A Phase 2 Multicenter Clinical Trial of Intraoperative Molecular Imaging of Lung Cancer with a pH-Activatable Nanoprobe.

PURPOSE: Intraoperative molecular imaging (IMI) uses tumor-targeted optical contrast agents to improve identification and clearance of cancer. Recently, a probe has been developed that only fluoresces when activated in an acidic pH, which is common to many malignancies. We report the first multicenter Phase 2 trial of a pH-activatable nanoprobe (pegsitacianine, ONM-100) for IMI of lung cancer. METHODS: Patients with suspected or biopsy-confirmed lung cancer scheduled for sublobar resection were administered a single intravenous infusion of pegsitacianine (1 mg/kg) one to three days prior to surgery. Intraoperatively, the patients underwent a white light thoracoscopic evaluation, and then were imaged with an NIR thoracoscope to detect tumor fluorescence. The primary study endpoint was the proportion of patients with a clinically significant event (CSE) which was defined as an intraoperative discovery during IMI that led to a change in the surgical procedure. Possible CSEs included (i) localizing the index lung nodule that could not be located by white light, (ii) identifying a synchronous malignant lesion, or (iii) recognizing a close surgical margin (< = 10 mm). Secondary endpoints were sensitivity, specificity, NPV, and PPV of pegsitacianine in detecting tumor-containing tissue. The safety evaluation was based on adverse event reporting, clinical laboratory parameters, and physical examinations. RESULTS: Twenty patients were confirmed as eligible and administered pegsitacianine. Most of the patients were female (n = 12 [60%]), middle-aged (mean age 63.4 years), and former smokers (n = 13 [65%], 28.6 mean pack years). Mean lesion size was 1.9 cm, and most lesions (n = 17 [85%]) were malignant. The most common histologic subtype was adenocarcinoma (n = 9). By utilizing IMI with pegsitacianine, one patient had a CSE in the detection of a close margin and another had localization of a tumor not detectable by traditional surgical means. Six of 19 (31.6%) malignant lesions fluoresced with mean tumor-to-background ratio (TBR) of 3.00, as compared to TBR of 1.20 for benign lesions (n = 3). Sensitivity and specificity of pegsitacianine-based IMI for detecting malignant tissue was 31.6% and 33.3%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) of pegsitacianine-based IMI was 75% and 7.1%, respectively. Pegsitacianine-based imaging was not effective in differentiating benign and malignant lymph nodes. From a safety perspective, no drug-related serious adverse events occurred. Four patients experienced mild pegsitacianine-related infusion reactions which required discontinuing the study drug with complete resolution of symptoms. CONCLUSIONS: Pegsitacianine-based IMI, though well tolerated from a safety perspective, does not consistently label lung tumors during resection and does not provide significant clinical benefit over existing standards of surgical care. The biology of lung tumors may not be as acidic as other solid tumors in the body thereby not activating the probe as predicted.

Non-Small Cell Lung Cancer Imaging Using a Phospholipase A2 Activatable Fluorophore.

Lung cancer, the most common cause of cancer-related death in the United States, requires advanced intraoperative detection methods to improve evaluation of surgical margins. In this study we employed DDAO-arachidonate (DDAO-A), a phospholipase A2 (PLA2) activatable fluorophore, designed for the specific optical identification of lung cancers in real-time during surgery. The in vitro fluorescence activation of DDAO-A by porcine sPLA2 was tested in various liposomal formulations, with 100 nm extruded EggPC showing the best overall characteristics. Extruded EggPC liposomes containing DDAO-A were tested for their stability under various storage conditions, demonstrating excellent stability for up to 4 weeks when stored at -20 °C or below. Cell studies using KLN 205 and LLC1 lung cancer cell lines showed DDAO-A activation was proportional to cell number. DDAO-A showed preferential activation by human recombinant cPLA2, an isoform highly specific to arachidonic acid-containing lipids, when compared to a control probe, DDAO palmitate (DDAO-P). In vivo studies with DBA/2 mice bearing KLN 205 lung tumors recapitulated these results, with preferential activation of DDAO-A relative to DDAO-P following intratumoral injection. Topical application of DDAO-A-containing liposomes to human (n = 10) and canine (n = 3) lung cancers ex vivo demonstrated the preferential activation of DDAO-A in tumor tissue relative to adjacent normal lung tissue, with fluorescent tumor-to-normal ratios (TNR) of up to 5.2:1. The combined results highlight DDAO-A as a promising candidate for clinical applications, showcasing its potential utility in intraoperative and back-table imaging and topical administration during lung cancer surgeries. By addressing the challenge of residual microscopic disease at resection margins and offering stability in liposomal formulations, DDAO-A emerges as a potentially valuable tool for advancing precision lung cancer surgery and improving curative resection rates.

