven patients with benign diseases.
Expression levels of integrin α v β 3 were signi cantly higher in tissues from patients with lung cancer (OD: 15 Integrin α v β 3 was also highly expressed not only in endothelial cells in the neovasculature, re ected by CD31 expression, but also in tumour cells (Fig. 5), with a higher density of neovasculature and integrin α v β 3 expression in the primary tumour. Integrin α v β 3 was also signi cantly correlated with CD31 expression in lung cancer (r = 0.30, p = 0.016). However, there was no correlation between tumour uptake of 99m Tc-Galacto-RGD 2 and integrin α v β 3 expression in the primary tumour in this study (Fig. supplemewnt-1). Squamous lung cancer usually showed higher level of α v β 3 in the tumor cell and the higher density of microvessel, which was consistent with RGD imaging as shown in the (Fig. Supplement-2). Aggressive LAC tends to higher express integrin α v β 3 in the tumour cell and has more dense microvessel, which showed focal uptake in the RGD image, as shown in the ( Fig.   Supplement-3). 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 = 0.08). In addition, the proliferation index (Ki-67) in LSC and SCLC (27.45 (11.88, 42.00) and 70.00 (55.13, 73.48), respectively) were both signi cantly higher than in LAC (10.15 (2.98, 27.89)) ( Table 3).  and 70 patients had bone metastasis. The metastatic lymph nodes and remote metastases showed high focal uptake of 99m Tc-Galacto-RGD 2 . However, although lymphadenopathy was evaluated by imaging follow-up, the nal diagnosis was not con rmed, and we were therefore unable to evaluate the diagnostic value of 99m Tc-Galacto-RGD 2 imaging for lymphadenopathy and remote metastasis in this study.

Discussion
Targeted therapy has signi cantly improved the outcome for patients with lung cancer in the last decade [32]. For example, EGFR is a major driver of NSCLC tumorigenesis [33], 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 noninvasive) means of patient selection. We previously validated the ability of 99m Tc-Galacto-RGD 2 to identify iodine-refractory status in patients with thyroid cancer [36]. In a rare case with a solitary brous tumour located in the main pulmonary artery, 99m Tc-Galacto-RGD 2 imaging played an important role in detecting the primary tumour and predicting the metastatic potential [27]. In the current study, we evaluated the use of 99m 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. We also examined the correlations between tumour uptake of 99m Tc-Galacto-RGD 2 and integrin α v β 3 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 99m Tc-Galacto-RGD2 showed a signi cantly 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 signi cantly lower uptake. 99m Tc-Galacto-RGD 2 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 99m Tc-Galacto-RGD 2 SPECT/CT, using a cut-off value of 2.5. However, the speci city 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 in ammatory pseudo-tumours, which usually show higher uptake of 99m Tc-Galacto-RGD 2 than other types of benign diseases, such as pneumonia [18].
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 signi cantly 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 speci c 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 signi cant. 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 ndings validate our hypothesis that lymphadenopathy and remote metastasis are mediated by speci c biological molecules. Integrin α v β 3 and CXCR4 may mediate angiogenesis, which may further promote lymph node and remote metastases. Imaging targeting integrin α v β 3 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 speci cally targeting cancerinduced invasion and metastases. This information is signi cant in light of the correlations of integrin α v β 3 overexpression with recurrence and poor prognosis, and in relation to early diagnosis and treatment-response monitoring. These ndings demonstrated that expression levels of integrin α v β 3 were strongly correlated with tumorigenic and aggressive behaviours in lung cancer cells. CXCR4 has been implicated in the chemotactic migration of cancer cells [16]. CXCR4 and integrin might synergistically promote lymphatic metastasis in lung cancer, and might act as clinical predictors of lymph node metastasis in NSCLC [38][39][40]. High expression levels of chemokines are related to a poor prognosis and chemotherapy tolerance in cancer patients [41][42][43][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 [45].
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-Galacto-RGD 2 and integrin α v β 3 expression because of the heterogenicity of lung cancer, however, both 99m Tc-Galacto-RGD 2 imaging and integrin α v β 3 expression behaved well in distinguishing lung cancer and benign lung disease. We supposed that tumour uptake of 99m Tc-Galacto-RGD 2 was related to integrin α v β 3 expression, neovascularization, and tumour stage. Integrin α v β 3 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 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-Galacto-RGD 2 depended on the neovasculature and integrin α v β 3 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 α v β 3 expression. Regarding the different subtypes of lung cancer, LSC usually had more neovascularization and higher integrin α v β 3 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 signi cantly higher than that in LAC and SCLCs (6.84 and 4.73, respectively) (Fig. 2). RGDtargeted 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 rst extensive longitudinal study to investigate the expression of integrin α v β 3 in lung cancer. 99m Tc-Galacto-RGD 2 imaging showed high sensitivity for the detection of primary lung cancer, but limited speci city. 99m Tc-Galacto-RGD 2 uptake in the primary tumour was attributed to integrin α v β 3 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 α v β 3 in the tumour cells. LSC had a higher density of neo-vessels and higher integrin α v β 3 expression, followed by LAC and then SCLC.
Furthermore, advanced lung cancer showed higher expression levels of integrin α v β 3 compared with early stages, and higher integrin α v β 3 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 ndings suggest that RGD based imaging might be a useful tool for lung cancer phenotyping and for evaluating tumour biological behaviours, such as aggressiveness. integrin α v β 3 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 ndings and to address the issue that tumour specimens suitable for IHC are not obtained from all patients.

Declarations
All procedures performed in this study involving human participants were carried out in accordance with the ethical standards of the Nanjing Medical University and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Informed Nanjing Clinical Medical Center "Nanjing Nuclear Medicine Centre". There is no con icts of interest.