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Center for Precision Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PennsylvaniaDivision of Thoracic Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Center for Precision Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PennsylvaniaDivision of Thoracic Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Center for Precision Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PennsylvaniaDivision of Thoracic Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Center for Precision Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PennsylvaniaDivision of Thoracic Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Center for Precision Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PennsylvaniaDivision of Thoracic Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Center for Precision Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PennsylvaniaDepartment of Radiology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania
Center for Precision Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PennsylvaniaDivision of Thoracic Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Corresponding author. Address for correspondence: Sunil Singhal, MD, Perelman School of Medicine, University of Pennsylvania, Philadelphia 6 White Building; 3400 Spruce St., Philadelphia, PA 19104.
Center for Precision Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PennsylvaniaDivision of Thoracic Surgery, Department of Surgery, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania
Intraoperative localization and resection of ill-defined pulmonary ground-glass opacities (GGOs) during minimally invasive pulmonary resection is technically challenging. Current preoperative techniques to facilitate localization of GGOs include microcoil and hook wire placement, both of which have logistic limitations, carry safety concerns, and do not help with margin assessment. In this clinical trial, we explored an alternative method involving near-infrared molecular imaging with a folate receptor–targeted agent, OTL38, to improve localization of GGOs and confirmation of resection margins.
Methods
In a human trial, 20 subjects with pulmonary GGOs who were eligible for video-assisted thoracoscopic surgery (VATS) resection received 0.025 mg/kg of OTL38 before the resection. The primary objectives were to (1) determine whether use of OTL38 allows safe localization of GGOs and assessment of margins during VATS and (2) determine patient, radiographic, and histopathologic variables that predict the amount of fluorescence during near-infrared imaging.
Results
We observed no toxicity. Of the 21 GGOs, 20 accumulated OTL38 and displayed fluorescence upon in situ or back table evaluation. Intraoperatively, near-infrared imaging localized 15 of 21 lesions whereas VATS alone localized 10 of 21 (p = 0.05). The addition of molecular imaging affected care of nine of 21 subjects by improving intraoperative localization (n = 6) and identifying close margins (n = 3). This approach was most effective for subpleural lesions measuring less than 2 cm. For lesions deeper than 1.5 cm from the pleural surface, intraoperative localization using fluorescent feedback was limited.
Conclusions
This approach provides a safe alternative for intraoperative localization of small, peripherally located pulmonary lesions. In contrast to alternative localization techniques, use of OTL38 also allows confirmation of adequate margins. Future studies will compare this approach to alternative localization techniques in a clinical trial.
Routine use of high-resolution computed tomography (CT) and implementation of lung cancer screening protocols have been linked to an increased incidence of indeterminate pulmonary nodules and ill-defined ground-glass opacities (GGOs). GGOs are radiographic findings that can represent abnormalities ranging from benign inflammatory processes to invasive primary lung cancers. Although nomograms predicting invasive pulmonary carcinoma have been proposed, resection remains the only approach to unequivocally determine GGO histology.
Unfortunately, intraoperative identification and confirmation of negative margins remain intraoperative challenges and are the result of GGOs’ subtle parenchymal changes and soft architecture, which complicate conventional visual and tactile localization. Difficulties are amplified during minimally invasive pulmonary resection, when manual palpation is further limited.
Several preoperative and intraoperative tumor marking techniques have been proposed to improve identification of GGOs. These approaches include tumor tattooing with indocyanine green, methylene blue, ethiodized oil, or technetium 99; intraoperative CT or fluoroscopy; and placement of hook wires, fiducials, or microcoils (reviewed in Keating and Singhal
). Perhaps the most universal approach involves CT-guided hook wire needle localization. According to a meta-analysis, successful localization by hook wire occurs in 95% of patients; however, the technique's limitations are well documented.
Logistically, hook wire deployment requires planning, coordination of scheduling, and close safety monitoring. Second, hook wire placement is associated with complications, including pneumothorax (in 35% of patients) and pulmonary hemorrhage (in 15% of patients).
Although most complications can be controlled during video-assisted thoracoscopic surgery (VATS), significant morbidity has been documented. Third, hook wire dislodgement occurs approximately 5% of patients, which decreases success rates by 50%.
In patients undergoing video-assisted thoracoscopic surgery excision, what is the best way to locate a subcentimetre solitary pulmonary nodule in order to achieve successful excision?.