Inhibition of glioblastoma cell proliferation and invasion by the choline-kinase inhibitor JAS239 varies with cell type and hypoxia.

Background: Elevated choline kinase alpha (ChoK) is observed in most solid tumours including glioblastomas (GBM), yet until recently, inhibitors of ChoK have demonstrated limited efficacy in GBM models. Given that hypoxia is associated with GBM therapy resistance, we hypothesised that tumour hypoxia could be responsible for such limitations. We therefore evaluated in GBM cells, the effect of hypoxia on the function of JAS239, a potent ChoK inhibitor. Methods: Rodent (F98 and 9L) and human (U-87 MG and U-251 MG) GBM cell lines were subjected to 72 hours of hypoxia conditioning and treated with JAS239 for 24 hours. NMR metabolomic measurements and analyses were performed to evaluate the signalling pathways involved. In addition, cell proliferation, cell cycle progression and cell invasion were measured in cell monolayers and 3D spheroids, with or without JAS239 treatment in normoxic or hypoxic cells to assess how hypoxia affects JAS239 function. Results: Hypoxia and JAS239 treatment led to significant changes in the cellular metabolic pathways, specifically the phospholipid and glycolytic pathways associated with a reduction in cell proliferation via induced cell cycle arrest. Interestingly, JAS239 also impaired GBM invasion. However, JAS239 effects were variable depending on the cell line, reflecting the inherent heterogeneity observed in GBMs. Conclusion: Our findings indicate that JAS239 and hypoxia can deregulate cellular metabolism, inhibit proliferation and alter cell invasion. These results may be useful for the design of new therapeutic strategies based on ChoK inhibition that can act on multiple pro-tumorigenic features.

A Biodegradable "One-For-All" Nanoparticle for Multimodality Imaging and Enhanced Photothermal Treatment of Breast Cancer.

Silver sulfide nanoparticles (Ag2S-NP) hold promise for various optical-based biomedical applications, such as near-infrared fluorescence (NIRF) imaging, photoacoustics (PA), and photothermal therapy (PTT). However, their NIR absorbance is relatively low, and previous formulations are synthesized using toxic precursors under harsh conditions and are not effectively cleared due to their large size. Herein, sub-5 nm Ag2S-NP are synthesized and encapsulated in biodegradable, polymeric nanoparticles (AgPCPP). All syntheses are conducted using biocompatible, aqueous reagents under ambient conditions. The encapsulation of Ag2S-NP in polymeric nanospheres greatly increases their NIR absorbance, resulting in enhanced optical imaging and PTT effects. AgPCPP nanoparticles exhibit potent contrast properties suitable for PA and NIRF imaging, as well as for computed tomography (CT). Furthermore, AgPCPP nanoparticles readily improve the conspicuity of breast tumors in vivo. Under NIR laser irradiation, AgPCPP nanoparticles significantly reduce breast tumor growth, leading to prolonged survival compared to free Ag2S-NP. Over time, AgPCPP retention in tissues gradually decreases, without any signs of acute toxicity, providing strong evidence of their safety and biodegradability. Therefore, AgPCPP may serve as a "one-for-all" theranostic agent that degrades into small components for excretion after fulfilling diagnostic and therapeutic tasks, offering good prospects for clinical translation.

A pH-Activatable Nanoprobe Labels Diverse Histologic Subtypes of Human Lung Cancer During Resection.

