Development of a Fluorescent Visualization System for Non-visible Lung Cancer Nodules

Lung cancer is the leading cause of cancer deaths in the European Union (EU) (267,000 deaths/year) and the fourth most common cancer (321,000 new cases/year). To date, radical surgery remains the best curative option in patients with early-stage lung cancer. Moreover, cancer screening programs have led to frequent diagnoses of indeterminate lung lesions, many requiring surgical biopsy for diagnosis and intervention.

For example, available data showed that 5.9 % of the European population over 15 years of age consumed at least 20 cigarettes per day (8.4 % in the male population), and around 12.6 % consumed less than 20. The recent lung cancer screening studies documented a prevalence of indeterminate pulmonary nodules as high as 50% in high-risk smokers, with a cancer detection rate in the overall screened population of 1%. Fascinatingly, 69% of screen-detected lung cancers were detected at early stage IA or IB.

Finally, studies on lung cancer screening, like the NELSON trial, showed a 26% reduction in lung cancer deaths at 10 years. The potential social impact of the present project is linked to establishing this screening program in Europe. One could estimate that if applied in the high-risk European population (i.e., high smokers), the screening could identify 3.5 million indeterminate pulmonary nodules, of which 250,000 are early-stage lung cancer. [2] Therefore, using the NIR-tracker the investigators propose could consent to a minimally invasive surgery in this scenario as a diagnostic and therapeutic procedure. Consequently, it could determine the reduction of time to diagnosis, morbidity, mortality, and costs of postoperative care associated with more invasive surgical procedures.

To date, lung anatomical resection with lymphadenectomy remains the best curative option in patients with early-stage non-small cell lung cancer. Moreover, cancer screening programs have led to frequent diagnoses of indeterminate lungs, many requiring surgical biopsy for diagnosis and intervention.

Nevertheless, the increase in the utilization of minimally invasive procedures (e.g., video-assisted thoracic surgery -VATS- and robotic-assisted thoracic surgery -RATS- ) remains mandatory in order to reduce the significant morbidity of classic surgery, the surgical trauma, to preserve organs function and to improve patient's quality of life. Nevertheless, minimally invasive surgery represents the surgical approach of choice in less than 40% of lung anatomical resections conducted in Europe. One of the significant issues that hinder the application of VATS and RATS to most early-stage NSCLC patients is the difficulty of recognizing lung nodules located deep in the lung parenchyma and, consequently, not visible with the traditional camera system. Indeed, VATS and RATS do not consent to manual lung palpation, making localizing the not superficial lung nodule problematic.

Several approaches have been developed to enhance the localization of indeterminate lung nodules and decrease the time to diagnosis and rate of conversion to open surgery. Nevertheless, none of these is 100% sensitive or without complications, also of severe grade.

Numerous pre-operative methods are being employed, including percutaneous CT-guided placement techniques, encompassing the use of microcoils, hook-wires, and spiral-wires. These devices can support nodule localization during minimally invasive lung procedures; nevertheless, they could be easily displaced during patient transport and positioning, intraoperative atelectasis, single lung ventilation, and surgeon manipulation. Moreover, some locations of the lung as the apex, near the diaphragm, and the proximity of mediastinum and great vessels. Furthermore, all these pre-operative localization techniques require two different procedures, one for the CT-guided referral placement and one for surgical treatment. Finally, the rate of pneumothorax, hemorrhage, and subcutaneous emphysema are not insignificant, and these complications are mandatory to avoid in several sub-groups of patients. Other pre-operative methods encompass the use of dye marking by methylene blue or fluorescent. [8] Nevertheless, the accuracy of the staining of the targeted area is greatly affected by the time between tumor marking and thoracoscopy. In particular, the significant impact is on the difficulty in dye visualization during operation, limited information on lesion depth, and rapid diffusion of dye into surrounding lung parenchyma between the time of injection and surgery. Of note, methylene blue has limited application in patients with anthracotic pigmentation. Moreover, also these techniques require two procedures for diagnosis and treatment. Lastly, these procedures remain complicated by the risk of pneumothorax, hemorrhage, dye air embolism, and cerebrovascular accident, and cases of lethal anaphylaxis to the dye of choice.

