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A novel technique for tumor localization and targeted lymphatic mapping in early-stage lung cancer

Open ArchivePublished:February 10, 2017DOI:https://doi.org/10.1016/j.jtcvs.2016.12.058

      Abstract

      Objective

      To investigate safety and feasibility of navigational bronchoscopy (NB)-guided near-infrared (NIR) localization of small, ill-defined lung lesions and sentinel lymph nodes (SLN) for accurate staging in patients with non–small cell lung cancer (NSCLC).

      Methods

      Patients with known or suspected stage I NSCLC were enrolled in a prospective pilot trial for lesion localization and SLN mapping via NB-guided NIR marking. Successful localization, SLN detection rates, histopathologic status of SLN versus overall nodes, and concordance to initial clinical stage were measured. Ex vivo confirmation of NIR+ SLNs and adverse events were recorded.

      Results

      Twelve patients underwent NB-guided marking with indocyanine green of lung lesions ranging in size from 0.4 to 2.2 cm and located 0.1 to 3 cm from the pleural surface. An NIR+ “tattoo” was identified in all cases. Ten patients were diagnosed with NSCLC and 9 SLNs were identified in 8 of the 10 patients, resulting in an 80% SLN detection rate. SLN pathologic status was 100% sensitive and specific for overall nodal status with no false-negative results. Despite previous nodal sampling, one patient was found to have metastatic disease in the SLN alone, a 12.5% rate of disease upstaging with NIR SLN mapping. SLN were detectable for up to 3 hours, allowing time for obtaining a tissue diagnosis and surgical resection. There were no adverse events associated with NB-labeling or indocyanine green dye itself.

      Conclusions

      NB-guided NIR lesion localization and SLN identification was safe and feasible. This minimally invasive image-guided technique may permit the accurate localization and nodal staging of early stage lung cancers.

      Key Words

      Abbreviations and Acronyms:

      CT (computed tomography), GGO (ground-glass opacity), HSA (human serum albumin), ICG (indocyanine green), LAD (lymphadenectomy), NB (navigational bronchoscopy), NIR (near-infrared), NSCLC (non–small cell lung cancer), PET (positron emission tomography), SLN (sentinel lymph node)
      Figure thumbnail fx1
      Level 7 near-infrared positive sentinel lymph node after navigational bronchoscopy-guided perilesional “tattoo” with indocyanine green.
      Navigational bronchoscopy-guided near-infrared tumor marking is feasible and allows localization and nodal staging of early stage lung cancers.
      Two major barriers to curative treatment of early stage lung cancer are surgical: (1) challenge in identifying small, ill-defined lesions intraoperatively for resection and (2) inadequate sampling and histologic scrutiny of tumor-associated lymph nodes. Navigational bronchoscopy-guided near-infrared marking is a novel technique that permits tumor localization and targeted staging of early lung cancers.
      See Editorial Commentary page 1119.
      See Editorial page 1077.
      Lung cancer is the leading cause of cancer-related mortality in the United States, with an estimated 158,080 deaths expected to occur in 2016.
      American Cancer Society
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      Despite “curative” resection and lymphadenectomy (LAD), there is a 30% recurrence rate and a 60%-80% 5-year survival in early stage non–small cell lung cancer (NSCLC).
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      Attempts to improve these outcomes have led to the initiation of national lung cancer screening, where an estimated 2 million Americans will be diagnosed with a new pulmonary nodule, and more than 80,000 will require surgery for diagnosis and treatment.
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      Unfortunately, many very early cancers discovered with screening are too small or ill-defined, as with ground-glass opacities (GGO), preventing localization for limited surgical resection or biopsy. Therefore, these small lesions are followed with imaging surveillance until they are large enough for diagnostic percutaneous biopsy or amenable to intraoperative localization. The concern of the surgeon and patient is that disease progression can occur during surveillance, thus potentially undermining the benefit of early detection. If a small or ill-defined lesion can be localized intraoperatively, limited surgical resection may not only offer a diagnosis but is also potentially curative for some of these small, peripheral lesions, if nodal disease is in fact absent.
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      Furthermore, many patients with poor cardiopulmonary reserve may only be candidates for limited resection, and thus precise intraoperative localization for lung parenchyma preservation with a negative surgical resection margin is critical for maximizing long-term outcomes.
      Despite negative preoperative imaging to rule out occult locoregional lymph node metastases, pathologic upstaging found at the time of surgical resection occurs in up to 18% of patients.
      • Licht P.B.
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      • Jakobsen E.
      A national study of nodal upstaging after thoracoscopic versus open lobectomy for clinical stage I lung cancer.
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      • et al.
      Nodal upstaging is more common with thoracotomy than with VATS during lobectomy for early-stage lung cancer: an analysis from the national cancer data base.
      Furthermore, nodal staging is inadequate in more than 50% of limited resections performed for early-stage cancers, and standard hematoxylin and eosin analysis of all lymph nodes in the specimen identifies occult metastases 16%-18% less often than when individual nodes are scrutinized histologically.
      • Osarogiagbon R.U.
      • Yu X.
      Mediastinal lymph node examination and survival in resected early-stage non-small lung cancer in the surveillance, epidemiology, and end results database.
      • Rusch V.W.
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      • et al.
      Occult metastases in lymph nodes predict survival in resectable non-small-cell lung cancer: report of the ACOSOG Z0040 trial.
      Occult nodal disease leads to understaging and missed opportunities for adjuvant therapy and may result in poor clinical outcomes.
      • Kubuschok B.
      • Passlick B.
      • Izbicki J.R.
      • Thetter O.
      • Pantel K.
      Disseminated tumor cells in lymph nodes as a determinant for survival in surgically resected non-small-cell lung cancer.
      • Martin L.W.
      • D'Cunha J.
      • Wang X.
      • Herzan D.
      • Gu L.
      • Abraham N.
      • et al.
      Detection of occult micrometastases in patients with clinical stage I non-small-cell lung cancer: a prospective analysis of mature results of CALGB 9761 (Alliance).
      • Ludwig M.S.
      • Goodman M.
      • Miller D.L.
      • Johnstone P.A.
      Postoperative survival and the number of lymph nodes sampled during resection of node-negative non-small cell lung cancer.
      • Osarogiagbon R.U.
      • Allen J.W.
      • Farooq A.
      • Berry A.
      • O'Brien T.
      Pathologic lymph node staging practice and stage-predicted survival after resection of lung cancer.
      • Varlotto J.M.
      • Recht A.
      • Nikolov M.
      • Flickinger J.C.
      • Decamp M.M.
      Extent of lymphadenectomy and outcome for patients with stage I nonsmall cell lung cancer.
      Thus, 2 major barriers facing the curative treatment of early-stage lung cancer are surgical: the current challenge in identifying small, ill-defined lesions intraoperatively for parenchyma-sparing resections and the inadequate sampling and histologic scrutiny of tumor-associated lymph nodes.
      Our previous phase I dose-titration trial using transpleural, peritumoral injection of the Food and Drug Administration-approved near-infrared (NIR) dye indocyanine green (ICG) in 41 patients demonstrated no adverse events, excellent real-time imaging via minimally invasive NIR thoracoscopy, and a dose-dependent increase in NIR signal within sentinel lymph nodes (SLNs).
      • Gilmore D.M.
      • Khullar O.V.
      • Jaklitsch M.T.
      • Chirieac L.R.
      • Frangioni J.V.
      • Colson Y.L.
      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.
      Although spillage of dye can occur after transpleural injection, SLNs were identified in 100% of patients at the optimized ICG dose. Importantly, transpleural injection required intraoperative palpation of the lesion for peritumoral injection, limiting this technique to superficial and/or relatively large lesions.
      We conducted the first-in-human pilot trial of patients with suspected or known early-stage NSCLC undergoing intraoperative navigational bronchoscopy (NB)-guided transbronchial, peritumoral ICG injection, subsequent NIR lesion localization, resection, and SLN mapping via minimally invasive thoracoscopic surgery. We aimed to show this comprehensive image-guided technique is both safe and feasible for both localization of early-stage lung cancers and targeting of tumor-associated SLNs.