Finally, although hook wire placement provides information useful for localization, it does not help confirm margins in real time.
As an alternative approach, our group has spent the last decade utilizing systemically delivered, near-infrared (NIR) contrast agents that selectively accumulate in pulmonary nodules. Our most recent and promising tracer is OTL38, which is an NIR agent that targets the folate receptor.
Initial studies involving folate receptor–targeted intraoperative molecular imaging (FR-IMI) have demonstrated fluorescence in 98% of solitary pulmonary nodules and have shown excellent safety.
These proof-of-concept studies have set the stage for studies to better understand how FR-IMI can reconcile the intraoperative challenges faced by the thoracic surgeon in relation to GGOs. In this study, we explored FR-IMI with OTL38 in 20 subjects who presented to our imaging center for resection of incidentally identified GGOs. The objectives were to (1) determine whether OTL38 safely improves minimally invasive localization of GGOs and confirmation of GGO margin and (2) determine patient, radiographic, and histopathologic variables that predict the fluorescence during NIR imaging.
Materials and Methods
Study Drug
OTL38 (C61H63N9Na4O17S4) (molecular weight 1414.42) is a folate analogue conjugated to the NIR dye S0456. OTL38 excites at 774 to 776 nm and emits at 794 to 796 nm.
The OTL38 for this study was provided by OnTarget Laboratories (West Lafayette, IN).
Study Design
This study was approved by the University of Pennsylvania’s institutional review board and registered at clinicaltrials.gov (NCT02602119). The primary objectives were to establish safety and feasibility. Secondary objectives were to (1) determine whether OTL38 improves minimally invasive localization and resection of GGOs and (2) to determine patient, radiographic, and histopathologic variables that predict fluorescence during FR-IMI.
A total of 20 subjects were recruited between July 2015 and October 2017. The included subjects presented with pure GGOs or GGOs with a solid component that were suggestive of lung neoplasms. We selected subjects with GGOs within 3 cm of the pleural surface given the known limitations of NIR imaging.
All subjects were scheduled for VATS wedge resection.
Participants received OTL38 (intravenously in a dose of 0.025 mg/kg) 3 to 6 hours before resection. During VATS, surgeons first utilized traditional thoracoscopic visualization and finger palpation to identify known lesions. Next, iridium was used to confirm lesion fluorescence (iridium described in Predina et al.
). If lesions were unidentifiable by traditional methods, NIR localization was attempted. All lesions were wedge-resected and imaged ex vivo before being submitted for pathologic evaluation. Margins were assessed ex vivo and compared by permanent histopathologic analysis. If close margins (defined as <1 cm) were identified, additional parenchyma was resected by wedge resection or segmentectomy.
Quantification of TBRs
Mean fluorescence intensity (MFI) of the tumor (MFItumor) was obtained by analyzing monochromatic NIR images with ImageJ software (http://rsb.info.nih.gov/ij) and measuring the region of interest, which correlated with the GGO. Background fluorescence (0.5–1.0 cm from the margin) was also obtained (MFIbackground). A minimum of 1000 pixels were included in each measurement. Calculations were repeated in triplicate from varying angles; tumor-to-background fluorescence ratios (TBRs) were calculated by using the following equation: TBR = MFItumor/MFIbackground. TBR assessments were averaged, and a mean TBR greater than 2.0 was considered fluorescent.
Statistical Analysis
Because of the exploratory nature of this study, logistic regression was used to determine patient and histopathologic variables that predicted in situ tumor fluorescence. Results were expressed as medians (interquartile ranges [IQRs]). Comparisons were made using Stata software (release 14, College Station, TX). A p value less than 0.05 was considered statistically significant.
Results
Subject and Safety Data
A total of 20 subjects with suspicious GGOs were enrolled and underwent VATS wedge resection with FR-IMI. Seven of the 20 subjects were male and 13 were female. The median age subjects at the time of resection was 71 years (IQR 63–73 years). A total of 21 lesions were identified by preoperative imaging. The median size was 1.3 cm (IQR 0.9–1.7 cm), with nine lesions (42.8%) being pure GGOs and 12 (57.1%) being GGOs with a solid component. Of the 16 lesions that were imaged by preoperative positron emission tomography, eight were fludeoxyglucose F 18PET–avid. Pathologic evaluation of the lesions revealed adenocarcinoma in situ (n = 2), minimally invasive adenocarcinoma (n = 7), invasive pulmonary adenocarcinoma (n = 11), and granuloma (n = 1).
