1. 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
American Association for Thoracic Surgery
95th Annual Meeting
April 25-29th, 2015

2. 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

3. 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

4. Near Infrared (NIR) Lymphatic Mapping
Video of Real-time Merged Images
NIR dye Indocyanine Green (ICG) emission range: nm
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.

5. 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.

6. 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.

7. 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

8. 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

9. 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.

10. 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.

11. 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

12. 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 < ]). 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:

13. 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 < ]). 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:

14. 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

15. 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.

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

17. Thank you