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

Abstract

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.

Introduction

Lung cancer remains the leading cause of cancer-related death in the United States, and is expected to account for over 125,000 deaths in 2024 [1]. For early stage disease, surgical resection can be curative. Traditionally, surgeons have had two tools—manual palpation and visual inspection—to identify and remove cancer during surgery. [2] However, there are significant limitations to these modalities, particularly with small tumors located beneath the lung’s surface.

Intraoperative molecular imaging (IMI) is an emerging technology to complement and enhance surgeons’ ability to identify malignant tissue during resection. IMI involves systemically administering a tumor-targeted fluorescent contrast agent in the preoperative period [3]. The tracer accumulates in malignant tissues and can be detected by wavelength-specific cameras to allow real-time visual identification of cancer tissue in situ or in specimens after surgical excision [4].

Over the last decade, our group has tested many different tumor-specific fluorescent contrast agents in the visible (380–700 nm) and near-infrared (NIR, 700–1000 nm) light spectra to guide resection of lung cancer, mesothelioma, and other cancers of the chest and mediastinum [5, 6]. These include non-receptor targeted agents such as high-dose indocyanine green (TumorGlow), which accumulates in sites of increased vascular permeability such as the tumor microenvironment via the enhanced permeability and retention (EPR) effect. [7–12] Other imaging agents that have been studied are targeted to cell surface receptors that are over-expressed on primary lung cancers and metastases to the lung, such as folate receptor alpha (FRα) [13–20], prostate specific membrane antigen (PSMA) [21], and carcinoembryonic antigen (CEA). [22, 23] Most recently, pafolacianine (Cytalux) was FDA approved for lung cancer surgery.

Recently, there has been interest in developing next generation “smart” optical probes to highlight tumors in a highly selective manner that reduces background fluorescence that occurs with other tracers [24]. These tracers do not fluoresce until they are processed by an enzyme or in response to a physiological change common to different cancer types. [24–27] A promising target for these probes is the acidic extracellular pH in tumors due to the Warburg effect, whereby cancer cells preferentially take up glucose and convert it into lactic acid. [28] Unlike the tightly regulated pH of the extracellular compartment of normal tissues, the tumor microenvironment of virtually all tumors is characterized by decreased extracellular pH compared with normal tissues.

Pegsitacianine is one such pH-activatable tracer that has recently been developed. It consists of polymeric micelles labeled with indocyanine green (ICG). In a neutral pH, the tracer is quenched, but upon localization to the acidic tumor microenvironment, pegsitacianine disassembles into individual polymers and the quenching of ICG is released. Preliminary work from our group showed efficacy of pegsitacianine in guiding resection of lung cancers, in both murine studies and a pilot human trial. On the basis of these results, we initiated a multicenter Phase 2 clinical trial, the results of which we report here.

Methods

Study Design and Procedure

This Phase 2 study (NCT05048082) was an interventional, open-label, single-arm trial conducted at the Hospital of the University of Pennsylvania and MD Anderson Cancer Center and was approved by both Institutional Review Boards. Patients provided informed consent and underwent computed tomographic (CT) scanning with 1-mm slice thickness and standard evaluations at the discretion of the surgeon (eg, positron emission tomography [PET], pulmonary function tests, biopsy).

Study participants received 1 mg/kg of intravenous pegsitacianine through a peripheral vein 24–72 h before resection. Patients were assessed sequentially during (1) lung inspection, (2) tumor resection, and (3) margin check. In each situation, the surgeon first performed the standard of care. Then, the surgeon used pegsitacianine-based IMI to look for additional cancer. Thus, each patient served as his or her own “intra-patient” control. If the surgeon changed the operation because of a finding discovered by IMI, the finding was designated a clinically significant event (CSE). There were three prespecified clinically significant events: (1) detection of synchronous disease undetected during pre-operative imaging (PET, CT, MRI) or by the surgeon during standard-of-care surgery; (2) detection of a positive surgical margin following standard of care resection; or (3) localization of a primary tumor unable to be located by standard surgical methods.