BACKGROUND: Intraoperative molecular imaging (IMI) uses tumor-targeted optical contrast agents to improve identification and clearance of cancer during surgery. Recently, pH-activatable contrast agents have been developed but none has been tested in lung cancer. Here, we report the successful clinical translation of pegsitacianine (ONM-100), a pH-activatable nanoprobe, for fluorescence-guided lung cancer resection. METHODS: We first characterized the pH setpoint for pegsitacianine fluorescence activation in vitro. We then optimized the specificity, dosing, and timing of pegsitacianine in murine flank xenograft models of lung adenocarcinoma and squamous cell carcinoma. Finally, we tested pegsitacianine in humans undergoing lung cancer surgery as part of an ongoing phase 2 trial. RESULTS: We found that the fluorescence activation of pegsitacianine occurred below physiologic pH in vitro. Using preclinical models of lung cancer, we found that the probe selectively labeled both adenocarcinoma and squamous cell carcinoma xenografts (mean tumor-to-background ratio [TBR] > 2.0 for all cell lines). In the human pilot study, we report cases in which pegsitacianine localized pulmonary adenocarcinoma and pulmonary squamous cell carcinoma (TBRs= 2.7 and 2.4) in real time to illustrate its successful clinical translation and potential to improve surgical management. CONCLUSIONS: This translational study demonstrates the feasibility of pegsitacianine as an IMI probe to label the two most common histologic subtypes of human lung cancer.

Intraoperative molecular imaging: 3rd biennial clinical trials update.

Significance: This third biennial intraoperative molecular imaging (IMI) conference shows how optical contrast agents have been applied to develop clinically significant endpoints that improve precision cancer surgery. Aim: National and international experts on IMI presented ongoing clinical trials in cancer surgery and preclinical work. Previously known dyes (with broader applications), new dyes, novel nonfluorescence-based imaging techniques, pediatric dyes, and normal tissue dyes were discussed. Approach: Principal investigators presenting at the Perelman School of Medicine Abramson Cancer Center's third clinical trials update on IMI were selected to discuss their clinical trials and endpoints. Results: Dyes that are FDA-approved or currently under clinical investigation in phase 1, 2, and 3 trials were discussed. Sections on how to move benchwork research to the bedside were also included. There was also a dedicated section for pediatric dyes and nonfluorescence-based dyes that have been newly developed. Conclusions: IMI is a valuable adjunct in precision cancer surgery and has broad applications in multiple subspecialties. It has been reliably used to alter the surgical course of patients and in clinical decision making. There remain gaps in the utilization of IMI in certain subspecialties and potential for developing newer and improved dyes and imaging techniques.

Prospective validation of tumor folate receptor expression density with the association of pafolacianine fluorescence during intraoperative molecular imaging-guided lung cancer resections.

PURPOSE: Pafolacianine, a folate receptor alpha-targeted NIR tracer, has demonstrated clear efficacy in intraoperative molecular imaging-guided (IMI) lung cancer surgery. However, the selection of patients who would benefit from IMI remains challenging given the variability of fluorescence with patient-associated and histopathologic factors. Our goal in this study was to prospectively evaluate whether preoperative FRα/FRß staining can predict pafolacianine-based fluorescence during real-time lung cancer resections. METHODS: This was a prospective study conducted between 2018 and 2022 that reviewed core biopsy and intraoperative data from patients with suspected lung cancer. A total of 196 patients were deemed eligible, of whom core biopsies were taken from 38 patients and assessed for FRα and FRß expression by immunohistochemistry (IHC). All patients underwent infusion of pafolacianine 24 h prior to surgery. Intraoperative fluorescence images were captured with the VisionSense bandpass filter-enabled camera. All histopathologic assessments were performed by a board-certified thoracic pathologist. RESULTS: Of the 38 patients, 5 (13.1%) were found to have benign lesions (necrotizing granulomatous inflammation, lymphoid aggregates) and 1 had metastatic non-lung nodule. Thirty (81.5%) had malignant lesions, with the vast majority (23, 77.4%) being lung adenocarcinoma (7 (22.5%) SCC). None of the benign tumors (0/5, 0%) exhibited in vivo fluorescence (mean TBR of 1.72), while 95% of the malignant tumors fluoresced (mean TBR of 3.11 ± 0.31) compared to squamous cell carcinoma (1.89 ± 0.29) of the lung and sarcomatous lung metastasis (2.32 ± 0.09) (p < 0.01). The TBR was significantly higher in the malignant tumors (p = 0.009). The median FRα and FRß staining intensities were both 1.5 for benign tumors, while the FRα and FRß staining intensities were 3 and 2 for malignant tumors, respectively. Increased FRα expression was significantly associated with the presence of fluorescence (p = 0.01), CONCLUSION: This prospective study sought to determine whether preoperative FRα and FRß expression on core biopsy IHC correlates with intraoperative fluorescence during pafolacianine-guided surgery. These results, although of small sample size, including limited non-adenocarcinoma cohort, suggest that performing FRα IHC on preoperative core biopsies of adenocarcinomas as compared to squamous cell carcinomas could provide low-cost, clinically useful information for optimal patient selection which should be further explored in advanced clinical trials.

