Molecular Imaging in Precision-Cut Non-Small Cell Lung Cancer Slices.

BACKGROUND: Small animal models remain invaluable for the preclinical study of emerging molecular imaging agents. However, the data obtained in this setting are generated in genetically homogenous animals that do not mimic human pathophysiology. The purpose of this study was to prospectively validate precision-cut lung slices (PCLSs) obtained from patients with lung cancer as a translational tool for the development of targeted fluorophores. METHODS: The lung tissue was gently inflated with 2% Low-Melt Agarose (Fisher, 16520050) to avoid lung damage and minimize inflation pressure. The slices were then loaded into specialized cylindrical cartridges and inserted into a compressotome, and slices 150 to 350 µm thick were cut. Samples were incubated with fluorophore conjugates for ex vivo validation and immunohistochemical staining for receptor expression. RESULTS: A total of 184 unique 3-dimensional, architecturally preserved normal lung and non-small cell lung cancer samples were obtained between 2020 and 2022. The median nodule size was 1.1 ± 0.21 cm for benign lesions and 2.1 ± 0.19 cm for malignant nodules. A total of 101 of 135 (74.8%) malignant lesions were adenocarcinoma spectrum lung cancers. The median viability was 9.78 ± 1.86 days, and 1 µM of FAPL-S0456 (high-affinity fibroblast activation protein [FAP] targeting ligand linked to the near-infrared fluorophore S0456, On Target Laboratories)-targeted near-infrared fluorochrome localization demonstrated correlative labeling of FAP-positive tumor areas with a correlation coefficient of +0.94 (P < .01). There was no FAP fluorochrome uptake in normal lungs (r = -1; P < .001). CONCLUSIONS: PCLSs comprise a novel human tissue-based translational model that can be used to validate the efficacy of molecular imaging fluorochromes. PCLSs preserve the tumor microenvironment and parenchymal architecture that closely resemble the interactions of the immune and stromal components in humans.

Single-institution experience of 500 pulmonary resections guided by intraoperative molecular imaging.

OBJECTIVE: Intraoperative molecular imaging (IMI) using tumor-targeted optical contrast agents can improve thoracic cancer resections. There are no large-scale studies to guide surgeons in patient selection or imaging agent choice. Here, we report our institutional experience with IMI for lung and pleural tumor resection in 500 patients over a decade. METHODS: Between December 2011 and November 2021, patients with lung or pleural nodules undergoing resection were preoperatively infused with 1 of 4 optical contrast tracers: EC17, TumorGlow, pafolacianine, or SGM-101. Then, during resection, IMI was used to identify pulmonary nodules, confirm margins, and identify synchronous lesions. We retrospectively reviewed patient demographic data, lesion diagnoses, and IMI tumor-to-background ratios (TBRs). RESULTS: Five hundred patients underwent resection of 677 lesions. We found that there were 4 types of clinical utility of IMI: detection of positive margins (n = 32, 6.4% of patients), identification of residual disease after resection (n = 37, 7.4%), detection of synchronous cancers not predicted on preoperative imaging (n = 26, 5.2%), and minimally invasive localization of nonpalpable lesions (n = 101 lesions, 14.9%). Pafolacianine was most effective for adenocarcinoma-spectrum malignancies (mean TBR, 2.84), and TumorGlow was most effective for metastatic disease and mesothelioma (TBR, 3.1). False-negative fluorescence was primarily seen in mucinous adenocarcinomas (mean TBR, 1.8), heavy smokers (>30 pack years; TBR, 1.9), and tumors greater than 2.0 cm from the pleural surface (TBR, 1.3). CONCLUSIONS: IMI may be effective in improving resection of lung and pleural tumors. The choice of IMI tracer should vary by the surgical indication and the primary clinical challenge.

Carcinoembryonic Antigen-Related Cell Adhesion Molecule Type 5 Receptor-Targeted Fluorescent Intraoperative Molecular Imaging Tracer for Lung Cancer: A Nonrandomized Controlled Trial.

