Staging of Superficial Esophageal Adenocarcinoma (EAC) Using Volumetric Laser Endomicroscopy (VLE)
Phase: NA
Conditions: Esophageal Cancer
Sponsor: Mayo Clinic

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Staging of Superficial Esophageal Adenocarcinoma using Volumetric Laser Endomicroscopy 
 
 
 
 
March 12, 2021 
 
 
Principal Investigator: 
 
Cadman Leggett, M.D. 
Barrett’s Esophagus Unit 
Division of Gastoenterology and Hepatology 
Mayo Clinic 
200 First Street SW Rochester, Minnesota 55905 
Email: leggett.cadman@mayo.edu 
Telephone: 507-284-5255 
 
 
Research Preceptor: 
 
Kenneth K Wang MD 
Kathy and Russ Van Cleve Professor of Gastroenterology 
Barrett’s Esophagus Unit 
Division of Gastroenterology and Hepatology 
Mayo Clinic 
200 First St SW Rochester MN 55905 
Email: wang.kenneth@mayo.edu 
Tel: 507-284-2174 

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A. Background, Innovation and Significance: 
The prognosis and management of esophageal adenocarcinoma (EAC) is strongly associated with the 
stage of disease. Accurate staging is particularly important for superficial EAC given that T1a disease can be 
treated effectively with endoscopic therapy while T1b disease generally requires esophagectomy due to a 
higher risk of lymph node metastasis.1 Our current practice relies on histologic assessment of endoscopic 
mucosal resection (EMR) specimens to determine depth of invasion. Endoscopic resection and in particular 
endoscopic submucosal dissection may place patients with T1b disease at an unnecessary risk. Complications 
such as bleeding have been reported in up to 33%, perforation in up to 5% and esophageal stenosis in up to 
30% of patients undergoing resection.2 Furthermore, up to 15% of patients who undergo EMR will eventually 
require surgery.2 Volumetric laser endomicroscopy (VLE) is an imaging platform that uses infrared light to 
generate cross-sectional views of the human esophagus with microscopic resolution.3 VLE has been used to 
detect dysplasia associated with Barrett’s esophagus (BE) but its use in cancer staging has not been  
previously explored. 4, 5 We propose that VLE can accurately and comprehensively stage T1 EAC. The use of 
this technology may help select patients with invasive disease for esophagectomy while avoiding complications 
associated with endoscopic resection. 
 
B. Specific Aims: 
Accurate staging of superficial EAC (T1a vs. T1b) is critical in guiding treatment of early luminal cancer. 
We hypothesize that VLE can accurately and comprehensively stage T1 EAC. Thus the specific aims of this 
proposal are: 
1. To correlate depth of cancer invasion measured using VLE to depth of invasion established by EMR 
(clinical gold-standard) in a retrospective cohort of T1 EAC patients. 
2. To determine the diagnostic performance of VLE in staging T1 EAC in a prospective cohort of patients 
undergoing endoscopic evaluation. 
 
C. Technology Review: 
The VLE imaging system (Nine Point 
Medical, MA) consists of an imaging console, 
monitor and optical probe. The system’s  
optical probe is centered by a balloon 
(diameter: 14 mm, 17 mm, 20 mm; length: 6 
cm) that is deployed through a gastroscope’s 
instrument channel. Imaging is performed by 
automatic helical pullback of the probe from  
the distal to the proximal end of the balloon 
over 90 seconds. A total of 1200 single cross- 
sectional frames are generated per scan. VLE 
images have an axial resolution of 7 µm and 
can reach an imaging depth of up to 3 mm, 
providing 
surface 
and 
subsurface 
microstructural detail. These characteristics 
make VLE an ideal imaging modality for 
staging T1 EAC. 
The latest generation of VLE platform 
allows users to identify a region of interest and 
perform 
laser-markings 
that 
appear 
as 
superficial cautery marks under white-light 
endoscopy. The advantages of this approach 
are the ability to perform direct VLE  to 
histology correlation and to demarcate an area 
for endoscopic resection. Imaging enhancing 
software provides an en-face VLE view, where 
anatomical landmarks can be easily identified. 
This    software    also    superimposes    signal 
Figure 1: (A) Cross-sectional view of esophagus using VLE in patient with 
non-dysplastic BE (B) Three-dimensional rendering of VLE scan, (C) En-face 
view of VLE scan with labeled anatomical landmarks. (D) En-face view of VLE 
scan of patient with T1 EAC with image enhancing software. Area of increased 
signal intensity demarcated by laser markings corresponding to area of 
neoplasia. Gastric epithelium shows normal increased signal intensity. 