All subjects completed OTL38 infusions with a median time of 3.3 hours (IQR 3.1–4.8 hours) elapsing before the start of molecular imaging. No drug-related adverse events were observed perioperatively or during 30 days of follow-up.
A summary of patient, lesion, and safety data is provided in Table 1.
Table 1Patient and Tumor Characteristics
ID
Age, y
Sex
Size, cm
Pure vs. Mixed GGO
SUV
Location
Depth, cm
Time to Imaging, h
In Situ TBR
Lesion Diagnosis
Impact of IMI
1
70
F
1.3
Pure GGO
0.8
RLL
2.0
2.97
2.6
Well differentiated, minimally invasive pulmonary adenocarcinoma, lepidic predominant
Close margin detected, tumor within 4 mm of stable line
Of the 21 preoperatively identified GGOs, 16 displayed lesion-specific fluorescence during intrathoracic NIR imaging (Fig. 1), whereas 20 of 21 displayed a signal upon back table NIR evaluation (Fig. 2). The median GGO (tumoral) fluorescence of the 20 fluorescent lesions was 108.2 arbitrary units (AU) (IQR 84.3–136.6 AU), which was significantly higher than the background intensity of benign lung (28.6 AU [IQR, 26.4–42.3 AU]) (Fig 1F) (p < 0.001). A median TBR of 3.8 (IQR 2.9–5.5) was noted. Of note, each fluorescent lesion displayed folate receptor-α expression, whereas the single nonfluorescent adenocarcinoma (of subject 9) lacked folate receptor-α expression (Supplementary Fig. 1).
Figure 1Pulmonary ground-glass opacities (GGOs) display in situ fluorescence during folate receptor–targeted intraoperative molecular imaging. In a representative example subject 11 presented with a 0.6-cm left lower lobe pure GGO by preoperative computed tomography (CT) (A). The lesion displayed no parenchymal abnormalities or tactile irregularities during traditional thoracoscopy (B); however, such lesions became obvious with the addition of real-time fluorescent feedback provided by intraoperative molecular imaging (C). Red circle indicates a pulmonary GGO. NIR, near-infrared.
Figure 2Ground-glass opacities (GGOs) display strong fluorescence upon ex vivo tumor inspection. In a representative example subject 12 presented with a 0.9-cm left upper lobe GGO by preoperative computed tomography (CT) (A). The GGO was unidentifiable during traditional white-light views (B) and during in situ folate receptor–targeted intraoperative molecular imaging (FR-IMI). Upon ex vivo tumor bisection, the nodule was identified (D) and displayed robust fluorescence during ex vivo fluorescent evaluation (E). Red circle indicates pulmonary GGO. NIR, near-infrared.
Applications of FR-IMI: Minimally Invasive Localization
After confirming selective accumulation of OTL38 within GGOs, we focused on the clinical utility of FR-IMI for GGOs (Fig. 3). A total of 10 of 21 nodules were localized by traditional VATS techniques, whereas 15 of 21 were localized with FR-NIR imaging (p = .05) (example provided in Supplementary Video 1). One nodule was localized by VATS alone, whereas six were localized by FR-IMI alone. The six nodules that were localized only by FR-IMI were smaller than the 10 nodules that were localized with VATS (0.9 cm versus 1.4 cm [p = 0.04]). With the exception of the adenocarcinoma in subject 10, all lesions identified by VATS were identifiable by NIR imaging. For the five lesions that were not localized by VATS or FR-NIR imaging, a generous wedge resection was required for successful removal.
Figure 3Intraoperative localization of 21 ground-glass opacities. Of the 21 preoperatively identified lesions, one was localized by video-assisted thoracoscopic surgery (VATS) alone whereas six were localized by folate receptor–targeted intraoperative molecular imaging (FR-IMI) alone. Nine lesions were localized by both VATS and FR-IMI, and five were not identified by any method.