Importance: Localization of subcentimeter ground glass opacities during minimally invasive thoracoscopic lung cancer resections is a significant challenge in thoracic oncology. Intraoperative molecular imaging has emerged as a potential solution, but the availability of suitable fluorescence agents is a limiting factor. Objective: To evaluate the suitability of SGM-101, a carcinoembryonic antigen-related cell adhesion molecule type 5 (CEACAM5) receptor-targeted near-infrared fluorochrome, for molecular imaging-guided lung cancer resections, because glycoprotein is expressed in more than 80% of adenocarcinomas. Design, Setting, and Participants: For this nonrandomized, proof-of-principal, phase 1 controlled trial, patients were divided into 2 groups between August 1, 2020, and January 31, 2022. Patients with known CEACAM5-positive gastrointestinal tumors suggestive of lung metastasis were selected as proof-of-principle positive controls. The investigative group included patients with lung nodules suggestive of primary lung malignant neoplasms. Patients 18 years or older without significant comorbidities that precluded surgical exploration with suspicious pulmonary nodules requiring surgical biopsy were included in the study. Interventions: SGM-101 (10 mg) was infused up to 5 days before index operation, and pulmonary nodules were imaged using a near-infrared camera system with a dedicated thoracoscope. Main Outcomes and Measures: SGM-101 localization to pulmonary nodules and its correlation with CEACAM5 glycoprotein expression by the tumor as quantified by tumor and normal pulmonary parenchymal fluorescence. Results: Ten patients (5 per group; 5 male and 5 female; median [IQR] age, 66 [58-69] years) with 14 total lesions (median [range] lesion size, 0.91 [0.90-2.00] cm) were enrolled in the study. In the control group of 4 patients (1 patient did not undergo surgical resection because of abnormal preoperative cardiac clearance findings that were not deemed related to SGM-101 infusion), the mean (SD) lesion size was 1.33 (0.48) cm, 2 patients had elevated serum CEA markers, and 2 patients had normal serum CEA levels. Of the 4 patients who underwent surgical intervention, those with 2+ and 3+ tissue CEACAM5 expression had excellent tumor fluorescence, with a mean (SD) tumor to background ratio of 3.11 (0.45). In the patient cohort, the mean (SD) lesion size was 0.68 (0.22) cm, and no elevations in serum CEA levels were found. Lack of SGM-101 fluorescence was associated with benign lesions and with lack of CEACAM5 staining. Conclusions and Relevance: This in-human proof-of-principle nonrandomized controlled trial demonstrated SGM-101 localization to CEACAM5-positive tumors with the detection of real-time near-infrared fluorescence in situ, ex vivo, and by immunofluorescence microscopy. These findings suggest that SGM-101 is a safe, receptor-specific, and feasible intraoperative molecular imaging fluorochrome that should be further evaluated in randomized clinical trials. Trial Registration: ClinicalTrials.gov identifier: NCT04315467.

Sodium Multivitamin Transporter-Targeted Fluorochrome Facilitates Enhanced Metabolic Evaluation of Tumors Through Coenzyme-R Dependent Intracellular Signaling Pathways.

