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Gastrointest Endosc. Author manuscript; available in PMC 2020 September 16.
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Published in final edited form as:

Gastrointest Endosc. 2018 September ; 88(3): 413–426. doi:10.1016/j.gie.2018.04.2352.

Screening for Esophageal Squamous Cell Carcinoma: Recent

Advances
DC Codipilly*,1, Y Qin*,1, Sanford M. Dawsey2, John Kisiel1, Mark Topazian1, David
Ahlquist1, PG Iyer1
1:Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester
2:Division of Cancer Epidemiology and Genetics, National Cancer Institute
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Abstract
Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal cancer
worldwide, with a high mortality due to advanced stage at diagnosis. Although most common in
an area known as the Asian Esophageal Cancer Belt, which extends from the Caspian Sea to
northern China, and in parts of Africa, high-risk populations also exist elsewhere in the world.
Screening for ESCC has been practiced in a few geographic areas and high-risk populations, with
varying levels of success. Esophageal squamous dysplasia is recognized as the precursor lesion for
ESCC. Endoscopic screening for ESCC/esophageal squamous dysplasia is expensive and not
sufficiently available in many high-risk regions. Recent advances in non-endoscopic screening
enhanced by biomarker-based disease detection have raised the prospect of improved accuracy and
availability of screening for esophageal squamous dysplasia and early stage ESCC. Development
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of a cost-effective, accurate, and well-tolerated screening test, if applied in endemic areas and
high-risk populations, has the potential to reduce mortality from this deadly disease worldwide. In
this review, we summarize recent developments in endoscopic and non-endoscopic screening
modalities.

Introduction:
Esophageal cancer ranks as the 6th most common cause of cancer death worldwide, with
456,000 new cases and 400,000 deaths annually1, 2. Nearly 90% of esophageal cancer cases
worldwide are esophageal squamous cell carcinoma (ESCC)2–4. The highest risk areas for
ESCC are found in two geographic belts, the Asian Esophageal Cancer Belt across central
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Asia, from the Caspian Sea to northern China, and a belt on the eastern coast of Africa, from
Ethiopia to South Africa (Figure 1).

Corresponding author: Prasad G. Iyer MD MSc, Professor of Medicine, Division of Gastroenterology and Hepatology, Mayo Clinic,
Rochester, MN, Telephone: 5072660338, iyer.prasad@mayo.edu.
*Co-first authors
Conflicts of Interest:
Prasad G. Iyer: Research funding from Exact Sciences, C2 Therapeutics, Medtronic, Nine Point Medical
John B. Kisiel and David A. Ahlquist: Listed as co-inventors in an intellectual property development agreement with Exact Sciences
and could receive future royalties.
 Codipilly et al. Page 2

Unfortunately, most patients with ESCC present with advanced disease: 5-year survival rates
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are less than 20% in developed countries and less than 5% in many developing countries
where most of the disease occurs3, 5, 6. Additionally, the outcomes of patients diagnosed
after the onset of symptoms have not changed in the last several decades despite advances in
therapy5. Early stage carcinoma, however, is associated with a substantially improved
survival of 80–90% at 5 years when treated endoscopically or surgically3, 7. Therefore, early
detection strategies are needed to improve outcomes of patients with ESCC.

Analogous to the association of Barrett’s esophagus (BE) and its related dysplasia to
esophageal adenocarcinoma (EAC), esophageal squamous dysplasia has been shown to be a
precursor to ESCC based on natural history studies conducted in China8, 9. These studies
have shown that the risk of progression to cancer increases depending on the degree of
dysplasia8. This has led to endoscopic treatment of high grade dysplasia (HGD). Given the
considerable time needed in the transformation from dysplastic epithelium to malignancy10,
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a window exists in which detection and treatment of esophageal squamous dysplasia can
reduce ESCC incidence.

In this review, we summarize and evaluate both conventional and emerging modalities to
screen for this lethal disease and its precursor.