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intensity attenuation measurements allowing for recognition of areas of concern and quantification of degree of 
signal intensity. 
 
A recent advancement in the field of volumetric laser endomicroscopy (VLE) is the release of an FDA approved 
image enhancement software termed Intelligent real-time image segmentation (IRIS) (Nine Point Medical, 
Bedford, MA). IRIS highlights established VLE features using a color-graded scale superimposed over cross-
sectional and reconstructed en-face views. VLE IRIS features include (1) surface hyper-reflectivity, a pink 
graded color scale at the epithelial surface that represents the degree of signal decay over depth (signal 
attenuation), (2) hypo-reflective structures (epithelial glands), filled by a solid blue color and (3) lack of layered 
architecture represented by an orange graded color bar at the external rim of the VLE image. Our VLE console 
was recently upgraded with the IRIS software package. Following the study methodology of this IRB protocol we 
plan to use IRIS to locate and delineate neoplastic regions prior to biopsy or resection. This is a continuum of 
our work using a previous offline version of IRIS to measure VLE signal attenuation to determine stage (T1a vs 
T1b) of early esophageal adenocarcinoma. (Endosc Int Open. 2019 Apr;7(4):E462-E470) The use of IRIS is 
meant to facilitate VLE interpretation and does not impact patient care. VLE scans will be reviewed with and 
without IRIS enhancement. 
 
D. Research Strategy: 
 
a. Rationale: 
We propose that the use of an advanced endoscopic imaging technology designed to image the 
esophagus in cross-section at microscopic resolution can accurately predict depth of invasion in patients with 
T1 EAC. An advantage of cancer staging using VLE is that it could prevent unnecessary endoscopic resection 
and associated complications. This technology also provides comprehensive assessment of the esophagus as 
compared to the current standard of practice that relies on a discrete area chosen for endoscopic resection to 
determine depth of invasion. 
 
b. Preliminary Data and Published Data: 
Our group has performed extensive work aimed at understanding the 
VLE features associated with BE dysplasia. These features include 
effacement of layered architecture, atypical gland conformations and 
increased epithelial (surface) VLE signal intensity.4, 5 More recently we 
validated 
a 
computer 
algorithm 
that 
performs 
segmentation 
and 
reconstruction of a VLE scan into an en-face view with superimposed signal 
intensity profiles. (Figure 1) VLE signal decay is dependent upon several 
tissue optical characteristics.6 This algorithm can be applied to estimate the 
depth of invasion based on degree of signal attenuation. Figure 2 highlights 
an example of how depth of invasion can be correlated with VLE signal 
attenuation. We also reported on the use of this technology to detect 
subquamous esophageal adenocarcinoma.7 
Our group has also performed work on histologic determinants of 
mortality for patients with T1 EAC and described that evidence of 
lymphovascular invasion and deep margin involvement are significantly 
associated with reduced overall survival.8 Ultimately, the data obtained from 
the present study will help determine if histologic features such as 
lymphovascular invasion can also be quantified with VLE. 
 
c. Study Design: 
 
I. 
Aim1: VLE to histology correlation of T1 EAC depth of invasion 
 
a. 
Endoscopic Mucosal Resection Database: Our group 
has developed an ex-vivo imaging platform for imaging EMR specimens using VLE.4 Patients with BE 
and early neoplasia undergo a clinically indicated EMR after participating in informed consent (IRB 
Figure 2: (A) endoscopic mucosal 
resection specimen of patient with T1a 
EAC and imaged with (B) VLE (C) 
Depth of invasion estimated by signal 
attenuation decay at 340 µm. 