BACKGROUND: Intraoperative molecular imaging (IMI)-guided resections have been shown to improve oncologic outcomes for patients undergoing surgery for solid malignancies. The technology utilizes fluorescent tracers targeting cancer cells without the use of any ionizing radiation. However, currently available targeted IMI tracers are effective only for tumors with a highly specific receptor expression profile, and there is an unmet need for IMI tracers to label a broader range of tumor types. Here, we describe the development and testing of a novel tracer (CR)-S0456) targeted to the sodium multivitamin transporter (SMVT). METHODS: Preclinical models of fibrosarcoma (HT-1080), lung (A549), breast (4T1), and renal cancers (HEK-293 T) in vitro and in vivo were used for assessment of (CR)-S0456 specific tumor labeling via sodium-mediated SMVT uptake in dipotassium phosphate or choline chloride-containing media buffer. Additionally, pharmacologic inhibition of multiple intracellular coenzyme-R obligate signaling pathways, including holocarboxylase synthetase (sulconazole nitrate), PI3K/AKT/mTOR (omipalisib), and calmodulin-dependent phosphatase (calmidazolium), were investigated to assess (CR)-S0456 uptake kinetics. Human fibrosarcoma-bearing xenografts in athymic nude mice were used for tumor and metabolic-specific labeling. Novel NIR needle confocal laser endomicroscopic (nCLE) intratumoral sampling was performed to demonstrate single-cell specific labeling by CR-S0456. RESULTS: CR-S0456 localization in vitro correlated with highly proliferative cell lines (MTT) and doubling time (p < 0.05) with the highest microscopic fluorescence detected in aggressive human fibrosarcomas (HT-1080). Coenzyme-R-specific localization was demonstrated to be SMVT-specific after competitive inhibition of internal localization with excess administration of pantothenic acid. Inhibiting the activity of SMVT by affecting sodium ion hemostasis prevented the complete uptake of CR-S0456. In vivo validation demonstrated (CR)-S0456 localization to xenograft models with accurate identification of primary tumors as well as margin assessment down to 1 mm3 tumor volume. Systemic treatment of xenograft-bearing mice with a dual PI3K/mTOR inhibitor suppressed intratumoral cell signaling and (CR)-S0456 uptake via a reduction in SMVT expression. Novel analysis of in vivo intratumoral cytologic fluorescence using near-infrared confocal laser endomicroscopy demonstrated the absence of coenzyme-R-mediated NIR fluorescence but not fibroblast activation protein (FAP)-conjugated fluorochrome, indicating specific intracellular inhibition of coenzyme-R obligate pathways. CONCLUSION: These findings suggest that a SMVT-targeted NIR contrast agent can be a suitable tracer for imaging a wide range of malignancies as well as evaluating metabolic response to systemic therapies, similar to PET imaging with immune checkpoint inhibitors.

Three-Dimensional Near-Infrared Specimen Mapping Can Identify the Distance from the Tumor to the Surgical Margin During Resection of Pulmonary Ground Glass Opacities.

BACKGROUND: Lung cancers can recur locally due to inadequate resection margins. Achieving adequate margin distances is challenging in pulmonary ground glass opacities (GGOs) because they are not easily palpable. To improve margin assessment during resection of GGOs, we propose a novel technique, three-dimensional near-infrared specimen mapping (3D-NSM). METHODS: Twenty patients with a cT1 GGO were enrolled and received a fluorescent tracer preoperatively. After resection, specimens underwent 3D-NSM in the operating room. Margins were graded as positive or negative based upon fluorescence at the staple line. Images were analyzed using ImageJ to quantify the distance from the tumor edge to the nearest staple line. This margin distance calculated by 3D-NSM was compared to the margin distance reported on final pathology several days postoperatively. RESULTS: 3D-NSM identified 20/20 GGOs with no false positive or false negative diagnoses. Mean fluorescence intensity for lesions was 110.92 arbitrary units (A.U.) (IQR: 77.77-122.03 A.U.) compared to 23.68 A.U. (IQR: 19.60-27.06 A.U.) for background lung parenchyma (p < 0.0001). There were 4 tumor-positive or close margins in the study cohort, and all 4 (100%) were identified by 3D-NSM. 3D-NSM margin distances were nearly identical to margin distances reported on final pathology (R2 = 0.9362). 3D-NSM slightly under-predicted margin distance, and the median difference in margins was 1.9 mm (IQR 0.5-4.3 mm). CONCLUSIONS: 3D-NSM rapidly localizes GGOs by fluorescence and detects tumor-positive or close surgical margins. 3D-NSM can accurately quantify the resection margin distance as compared to formal pathology, which allows surgeons to rapidly determine whether sublobar resection margin distances are adequate.

Glycoprotein Receptor CEACAM5-Targeted Intraoperative Molecular Imaging Tracer in Non-Small Cell Lung Cancer.