In addition to localization, utilization of FR-IMI provided reliable feedback that allowed for rapid margin assessment by ex vivo evaluation. Margins assessed by FR-IMI and final pathology were similar (p = .67), and the median difference in margin was within 1 mm (IQR 3 mm–1 mm) (Fig. 4). Incorporation of this information into the resection algorithm provided reliable, real-time feedback that resulted in immediate re-resection in three subjects. In these subjects, fluorescent feedback during FR-IMI accurately demonstrated tumor within 6 mm in the stable line (Fig. 4). Feedback was most useful in subjects 1 and 18, in whom tumor was not obviously involving the tumor margin. The margins assessed by FR-IMI were 100% accurate, with no false-positive or false-negative results identified. The median time for margin assessment by FR-IMI was 2.2 minutes, whereas 28.2 minutes was required with frozen sections (p = 0.02).
Figure 4Folate receptor–targeted intraoperative molecular imaging (FR-IMI) accurately determines tumor margins by ex vivo analysis. For subjects with fluorescent ground-glass opacities (n = 20), tumor margins were assessed by FR-IMI by using the iridium’s exoscopic configuration and measurements compared by pathologic assessment (the criterion standard). The margins obtained from both approaches were strongly correlated, with the median differences being less than 1 mm (the measures were similar [p = 0.42]) (A). In a representative example (subject 8) of clear margin identification, margin assessment after resection of a ground-glass opacity is challenging by white-light–only views (B); however, an adequate margin (2.3 cm) was confirmed with use of FR-IMI (C). In two subjects, utilization of FR-IMI allowed identification of close tumor margins that required reexcision. In a representative example of inadequate margin (in subject 5), tumor margins were unclear during white-light evaluation (D); however, FR-IMI reliably identified tumor within 1 mm of the stable line (E). Yellow arrow indicates positive staple line. NIR, near-infrared.
Clinically significant events are noted in Table 1.
Histopathologic and Clinical Variables Predicting Fluorescence of GGOs during FR-IMI
To maximize the utility of FR-IMI for localization and margin assessment, in situ intraoperative fluorescence is critical. To predict which GGOs would display in situ fluorescence during FR-IMI, several clinical, radiographic, and histopathologic variables were assessed: depth from the pleural surface on the preoperative CT scan, subtype of the invasive pulmonary adenocarcinoma, tumor differentiation, GGO type (pure versus mixed), time from drug delivery to imaging, preoperative standardized uptake value, GGO size, patient sex, patient age, and tumor location (Table 2). We found that only GGO depth from the pleural surface predicted in situ fluorescence. To this end, 100% of the fluorescent lesions within 1.0 cm of the pleural were identified during thoracoscopic NIR imaging.
Table 2Logistic Regression Analysis of Factors Predicting In Situ Fluorescence
Reliable localization and complete resection of pulmonary GGOs during minimally invasive pulmonary resection represents a major clinical challenge. In this study, we have evaluated FR-IMI as an intraoperative adjunct to improve the localization and resectability of GGOs. We demonstrated excellent safety and feasibility. FR-IMI affected clinical care in nine of 20 subjects by improving minimally invasive lesion localization and providing accurate margin assessment. This approach was most useful in subjects with small and peripheral lesions. Additionally, FR-IMI provides the opportunity to perform real-time margin assessment before submitting specimens for pathologic analysis. FR-IMI may ultimately serves as a safe and practical localization technique that can complement invasive localization techniques such as preoperative microcoil or hook wire placement.
A number of preoperative approaches have been proposed to improve intraoperative localization of GGOs; however, almost all of them require access to image-guided therapeutic operating rooms with CT, navigational bronchoscopy, or fluoroscopy. Such operating rooms are not universal and may not be readily available for thoracic surgeons. In addition to logistic limitations, endoscopic and transthoracic placement of microcoils, fiducials, and hook wires are invasive and associated with well-documented patient risks, including radiation exposure, dislodgment, pneumothorax, and bleeding.
As an alternative to improve safety, direct intratumoral delivery of indocyanine green with intraoperative NIR imaging has been proposed by several groups.
Identification of metastatic nodal disease in a phase 1 dose-escalation trial of intraoperative sentinel lymph node mapping in non-small cell lung cancer using near-infrared imaging.
In these studies, excellent safety and reliable detection of lesions as deep as 2 to 3 cm have been observed. Despite safety improvements, NIR tumor tattooing with indocyanine green still remains dependent on direct tumor injection with use of advanced imaging platforms. Furthermore, direct tumor marking with any of the aforementioned approaches provides minimal information for those subjects presenting with multifocal disease.
