American Association for Thoracic Surgery Near Infrared (NIR) Image-guided Lymphatic Mapping for Adequate Nodal Staging in Esophageal Cancer Krista J. Hachey MD, Denis M. Gilmore MD, Katherine W. Armstrong MPH, Sean E. Harris MA, Jon O. Wee MD, Yolonda L. Colson MD, PhD http://www.emdmillipore.com/US/en/product/Anti-phospho-Histone-H3%28Ser10%29-Antibody,MM_NF-09-797#documentation American Association for Thoracic Surgery 95th Annual Meeting April 25-29th, 2015

Disclosures I have no equity, ownership, stock, options or any interest in any company I do not consult for any company We gratefully acknowledge Novadaq for the generous loan of the Pinpoint system. There has been no data preview or monetary sponsorship for this collaboration Use of ICG for NIR-imaging of SLN is NOT FDA-approved

Introduction Why is this clinical trial important? 5-year survival: <40% (node positive) High prevalence of nodal disease even with T1 lesions (16-36%) Locoregional nodal recurrence rates: ~5-20% Robust lymphadenectomy (LAD) for prognostic staging Esophageal lymphatics: local (en bloc with the specimen) and regional (separate from the specimen) No established techniques to determine which regional nodes are most critical for removal and analysis Esophageal cancer is an aggressive disease with high rates of nodal metastasis and even locoregional nodal recurrence As a result, extensive LAD is recommended. Clearly esophageal lymphatics are complex – there are numerous LNs directly on the esophagus. We call these “local” in this trial. Disease in these nodes is removed en bloc with specimen at the time of surgery. Addditionally, there are regional LN stations separate from the esophagus throughout 3 compartments. Given our groups experience with Near infrared lymphatic mapping in lung and other cancer, we hypothesized the NIR mapping of tumor-associated regional LNs could be safe and feasible with potential to improve staging in this disease. Locoregional recurrence rates: Dresner Mariette Altorki Swanson Hypothesis We hypothesize that identifying regional LNs at greatest risk for metastases for focused histologic analysis is safe and feasible via NIR lymphatic mapping with potential to improve clinical outcomes

Near Infrared (NIR) Lymphatic Mapping Video of Real-time Merged Images NIR dye Indocyanine Green (ICG) emission range: 750-950nm ICG signal detection using a near-infrared videoscopic camera Visualization of merged and single channel real-time images Color Video Camera NIR Camera Light Source (color, NIR fluorescence excitation) Indocyanine Green is the only FDA approved near infrared dye. Normal human tissues have minimal autofluorescence in that range so the signal to noise ratio is favorable with this tracer. Intraoperatively, ICG uptake is detected using a NIR videoscopic camera and realtime merged images demonatrate ICG signal in green with a familiar thoracoscopic background. Surgical Field Courtesy of J Frangioni at BIDMC.

Trial Aims Assess the safety and feasibility of intra-operative, minimally invasive, Near Infrared (NIR) image-guided lymphatic mapping of regional LNs in esophageal cancer Compare the feasibility of lymphatic mapping with Near Infrared dye Indocyanine Green (ICG) alone or premixed with human serum albumin (ICG:HSA) As a protein carrier has been shown to improve dye retention in lymphatics in other trials.

Methods: Patient Selection Pilot “first-in-human” clinical trial Ten patients with early stage adenocarcinoma T1 (n=2); T2 (n=2); T3 (n=6) Standard preoperative clinical work up: EUS, CT, PET/CT 8 of 10 patients had preoperative chemoradiation Five patients had EUS findings concerning for enlarge paraesophageal nodes (pts: 2, 3, 4, 6, 8 Three patients had positive pre-treatment PET/CT findings: 4 (mediastinal paraesoph), 8 (retrocardiac, root of mesentary – bx neg), 10 (avid gastrohepatic) Two patients had positive post-treatment PET/CT: patient 1: Left mediastinum and patient 10: less prominent gatrohepatic CROSS trial: carboplatin, paclitaxel, radiotherapy. No good data to support adjuvant.