      Methods

      Study Design and Patients

      This prospective pilot trial evaluates the feasibility of NB-guided peritumoral ICG injection for NIR “tattoo” marking of suspected or known lung cancers and NIR+ SLN retrieval for nodal staging (Figure 1). The trial protocol was approved by the Partners Internal Review Board (Boston, Mass), and all cases were conducted at Brigham and Women's Hospital (Boston, Mass). Primary endpoints included visualization of the NIR+ “tattoo,” complete resection of the lesion, identification of NIR+ SLNs, and histopathologic status of SLNs versus overall LNs in the LAD specimen. In addition, adverse events associated with NB-labeling or to ICG dye itself were recorded.
      Figure thumbnail gr1
      Figure 1NB-guided NIR imaging technique. NB-guided lesion localization for NIR+ “tattoo,” NIR lymphatic migration, and in situ NIR+ SLN identification for in-depth pathologic analysis. NB, Navigational bronchoscopy; NIR, near-infrared; SLN, sentinel lymph node.
      Twelve patients with clinically suspected or biopsy-proven T1N0 NSCLC scheduled to undergo minimally invasive lung resection were enrolled between March and December 2015 following informed consent for surgery and independently for the study protocol. Exclusion criteria included age <18 years old, pregnancy or breastfeeding, history of iodide or seafood allergy, suspected or known metastatic nodal disease, and previous neoadjuvant chemotherapy or radiation therapy to the lung. Although lobectomy is the standard of care for lung cancer, initial patients selected for this trial were those undergoing minimally invasive wedge resection for diagnosis or treatment either due to patient comorbidity, lesion characteristics,
      • Ettinger D.S.
      • Wood D.E.
      • Akerley W.
      • Bazhenova L.A.
      • Borghaei H.
      • Camidge D.R.
      • et al.
      NCCN guidelines insights: non-small cell lung cancer, version 4.2016.
      or patient preference as the aim of this trial was to test our technique for both lesion localization and targeted lymphatic mapping. All study patients underwent standard preoperative chest computed tomography (CT) ± positron emission tomography (PET)/CT for staging. Four patients with enlarged or suspicious lymph nodes had documented negative nodal pathology via cervical mediastinoscopy or endobronchial ultrasound prior to lung resection. All patients found to have NSCLC underwent LAD at the time of surgery.