In this study, we have provided preliminary data suggesting that FR-IMI with OTL38 can serve as an alternative approach for localization of GGOs. OTL38 is a folate analogue that is linked to a NIR fluorophore and thus binds the folate receptor, after which it is quickly internalized. The folate receptor is an appealing target for localization of a pulmonary lesion given the high expression patterns in primary lung cancers, pulmonary metastases, and benign inflammatory processes such as granulomas.
In normal lung parenchyma, the folate receptor is expressed only at low levels on the apical surface and is therefore not exposed to systemic OTL38. In addition to widespread applicability for pulmonary lesions, FR-IMI with OTL38 offers several advantages over current localization approaches: (1) FR-IMI requires no additional invasive procedures, which contrasts with other localization approaches, including microcoil or hook wire placement and tumor tattooing
Identification of metastatic nodal disease in a phase 1 dose-escalation trial of intraoperative sentinel lymph node mapping in non-small cell lung cancer using near-infrared imaging.
; (2) FR-IMI is efficient, can be delivered while the patient is in the preoperative holding area, and requires fewer than 5 minutes for intraoperative interpretation; (3) FR-IMI is associated with no toxicity, which provides important patient safety advantages over previously mentioned localization techniques
; (4) systemic drug delivery provides an opportunity for detection multiple lesions and margin assessment; and (5) FR-IMI can provide information valuable for margin assessment.
In this trial we have noted that 20 of 21 preoperatively identified lesions accumulated OTL38 in a folate receptor-dependent manner. FR-IMI localized GGOs representing inflammatory processes, adenocarcinoma in situ, minimally invasive adenocarcinoma, and invasive pulmonary adenocarcinoma. This fits nicely with our previous work demonstrating successful localization of solid pulmonary masses, including pulmonary adenocarcinomas, pulmonary squamous cell carcinomas,
Intraoperatively, FR-IMI affected care of nine of 20 subjects by improving minimally invasive localization (n = 6) and identification of close margins (n = 3). Localization was superior to traditional visualization and finger palpation alone. These localization benefits were most obvious for small GGOs situated within 1.5 cm of the pleural surface. We acknowledge limitations in depth of detection; however, we believe that the advantages in terms of safety, feasibility, and utility for multifocal disease suggest a potential role for FR-IMI for subpleural small GGOs. Because current imaging systems for FR-IMI are unreliable in detecting deeper lesions, traditional preoperative localization (e.g., hook wire or microcoil) remain superior in these scenarios. In the future, NIR bronchoscopy or photoacoustic detection may improve depth of detection during NIR imaging, as has been described for melanoma.
With regard to FR-IMI for margin assessment, the data demonstrate that margin assessment by NIR imaging is similar to that by pathologic evaluation. We acknowledge that in certain circumstances, this information may not add to standard-of-care approaches involving visualization and frozen section analysis; however, FR-IMI provides real-time data that are helpful for those cases of margin assessments in which the status is not obvious by the naked eye or palpation alone.
In summary, in this exploratory trial we have provided data demonstrating that FR-IMI with OTL38 can be a safe adjunct capable of intraoperative localization and margin assessment for a subset of small GGOs. Future studies will focus on better understanding the potential role for FR-IMI in relation to alternative localization techniques. This approach is currently being evaluated in a multicenter phase II trial (NCT02872701).
Acknowledgments
Dr. Predina was supported by the American Philosophical Society, the National Institutes of Health (grant F32CA210409), and an Association for Academic Surgery research grant. Dr. Singhal was supported by the National Institutes of Health (grant R01 CA193556). Dr. Predina and Dr. Singhal participated in study design, data collection, data analysis, manuscript preparation, and manuscript review. Dr. Newton, Mr. Corbett, Ms. Sulyok, Mr. Shin, and Dr. Kucharczuk participated in data collection, data analysis, and manuscript review. Dr. Deshpande, Dr. Barbosa, and Dr. Low assisted in data collection, data analysis, manuscript preparation, and manuscript review.
In patients undergoing video-assisted thoracoscopic surgery excision, what is the best way to locate a subcentimetre solitary pulmonary nodule in order to achieve successful excision?.
Identification of metastatic nodal disease in a phase 1 dose-escalation trial of intraoperative sentinel lymph node mapping in non-small cell lung cancer using near-infrared imaging.
Disclosure: Dr. Low is a board member at OnTarget Laboratories, the producers of the study drug. The remaining authors declare no conflict of interest.
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