Metabolic changes in glioblastomas in response to choline kinase inhibition: In vivo MRS in rodent models.

Changes in glioblastoma (GBM) metabolism was investigated in response to JAS239, a choline kinase inhibitor, using MRS. In addition to the inhibition of phosphocholine synthesis, we investigated changes in other key metabolic pathways associated with GBM progression and treatment response. Three syngeneic rodent models of GBM were used: F98 (N = 12) and 9L (N = 8) models in rats and GL261 (N = 10) in mice. Rodents were intracranially injected with GBM cells in the right cortex and tumor growth was monitored using T2 -weighted images. Animals were treated once daily with intraperitoneal injections of 4 mg/kg JAS239 (F98 rats, n = 6; 9L rats, n = 6; GL261 mice, n = 5) or saline (control group, F98 rats, n = 6; 9L rats, n = 2; GL261 mice, n = 5) for five consecutive days. Single voxel spectra were acquired on Days 0 (T0, baseline) and 6 (T6, end of treatment) from the tumor as well as the contralateral normal brain using a PRESS sequence. Changes in metabolite ratios (tCho/tCr, tCho/NAA, mI/tCr, Glx/tCr and (Lip + Lac)/Cr) were used to assess metabolic pathway alterations in response to JAS239. Tumor growth arrest was noted in all models in response to JAS239 treatment compared with saline-treated animals, with a significant reduction (p < 0.05) in the F98 model. A reduction in tCho/tCr was observed with JAS239 treatment in all GBM models, indicating reduced phospholipid metabolism, with the highest reduction in 9L followed by GL261 and F98 tumors. A significant reduction (p < 0.05) in the tCho/NAA ratio was observed in the 9L model. A significant reduction in mI/tCr (p < 0.05) was found in JAS239-treated F98 tumors compared with the saline-treated animals. A non-significant trend of reduction in Glx/tCr was observed only in F98 and 9L tumors. JAS239-treated F98 tumors also showed a significant increase in Lip + Lac (p < 0.05), indicating increased cell death. This study demonstrated the utility of MRS in assessing metabolic changes in GBM in response to choline kinase inhibition.

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.

Indocyanine green fluorescence-guided surgery in head and neck cancer: A systematic review.

OBJECTIVE: To assess the feasibility and effectiveness of indocyanine green (ICG) for image-guided resection of head and neck cancer (HNC). DATA SOURCES: PubMed, Embase, and Scopus databases. REVIEW METHODS: Searches were conducted from database inception to February 2022. Patient and study characteristics, imaging parameters, and imaging efficacy data were extracted from each study. RESULTS: Nine studies met inclusion criteria, representing 103 head and neck tumors. Weighted mean ICG dose and imaging time were 1.27 mg/kg and 11.77 h, respectively. Among the five studies that provided quantitative metrics of imaging efficacy, average ICG tumor-to-background ratio (TBR) was 1.56 and weighted mean ONM-100 TBR was 3.64. Pooled sensitivity and specificity across the five studies were 91.7 % and 71.9 %, respectively. CONCLUSION: FGS with ICG may facilitate real-time tumor-margin delineation to improve margin clearance rates and progression-free survival. Future studies with validated, quantitative metrics of imaging success are necessary to further evaluate the prognostic benefit of these techniques.

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.

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.

Presence of non-Newtonian fluid in invasive pulmonary mucinous adenocarcinomas impacts fluorescence during intraoperative molecular imaging of lung cancer.