BACKGROUND: Intraoperative molecular imaging has emerged as a potential tool in addressing challenges faced during lung cancer surgery by localizing small lesions, ensuring negative margins, and identifying synchronous cancers. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) glycoprotein has emerged as a potential target in fluorescent labeling of non-small cell lung cancer given the high antigen density in tumor cells and absence of expression in normal parenchyma. The goal of our study was to determine whether anti-CEACAM5 targeted near-infrared fluorochrome could be a suitable target in non-small cell lung cancer. METHODS: The CEACAM5 expression was evaluated in AB-12 (known negative control), HT29 (known positive control), and H460 (non-small cell lung cancer) cell lines by polymerase chain reaction. SGM-101, a CEACAM5 antibody, coupled with a BM-104 near-infrared fluorescent tracer was evaluated with dose escalation, in vitro cellular localization, and immunofluorescence microscopy. Subsequently, in vivo validation was performed in 52 athymic nude xenografts. RESULTS: Polymerase chain reaction analysis demonstrated 3000x relative expression of CEACAM5 in HT-29 cells compared with AB-12. The H460 cells showed 1000x relative expression compared with AB12 (P < .05). Both HT29 and H460 cells showed tracer internalization with signal to background ratio of 4.5 (SD 0.34) whereas there was minimal uptake by AB12 cells with signal to background ratio 1.1 (SD 0.1; P < .05). There was linear fluorescence increase with increasing tracer dosing in receptor expressing cell lines. In preclinical models, HT-29 and H460 cells lines produced near-infrared fluorescence with average tumor to background ratio of 3.89 (SD 0.25) irrespective of tumor size compared with no fluorescence by AB12 tumors (P < .05). The CEACAM5 expressing tumors had excellent dye uptake compared with AB12 tumors. CONCLUSIONS: CEACAM5 serves as a possible receptor for targeted intraoperative molecular imaging resections in lung cancer. This study sets a path for evaluation of CEACAM5 targets in future clinical trials.

Novel intraoperative near-infrared imaging strategy to identify abnormalities in the anterior mediastinum.

Thoracic surgeons are frequently asked to biopsy suspicious tissues in the anterior mediastinum to discriminate between a reactive versus malignant pathology such as lymph nodes. The most common benign cause of a mediastinal lymph node is a reactive lymph node from a prior infection or inflammatory process such as post-COVID or granulomatous disease. The most common malignant cause is a lymphoproliferative disorder but also metastatic disease from neck, breast and other regional cancers. Biopsies in this location are challenging because they are far from the trachea and the sternum is a barrier to most diagnostic procedures. Thus, a surgical biopsy is frequently required and a common procedure for Thoracic surgeons. Technically, identifying these lesions can be challenging, particularly for small lesions or those in patients with high body mass index. In order to improve contrast between diseased tissue in the anterior mediastinum and surrounding adipose tissue, we have been studying near-infrared imaging during surgery using indocyanine green (ICG) to give contrast to the abnormal tissues and to avoid an unnecessary extended resection. We developed a modified technique to give ICG to a patient during a biopsy in the anterior mediastinum to specifically highlight abnormal tissues. As a proof-of-principle, we present a case of a young woman with a suspicious 2 cm mediastinal lymph node that required surgical biopsy.

First-in-human results of targeted intraoperative molecular imaging for visualization of ground glass opacities during robotic pulmonary resection.