We performed the operations in three standard steps. First, during lung inspection, the surgeon entered the chest by robotic or video-assisted thoracoscopic surgery and then surveyed for additional tumors by visual inspection or palpation. Then, IMI was used to find additional nodules. If they were discovered, the surgeon decided whether the nodules could be resected safely without significantly changing the operation. Second, during tumor resection, the surgeon performed the standard-of-care removal of the known primary pulmonary nodule and lymph node dissection. The surgeon used white-light and manual palpation through the port-site incisions to confirm the location of the lesion. Only if the surgeon could not find the nodule, IMI was used to help locate the lesion. Third, after the specimen was removed, it was examined on the back table to assess the margins. A positive margin was defined as fluorescence within 5 mm of the parenchymal staple line or residual pegsitacianine fluorescence in the surgical bed. The specimen was submitted for pathologic examination by a specialized lung pathologist.

All excised specimens were examined using standard hematoxylin and eosin staining. Only nodules or margins that were fluorescent and contained cancer cells were “true positives.” Any nodules or margins that were fluorescent but did not contain cancer cells were “false positives.”

Molecular Contrast Agent

Pegsitacianine consists of polymeric micelles labeled with indocyanine green (ICG). The pegsitacianine chemical structure consists of a diblock copolymer of polyethyleneglycol (PEG) (~ 113 repeating units) and a polymethyl methacrylate (PMMA) derivative covalently conjugated to an ICG fluorophore. Covalent attachment of ICG to the PMMA portion of the polymer results in fluorescence quenching when present in the micellar state. Upon micelle localization to the acidic tumor microenvironment, pegsitacianine disassembles into individual polymers and the quenching of ICG is released. For clinical trial patients, vials containing 9 mg pegsitacianine formulated in sterile water for injection with 10% trehalose were used to prepare infusions at a concentration of 1 mg/ml. After the final product was prepared, the imaging agent was intravenously administered to the subjects.

Near‑Infrared Cameras

The surgical field was imaged with a clinical grade optical imaging device designed for thoracoscopic imaging (Vision-Sense, Medtronic) or robotic imaging (daVinci Firefly) with an external light source providing both white light for visible light imaging and 805-nm laser illumination for optimal excitation of ICG fluorescence. Fluorescence was assessed in the primary lesion in situ and the resection specimens were also imaged ex vivo before being sent for pathologic analysis. In this trial, surgeons adhered to standard-of-care principles of oncologic resection as guided by frozen section pathology. There were no alterations in resection margins based solely upon fluorescence imaging, given that the efficacy of this technology has not yet been confirmed.

Histopathologic and Fluorescent Microscopic Review of Specimens

Specimens were formalin fixed and paraffin embedded. Sequential 5 μm sections were obtained and underwent comprehensive histopathologic analysis by a board-certified thoracic pathologist. Sections were stained using standard hematoxylin/eosin (H&E) staining. To understand pegsitacianine accumulation patterns in tumor sections, unstained sections were evaluated using a NIR fluorescence scanner (Leica Microsystems, Buffalo Grove, IL).

Post Hoc Image Analysis and Statistics

Post hoc image analysis was performed to quantify the amount of fluorescence using ROI software within ImageJ (NIH; https://imagej.nih.gov/ij). A background fluorescence level was similarly obtained, and target-or tumor-to-background (TBR) was calculated.

Imaging assessments included the calculation of mean fluorescence intensity (MFI) of all collected surgical specimens (eg, primary tumor, background tissue, lymph nodes). Specimens were imaged both prior to and immediately following excision on a piece of representative normal tissue outside of the surgical field (all specimens). MFI values were used to compute TBR values for the collected specimens. Correlation of imaging observations to pathological outcomes of each specimen was used to understand pegsitacianine performance (ie, sensitivity, specificity, NPV, PPV).

All comparisons were made using Stata Statistical Software release 14 (StataCorp, College Station, TX). Student’s t-tests were utilized for statistical comparison unless otherwise noted. A p value of 0.05 or less was considered statistically significant.

Adverse Event Monitoring

Safety assessments included adverse events (AEs), clinical laboratory test results (hematology and serum chemistry), vital sign measurements (blood pressure, pulse, respiratory rate, and temperature), physical examination findings, and 12-lead ECG results. An AE was defined as any untoward medical occurrence in a subject enrolled into the study regardless of its causal relationship to study treatment. Per the study protocol, a treatment emergent adverse event (TEAE) was defined as an adverse event (AE) that occurred (or worsened) during study drug administration through Day 21 (± 10 days) after administration of study drug. All AEs that occurred beginning on Day 0 through the end of the study (Day 21 ± 10 days) were recorded, whether they were observed by the Investigator, reported by the subject, observed from laboratory findings, or collected by other means. The description of the AE included the onset date, duration, date of resolution, severity, seriousness, etiology, and the likelihood of relationship of the AE to study drug. All AEs were to be followed to satisfactory resolution or until the Investigator deemed the event to be chronic or the subject to be stable.