BACKGROUND: Intraoperative molecular imaging (IMI) with folate-targeted NIR tracers has been shown to improve lesion localization in more than 80% of lung adenocarcinomas. However, mucinous adenocarcinomas (MAs) and invasive mucinous adenocarcinomas (IMAs) of the lung, which are variants of adenocarcinoma, appear to have decreased fluorescence despite appropriate folate receptor expression on the tumor surface. We hypothesized that the etiology may be related to light excitation and emission through non-Newtonian fluid (mucin) produced by goblet and columnar cancer cells. METHODS: Intraoperative data for 311 subjects were retrospectively reviewed from a prospectively collected 6-year database. For standardization, all patients underwent infusion of the same targeted molecular optical contrast agent (pafolacianine, folate receptor-targeted NIR fluorochrome) for lung cancer resections. Then, the ratio of the mean fluorescence intensity of the tumors and background tissues (TBR) was calculated. Tumors were examined for mucin, FRa, FRb, and immunofluorescent tracer uptake by a board-certified pathologist. The optical properties of mucin analyzed by imaging software were used to create in vitro gel models to explore the effects on NIR tracer fluorescence intensity. RESULTS: A large proportion (192, 62%) of the patients were female, with an average of 62.8 years and a 34-year mean pack smoking history. There were no severe (Clavien-Dindo > III) complications related to pafolacianine infusion. A total of 195 lesions in the study were adenocarcinomas, of which 19 (6.1%) were of the mucinous subtype. A total of 14/19 of the patients had a smoking history, and more than 74% of the IMA lesions were in the lower lobes. IMA lesions had a lower in situ TBR than nonmucinous adenocarcinomas (2.64 SD 0.23) vs (3.45 SD 0.11), respectively (p < 0.05). Only 9/19 (47%) were localized in situ. Tumor bisection and removal of mucin from IMAs significantly increased pafolacianine fluorescence, with resultant TBR not being significantly different from the control group (4.67 vs 4.89) (p = 0.19). Of the 16 lesions that underwent FR expression analysis, 15/16 had FR presence on cancer cells or tumor-associated macrophages in the tumor microenvironment. There was no statistically significant difference in fluorescence intensity during immunofluorescence analysis (4.99 vs 5.08) (p = 0.16). Physical removal of mucin from IMAs improved the TBR from 3.11 to 4.67 (p < 0.05). In vitro analysis of the impact of synthetic non-Newtonian fluid (agarose 0.5%) on NIR tracer fluorescence showed a decrease in MFI by a factor of 0.25 regardless of the concentration for each 5 mm thickness of mucin. CONCLUSION: The mucinous subtype of lung adenocarcinomas presents a unique challenge in pafolacianine-targeted IMI-guided resections. The presence of non-Newtonian fluids presents a physical barrier that dampens the excitation of the tracer and fluorescence emission detected by the camera. Knowledge of this phenomenon can allow the surgeon to critically analyze lesion fluorescence parameters during IMI-guided lung cancer resections.

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.

Assessing Tumour Haemodynamic Heterogeneity and Response to Choline Kinase Inhibition Using Clustered Dynamic Contrast Enhanced MRI Parameters in Rodent Models of Glioblastoma.

To investigate the utility of DCE-MRI derived pharmacokinetic parameters in evaluating tumour haemodynamic heterogeneity and treatment response in rodent models of glioblastoma, imaging was performed on intracranial F98 and GL261 glioblastoma bearing rodents. Clustering of the DCE-MRI-based parametric maps (using Tofts, extended Tofts, shutter speed, two-compartment, and the second generation shutter speed models) was performed using a hierarchical clustering algorithm, resulting in areas with poor fit (reflecting necrosis), low, medium, and high valued pixels representing parameters Ktrans, ve, Kep, vp, τi and Fp. There was a significant increase in the number of necrotic pixels with increasing tumour volume and a significant correlation between ve and tumour volume suggesting increased extracellular volume in larger tumours. In terms of therapeutic response in F98 rat GBMs, a sustained decrease in permeability and perfusion and a reduced cell density was observed during treatment with JAS239 based on Ktrans, Fp and ve as compared to control animals. No significant differences in these parameters were found for the GL261 tumour, indicating that this model may be less sensitive to JAS239 treatment regarding changes in vascular parameters. This study demonstrates that region-based clustered pharmacokinetic parameters derived from DCE-MRI may be useful in assessing tumour haemodynamic heterogeneity with the potential for assessing therapeutic response.

3D Specimen Mapping Expedites Frozen Section Diagnosis of Nonpalpable Ground Glass Opacities.