On the other hand, clinical pre-operative staging and surgical planning are based on pre-operative images taken before surgery, either by computed tomography (CT), positron emission tomography (PET), or magnetic resonance imaging (MRI). These pre-operative imaging assessments frequently underestimate lymph node involvement and secondary localizations. This results in an upstaging after surgical resection ranging from 9 to 24 % in clinical Stage I lung cancer. [10, 11] Nevertheless, the current system of intraoperative imaging, based on direct injection of a tracker in the principal tumoral mass, demonstrated a substantial limitation in lung cancer. This is principally due to the deeper location of the lymph node, usually profoundly engaged in normal fat tissue, and to the irregular lymph node drain system in the respiratory region.

In this context, intraoperative fluorescence imaging can enhance the real-time identification of cancer cells during minimally invasive surgical procedures. This could overcome the difficulty of finding cancer nodules located deep in the lung parenchyma, not visible on the surface of normal, uninvolved tissue. The Near-infrared (NIR) fluorescence (700-1,000 nm) detection avoids the natural background fluorescence interference of biomolecules, which provides high contrast between the target and background tissues in small animals. NIR fluorophores have a more comprehensive dynamic range and minimal background fluorescence because of reduced scattering compared with visible fluorescence detection. However, the conventional near-infrared (NIR) indocyanine green (ICG) method demonstrated a significant limitation in deep cancer recognition, principally due to its intrinsic low-depth tissue penetration. Similarly, the lymph-node sentinel approach conducted by the ICG method proved to be inefficient, mainly due to the non-specificity of the tracker and the irregular pathway of pulmonary lymph node drainage.

The IRDye® 800CW is an indocyanine-type NIR fluorophore with peak absorption at 775 nm and peak excitation emission at 796 nm. It provides a quantum yield of 9% with an extinction coefficient of 242,000 M-1cm-1. It has a molecular weight of 962 Da. The IRDye® 800CW demonstrated enhanced tissue penetration compared to other NIR dyes.

Epidermal growth factor (EGF) is a 53-amino acid cytokine (6.2 kDa) that is secreted by ectodermic cells, monocytes, kidneys, and duodenal glands. EGF stimulates the growth of epidermal and epithelial cells. EGF and at least seven other growth factors and their transmembrane receptor kinases play essential roles in cell proliferation, invasion, metastasis, neovascularization, adhesion, migration, differentiation, and inhibition of apoptosis. The EGF receptor (EGFR) family consists of four transmembrane receptors, which include EGFR (HER1/erbB-1), HER2 (erbB-2/neu), HER3 (erbB-3), and HER4 (erbB-4); and is commonly overexpressed in lung cancer. Cetuximab is a monoclonal antibody able to inhibit and degrade the EGFR. Given by intravenous infusion (IV), Cetuximab binds to the EGFR and stops the binding and activation of the downstream signaling pathways. Moreover, as the investigators previously published, EGFR mutation is linked with skip-metastasis phenomena (i.e., pathologically proved mediastinal lymph node involvement in the absence of intrapulmonary or hilar lymph node disease).

The combination with the clinical approved monoclonal antibody anti-epidermal growth factor EGFR Cetuximab (Cetuximab-IRDye800) has shown promising results as a specific tracker in other cancer types (i.e., brain, pancreas, head, and neck). The investigators hypothesize that using Cetuximab-IRDye800 during minimally invasive surgical procedures for lung cancer could overcome the limitation demonstrated by ICG and the traditional localization strategies (e.g., coil, hook, dye intra-tumoral injection). The investigators expect to validate using an optimal tracker that can be easily applied intraoperatively during minimally invasive lung surgical procedures. The investigators expect to define the optimal time window and the optimal dose of administration of the tracker. The investigators expect to discover neoplastic localization in lymph nodes and lung parenchyma not predictable pre-operatively.