Clinical studies have demonstrated that intraoperative molecular imaging (IMI) with pafolacianine identifies occult pulmonary lesions that are not identified by preoperative CT or by intraoperative inspection techniques in ∼20% of patients. In this study we provide a description of occult lesion clinical data and evaluate characteristics so that surgeons can better incorporate this emerging technology into clinical decision making. Participants (n=100) enrolled in a Phase 3 trial of IMI with pafolacianine during pulmonary resection (ELUCIDATE, NCT04241315) were identified. Participants underwent preoperative computed tomography (CT)with 1.25mm slices. Variables and lesion characteristics were analyzed. Positive predictive value and false positive rates were tabulated for IMI fluorescent lesions with predictors of malignant versus benign occult lesions described. IMI identified 29 occult lesions in 23 (23%) participants. Seventeen of 29 (58%) lesions were identified within the same lobe as known lesions; 12 of 29 (42%) identified in a different lobe from the suspicious nodule known by preoperative assessment. Twenty-three of 29 (79%) of occult lesions found by IMI were resected with an additional wedge resection. Ten of 29 (34%) lesions identified by IMI were malignant. There was no additional morbidity in participants with lesions resected. With pafolacianine, 7 participants had a synchronous primary stage I lung cancer identified and one subject had additional metastases identified. . IMI with pafolacianine identifies occult malignant lesions during pulmonary resection despite thorough preoperative imaging and intraoperative assessment by experienced surgeons.

Background: In situ cancer vaccination is a therapeutic approach that involves stimulating the immune system in order to generate a polyclonal, anti-tumor response against an array of tumor neoantigens. Traditionally, in situ vaccination approaches have utilized adenoviral vectors to deliver immune-stimulating genes directly to the tumor microenvironment. Lipid nanoparticle (LNP)-mediated delivery methods offer several advantages over adenoviral delivery approaches, including increased safety, repeated administration potential, and enhanced tumor microenvironment activation. Methods: To explore in situ vaccination using LNPs, we evaluated LNP-mediated delivery of a reporter gene, mCherry, and an immune-stimulating gene, IFNβ, in several in vitro and in vivo models of lung cancer. Results: In vitro experiments demonstrated successful transfection of murine cancer cell lines with LNPs carrying both mCherry and IFN-β mRNA, resulting in high expression levels and IFNβ production. In vivo studies using LLC.ova flank tumors showed that intratumoral injection of IFNβ-mRNA LNPs led to significant IFNβ production within the tumor microenvironment, with minimal systemic exposure. Therapeutic efficacy was evaluated by injecting established LLC.ova flank tumors with IFNβ-mRNA LNPs bi-weekly for two weeks. Treated tumors showed significant growth inhibition compared to controls. Flow cytometric analysis of tumor-infiltrating leukocytes revealed that tumors injected with IFNβ-mRNA LNPs were associated with an increased CD8:CD4 T-cell ratio among lymphocytes, more CD69-expressing CD8 T-cells, and an increased presence of M1 macrophages. Efficacy and an abscopal effect were confirmed in a squamous cell carcinoma model, MOC1. No toxicity was observed. Conclusions: These findings show that intratumoral LNP delivery of immune-stimulating mRNA transcripts, such as IFNβ, can effectively stimulate local anti-tumor immune responses and warrants further investigation as a potential immunotherapeutic approach for cancer.

As thoracic surgeons more frequently address smaller lung lesions and perform lung-sparing resections, their objective is to resect an adequate specimen and margin without removing excess healthy lung tissue. Although perioperative lung nodule localization has been in practice for decades, the existing and emerging techniques used for the identification of targeted and occult lesions are more widely utilized today than they were in the past. In this review, we detail the logic behind this increase in use, classify the techniques into preoperative and intraoperative categories, and define the specific modalities available. Where applicable, we review the published data comparing techniques, detailing efficacy and safety. In the preoperative space, we describe standard computed tomography (CT)-guided localization, virtual-assisted lung mapping, electromagnetic navigation bronchoscopy, robotic-assisted bronchoscopy, and novel fiducial markers. In the intraoperative space, we describe classical localization techniques, novel applications of intraoperative cone-beam CT, and fluorescence-guided surgery and intraoperative molecular imaging (IMI). Lastly, we review emerging approaches for intraoperative molecular imaging including a report on agents in early-stage clinical trials and a brief survey of promising preclinical models. With each approach mentioned, we analyze the potential benefits and hazards, and appraise the evidence for (or against) the use of any specific modality.

Safety and immunologic priming using neoadjuvant, intratumoral gene-mediated cytotoxic immunotherapy (GMCI) has been established in a phase 1 clinical trial involving patients with resectable non−small cell lung cancer (NSCLC). Here we examine long-term clinical outcomes of this cohort and compare the results with a comparable contemporaneous control group. We performed a retrospective review of a prospective database involving our trial cohort. We then identified comparable patients in a ratio of 5:1 considering pathologic stage, histology, resection extent, and age. Kaplan-Meier curves were generated. Differences in disease-free and overall survival were measured by log-rank test. Intratumoral GMCI was delivered to 12 patients with NSCLC by endobronchial ultrasound (n = 11) or video-assisted thoracic surgery (n = 1). Among patients receiving GMCI and control patients, more than 70% had stage II or stage III disease. Median follow-up for the surviving patients enrolled was 6.0 years (interquartile range, 5.2-6.7 years). Median diseases-free survival was significantly increased among patients receiving GMCI relative to control (2.4 years vs 1.0 year; P = .04). Median OS in the GMCI group was not reached (8 of 12 patients remain alive) versus 3.0 years in controls and was significant by log-rank at 2-7 years (P = .013-.035). Neoadjuvant immune stimulation induces local and systemic immune activation, is safe when combined with modern systemic therapies, and may confer long-term oncologic advantages. These data provide a foundation for additional trials exploring intratumoral immunotherapy for patients with NSCLC, especially in conjunction with immune checkpoint inhibitors.