BACKGROUND: Pulmonary ground glass opacities (GGOs) are early-stage adenocarcinoma spectrum lesions that are not easily palpable. Challenges in localizing GGOs during intraoperative pathology can lead to imprecise diagnoses and additional time under anesthesia. To improve localization of GGOs during frozen section diagnosis, we evaluated a novel technique, 3-dimensional near-infrared specimen mapping (3D-NSM). METHODS: Fifty-five patients with a cT1 GGO were enrolled and received a fluorescent tracer preoperatively. After resection, specimens were inspected to identify lesions. Palpable and nonpalpable nodules underwent 3D-NSM and the area of highest fluorescence was marked with a suture. Time for 3D-NSM, time for frozen section diagnosis, and number of tissue sections examined were recorded. To compare 3D-NSM with standard-of-care techniques, a control cohort of 20 subjects with identical inclusion criteria were enrolled. Specimens did not undergo 3D-NSM and were sent directly to pathology. RESULTS: 3D-NSM localized 54 of 55 lesions with 1 false negative. All 41 palpable lesions were identified by 3D-NSM. Thirteen (92.8%) of 14 nonpalpable lesions were located by 3D-NSM. Time to diagnosis for the 3D-NSM cohort was 23.5 minutes, compared with 26.0 minutes in the control cohort (P = .04). 3D-NSM did not affect time to diagnosis of palpable lesions (23.2 minutes vs 21.4 minutes; P = .10). 3D-NSM significantly reduced time to diagnosis for nonpalpable lesions (23.3 minutes vs 34.4 minutes; P < .0001). 3D-NSM also reduced the number of tissue sections analyzed in nonpalpable lesions (4.50 vs 11.00; P < .0001). CONCLUSIONS: 3D-NSM accurately localizes GGOs and expedites intraoperative diagnosis by reducing the number of tissue sections analyzed for nonpalpable GGOs.

Second window ICG predicts gross-total resection and progression-free survival during brain metastasis surgery.

OBJECTIVE: Metastases are the most common intracranial malignancies and complete resection can provide relief of neurological symptoms and reduce recurrence. The authors' prospective pilot study in 2017 demonstrated promising results for the application of high-dose, delayed imaging of indocyanine green (ICG), known as second window ICG (SWIG), in patients undergoing surgery for brain metastases. In this prospective cohort study, the authors evaluated intraoperative imaging and clinical outcomes of treatment using SWIG. METHODS: Patients were prospectively enrolled in an approved study of high-dose, delayed ICG (SWIG) and received 5 mg/kg (2014-2018) or 2.5 mg/kg (2018-2019) ICG 24 hours preoperatively. Intraoperatively, near-infrared (NIR) imaging was performed using a dedicated NIR exoscope. NIR images were analyzed and the signal-to-background ratio (SBR) was calculated to quantify fluorescence. Residual fluorescence on the postresection NIR view was compared and correlated to the residual gadolinium enhancement on postoperative MRI. Patient survival and predictive factors were analyzed. RESULTS: In total, 51 intracranial metastases were surgically treated in 47 patients in this cohort. All 51 metastatic tumors demonstrated strong NIR fluorescence (mean SBR 4.9). In tumors ≤ 10 mm from the cortical surface, SWIG with 5 mg/kg ICG produced enhanced transdural tumor visibility (91.3%) compared to 2.5 mg/kg (52.9%; p = 0.0047). Neoplastic margin detection using NIR fluorescence compared to white light improved sensitivity, albeit lowered specificity; however, increasing the SBR cutoff for positive fluorescence significantly improved specificity without sacrificing sensitivity, increasing the overall accuracy from 57.5% to 72.5%. A lack of residual NIR fluorescence after resection was closely correlated with a lack of residual enhancement on postoperative MRI (p = 0.007). Among the 16 patients in whom tumor recurred at the site of surgery, postoperative MRI successfully predicted 8 cases, whereas the postresection NIR view predicted 12 cases. Progression-free survival rate at 12 months was greater for patients without residual NIR fluorescence (38%) than for those without residual enhancement on postoperative MRI (29%). CONCLUSIONS: The current study demonstrates the clinical benefits of the SWIG technique in surgery for patients with brain metastases. Specifically, this technique allows for dose-dependent, transdural localization of neoplasms and improved sensitivity in neoplastic margin detection. Postresection residual fluorescence can be a powerful tool to evaluate extent of resection in conjunction with MRI, and it may guide decisions on brain metastasis management.

Combined fluorescence-guided surgery and photodynamic therapy for glioblastoma multiforme using cyanine and chlorin nanocluster.