Methods: Intraoperative Protocol Eligible pt in OR Endoscopy Endoscopic peritumoral, submucosal injection of ICG (2.5mg ICG in 1cc of sterile water or albumin) Minimally invasive esophagectomy + NIR imaging (during laparoscopy + VATS) There was no intraoperative staging in this pilot study Identification of NIR+ LN in vivo Ex vivo NIR imaging of all specimens Routine LN Pathologic analysis

Results In vivo NIR imaging was feasible in all cases (n=10) NIR imaging revealed 2-6 NIR+ tumor-draining regional LNs per patient (n=6) (fig). NIR+ LNs detected from 30 mins – 3.5 hrs after ICG injection. 4 didn’t have identified node – one was stage IV and the other 3 were ICG alone cases Signal stays in the nodes throughout the duration of the operation. NIR image-guided identification of a tumor-associated paraesophageal LN

NIR+ LNs as a Function of ICG Carrier Results NIR+ LNs as a Function of ICG Carrier Pt # ICG carrier Regional LNs (NIR+/total) Total LNs sampled 1 ICG:HSA 6/10 40 2 2/2 22 3 ICG alone 0/11 26 4 0/3 28 5 0/2 18 6 N/A 7 1/5 33 8 2/8 25 9 3/7 10 4/6 31 ICG:HSA – superior tracer NIR+ regional LNs identified: 4/4 ICG:HSA 2/5 ICG alone Although this is a small trial, we found that ICG:HSA appears to be a superior tracer as evidenced by the NIR+ LN yield.

NIR+ LNs as a Function of ICG Carrier Results NIR+ LNs as a Function of ICG Carrier Pt # ICG carrier Regional LNs (NIR+/total) Total LNs sampled 1 ICG:HSA 6/10 40 2 2/2 22 3 ICG alone 0/11 26 4 0/3 28 5 0/2 18 6 N/A 7 1/5 33 8 2/8 25 9 3/7 10 4/6 31 ICG:HSA – superior tracer NIR+ regional LNs identified: 4/4 ICG:HSA 2/5 ICG alone Mean total number of LNs resected: T1: 21.5 (+/- 4.95) T2/T3: 29.3 (+/- 5.99) Although this is a small trial, we found that ICG:HSA appears to be a superior tracer as evidenced by the NIR+ LN yield.

Results High NIR background on the specimen limits identification of distinct NIR+ “local” nodes. No adverse events. Image is from patient 7 – who had neoadj chemoradiation and yet we still see the ICG migration throughout the esophagus