      NB-Guided ICG Peritumoral Injection

      NB is a real-time localization system that uses 3-dimensional images generated from a preoperative NB-protocol chest CT or PET/CT scan with 1 × 0.8-mm slice intervals. The superDimension system(Covidien, Minneapolis, Minn) used creates a “road map” through the airway anatomy to guide a NB catheter to the lesion of interest for intraoperative biopsy and peritumoral ICG injection. ICG (Novadaq Technologies, Bonita Springs, Fla) was diluted to 2.5 mg/mL using 25% human serum albumin (ICG:HSA), as albumin has been shown to increase the effective hydrodynamic diameter of ICG almost 6-fold, resulting in increased lymphatic retention.
      • Gilmore D.M.
      • Khullar O.V.
      • Colson Y.L.
      Developing intrathoracic sentinel lymph node mapping with near infrared fluorescent imaging in non-small cell lung cancer.
      Based on our group's previous transpleural NIR dose escalation trial for lung cancer,
      • Ludwig M.S.
      • Goodman M.
      • Miller D.L.
      • Johnstone P.A.
      Postoperative survival and the number of lymph nodes sampled during resection of node-negative non-small cell lung cancer.
      a dose of 1.25-2.5 mg (0.5-1 mL) ICG was used. Using the NB catheter location registration, access to within a centimeter of the lesion was obtained via the airway. A needle was then advanced towards the lesion to inject ICG just deep to the lesion of interest. This allowed ICG marking of the deep margin of the lesion, ensuring the entire lesion was removed with resection of the ICG labeled parenchyma. The number of peritumoral injections used in each case ranged from 1 to 3 discrete injections, depending on lesion location, feasibility of multiple injection sites, and the number of lesions to be localized. The “tattoo” was visualized from the pleural surface due to the property of NIR light to penetrate tissue.
      • Frangioni J.V.
      In vivo near-infrared fluorescence imaging.
      NB-guided lesion biopsy with fluoroscopy was performed at the discretion of the operating surgeon.

      NIR Image-Guided Resection and Lymphatic Mapping

      After NB-guided peritumoral injection, patients were repositioned to the appropriate lateral decubitus position for resection via video-assisted thoracoscopic surgery. Real-time NIR fluorescence images were obtained intraoperatively using a minimally invasive NIR PINPOINT imaging system (Novadaq) including a 10-mm, 30-degree NIR thoracoscopic camera. ICG signal was detected in vivo at the site of peritumoral “tattoo” in all patients, with minimal NIR autofluorescence or background signal from normal human tissue. All patients underwent lung resection, and those with confirmed NSCLC underwent lymph node sampling comprising the LAD specimen with NIR imaging for SLN identification and resection. Specimens were imaged ex vivo to confirm NIR signal. The decision for definitive wedge resection versus lobectomy was based on lesion characteristics (identification of benign disease, multifocal adenocarcinoma, or metastatic disease), limited pulmonary status, or patient preference. For diagnoses other than NSCLC (benign lesions or metastases from another primary site), LAD and NIR SLN imaging were not performed.

      Pathologic Analysis

      The surgical specimen and the individually labeled nodal specimens, including NIR+ SLNs, were sent for routine histopathologic analysis. All LNs were fixed in formalin and embedded in paraffin for hematoxylin and eosin staining. Pathologic status was determined by an experienced lung pathologist without knowledge of which node was identified as NIR+ intraoperatively.

      Statistical Analysis

      The data reported is descriptive of the events that occurred in the trial with mean and standard deviation reported where appropriate. Specificity and sensitivity of the SLN are calculated based on the pathologic status of the NIR+ SLN in comparison with all nodes sampled along with the LAD.