INTRODUCTION: Glioblastoma multiforme (GBM) is the most common primary intracranial malignancy; survival can be improved by maximizing the extent-of-resection. METHODS: A near-infrared fluorophore (Indocyanine-Green, ICG) was combined with a photosensitizer (Chlorin-e6, Ce6) on the surface of superparamagnetic-iron-oxide-nanoparticles (SPIONs), all FDA-approved for clinical use, yielding a nanocluster (ICS) using a microemulsion. The physical-chemical properties of the ICS were systematically evaluated. Efficacy of photodynamic therapy (PDT) was evaluated in vitro with GL261 cells and in vivo in a subtotal resection trial using a syngeneic flank tumor model. NIR imaging properties of ICS were evaluated in both a flank and an intracranial GBM model. RESULTS: ICS demonstrated high ICG and Ce6 encapsulation efficiency, high payload capacity, and chemical stability in physiologic conditions. In vitro cell studies demonstrated significant PDT-induced cytotoxicity using ICS. Preclinical animal studies demonstrated that the nanoclusters can be detected through NIR imaging in both flank and intracranial GBM tumors (ex: 745 nm, em: 800 nm; mean signal-to-background 8.5 ± 0.6). In the flank residual tumor PDT trial, subjects treated with PDT demonstrated significantly enhanced local control of recurrent neoplasm starting on postoperative day 8 (23.1 mm3 vs 150.5 mm3, p = 0.045), and the treatment effect amplified to final mean volumes of 220.4 mm3 vs 806.1 mm3 on day 23 (p = 0.0055). CONCLUSION: A multimodal theragnostic agent comprised solely of FDA-approved components was developed to couple optical imaging and PDT. The findings demonstrated evidence for the potential theragnostic benefit of ICS in surgical oncology that is conducive to clinical integration.

Evaluation of Diagnostic Accuracy Following the Coadministration of Delta-Aminolevulinic Acid and Second Window Indocyanine Green in Rodent and Human Glioblastomas.

PURPOSE: Fluorescence-guided-surgery offers intraoperative visualization of neoplastic tissue. Delta-aminolevulinic acid (5-ALA), which targets enzymatic abnormality in neoplastic cells, is the only approved agent for fluorescence-guided neurosurgery. More recently, we described Second Window Indocyanine Green (SWIG) which targets neoplastic tissue through enhanced vascular permeability. We hypothesized that SWIG would demonstrate similar clinical utility in identification of high-grade gliomas compared with 5-ALA. PROCEDURES: Female C57/BL6 and nude/athymic mice underwent intracranial implantation of 300,000 GL261 and U87 cells, respectively. Tumor-bearing mice were euthanized after administration of 5-ALA (200 mg/kg intraperitoneal) and SWIG (5 mg/kg intravenous). Brain sections were imaged for protoporphyrin-IX and ICG fluorescence. Fluorescence and H&E images were registered using semi-automatic scripts for analysis. Human subjects with HGG were administered SWIG (2.5 mg/kg intravenous) and 5-ALA (20 mg/kg oral). Intraoperatively, tumors were imaged for ICG and protoporphyrin-IX fluorescence. RESULTS: In non-necrotic tumors, 5-ALA and SWIG demonstrated 90.2 % and 89.2 % tumor accuracy (p value = 0.52) in U87 tumors and 88.1 % and 87.7 % accuracy (p value = 0.83) in GL261 tumors. The most distinct difference between 5-ALA and SWIG distribution was seen in areas of tumor-associated necrosis, which often showed weak/no protoporphyrin-IX fluorescence, but strong SWIG fluorescence. In twenty biopsy specimens from four subjects with HGG, SWIG demonstrated 100 % accuracy, while 5-ALA demonstrated 75-85 % accuracy; there was 90 % concordance between SWIG and 5-ALA fluorescence. CONCLUSION: Our results provide the first direct comparison of the diagnostic utility of SWIG vs 5-ALA in both rodent and human HGG. Given the broader clinical utility of SWIG compared with 5-ALA, our data supports the use of SWIG in tumor surgery to improve the extent of safe resections. CLINICAL TRIAL: NCT02710240 (US National Library of Medicine Registry; https://www.clinicaltrials.gov/ct2/show/NCT02710240?id=NCT02710240&draw=2&rank=1 ).