Background: Identifying ground glass opacities (GGOs) is challenging during robot-assisted thoracic surgery (RATS). Intraoperative molecular imaging (IMI) using tumor-targeted fluorescent tracers may address this clinical problem, but has never been evaluated in RATS. In a pilot study, we sought to determine whether IMI during RATS (RIMI) can localize GGOs. Methods: Ten patients with a cT1 GGO were enrolled. Prior to resection, participants received a folate-receptor targeted fluorescent tracer (OTL38). During RATS, a white-light robotic scope was utilized to identify tumors. RIMI was then conducted using a RATS thoracoscope with a wavelength-specific camera. Finally, a video-assisted thoracic surgery (VATS) thoracoscope designed to detect OTL38 was used as a control to compare to RIMI. The lesions were then resected under RIMI guidance. Results: By white-light robotic scope, 7/10 (70%) GGOs were visually identifiable by pleuroparenchymal distortions. RIMI identified tumor-specific fluorescence in all (100%) subjects. RIMI clearly located the three nodules that could not be seen by robotic white-light imaging. The mean fluorescence intensity (MFI) of tumors was 99.48 arbitrary units (A.U.) (IQR, 75.72-130.49 A.U.), which was significantly higher than background tissue with mean MFI 20.61 A.U. (IQR, 13.49-29.93 A.U., P<0.0001). Mean signal-to-background ratio was 5.71 (range, 2.28-10.13). When compared to VATS-IMI as a control, there were no significant differences in MFI of tumors, background tissue, or signal-to-background ratios. In summary, RIMI compared favorably to VATS-IMI by all measured imaging characteristics. Conclusions: RIMI is feasible for identification of GGOs during robotic resection as compared to white light thoracoscopy and compares favorably to VATS-IMI.

Peripheral blood leukocyte mitochondrial DNA content and risk of lung cancer.

Background: Previous studies of peripheral blood leukocyte mitochondrial DNA (mtDNA) content and risk of lung cancer have yielded inconsistent results, and no studies have evaluated the association between mtDNA content and post-resection lung cancer outcomes. Methods: Using a case-control study design, we evaluated the association between mtDNA content and risk of lung cancer in 465 cases and 378 controls. We also evaluated the association between mtDNA content and survival in 189 cases with surgically resected non-small cell lung cancer (NSCLC). Relative mtDNA content was measured using a quantitative real-time polymerase chain reaction (PCR) assay in peripheral blood genomic DNA. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) using multivariable logistic regression, adjusting for age, gender, race, and smoking history. Results: mtDNA content was lower in cases compared to controls, with medians of 1.26 [interquartile range (IQR), 0.98-1.70)] and 1.79 (IQR, 1.34-2.10; P<0.001), respectively. Compared to the quartile of subjects with the highest mtDNA content, there was significantly higher likelihood of lung cancer in the second lowest quartile (OR 3.44; 95% CI: 2.06-5.75) and the lowest quartile (OR 6.36; 95% CI: 3.86-10.47). In patients with resected NSCLC, there was no association between lower mtDNA content and recurrence-free survival (RFS) [hazard ratio (HR) 0.89; 95% CI: 0.47-1.66] or overall survival (OS) (HR 0.71; 95% CI: 0.35-1.46). Conclusions: Thus, our results counter previous studies and find that lower mtDNA content is associated with lung cancer risk. Our results suggest that mtDNA content could potentially serve as a risk biomarker, but is not associated with survival outcomes in NSCLC.

Precision Surgery Guided by Intraoperative Molecular Imaging.

Intraoperative molecular imaging (IMI) has recently emerged as an important tool in the armamentarium of surgical oncologists. IMI allows real-time assessment of oncologic resection quality, margin assessment, and occult disease detection during real-time surgery. Numerous tracers have now been developed for use in IMI-guided tissue sampling. Fluorochromes localize to the tumor by taking advantage of their disorganized capillary milieu, overexpressed receptors, or upregulated enzymes. Although fluorescent tracers can suffer from issues of autofluorescence and lack of depth penetration, these challenges are being addressed through hybrid radioactive/fluorescent tracers and new tracers that fluoresce in the near-infrared (NIR-II [wavelength > 1,000 nm]) range. IMI is already being used to treat numerous cancers, with demonstrated improvement in cancer recurrence and patient outcomes without incurring significant burden on either clinicians or patients. In this comprehensive review, we discuss history, mechanism, current oncologic applications, and future directions of IMI-guided optical biopsy.

Preclinical Evaluation of an Activity-Based Probe for Intraoperative Imaging of Esophageal Cancer.