Location of NIR+ Regional LNs NIR+ Regional LN Locations and Prevalence in GEJ and Lower Esophageal Cancers LN Station (#) % Cases (N=6) Subcarinal (7) 16.7% (1) Lower paraesophageal (8L) Paracardial (16) 83.3% (5) Left gastric (17) 33.3% (2) Celiac Axis (20) Frequency of NIR+ LN by Station 16.7% 16.7% The location of NIR+ regional nodes was consistent with metastatic patterns described for lower esophageal and GE junction adenocarcinomas with the majority of cases having NIR+ paracardial and gastric lymph nodes, as well as celiac axis and lower mediastinal nodes. In the two patients with PET/CT avidity: the left gastric nodes were NIR+ (pt 10) and left lower paraesophageal LN in mediastinum was NIR+ (pt 1). AJCC 6th edition: node stations 2 through 17 were considered regional nodes (N1). Lymph node stations 18 and 19 and retroperitoneal nodes such as retrocaval or retroaortic lymph nodes were designated as nonregional (M1b) nodes. For esophageal cancers of the distal esophagus or gastroesophageal junction, nodal station 20 (celiac node) was considered metastatic (M1a) disease. Nonregional nodal involvement (n = 17) was associated with decreased survival compared with regional (n = 441) or celiac nodal (n = 73) involvement (3-year: 0% versus 24% and 23%; p < 0.001). The number of involved lymph nodes was strongly associated with survival (3-year: 0 nodes = 63%, 1 to 3 nodes = 31%, more than 3 nodes = 13%; p < 0.001), and multivariable Cox proportional-hazards analysis suggested that the location and number of involved lymph nodes were independent predictors of survival (p < 0.001). We propose a modified nodal staging system that designates celiac nodes as regional and includes number of involved nodes: pN0, no nodes (3 years = 63%, n = 496); pN1-regional, 1 to 3 nodes (3 years = 32%, n = 292); pN2-regional, more than 3 nodes (3 years = 14%, n = 222); pN3-nonregional node (3 years = 0%, n = 17 [p < 0.0001]). This modified nodal staging system better predicts survival than the current AJCC nodal staging system in which survival for pN1 (3 years = 24%) and pM1a (3 years = 23%) do not differ (p = 0.67). The use of induction before surgical resection did not alter the predictive effect of the new nodal staging system. Regional Lymph Node Stations for Staging Esophageal Cancer, From Front (A) and Side (B) 1 Supraclavicular nodes Above suprasternal notch and clavicles 2R Right upper paratracheal nodes Between intersection of caudal margin of innominate artery with trachea and the apex of the lung 2L Left upper paratracheal nodes Between top of aortic arch and apex of the lung 3P Posterior mediastinal nodes Upper paraesophageal nodes, above tracheal bifurcation 4R Right lower paratracheal nodes Between intersection of caudal margin of innominate artery with trachea and cephalic border of azygous vein 4L Left lower paratracheal nodes Between top of aortic arch and carina 5 Aortopulmonary nodes Subaortic and para-aortic nodes lateral to the ligamentum arteriosum 6 Anterior mediastinal nodes Anterior to ascending aorta or innominate artery 7 Subcarinal nodes Caudal to the carina of the trachea 8M Middle paraesophageal lymph nodes From the tracheal bifurcation to the caudal margin of the inferior pulmonary vein 8L Lower paraesophageal lymph From the caudal margin of the inferior pulmonary vein to the esophagogastric junction 9 Pulmonary ligament nodes Within the inferior pulmonary ligament 10R Right tracheobronchial nodes From cephalic border of azygous vein to origin of RUL bronchus 10L Left tracheobronchial nodes Between carina and LUL bronchus 15 Diaphragmatic nodes Lying on the dome of the diaphragm, and adjacent to or behind the crura 16 Paracardial nodes Immediately adjacent to the gastroesophageal junction 17 Left gastric nodes Along the course of the left gastric artery 18 Common hepatic nodes Along the course of the common hepatic artery 19 Splenic nodes Along the course of the splenic artery 20 Celiac nodes At the base of the celiac artery 366 HOFSTETTER ET AL Ann Thorac Surg PROPOSED MODIFICATION OF NODAL STATUS 2007;84:365–75 GENERAL 83.3% 33.3% 16.7% *Castoro C et al. Ann Surg Oncol (2011)18:3743-3754