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ID:12-006665). The fresh EMR specimen is imaged with VLE and inked for orientation. Through an 
established pathology core lab protocol (Proc 053417.002) for sectioning of the EMR specimens we 
are able to obtain direct imaging-to- histology correlation Using this imaging platform we have imaged 
over 200 EMR specimens since 2012 of which 60 specimens  were obtained from patients with T1 
EAC (T1a EAC, N=45; T1b EAC, N=15). 
 
b. 
Histopathologic 
Review: 
EMR 
histology 
will 
be  
reviewed systematically to assess grade of tumor differentiation, depth of invasion, and presence or 
absence of lymphovascular invasion.     Depth   of   invasion   will  be   manually  quantified by 

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measuring distance (µm) from mucosal surface to deepest point of invasion. (Figure 3) The Paris 
classification suggests a cut-off of 500 µm to divide depth of submucosal invasion into superficial (sm1) 
and deep (sm2). This classification has been correlated to depth of invasion defined by layer 
involvement: (1) T1a EAC: tumor invasion into the lamina propria or muscularis mucosa and (2) T1b 
EAC: tumor invading into the submucosa.9 
 
c. Correlation of VLE Signal Attenuation to Depth of Cancer Invasion: A validated computer  algorithm 
will be used to quantify the degree of signal attenuation in the T1 EAC EMR specimen database 
identified above. VLE signal attenuation measurements will be correlated to  depth  of invasion as 
determined by histology (dB/µm). Based on these measurements, a cutoff signal attenuation value will be 
established to distinguish between superficial and deep invasion. 
 
II. 
Aim 2: Diagnostic performance of VLE in staging T1 EAC 
 
Patient Selection: 
 a. Patients referred to our institution for management of BE and early neoplasia will be prospectively enrolled in 
this study. Inclusion criteria include patients with T1 EAC who are undergoing endoscopic evaluation including 
EMR.  
      Inclusion: 
• 
Patient over the age of 18 
• 
Patient undergoing an upper endoscopy  
    with prior-biopsy confirmed Barrett’s Esophagus. 
• 
Ability to provide written, informed consent 
• 
No significant esophagitis (LA grade <B, C, and D)              
 
 
Figure 4: Study flow diagram  
Exclusion:  
• 
Patients who have achieved complete  
    remission of intestinal metaplasia (CR-IM) 
• 
Patients without visible BE at the time of study EGD 
• 
Patients for whom use of the NvisionVLE device 
     would be in conflict with the instruction for use.  
• 
Prior esophageal or gastric surgical resection 
• 
Significant esophageal stricture requiring dilatation 
• 
Patients who require anticoagulation for who 
     biopsy would be contraindicated 
• 
Patients who are known to be pregnant                                                                  
 
     b. Endoscopic Evaluation: Patients will  
undergo 
high-definition 
white 
light 
endoscopy/narrow band imaging with detailed 
inspection of the BE mucosa. The length of the 
BE segment will be measured using Prague 
criteria (circumferential length, maximal length). 
Area(s) of mucosal nodularity/irregularity will be 
classified (Paris classification) and their location 
recorded 
(o’clock 
position, 
distance 
from 
incisors). If clinically indicated, an area(s) will be 
chosen for EMR.   
 
  
 
 
 
 
 
 
 
  
 
 
Figure4:Studyflow diagram 
 
d. Volumetric Laser Endomicroscopy: A VLE 
Figure 4: Study flow diagram scan will be performed. 
The VLE scan will be systematically reviewed          
based on a previously established protocol. If 
an 
area(s) 
is 
chosen 
for 
EMR 
during 

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endoscopic  evaluation it will be identified and 
demarcated using VLE laser markings. If  
additional areas of   
concern are identified under VLE, they will be 
demarcated using VLE laser marking. All 
area(s) demarcated with VLE but not identified 
on endoscopic evaluation will undergo targeted 
biopsies. Area(s) identified under endoscopic 
evaluation and demarcated with VLE will 
undergo EMR as outlined below. Standard 
surveillance biopsies will be obtained across the 
remaining BE segment. 
 
e. Assessment of Cancer Depth of Invasion: VLE scans will be analyzed using an automated computer 
algorithm to measure degree of signal attenuation over areas demarcated for EMR. The signal attenuation 
cut-off established from aim1 will be used to classify cancers as superficial vs. deep. EMR specimens will be 
reviewed for grade of tumor differentiation, depth of invasion, and presence or absence of lymphovascular 
invasion. Depth of tumor invasion will be measured as outlined above and used to classify cancers as 
superficial vs. deep. 
 
f. Statistical Considerations and Data Analysis: This is a prospective cohort pilot study to determine the 
diagnostic performance of VLE in staging T1 EAC. To achieve a specificity of 90% with a 95% confidence 
interval, we estimate that a total of 60 patients with T1 EAC will need to be enrolled in this study taking into 
account that approximately 75% of patients will be staged as having T1a disease on histology. Sensitivity, 
specificity, negative predictive value, positive predictive value and diagnostic accuracy will be calculated for 
VLE compared to histology as the gold-standard in staging T1 EAC. Kappa statistics will be performed to 
test for agreement of the diagnostic methods. 
 