Background: Early detection and complete resection are important prognostic factors for esophageal cancer (EC). Intraoperative molecular imaging (IMI) using tumor-targeted tracers is effective in many cancer types. However, there are no EC-specific IMI tracers. We sought to test a cathepsin activity-based tracer (VGT-309) for EC resection. Methods: Murine (AKR, HNM007) and human (OE19) EC cell lines were screened for cathepsin expression by western blotting. In vitro binding affinity of VGT-309 was evaluated by fluorescence microscopy. Flank tumor models were developed by injecting EC cells into the flanks of BALB/c or athymic nude mice. Mice pretreated with a cathepsin inhibitor (JPM-OEt) were used to confirm on target binding. Animals were injected with 2 mg/kg VGT-309, underwent IMI, and were sacrificed 24 hours after injection. Results: Cathepsins B, L, S, and X were expressed by EC cell lines, and all cell lines were labeled in vitro with VGT-309. Fluorescent signal was eliminated when cells were pretreated with JPM-OEt. On biodistribution analysis, VGT-309 accumulated in the liver, kidneys, and spleen without other organ involvement. VGT-309 selectively accumulated in flank allografts and xenografts, with mean signal-to-background ratio of 5.21 (IQR: 4.18-6.73) for flank allografts and 4.34 (IQR: 3.75-5.02) for flank xenografts. Fluorescence microscopy and histopathological analysis confirmed the selective accumulation of the tracer in tumors compared to background normal tissues. Conclusions: VGT-309 is an effective tracer for IMI of esophageal cancer. There is potential for clinical translation both as an adjunct to endoscopic detection and for complete removal of disease during esophagectomy.

Targeted detection of cancer cells during biopsy allows real-time diagnosis of pulmonary nodules.

BACKGROUND: The diagnostic yield of biopsies of solitary pulmonary nodules (SPNs) is low, particularly in sub-solid lesions. We developed a method (NIR-nCLE) to achieve cellular level cancer detection during biopsy by integrating (i) near-infrared (NIR) imaging using a cancer-targeted tracer (pafolacianine), and (ii) a flexible NIR confocal laser endomicroscopy (CLE) system that can fit within a biopsy needle. Our goal was to assess the diagnostic accuracy of NIR-nCLE ex vivo in SPNs. METHODS: Twenty patients with SPNs were preoperatively infused with pafolacianine. Following resection, specimens were inspected to identify the lesion of interest. NIR-nCLE imaging followed by tissue biopsy was performed within the lesion and in normal lung tissue. All imaging sequences (n = 115) were scored by 5 blinded raters on the presence of fluorescent cancer cells and compared to diagnoses by a thoracic pathologist. RESULTS: Most lesions (n = 15, 71%) were adenocarcinoma-spectrum malignancies, including 7 ground glass opacities (33%). Mean fluorescence intensity (MFI) by NIR-nCLE for tumor biopsy was 20.6 arbitrary units (A.U.) and mean MFI for normal lung was 6.4 A.U. (p < 0.001). Receiver operating characteristic analysis yielded a high area under the curve for MFI (AUC = 0.951). Blinded raters scored the NIR-nCLE sequences on the presence of fluorescent cancer cells with sensitivity and specificity of 98% and 97%, respectively. Overall diagnostic accuracy was 97%. The inter-observer agreement of the five raters was excellent (κ = 0.95). CONCLUSIONS: NIR-nCLE allows sensitive and specific detection of cancer cells in SPNs. This technology has far-reaching implications for diagnostic needle biopsies and intraprocedural decision-making.

A Cathepsin-Targeted Quenched Activity-Based Probe Facilitates Enhanced Detection of Human Tumors during Resection.