Results

Patient Characteristics

Between April and August 2022, 20 patients scheduled for pegsitacianine-guided resection of lung lesions suspicious for malignancy were enrolled. Within the study cohort (Table 1), patients tended to be female (n = 13, 65%), white (n = 16, 80%), middle-aged (mean age: 63.9 years), and former smokers (n = 13, mean pack years = 26.3). Mean lesion size was 1.9 cm (IQR: 1.3–2.4 cm) and mean depth from the pleural surface was 0.8 cm. Most lesions were FDG-avid by preoperative PET scan (mean SUV = 3.7), and 6 (30%) lesions were sub-solid ground glass opacities as determined by a board-certified thoracic radiologist. By final histopathologic analysis, the most common diagnosis (n = 9, 41%) was an adenocarcinoma-spectrum malignancy. The other lesions in the study were carcinoid tumors (n = 4, 18%), metastatic lesions (n = 3, 14%), squamous cell carcinoma (n = 1, 5%), or benign lesions (n = 3, 14%).

Table 1.

Patient and Lesion Characteristics

Patient Characteristics
	Number (%) or Mean [IQR]
Total Patients	20
Age	63.9 [60–70.5]
Sex	7 (35%)
Male	13 (65%)
Female
Race	16 (80%)
White	3 (15%)
Black	1 (5%)
Other/Unknown
Former Smokers	13 (65%)
Pack Years	26.3 [15 – 35]
Lesion Characteristics
Total Lesions	22
Size of Lesion (cm)	1.9 [1.3–2.4]
Depth from Pleural Surface (cm)	0.8 [0–1.2]
Tumor Location	6 (27%)
RUL	0 (0%)
RML	4 (18%)
RLL	7 (30%)
LUL	5 (23%)
LLL
Final Pathology	14 (64%)
Primary Lung Cancer	9 (41%)
Invasive Adenocarcinoma	1 (5%)
Squamous Cell Carcinoma	4 (18%)
Carcinoid Tumor	5 (23%)
Metastatic Lesion	3 (14%)
Benign Lesion
Tumor Differentiation	10 (45%)
Well Differentiated	2 (9%)
Moderately Differentiated	6 (27%)
Poorly Differentiated	4 (18%)
Not Reported

Safety Profile

All patients were assessed for potential adverse effects following the start of pegsitacianine dosing through 28 days after dosing. In total, nineteen (95%) subjects experienced ≥ 1 TEAE with four (20%) subjects experiencing a TEAE that was deemed related to pegsitacianine infusion. One subject experienced mild hypotension requiring discontinuation of the infusion, two subjects experienced flushing and a rash that did not require discontinuation of the infusion, and one subject experienced nausea that did not require discontinuation of the infusion. Two Serious Adverse Events (SAEs) were reported, with both deemed unrelated to pegsitacianine. Overall, the use of pegsitacianine in this patient population was deemed safe and tolerable.

Primary Efficacy Outcome

Four patients in the study cohort were excluded from the primary efficacy outcome calculations due to either receiving an incomplete dose of pegsitacianine (n = 1) or due to inability to capture IMI images during the operation (n = 3). These exclusion criteria were prespecified prior to initiation of the study. Two of 16 (12.5%) patients had a clinically significant event (CSE) related to intraoperative imaging with pafolacianine. There was a single CSE for localization of a preoperatively identified lesion when standard techniques failed and one CSE for detection of a close margin on back table imaging. This patient was a 62-year-old woman with no history of tobacco use who presented with a 2.4 cm right upper lobe nodule found during CT chest to evaluate non-specific chest wall pain. The nodule was FDG avid on PET/CT (SUV 4.0) and was 0.8 cm deep to the pleural surface. Intraoperatively, the surgeon was unable to locate the primary tumor using white light and digital palpation alone. Without fluorescence imaging, the surgeon would have converted to an open thoracotomy to locate and remove the tumor. However, upon inspection with the NIR imaging system, the pegsitacianine-labeled tumor was immediately identified (Fig. 1, TBR = 2.7), allowing the surgeon to confidently remove the tumor by minimally invasive resection and spare the patient the morbidity of a thoracotomy. Final pathology confirmed pulmonary adenocarcinoma that was resected with negative margins.