Rationale and Outcomes of Screening and Surveillance Programs

The main rationale for ESCC screening is to detect asymptomatic esophageal squamous
dysplasia and early ESCC, to enable curative treatment. Several studies have demonstrated
increases in early detection, mortality reduction, and cost effectiveness by endoscopic
screening programs in high-risk populations.
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In endemic areas in China, a community assignment trial with 10 years of follow up

demonstrated a 33% reduction in the cumulative ESCC related mortality in intervention
communities where 40–69 year-old adults were screened once by Lugol’s
chromoendoscopy, compared to communities without screening.11 Similar results were
found in other large retrospective studies of high-risk Chinese populations.12, 13 Modeling
studies have shown that population-based screening of high risk populations in China can be
cost effective.14, 15

In non-endemic areas, evidence supports endoscopic screening in patients with a history of

head-and-neck cancers. In a case-control study, Su et al. found a higher prevalence of
secondary ESCC in head-and-neck cancer patients receiving routine endoscopies compared
to the non-screening group ( 4.5% vs. 3.0%, p=0.04), with earlier stage at diagnosis
(p=0.03).16 Screening and early diagnosis correlated with improved survival, as Murakami
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et al. reported significantly superior 5-year survival in patients whose secondary ESCC was
diagnosed on endoscopic screening versus symptomatically (60.4% versus 0.0%, p <0.01).17

For another high-risk population-tylosis, limited data from a small case series has shown that
annual screening endoscopy detects early ESCC, with improved outcomes compared to
those diagnosed after onset of symptoms.18

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Given these findings, screening programs have been proposed for high-risk populations
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around the world. However, effective screening programs must be accurate, safe, cost-
effective, and facilitate curative therapeutic intervention.19 With the advent of endoscopic
treatment of esophageal squamous dysplasia and early stage ESCC, screening programs
have gained additional impetus.

Current recommendations on ESCC Screening (Table 1)

Given the low burden of disease in the general population of the United States, guidelines
from the American Gastroenterology Association do not advocate population based
screening for ESCC.20 However, screening may be worthwhile in endemic areas for
individuals over a certain age, and screening may also be indicated in certain high-risk
groups such as patients with head-and-neck cancers, tylosis, or history of caustic ingestions.
Although smoking and alcohol abuse have a dose-dependent association with ESCC,
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screening for ESCC is not routinely recommended unless other risk factors are present. A
recently published risk-prediction model from an endemic population in China incorporating
more than 10 risk variables was able to predict severe squamous dysplasia with an AUC
between 0.62–0.85, with age being the single most significant risk factor.21 Further
refinement of similar risk-prediction models is crucial to identify the target individuals for
screening in most populations. Currently proposed screening guidelines are summarized in
Table 1.

Technologies currently utilized and in development for ESCC/Squamous Dysplasia

screening
Endoscopic screening methods (Table 2)—Traditionally, endoscopy has been the
modality of choice for ESCC screening. However, endoscopy encompasses a heterogeneous
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spectrum of technology: their applications in ESCC screening have been summarized in

Table 2.

Lugol’s Chromoendoscopy: Although conventional white light endoscopy (WLE) can

reliably detect invasive cancer of the esophagus, it lacks sensitivity for detecting esophageal
squamous dysplasia, the precursor to ESCC and the main target of screening, which often
appears similar to normal squamous mucosa.22, 23 While high-resolution WLE has been
used in the detection of dysplasia in colonic polyps and Barrett’s esophagus, its role has not
been formally evaluated in the detection of squamous dysplasia. Rather, Lugol’s
chromoendoscopy is the current standard to highlight areas of abnormality, significantly
increasing the ability to detect esophageal squamous dysplasia.24–27

In Lugol’s chromoendoscopy, iodine binds reversibly to glycogen, which is less abundant in

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immature and rapidly dividing cells in the squamous epithelium, such as those found in
esophageal squamous dysplasia or esophagitis. Dysplastic and inflammatory esophageal
mucosa remain unstained compared to normal epithelium, allowing for targeted biopsies.28
Figure 3 shows a dysplastic lesion on WLE, NBI, and Lugol’s chromoendoscopy.

In a study of patients with head and neck cancer undergoing WLE followed by Lugol’s, the
detection of advanced cancer was similar between WLE and Lugol’s, but WLE was only

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able to detect 55% of esophageal squamous dysplasia identified with chromoendoscopy.25

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Chromoendoscopy has been found to have a sensitivity ranging from 92%–100% and
specificity of 37%–82% for detecting esophageal squamous dysplasia.27, 29–32 The main
reason for the wide range in specificity is that esophagitis can appear as an unstained lesion,
but this is readily discernable on histology.

Lugol’s chromoendoscopy is inexpensive and relatively easy to perform and interpret.