PURPOSE: Fluorescence-guided surgery using tumor-targeted contrast agents has been developed to improve the completeness of oncologic resections. Quenched activity-based probes that fluoresce after covalently binding to tumor-specific enzymes have been proposed to improve specificity, but none have been tested in humans. Here, we report the successful clinical translation of a cathepsin activity-based probe (VGT-309) for fluorescence-guided surgery. EXPERIMENTAL DESIGN: We optimized the specificity, dosing, and timing of VGT-309 in preclinical models of lung cancer. To evaluate clinical feasibility, we conducted a canine study of VGT-309 during pulmonary tumor resection. We then conducted a randomized, double-blind, dose-escalation study in healthy human volunteers receiving VGT-309 to evaluate safety. Finally, we tested VGT-309 in humans undergoing lung cancer surgery. RESULTS: In preclinical models, we found highly specific tumor cell labeling that was blocked by a broad spectrum cathepsin inhibitor. When evaluating VGT-309 for guidance during resection of canine tumors, we found that the probe selectively labeled tumors and demonstrated high tumor-to-background ratio (TBR; range: 2.15-3.71). In the Phase I human study, we found that VGT-309 was safe at all doses studied. In the ongoing Phase II trial, we report two cases in which VGT-309 localized visually occult, non-palpable tumors (TBRs = 2.83 and 7.18) in real time to illustrate its successful clinical translation and potential to improve surgical management. CONCLUSIONS: This first-in-human study demonstrates the safety and feasibility of VGT-309 to label human pulmonary tumors during resection. These results may be generalizable to other cancers due to cathepsin overexpression in many solid tumors.

Comparative Experience of Short-wavelength Versus Long-wavelength Fluorophores for Intraoperative Molecular Imaging of Lung Cancer.

BACKGROUND: Intraoperative molecular imaging (IMI) using tumor-targeted optical contrast agents can improve cancer resections. The optimal wavelength of the IMI tracer fluorophore has never been studied in humans and has major implications for the field. To address this question, we investigated 2 spectroscopically distinct fluorophores conjugated to the same targeting ligand. METHODS: Between December 2011 and November 2021, patients with primary lung cancer were preoperatively infused with 1 of 2 folate receptor-targeted contrast tracers: a short-wavelength folate-fluorescein (EC17; λ em =520 nm) or a long-wavelength folate-S0456 (pafolacianine; λ em =793 nm). During resection, IMI was utilized to identify pulmonary nodules and confirm margins. Demographic data, lesion diagnoses, and fluorescence data were collected prospectively. RESULTS: Two hundred eighty-two patients underwent resection of primary lung cancers with either folate-fluorescein (n=71, 25.2%) or pafolacianine (n=211, 74.8%). Most tumors (n=208, 73.8%) were invasive adenocarcinomas. We identified 2 clinical applications of IMI: localization of nonpalpable lesions (n=39 lesions, 13.8%) and detection of positive margins (n=11, 3.9%). In each application, the long-wavelength tracer was superior to the short-wavelength tracer regarding depth of penetration, signal-to-background ratio, and frequency of event. Pafolacianine was more effective for detecting subpleural lesions (mean signal-to-background ratio=2.71 vs 1.73 for folate-fluorescein, P <0.0001). Limit of signal detection was 1.8 cm from the pleural surface for pafolacianine and 0.3 cm for folate-fluorescein. CONCLUSIONS: Long-wavelength near-infrared fluorophores are superior to short-wavelength IMI fluorophores in human tissues. Therefore, future efforts in all human cancers should likely focus on long-wavelength agents.

Targeted detection of cancer at the cellular level during biopsy by near-infrared confocal laser endomicroscopy.

Suspicious nodules detected by radiography are often investigated by biopsy, but the diagnostic yield of biopsies of small nodules is poor. Here we report a method-NIR-nCLE-to detect cancer at the cellular level in real-time during biopsy. This technology integrates a cancer-targeted near-infrared (NIR) tracer with a needle-based confocal laser endomicroscopy (nCLE) system modified to detect NIR signal. We develop and test NIR-nCLE in preclinical models of pulmonary nodule biopsy including human specimens. We find that the technology has the resolution to identify a single cancer cell among normal fibroblast cells when co-cultured at a ratio of 1:1000, and can detect cancer cells in human tumors less than 2 cm in diameter. The NIR-nCLE technology rapidly delivers images that permit accurate discrimination between tumor and normal tissue by non-experts. This proof-of-concept study analyzes pulmonary nodules as a test case, but the results may be generalizable to other malignancies.