Fig. 1.

Pegsitacianine labeling of a visually occult, nonpalpable tumor. The top row shows the representative preoperative CT and PET images of a right upper lobe lung nodule from a patient in the clinical trial. The bottom row shows paired white light, near infrared (NIR), and overlay intraoperative images showing pegsitacianine fluorescence. The nodule is not apparent on white light imaging, but is clearly demarcated by pegsitacianine

The second CSE was a close margin detected on NIR imaging. The patient is a 77-year-old male who was found to have a 5 cm right lower lobe mass identified as an incidental finding during a CT abdomen for intermittent abdominal pain. The mass was abutting the pleural surface and was FDG-avid on PET scan (SUV 16.2). The mass was removed as part of a bilobectomy. On the back table, the tumor was highly fluorescent on ex vivo imaging (Fig. 2, TBR = 3.4), and NIR imaging was concerning for tumor close (< 5 mm) to the staple line. Frozen section confirmed a close margin and an additional wider margin was resected to achieve adequate margins. Final pathology established the diagnosis as pulmonary squamous cell carcinoma.

Fig. 2.

Pegsitacianine labeling of a close resection margin. The top row shows the representative preoperative CT and PET images of a central right lung mass from a patient in the clinical trial. The bottom row shows paired white light, near infrared (NIR), and overlay images showing pegsitacianine fluorescence in the resection specimen ex vivo. Fluoroscence is noted in the very near proximity of the resection margin, and the close margin was confirmed by pathologic analysis

Secondary Efficacy Outcomes

The diagnostic performance of pegsitacianine for detection of cancerous tissue was the secondary efficacy outcome. Of the 19 cancerous lesions resected, 6 fluoresced in situ using NIR imaging for a calculated sensitivity of 0.32. Of the 3 benign lesions resected, 1 was not fluorescent for a calculated specificity of 0.33. The positive predictive value (PPV) of NIR fluorescence using pegsitacianine was 0.75 and the negative predictive value (NPV) was 0.07, given the 13 cancerous lesions that did not fluoresce. The false negative lesions included 6 adenocarcinomas, 4 metastatic lesions, and 3 carcinoid tumors (Fig. 3 for representative example). On back table ex vivo imaging, two of the cancerous lesions that did not fluoresce in vivo were found to be fluorescent, leading to modest improvements in diagnostic performance (sensitivity 0.47, specificity 0.33, PPV 0.82, NPV 0.09). Fluorescence was not a reliable predictor of lymph node positivity, as the single positive lymph node in the study population was non-fluorescent, and there were two false positive fluorescent nodes in a total of 39 imaged lymph node specimens.

Fig. 3.

Representative images of a nonfluorescent lesion (false negative) in the study. The top row shows the representative preoperative CT and PET images of a left upper lobe lung nodule from a patient in the clinical trial. The bottom row shows paired white light, near infrared (NIR), and overlay intraoperative images. Neither brightfield nor NIR imaging delineated the nodule in question, which required a thoracotomy for identification. Final pathology returned as adenocarcinoma

Subanalyses – Fluorescence Quantification and Patterns of Tracer Accumulation

Pegsitacianine based NIR imaging identified multiple subtypes of lung cancer including adenocarcinoma, squamous cell carcinoma, and carcinoid tumor. Colorectal lung metastases were also identified by IMI with pegsitacianine. Of the fluorescent nodules, average in situ TBR was 3.00 (IQR: 2.12–3.74). Mean tumor MFI was 101.24 arbitrary units (a.u.; IQR: 77.26–123.30 a.u.) and mean background MFI was 34.18 a.u. (IQR: 31.99–35.32 a.u.). On back table imaging, mean TBR of the fluorescent nodules was 4.23 (IQR: 1.94–4.16). Mean tumor MFI was 123.73 a.u. (IQR: 65.73–127.96 a.u.) and mean background MFI was 29.81 a.u. (IQR: 28.40–31.68 a.u.).