      Results

      Patient and Lesion Characteristics

      Twelve patients, 10 female and 2 male, underwent NB-guided NIR marking of lung lesions. Mean patient age was 62 years old with a standard deviation of 6.7 years. A total of 15 lung lesions, all malignant, were resected ranging in size from 0.4 to 2.2 cm, with a depth from the pleural surface of 0.1 to 3.0 cm (Table 1). Seven lesions were characterized as solid on preoperative imaging, 6 were semisolid, 1 was a pure GGO, and 1 was not evident on preoperative imaging. Lesions were identified in the right upper lobe (n = 4), right lower lobe (n = 2), left upper lobe (n = 6), and left lower lobe (n = 3). Wedge resection was performed for all lesions to establish a pathologic diagnosis with completion lobectomy in 1 case. Two patients were diagnosed with metastatic disease from another primary and 10 patients with NSCLC.
      Table 1Characteristics of pulmonary nodules and NB-guided NIR “tattoo” marking
      Patient no.Lesion locationFinal pathologic size, cmLesion typeDepth from pleural surface, cm
      Measured from lesion to nearest pleural surface on preoperative CT.
      Fluoro time for ICG injection, sTime from ICG injection to NIR tattoo ID, minMargin distance of initial wedge resection, cm
      Intraoperative margin.
      Lesion diagnosis
      ICG aloneTotal
      1RLL1.8Solid1.0NR
      Not recorded.
      19.7651.2Adenocarcinoma
      2LLL1.9Solid2.0NR26600.1
      Patient did undergo completion lobectomy.
      Adenocarcinoma
      3LLL2.0Solid0.5NR2552N/AAtypical spindle cell neoplasm
      4LUL1.3Mixed GGO/solid1.0380.7Adenocarcinoma
      5
      Two separate lesions were NIR marked with 1.25 mg of ICG (in 0.5 mL of ICG:HSA) per lesion. Patient 12 also had an incidentally found third focus of adenocarcinoma on final pathology.
      RUL1.6Solid3.02033550.5Metastatic leiomyosarcoma
      RUL0.4Solid0.8500.4
      6LUL1.5Solid2.11818161
      Case included needle localization under CT guidance after NIR “tattoo” and before conversion to VATS.
      1.5Adenocarcinoma
      7LLL0.7Mixed GGO/solid0.11417431.0Adenocarcinoma
      8LUL1.3Mixed GGO/solid0.4430.2Adenocarcinoma
      9LUL2.2Solid0.1614661.8Squamous cell carcinoma
      10LUL0.9Mixed GGO/solid0.11212481.1Adenocarcinoma
      11LUL1.1Mixed GGO/solid1.6310732.8Adenocarcinoma
      12
      Two separate lesions were NIR marked with 1.25 mg of ICG (in 0.5 mL of ICG:HSA) per lesion. Patient 12 also had an incidentally found third focus of adenocarcinoma on final pathology.
      RUL2.1Mixed GGO/solid0.3642.4Adenocarcinoma
      RLL1.5GGO1.0810.1
      NB, Navigational bronchoscopy; NIR, near-infrared; ICG, indocyanine green; RLL, right lower lobe; NR, not reported; LLL, left lower lobe; N/A, not available; LUL, left upper lobe; GGO, ground-glass opacity; RUL, right upper lobe.
      Measured from lesion to nearest pleural surface on preoperative CT.
      Intraoperative margin.
      Not recorded.
      § Patient did undergo completion lobectomy.
      Case included needle localization under CT guidance after NIR “tattoo” and before conversion to VATS.
      Two separate lesions were NIR marked with 1.25 mg of ICG (in 0.5 mL of ICG:HSA) per lesion. Patient 12 also had an incidentally found third focus of adenocarcinoma on final pathology.

      NB-Guided NIR Lesion Localization and Resection

      NB-guided NIR “tattoo” marking was performed under general anesthesia with the patient supine and immediately before lung resection in all 12 cases with all 14 known lesions NIR marked and intraoperatively identified. The size of the visualized pleural “tattoo” depended on the depth and location of injection of ICG. Two separate lesions were marked with 0.5 mL of ICG:HSA per lesion in patients 5 and 12. These patients underwent wedge resections for diagnosis of potential multifocal disease. Patient 12 was discovered on final pathology to have a third focus of adenocarcinoma not palpable or identified on preoperative imaging, but it was removed within the wedge resection of an adjacent NIR marked tumor.
      Total NB time for peritumoral injection, which included biopsy and on-site cytologic interpretation as requested by the surgeon in 7 cases, averaged 34.5 minutes with approximately 20 minutes being for the entire navigation, dye preparation, and lesion “tattoo” if biopsy was not required. Total fluoroscopy time for injection of ICG alone ranged from 3 to 20 seconds and for patients who also underwent NB-biopsy ranged from 19.7 to 26 seconds with a total dose of 0.93-3.94 mGy.
      Immediately after placement of the NIR thoracoscope, the NIR “tattoo” was identified successfully on the pleural surface corresponding to each lesion in all cases. The average time to visualization after injection was 65 minutes with a standard deviation of 31 minutes. Given the depth of the lesion in patient 6, needle localization under CT-guidance after ICG injection was unsuccessfully attempted as a backup strategy before video-assisted thoracoscopic surgery delaying the assessment of NIR marking which was still readily visible at 161 minutes (Figure 2). For each patient, the site of NIR+ “tattoo” colocalized with the primary nodule identified on all frozen section analyses and was associated with negative surgical margins in all cases (mean margin distance 0.9 cm ± 0.7 cm). The average lesion to lesion:margin ratio was 1.3 ± 0.8. No clinically significant adverse events after NB and the injection of ICG were noted including bleeding, pneumothoraces, or bronchospasm.
      Figure thumbnail gr2
      Figure 2NB-guided NIR “tattoo” of lesion 2.1 cm from the pleural surface. A, Navigational bronchoscopy guides the injection of 1 mL of ICG. B, Axial CT image demonstrating a 1.1-cm lesion (yellow marker) that is 2.1 cm from the pleural surface (blue arrows). C, NIR+ “tattoo” is easily identifiable from the pleural surface nearly 4 hours after ICG injection.