Impact of Intraoperative Molecular Imaging after Fluorescent-Guided Pulmonary Metastasectomy for Sarcoma.

BACKGROUND: Intraoperative molecular imaging (IMI) has been shown to improve lesion detection during pulmonary sarcomatous metastasectomy. Our goal in this study was to evaluate whether data garnered from IMI-guided resection of pulmonary sarcoma metastasis translate to improved patient outcomes. STUDY DESIGN: Fifty-two of 65 consecutive patients with a previous history of sarcomas found to have pulmonary nodules during screening were enrolled in a nonrandomized clinical trial. Patients underwent TumorGlow the day before surgery. Data on patient demographics, tumor biologic characteristics, preoperative assessment, and survival were included in the study analysis and compared with institutional historical data of patients who underwent metastasectomy without IMI. p values < 0.05 were considered significant. RESULTS: IMI detected 42 additional lesions in 31 patients (59%) compared with the non-IMI cohort where 25% percent of patients had additional lesions detected using tactile and visual feedback only (p < 0.05). Median progression-free survival (PFS) for patients with IMI-guided pulmonary sarcoma metastasectomy was 36 months vs 28.6 months in the historical cohort (p < 0.05). IMI-guided pulmonary sarcoma metastasectomy had recurrence in the lung with a median time of 18 months compared with non-IMI group at 13 months (p < 0.05). Patients with synchronous lesions in the IMI group underwent systemic therapy at a statistically higher rate and tended to undergo routine screening at shorter interval. CONCLUSIONS: IMI identifies a subset of sarcoma patients during pulmonary metastasectomy who have aggressive disease and informs the medical oncologist to pursue more aggressive systemic therapy. In this setting, IMI can serve both as a diagnostic and prognostic tool without conferring additional risk to the patient.

Anterior Mediastinal Neuroblastoma Associated with Syndrome of Inappropriate Antidiuretic Hormone Secretion: A Morphologic, Immunohistochemical, and Genetic Case Report and Review of the Literature.

We report a mediastinal neuroblastoma in an octogenarian with paraneoplastic syndrome of inappropriate antidiuretic hormone secretion (SIADH). Neuroblastomas are very rare tumors in adults, with thoracic or mediastinal locations being especially uncommon. These neoplasms have been occasionally associated with the SIADH. Given the rarity of incidence and paucity of diagnostic and outcomes data, the significance of standard neuroblastoma prognostic characteristics is unclear, and no treatment paradigms exist for these patients. Further studies are needed to inform future clinical guidelines.

A Prostate-Specific Membrane Antigen-Targeted Near-Infrared Conjugate for Identifying Pulmonary Squamous Cell Carcinoma during Resection.

Pulmonary squamous cell carcinoma is the second most common lung cancer subtype and has a low 5-year survival rate at 17.6%. Complete resection with negative margins can be curative, but a high number of patients suffer early postoperative recurrence due to inadequate disease clearance at the index operation. Intraoperative molecular imaging (IMI) with tumor-targeted optical contrast agents is effective in improving resection completeness for other tumor types, but there are no IMI tracers targeted to pulmonary squamous cell carcinoma. In this report, we describe the use of a novel prostate-specific membrane antigen (PSMA)-targeted near-infrared conjugate (OTL78) to identify pulmonary squamous cell carcinoma. We identified PSMA as a viable target by examining its expression in human lung tumor specimens from a surgical cohort. Ninety-four percent of tumors expressed PSMA in either the pulmonary squamous cells or the tumor neovasculature. Using in vitro and in vivo models, we found that OTL78 reliably localized pulmonary squamous cell carcinoma in a PSMA-dependent manner. Finally, we found that IMI with OTL78 markedly improved surgeons' ability to identify residual disease after surgery in a preclinical model. Ultimately, this novel optical tracer may aid surgical resection of pulmonary squamous cell carcinoma and potentially improve long-term outcomes.