Location of NIR+ Regional LNs NIR+ Regional LN Locations and Prevalence in GEJ and Lower Esophageal Cancers LN Station (#) % Cases (N=6) Subcarinal (7) 16.7% (1) Lower paraesophageal (8L) Paracardial (16) 83.3% (5) Left gastric (17) 33.3% (2) Celiac Axis (20) Frequency of NIR+ LN by Station Rate of LN Metastasis by Station* 16.7% ~5-12% 16.7% ~22-30% Suggesting that we are in fact mapping out a tumor-associated pathway The location of NIR+ regional nodes was consistent with metastatic patterns described for lower esophageal and GE junction adenocarcinomas with the majority of cases having NIR+ paracardial and gastric lymph nodes, as well as celiac axis and lower mediastinal nodes. In the two patients with PET/CT avidity: the left gastric nodes were NIR+ (pt 10) and left lower paraesophageal LN in mediastinum was NIR+ (pt 1). AJCC 6th edition: node stations 2 through 17 were considered regional nodes (N1). Lymph node stations 18 and 19 and retroperitoneal nodes such as retrocaval or retroaortic lymph nodes were designated as nonregional (M1b) nodes. For esophageal cancers of the distal esophagus or gastroesophageal junction, nodal station 20 (celiac node) was considered metastatic (M1a) disease. Nonregional nodal involvement (n = 17) was associated with decreased survival compared with regional (n = 441) or celiac nodal (n = 73) involvement (3-year: 0% versus 24% and 23%; p < 0.001). The number of involved lymph nodes was strongly associated with survival (3-year: 0 nodes = 63%, 1 to 3 nodes = 31%, more than 3 nodes = 13%; p < 0.001), and multivariable Cox proportional-hazards analysis suggested that the location and number of involved lymph nodes were independent predictors of survival (p < 0.001). We propose a modified nodal staging system that designates celiac nodes as regional and includes number of involved nodes: pN0, no nodes (3 years = 63%, n = 496); pN1-regional, 1 to 3 nodes (3 years = 32%, n = 292); pN2-regional, more than 3 nodes (3 years = 14%, n = 222); pN3-nonregional node (3 years = 0%, n = 17 [p < 0.0001]). This modified nodal staging system better predicts survival than the current AJCC nodal staging system in which survival for pN1 (3 years = 24%) and pM1a (3 years = 23%) do not differ (p = 0.67). The use of induction before surgical resection did not alter the predictive effect of the new nodal staging system. Regional Lymph Node Stations for Staging Esophageal Cancer, From Front (A) and Side (B) 1 Supraclavicular nodes Above suprasternal notch and clavicles 2R Right upper paratracheal nodes Between intersection of caudal margin of innominate artery with trachea and the apex of the lung 2L Left upper paratracheal nodes Between top of aortic arch and apex of the lung 3P Posterior mediastinal nodes Upper paraesophageal nodes, above tracheal bifurcation 4R Right lower paratracheal nodes Between intersection of caudal margin of innominate artery with trachea and cephalic border of azygous vein 4L Left lower paratracheal nodes Between top of aortic arch and carina 5 Aortopulmonary nodes Subaortic and para-aortic nodes lateral to the ligamentum arteriosum 6 Anterior mediastinal nodes Anterior to ascending aorta or innominate artery 7 Subcarinal nodes Caudal to the carina of the trachea 8M Middle paraesophageal lymph nodes From the tracheal bifurcation to the caudal margin of the inferior pulmonary vein 8L Lower paraesophageal lymph From the caudal margin of the inferior pulmonary vein to the esophagogastric junction 9 Pulmonary ligament nodes Within the inferior pulmonary ligament 10R Right tracheobronchial nodes From cephalic border of azygous vein to origin of RUL bronchus 10L Left tracheobronchial nodes Between carina and LUL bronchus 15 Diaphragmatic nodes Lying on the dome of the diaphragm, and adjacent to or behind the crura 16 Paracardial nodes Immediately adjacent to the gastroesophageal junction 17 Left gastric nodes Along the course of the left gastric artery 18 Common hepatic nodes Along the course of the common hepatic artery 19 Splenic nodes Along the course of the splenic artery 20 Celiac nodes At the base of the celiac artery 366 HOFSTETTER ET AL Ann Thorac Surg PROPOSED MODIFICATION OF NODAL STATUS 2007;84:365–75 GENERAL 83.3% ~13-59% 33.3% ~19-36% 16.7% ~14-19% *Castoro C et al. Ann Surg Oncol (2011)18:3743-3754

Histopathologic Analysis of LN Specimens Results Histopathologic Analysis of LN Specimens Pt # Regional LNs # metastatic/total Local en bloc LNs #metastatic/total 3* 0/11 1/15 7 0/5 1/28 8 0/8 1/17 9 0/10 10 0/6 7/25 None of the regional lymph nodes had disease, including NIR+ nodes. 5 cases had metastasis in local nodes which were removed on bloc with the specimen. * ICG alone cohort, all others ICG:HSA Pathologic status of NIR+ regional LNs is consistent with that of other regional LNs Metastatic LNs were found only en bloc with the esophageal specimen

Conclusions This is the first study to examine lymphatic mapping of regional LNs in esophageal cancer using intra-operative minimally invasive NIR imaging. NIR imaging is safe and feasible in esophageal cancer. Distribution of NIR+ LNs appears to mimic known LN metastatic patterns Targeted NIR-guided removal of tumor-associated nodes may serve as a minimally invasive staging tool and assist in adequate nodal sampling during lymphadenectomy.

Future Directions Optimize NIR imaging of local LNs: dose de-escalation to minimize background Other innovative imaging techniques for nodal assessment

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