      NB-Guided NIR Imaging for SLN Identification

      Figure thumbnail fx2
      Video 1A novel technique using navigational bronchoscopy for tumor localization and targeted lymphatic mapping in early-stage lung cancer. Video available at: http://www.jtcvsonline.org/article/S0022-5223(17)30190-3/addons.
      Table 2SLN yield and specimen pathology
      Patient no.SLN station (no. nodes)Time to SLN resection (min) (from injection)SLN pathologyOther lymph node stations (no. nodes)
      Additional nodes removed as part of the routine lymphadenectomy.
      Other lymph node pathologyPathologic stage
      1Level 7 (1)124PositiveLevel 4 (1); Level 7 (1); Level 10 (1); Level 11 (5)NegativeT1aN2
      2Level 10 (1)180NegativeLevel 5 (1); Level 10 (4); other hilar (6)NegativeT1aN0
      4Level 5 (1)155NegativeLevel 11 (2); Level 7 (1)NegativeT1aN0
      6Level 5 (2); Level 7 (5)NegativeT1aN0
      7Level 7 (3)64NegativeLevel 9 (3)NegativeT1aN0
      8Level 5 (1); Level 7 (1); Level 10L (1)NegativeT1aN0
      9Level 5 (1)134NegativeLevel 10L (1)NegativeT1bN0
      10Level 7 (1)114NegativeLevel 5 (1); Level 10 (1)NegativeT1aN0
      11Level 5 (1); Level 7 (1)102NegativeNegativeT1aN0
      12Level 11R (1)92NegativeLevel 4R (1); Level 7 (1)NegativeT1bN0
      T1aN0
      T1aN0
      SLN, Sentinel lymph node.
      Additional nodes removed as part of the routine lymphadenectomy.
      Figure thumbnail gr3
      Figure 3NB-guided NIR “tattoo” and SLN identification. A, NB-guided lesion localization for transbronchial, peritumoral ICG injection. B, NIR+ “tattoo” on pleural surface ∼1 hour after ICG injection (3 injections used). C, In vivo NIR+ level 7 SLN identification ∼2 hours after ICG injection. D, Axial CT of a 1.2-cm LLL lesion. E, NIR+ ICG “tattoo” is nearly identical to a concomitant methylene blue marking (blue arrow). F, In vivo NIR+ level 7 SLN (white arrow) identified in situ by ICG alone with no methylene blue. G, Axial CT of 1.5-cm ill-defined, pure ground-glass opacity. H, In vivo NIR lymphatic track from RLL peritumoral “tattoo” (blue arrow) to level 11R nodal station, RUL peritumoral “tattoo” (white arrow). I, In vivo NIR+ level 11R SLN (white arrow). NB, Navigational bronchoscopy; NIR, near-infrared; SLN, sentinel lymph node.

      Pathologic Analysis

      Both the lung and SLN specimens were imaged ex vivo before pathologic analysis to confirm NIR+ status. Pathologic analysis of resected lesions revealed primary lung adenocarcinoma (n = 9), squamous cell carcinoma (n = 1), atypical spindle cell neoplasm (n = 1), and metastatic leiomyosarcoma (n = 1). In all cases in which LAD was performed, a negative SLN was reflective of the status of all other nodes in the LAD specimen. Importantly, the NIR+ level 7 SLN identified in patient 1 was the only pathologic positive lymph node identified within the LAD specimen, resulting in upstaging from a negative nodal status (despite preoperative cervical mediastinoscopy) to stage IIIa disease. On the basis of these findings, the pathologic status of the 9 identified SLN was 100% sensitive and specific for overall nodal disease.