On back table imaging, after bisecting the tumor specimens, we noted that tracer distribution was not uniform throughout the specimen and tended to accumulate on the peripheral borders of tumors (Fig. 4). This was consistent with prior patterns observed in animal models [29]. Mean MFI at the borders of fluorescent tumors was 135.62 a.u. (IQR: 83.77–133.68), while mean MFI at the center of tumors was significantly lower at 67.24 (IQR: 45.49–89.24, p = 0.003).

Fig. 4.

Patterns of distribution of pegsitacianine fluorescence in resected tumors. A. Representative white light, near-infrared (NIR), and overlay images of an ex vivo tumor section showing pegsitacinine fluorescence at the tumor periphery (arrow) but minimal fluorescence in the center of the tumor. B. NIR heatmaps of tumor samples taken from the periphery and center of a tumor, again showing pegsitacianine fluorescence preferentially distributed a the tumor border. C. Mean fluorescence intensity of tissue taken from the peripheral borders of tumors compared with tissue taken from the center of tumors. ** represents p < 0.01

Overall Results

Pegsitacianine was safe and well-tolerated in this study; there were four mild infusion reactions and no drug-related SAEs. CSEs were observed in 2 of the 16 patients in the trial, but overall diagnostic performance was inconsistent.

Discussion

This study was the first multicenter trial of pegsitacianine, a pH-sensitive NIR nanoprobe, for identifying pulmonary tumors during resection. Overall, we found that the study drug was well tolerated from a safety perspective and identified tumors of a range of different histologies including pulmonary adenocarcinoma, squamous cell carcinoma, carcinoid tumor, and metastatic cancers from other organs. There were two CSEs based upon IMI with pegsitacianine: localization of a visually occult, nonpalpable tumor and identification of a close surgical margin. However, the overall diagnostic performance of the tracer was unreliable with sensitivity of 31.6% and specificity of 33.3%.

This study builds upon a significant body of work in IMI during resection of thoracic cancers. Our group and others have extensively studied several IMI tracers during surgery for lung cancer. Initial studies examined the utility of ICG for identifying pulmonary nodules via the enhanced permeability and retention (EPR) effect. This technique was highly sensitive but lacked specificity, and could not distinguish inflammatory tissue from cancer. To improve specificity, our group and others tested agents targeted to cell surface receptors, such as prostate specific membrane antigen (PSMA), folate receptor alpha (FRα), and the carcinoembryonic antigen (CEA) receptor. More recent efforts have focused on the development of activatable “smart” probes that become fluorescent in response to physiologic or enzymatic changes in the tumor milieu.

Pegsitacianine was safe and well tolerated in this study; however, we were disappointed that more tumors did not fluoresce. We believe there are several possibilities for this lack of fluorescence in pulmonary tumors. First, we noticed that in several tumors, the fluorescence was only in the outer rim of the cancer, but did not penetrate deep into the tumor (see Fig. 4). This was confirmed on cross-section molecular imaging. It is possible that the dye is immediately activated as it enters the tumor, thereby, not fluorescing by the time it reached the center of the tumor. Second, tumors are highly oxygenated. It is possible that the data on solid tumor acidity may not be applicable to lung cancers due to the higher oxygen tension that exists in this environment. The high levels of carbonic anhydrase in the lungs may rapidly alter tumor pHs. Further exploration is needed to identify subtypes of pulmonary cancers for which pegsitacianine-based IMI may be effective.

With the growth in number of tracers available for IMI-guided tumor resection, it will be increasingly important for surgeons to select the appropriate imaging agent for the tumor in question. This choice must consider multiple factors including the cancer type and suspected histology, depth from the organ surface, and the specific information sought from IMI (i.e. tumor localization, margin evaluation, detection of synchronous nodules). Surgeons must consider the evidence supporting each tracer in different types of cancer and patient populations. The publication of all trial results—including negative trials—will be important in understanding which patients may benefit most from certain tracers. For instance, although this study does not support the routine use of pegsitacianine for guidance of lung cancer resection, there is robust evidence for the use of this tracer for IMI in a range of other cancer types including head and neck cancer, esophageal cancer, and colon cancer. Furthermore, we showed that the probe clearly highlights the circumferential boundaries of the tumor and can thus aid surgeons’ evaluation of whether a tumor resection margin is adequate to prevent locoregional tumor recurrence. Further work is needed to continue to delineate the optimal indications for use of pegsitacianine in oncologic resections.

Data Availability

The data associated with this study are presented in the paper. Additional imaging data from this study are available upon request to the Corresponding Author.