Background: Near-infrared (NIR) imaging using the second time window of indocyanine green (ICG) allows localization of pulmonary, pleural, and mediastinal malignancies during surgery. Based on empirical evidence, we hypothesized that different histological tumor types fluoresce optimally at different ICG doses. Study Design: Patients with thoracic tumors biopsy-proven or suspicious for malignancy were enrolled in an NIR imaging clinical trial. Patients received a range of ICG doses one day prior to surgery: 1 mg/kg (n=8), 2 mg/kg (n=8), 3 mg/kg (n=13), 4 mg/kg (n=8), and 5 mg/kg (n=8). Intraoperatively, NIR imaging was performed. The endpoint was to identify the highest tumortobackground fluorescence ratio (TBR) for each tumor type at each dose. Final pathology confirmed tumor histology. Results: Of 45 patients, 41 had malignancies (18 non-small cell lung cancer (NSCLC), 3 pulmonary neuroendocrine tumors, 13 thoracic metastases, 3 thymoma, 3 mesothelioma). At doses of 4-5 mg/kg, the TBR from primary NSCLC versus other malignancies was no different (2.70 vs. 3.21, P=1.00). At doses of 1-3 mg/kg, the TBR was greater for the non-NSCLCs (3.19 vs. 1.49, P=0.0006). Background fluorescence from the heart or ribs was observed in 1/16 cases at 1-2

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Gene-mediated cytotoxic immunotherapy (GMCI) is an immuno-oncology approach involving local delivery of a replication-deficient adenovirus expressing herpes simplex thymidine kinase (AdV-tk) followed by anti-herpetic prodrug activation that promotes immunogenic tumor cell death, antigen-presenting cell activation, and T cell stimulation. This phase I dose-escalation pilot trial assessed bronchoscopic delivery of AdV-tk in patients with suspected lung cancer who were candidates for surgery. A single intra-tumoral AdV-tk injection in three dose cohorts (maximum 10 12 viral particles) was performed during diagnostic staging, followed by a 14-day course of the prodrug valacyclovir, and subsequent surgery 1 week later. Twelve patients participated after appropriate informed consent. Vector-related adverse events were minimal. Immune biomarkers were evaluated in tumor and blood before and after GMCI. Significantly increased infiltration of CD8 + T cells was found in resected tumors. Expression of activation, inhibitory, and proliferation markers, such as human leukocyte antigen (HLA)-DR, CD38, Ki67, PD-1, CD39, and CTLA-4, were significantly increased in both the tumor and peripheral CD8 + T cells. Thus, intratumoral AdV-tk injection into non-small-cell lung cancer (NSCLC) proved safe and feasible, and it effectively induced CD8 + T cell activation. These data provide a foundation for additional clinical trials of GMCI for lung cancer patients with potential benefit if combined with other immune therapies.

Background Intraoperative frozen section (FS) consultation is an important tool in surgical oncology that suffers from sampling error because the pathologist does not always know where to perform a biopsy of the surgical specimen. Intraoperative molecular imaging is a technology used in the OR to visualize lesions during surgery. We hypothesized that molecular imaging can address this pathology challenge in FS by visualizing the cancer cells in the specimen in the pathology suite. Here, we report the development and validation of a molecular-imaging capable cryostat called Smart-Cut. Methods A molecular imaging capable cryostat prototype was developed and tested using a murine model. Tumors grown in mice were targeted with a NIR contrast agent, indocyanine green (ICG), via tail vein injection. Tumors and adjacent normal tissue samples were frozen sectioned with Smart-Cut. Fluorescent sections and non-fluorescent sections were prepared for H&E and fluorescent microscopy. Fluorescent signal was quantified by tumor-to-background ratio (TBR). NIR fluorescence was tested in one patient enrolled in a clinical trial. Results The Smart-Cut prototype has a small footprint and fits well in the pathology suite. Fluorescence imaging with Smart-Cut identified cancerous tissue in the specimen in all 12 mice. No false positives or false negatives were seen, as confirmed by H&E. The mean TBR in Smart-Cut positive tissue sections was 6.8 (SD±3.8). In a clinical application in the pathology suite, NIR imaging identified two lesions in a pulmonary resection specimen, where traditional grossing only identified one. Conclusion Molecular imaging can be integrated into the pathology suite via the Smart-Cut device, and can detect cancer in frozen tissue sections using molecular imaging in a murine model.