      Discussion

      This is the first reported human pilot trial to demonstrate the safety and feasibility of NB-guided peritumoral NIR marking of lung lesions and mapping of tumor-associated SLNs. Twelve patients successfully underwent NB-guided marking, allowing for lung lesion localization and resection with a negative margin in all patients. SLNs were identified in 80% of the 10 patients found to have NSCLC. This minimally invasive technique allows for the accurate localization and staging of small, ill-defined pulmonary nodules and GGOs representing a significant advancement in the way we manage, treat, and stage early lung cancers.
      Nodal status is a significant prognostic indicator in lung cancer, yet many patients still do not undergo proper nodal staging leading to potentially understaged and undertreated disease, which may contribute to poor overall survival and relatively high recurrence rates.
      • Su S.
      • Scott W.J.
      • Allen M.S.
      • Darling G.E.
      • Decker P.A.
      • McKenna R.J.
      • et al.
      Patterns of survival and recurrence after surgical treatment of early stage non-small cell lung carcinoma in the ACOSOG Z0030 (ALLIANCE) trial.
      • Rusch V.W.
      • Hawes D.
      • Decker P.A.
      • Martin S.E.
      • Abati A.
      • Landreneau R.J.
      • et al.
      Occult metastases in lymph nodes predict survival in resectable non-small-cell lung cancer: report of the ACOSOG Z0040 trial.
      • Kubuschok B.
      • Passlick B.
      • Izbicki J.R.
      • Thetter O.
      • Pantel K.
      Disseminated tumor cells in lymph nodes as a determinant for survival in surgically resected non-small-cell lung cancer.
      • Martin L.W.
      • D'Cunha J.
      • Wang X.
      • Herzan D.
      • Gu L.
      • Abraham N.
      • et al.
      Detection of occult micrometastases in patients with clinical stage I non-small-cell lung cancer: a prospective analysis of mature results of CALGB 9761 (Alliance).
      • Ludwig M.S.
      • Goodman M.
      • Miller D.L.
      • Johnstone P.A.
      Postoperative survival and the number of lymph nodes sampled during resection of node-negative non-small cell lung cancer.
      • Osarogiagbon R.U.
      • Allen J.W.
      • Farooq A.
      • Berry A.
      • O'Brien T.
      Pathologic lymph node staging practice and stage-predicted survival after resection of lung cancer.
      • Varlotto J.M.
      • Recht A.
      • Nikolov M.
      • Flickinger J.C.
      • Decamp M.M.
      Extent of lymphadenectomy and outcome for patients with stage I nonsmall cell lung cancer.
      Although preoperative imaging, in particular CT and PET/CT, is necessary for clinical staging, histologic diagnosis is still required to ensure N0 status, given the relatively low sensitivity and specificity for detecting metastatic nodal disease in early-stage lung cancers.
      • Schmidt-Hansen M.
      • Baldwin D.R.
      • Zamora J.
      FDG-PET/CT imaging for mediastinal staging in patients potentially resectable non-small cell lung cancer.
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      • Um S.W.
      • et al.
      Pure ground glass nodular adenocarcinomas: are preoperative positron emission tomography/computed tomography and brain magnetic resonance imaging useful or necessary?.
      The high false-negative rate is evident by the nearly 18% incidence of pathologic upstaging reported in early-stage lung cancers, and, in fact, our study resulted in the upstaging to stage III disease in 12.5% (1/8) of patients in whom SLNs were analyzed.
      • Licht P.B.
      • Jorgensen O.D.
      • Ladegaard L.
      • Jakobsen E.
      A national study of nodal upstaging after thoracoscopic versus open lobectomy for clinical stage I lung cancer.
      • Medbery R.L.
      • Geilespie T.W.
      • Liu Y.
      • Nickleach D.C.
      • Lipscomb J.
      • Sancheti M.S.
      • et al.
      Nodal upstaging is more common with thoracotomy than with VATS during lobectomy for early-stage lung cancer: an analysis from the national cancer data base.
      Although nodal sampling has been shown to be equivalent to radical LAD, SLN mapping offers distinct advantages including targeting the tumor-draining node(s) that are at increased risk of harboring metastatic disease and also permitting focused in-depth histologic scrutiny.
      • Darling G.E.
      • Allen M.S.
      • Decker P.A.
      • Ballman K.
      • Malthaner R.A.
      • Inculet R.I.
      • et al.
      Randomized trial of mediastinal lymph node sampling versus complete lymphadenectomy during pulmonary resection in the patient with N0 or N1 (less than hilar) non-small cell carcinoma: results of the American College of Surgery Oncology Group Z0030 trial.
      In addition, patients with early-stage NSCLC are prone to developing a second primary NSCLC at a rate of 1%-2%/year. In the future, targeted nodal assessment could potentially allow for subsequent nodal staging of a second ipsilateral lung cancer, an opportunity that is currently not possible if a full LAD has previously been performed.
      • Boyle J.M.
      • Tandberg D.J.
      • Chino J.P.
      • D'Amico T.A.
      • Ready N.E.
      • Kelsey C.R.
      Smoking history predicts for increased risk of second primary lung cancer: a comprehensive analysis.
      Under the current NCCN guidelines, patients with small, peripheral semi-solid, or slow-growing lesions may not require a lobectomy (although an anatomic resection is still preferred),
      • Ettinger D.S.
      • Wood D.E.
      • Akerley W.
      • Bazhenova L.A.
      • Borghaei H.
      • Camidge D.R.
      • et al.
      NCCN guidelines insights: non-small cell lung cancer, version 4.2016.
      and some studies are indicating favorable long-term outcomes with limited resection.
      • Altorki N.K.
      • Yip R.
      • Hanaoka T.
      • Bauer T.
      • Aye R.
      • Kohman L.
      • et al.
      Sublobar resection is equivalent to lobectomy for clinical stage 1A lung cancer in solid nodules.
      • Schuchert M.J.
      • Kilic A.
      • Pennathur A.
      • Nason K.S.
      • Wilson D.O.
      • Luketich J.D.
      • et al.