 Anticipated Results, Interpretation and Potential Pitfalls: 
In the first aim of this study we explore the correlation between VLE signal attenuation and depth of 
cancer invasion. We use VLE scans from an EMR database of T1 EAC patients obtained using a validated ex- 
vivo imaging platform. A novel computer algorithm is used to measure the degree of signal attenuation. We 
anticipate establishing a cut-off of signal attenuation value that correlates with histologic depth of cancer 
invasion (superficial vs. deep). A potential setback to this approach is the ability to accurately measure  
absolute histologic depth of invasion, especially in cases with duplicated muscularis mucosa. This setback is 
mitigated by our prior experience with histological assessment of EMR specimens in which we established a 
standardized approach to specimen review that will involve two independent gastrointestinal pathologists. 
In the second aim of this study we validate the accuracy of VLE in staging T1 EAC compared to a 
histologic gold-standard. We anticipate that VLE will be able to distinguish between superficial and deep 
cancer with a >90% accuracy. A potential pitfall to this approach is that the signal attenuation cut-off will be 
established by imaging of EMR specimens. There is little variation in imaging characteristics between ex-vivo 
and in-vivo VLE imaging. However, if we find a broad distribution of signal attenuation measurement in EMR 
specimens we plan to perform preliminary validation of this approach using a cohort of T1 EAC patients (N=10) 
who have undergone in-vivo VLE and EMR as part of a previous research protocol. 
 
References: 
 
1. 
Shaheen NJ, Falk GW, Iyer PG, et al. ACG Clinical Guideline: Diagnosis and Management of Barrett's 
Esophagus. Am J Gastroenterol 2016;111:30-50; quiz 51. 
2. 
Conio M, Ponchon T, Blanchi S, et al. Endoscopic mucosal resection. Am J Gastroenterol 
2006;101:653-63. 
3. 
Wolfsen HC, Sharma P, Wallace MB, et al. Safety and feasibility of volumetric laser endomicroscopy in 
patients with Barrett's esophagus (with videos). Gastrointest Endosc 2015;82:631-40. 
4. 
Leggett CL, Gorospe EC, Chan DK, et al. Comparative diagnostic performance of volumetric laser 

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endomicroscopy and confocal laser endomicroscopy in the detection of dysplasia associated with 
Barrett's esophagus. Gastrointest Endosc 2016;83:880-888 e2. 
5. 
Evans JA, Poneros JM, Bouma BE, et al. Optical coherence tomography to identify intramucosal 
carcinoma and high-grade dysplasia in Barrett's esophagus. Clinical gastroenterology and hepatology : 
the official clinical practice journal of the American Gastroenterological Association 2006;4:38-43. 
6. 
Muppa P, Leggett CL, Chan DK, et al. Effacement of the Mucosal Layer Observed With Volumetric 
Laser Endomicroscopy Is Not Associated With Mucosal Density or Thickness of Barrett's Esophagus. 
Gastroenterology 2015;148:S347-S348. 
7. 
Leggett CL, Gorospe E, Owens VL, et al. Volumetric laser endomicroscopy detects subsquamous 
Barrett's adenocarcinoma. The American journal of gastroenterology 2014;109:298-9. 
8. 
Leggett CL, Lewis JT, Wu TT, et al. Clinical and histologic determinants of mortality for patients with 
Barrett's esophagus-related T1 esophageal adenocarcinoma. Clinical gastroenterology and hepatology 
 : the official clinical practice journal of the American Gastroenterological Association 2015;13:658-64 
 e1-3. 
 9. Fotis D, Doukas M, Wijnhoven BP, et al. Submucosal invasion and risk of lymph node invasion in early 
Barrett's cancer: potential impact of different classification systems on patient management. United 
 European gastroenterology journal 2015;3:505-13.