      Oncologic outcomes after surgical resection of subcentimeter non-small cell lung cancer.
      These small ill-defined lesions may be difficult to localize intraoperatively; however, with this technique, they can easily be identified and staged in a directed fashion and in a single operative setting. This is important for patients undergoing sublobar resection, as traditional rates of nodal sampling with sublobar resection have been highly variable.
      • Osarogiagbon R.U.
      • Yu X.
      Mediastinal lymph node examination and survival in resected early-stage non-small lung cancer in the surveillance, epidemiology, and end results database.
      Furthermore, this technique for targeted lymphatic mapping may offer an advantage for poor surgical candidates who cannot tolerate a large anatomic resection but would receive better staging than a patient undergoing focused definitive radiation such as stereotactic body radiation therapy.
      In this pilot study, the first patient evaluated was found to have nodal disease solely in the N2 level NIR+ SLN, which was not detected previously on PET/CT or cervical mediastinoscopy despite multiple LNs sampled including several nodes in the same station. This change in clinical stage significantly changed the prognosis and treatment of the patient resulting in the administration of adjuvant chemotherapy. This disease may have remained occult without the focused direction of the NIR signal as all of the other NIR nodes in the same nodal station were negative for metastatic disease. Similar upstaging was noted in 2 patients in our prior transpleural NIR trial, reinforcing the importance of targeting specific tumor-associated SLNs for focused pathologic analysis.
      • Gilmore D.M.
      • Khullar O.V.
      • Jaklitsch M.T.
      • Chirieac L.R.
      • Frangioni J.V.
      • Colson Y.L.
      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.
      Early porcine studies of NB-guided NIR imaging by Anayama and Wada demonstrated the significant potential for clinical translation of a transbronchial ICG injection technique.
      • Gilmore D.M.
      • Khullar O.V.
      • Colson Y.L.
      Developing intrathoracic sentinel lymph node mapping with near infrared fluorescent imaging in non-small cell lung cancer.
      • Anayama T.
      • Qiu J.
      • Chan H.
      • Nakajima T.
      • Weersink R.
      • Daly M.
      • et al.
      Localization of pulmonary nodules using navigation bronchoscope and a near-infrared fluorescence thoracoscopy.
      • Wada H.
      • Hirohashi K.
      • Anayama T.
      • Nakajima T.
      • Kato T.
      • Chan H.H.
      • et al.
      Minimally invasive electro-magnetic navigational bronchoscopy-integrated near infrared-guided sentinel lymph node mapping in the porcine lung.
      Anayama and colleagues
      • Anayama T.
      • Qiu J.
      • Chan H.
      • Nakajima T.
      • Weersink R.
      • Daly M.
      • et al.
      Localization of pulmonary nodules using navigation bronchoscope and a near-infrared fluorescence thoracoscopy.
      reported the feasibility of NB-guided ICG delivery adjacent to porcine agar pseudotumors, with NIR signal detection up to a depth of 2.4 cm and lasting as long as 6 hours in an inflated porcine lung. In the current study, the NIR “tattoo” was readily identifiable in all 12 patients with peritumoral NIR signal detection at depths of up to 3 cm from the pleural surface. Premixing ICG with HSA, which we have shown previously to be critical for successful SLN identification in humans, also resulted in a stable NIR+ signal without significant parenchymal diffusion for more than 4.5 hours after ICG injection. In the study by Wada and colleagues,
      • Wada H.
      • Hirohashi K.
      • Anayama T.
      • Nakajima T.
      • Kato T.
      • Chan H.H.
      • et al.
      Minimally invasive electro-magnetic navigational bronchoscopy-integrated near infrared-guided sentinel lymph node mapping in the porcine lung.
      attenuated nodal signal was noted with transbronchial ICG injection using ICG concentrations as low as 10 μg for lung marking, though previous studies have shown that these low ICG doses are inadequate for human translation of NIR techniques.
      • Gilmore D.M.
      • Khullar O.V.
      • Jaklitsch M.T.
      • Chirieac L.R.
      • Frangioni J.V.
      • Colson Y.L.
      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.
      • Gilmore D.M.
      • Khullar O.V.
      • Colson Y.L.
      Developing intrathoracic sentinel lymph node mapping with near infrared fluorescent imaging in non-small cell lung cancer.
      Thus, using our previously established dose of ICG:HSA, the current trial demonstrates the feasibility of SLN identification after transbronchial injection with persistence of NIR signal in SLN for at least 3 hours after injection, allowing sufficient time for completion of intraoperative diagnosis, resection, and staging.
      NB-guided NIR marking and directed surgical resection allows for diagnosis and treatment to be carried out in a single operative setting with the NIR “tattoo” placed via the same NB working channel used for biopsy of a lesion, thus streamlining care, reducing time to therapy, and potentially reducing costs This is in contrast to CT-guided percutaneous biopsy, which typically requires coordination with an interventional radiologist and access to an imaging suite before surgery. In addition, patients are exposed to radiation and are at risk of pneumothorax and possible chest tube placement for the management of complications that may delay definitive diagnosis and treatment.
      • Dale C.R.
      • Madtes D.K.
      • Fan V.S.
      • Gorden J.A.
      • Veenstra D.L.
      Navigational bronchoscopy with biopsy versus CT-guided biopsy for the diagnosis of a solitary pulmonary nodule: a cost-consequences analysis.
      • Wang Memoli J.S.
      • Neitert P.J.
      • Silvestri G.A.
      Meta-analysis of guided bronchoscopy for the evaluation of the pulmonary nodule.
      This risk is mitigated with NB as biopsy and ICG injection do not require traversing the pleural surface and may be irrelevant when performed immediately before surgical entry into the chest. The current study demonstrated that NB-guided ICG injection was safe without associated adverse events. Total procedure time including NB-guided peritumoral injection was similar to previous reports adding on average 20 minutes to the operative time for both diagnosis, when indicated, and labeling of the lesions.
      • Eberhardt R.
      • Anantham D.
      • Herth F.
      • Feller-Kopman D.
      • Ernst A.
      Electromagnetic navigation diagnostic bronchoscopy in peripheral lung lesions.
      Importantly, other approaches to intraoperative detection of pulmonary nodules have been described, including lesion localization with blue dyes, microcoils, percutaneous wire placement, and intravenous ICG, but none of these techniques permit concurrent SLN mapping for the lesion of interest.
      • Krimsky W.S.
      • Minnich D.J.
      • Cattaneo S.M.
      • Sarkar S.A.
      • Harley D.P.
      • Finley
      • et al.
      Thoracoscopic detection of occult indeterminate pulmonary nodule using bronchoscopic pleural dye marking.
      • Galetta D.
      • Bellomi M.
      • Grana C.
      • Spaggiari L.
      Radio-guided localization and resection of small or ill-defined pulmonary lesions.
      • Finley R.J.
      • Mayo J.R.
      • Grant K.
      • Clifton J.C.
      • English J.
      • Leo J.
      • et al.
      Preoperative computed tomography-guided microcoil localization of small peripheral pulmonary nodules: a prospective randomized controlled trial.
      • Chen S.
      • Zhou J.
      • Zhang J.
      • Hu H.
      • Luo X.
      • Zhang Y.
      • et al.
      Video-assisted thoracoscopic solitary pulmonary nodule resection after CT-guided hookwire localization: 43 cases report and literature review.
      • Okusanya O.T.
      • Holt D.
      • Heitjan D.
      • Deshpande C.
      • Venegas O.
      • Jiang J.
      • et al.
      Intraoperative near-infrared imaging can identify pulmonary nodules.
      • Rosenthal E.L.
      • Warram J.M.
      • de Boer E.
      • Basilion J.P.
      • Biel M.A.
      • Bogyo M.
      • et al.
      Successful translation of fluorescence navigation during oncologic surgery: a consensus report.
      Blue dyes and radiocolloid tracers have not demonstrated reproducible or accurate SLN detection even in multicenter studies, in large part due to pigmented anthracotic thoracic LNs, high tracer background signal, and rapid migration due to small particle size.
      • Liptay M.J.
      • D'Amico T.A.
      • Nwogu C.
      • Demmy T.L.
      • Wang X.F.
      • Gu L.
      • et al.
      Intraoperative sentinel node mapping with technitium-99 in lung cancer: results of CALGB 140203 multicenter phase II trial.
      • Rzyman W.
      • Hagen O.M.
      • Dziadziuszko R.
      • Kobierska-Gulida G.
      • Karmolinski A.
      • Lothe M.I.
      • et al.
      Blue-dye intraoperative sentinel lymph node mapping in early non-small cell lung cancer.
      The 1 patient in this trial who underwent concurrent methylene blue injection did demonstrate equivalent localization with lesional “tattoo”; however, there was no discernible migration to lymph nodes and the SLN was identified only by ICG. The translational success of other tumor-targeted approaches to fluorescence labeling, such as antibody-conjugated and enzyme-activated probes, appears to rely on the ability to predict tumor biology and histology, which can be difficult when evaluating ill-defined lung nodules, particularly when lesions are very small and preoperative histology or genomic sequencing is not available.
      • Nguyen Q.T.
      • Olson E.S.
      • Aguilera T.A.
      • Jiang T.
      • Scadeng M.
      • Ellies L.G.
      • Tsien R.Y.
      Surgery with molecular fluorescence imaging using activatable cell-penetrating peptides decreases residual cancer and improves survival.
      • Strong V.E.
      • Humm J.
      • Russo P.
      • Jungbluth A.
      • Wong W.D.
      • Daghighian F.
      • et al.
      A novel method to localize antibody-targeted cancer deposits intraoperatively using handheld PET beta and gamma probes.
      Limitations of the current prospective pilot trial include a small sample size, limited histologic and radiographic variability in tumor type, and restriction to NB-accessible lesions. Pathologic findings demonstrated the predictive ability of identified NIR+ SLNs scrutinized by routine pathologic analysis to identify occult nodal disease, and in the future, in-depth immunohistochemical evaluation may further improve the sensitivity of this approach. In addition, long-term recurrence and survival outcomes are necessary in larger, multicenter trials to determine whether cases identified as N0 based on SLN status behave as true node negative cases and exhibit better overall survival than patients deemed node-negative by standard hematoxylin and eosin analysis of routine LAD specimens.
      This novel first-in-human prospective pilot trial demonstrates that the NB-guided NIR technique for tumor localization and targeted lymphatic mapping is safe and feasible even for small ill-defined, stage I lung cancers. With continued development, this technology has the potential to significantly extend surgical resection and staging to include nonpalpable lesions in patients that are unable to tolerate a “blind lobectomy” and may alter the way early-stage NSCLC is identified and staged intraoperatively. Under a single anesthetic, patients may have an early-stage NSCLC accurately biopsied for diagnosis, definitively resected, and accurately staged, allowing surgeons to truly achieve the curative benefits purported with early-detection CT-screening programs.

      Conflict of Interest Statement

      Authors have nothing to disclose with regard to commercial support.
      We acknowledge Novadaq for the donation of ICG dye for the initial 10 patients as proof-of-concept for NB-labeling. The authors would also like to thank Marcia Williams for preparing the illustration used in Figure 1. There has been no preview, restrictions, or direct monetary sponsorship for this trial.

      Supplementary Data

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