Lugol’s solution contains iodine, potassium iodide and water; it can be made on site in
developing countries. Therefore, Lugol’s chromoendoscopy is currently the standard of care
for ESCC screening. Adverse effects of Lugol’s solution are infrequent but include irritant
esophagitis/gastritis, hypersensitivity reactions, aspiration pneumonia, and thyrotoxicosis in
patients with endemic goiters.33, 34 Furthermore, the interpretation of abnormal findings
depends on the clinician administering the test, and interobserver variability has not been
adequately described for ESCC screening.
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Optical enhancement techniques: Narrow Band Imaging: By utilizing specific “narrow

band” wavelengths (415 nm and 540 nm) that match the absorption peaks of hemoglobin,
narrow band imaging (NBI) allows for clearer depiction of capillary networks and
superficial mucosal patterns.35 esophageal squamous dysplasia or early ESCC is
characterized by a “brownish” appearance with abnormal vascular patterns of intracapillary
papillary loops (IPCLs) (Figure 2). Detailed classifications of these abnormalities have been
proposed and validated in the identification and staging of dysplasia and early carcinoma.36

In a prospective study with 202 subjects with high-risk factors for ESCC, where each subject
sequentially underwent WLE, NBI, and chromoendoscopy, Nagami, et al., found that the
accuracy, sensitivity, and specificity of NBI for detecting invasive ESCC and high grade
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intraepithelial neoplasia were 77.0, 88.3, and 75.2 %, respectively; compared to

chromoendoscopy with 68.0, 94.2, and 64.0%, respectively.31 NBI was not statistically
inferior to chromoendoscopy in sensitivity (p=0.67); but significantly superior in specificity
and overall accuracy (p=0.01).31 A recent meta-analysis of 18 studies including more than
1,900 patients also found that NBI had a significantly improved specificity compared to
chromoendoscopy, with no significant difference in sensitivity.32

Fujifilm Blue Laser Imaging (BLI) (LASEREO; Fujifilm Co., Tokyo, Japan) utilizes two
lasers in addition to white light to sharpen image quality.37 A small retrospective study
showed that endoscopists preferred BLI for detection of ESCC38 but no objective evidence
exists in its application as a screening tool for ESCC. Furthermore, the interobserver
variability of narrow band imaging for esophageal pathology is not clear, but promising
results have been reported for upper aerodigestive tract lesions.39
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Post Processing Imaging technology (i-SCAN, FICE): Several software driven advances
have allowed for increased mucosal sharpness, clarity, and image processing in real-time.
The i-SCAN (Pentax, Tokyo, Japan) technology utilizes surface, contrast, and tone
enhancement.40 FICE (Fuji Intelligent Chromoendoscopy) (Fujinon, Fujifilm Medical Co,
[Saitama, Japan]) enhances image quality through the use of spectral estimation technology.
41

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Using Lugol’s and FICE, Li et al 44 examined 257 patients with suspicious esophageal
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lesions (27 ESCC, 22 HGIN, 40 LGIN, and 12 normal esophagus). Compared to Lugol’s,
which detected ESCC, HGIN, LGIN with a sensitivity of 88.9%, 77.3%, and 77.5%
respectively, FICE’s sensitivity was 92.6%, 86.4%, 87.5%, respectively. However, these
improvements were not statistically significant (p=0.64).

When combined with transnasal endoscopy (TNE) in a study of 99 patients with history of
head and neck cancer, FICE was found to be more sensitive than TNE with WLE alone for
detection of superficial ESCC (100% vs. 25%, respectively), with comparable specificities
(96.8% vs. 97.8%).42

Transnasal endoscopy: Ultrathin transnasal endoscopy (TNE) allows for visualization of

the esophagus without sedation, abrogating the risks of sedation and reducing costs. Huang,
et al., found TNE to be well-tolerated in over 98% of patients.43 Wang, et al., demonstrated a
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reasonable safety profile with only 2 of 441 patients undergoing TNE having complications
treated conservatively.44 When combined with FICE, Arantes, et al., observed sensitivities
and specificities of over 90% for detecting ESCC.45 Further study with this modality in the
identification of esophageal squamous dysplasia is required, but without the risks and costs
of sedation, TNE could become highly utilized in endemic areas.

Despite extensive data on patient acceptability, TNE is not widely used. This may reflect
lack of physician training or experience, and perhaps lack of patient and physician
preference for unsedated transnasal procedures.

Endocytoscopy: The ability to combine a microscope with a flexible endoscope was

introduced in the early 2000s.46 Endocytoscopy can diagnose dysplastic lesions without
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biopsy, reducing the wait for pathology and the costs for another procedure. The
endocytoscope (Olympus GIF-1T240, Olympus Medical Systems Corp. Tokyo, Japan) can
be passed through the instrument channel of a standard endoscope, where a soft hood at the
end of the endoscope allows for continuous contact with the mucosa (Figure 4).41 Methylene
or toluene blue spraying is required for staining to enhance visualization of dysplastic
cellular elements.

An early study of 75 patients by Inoue, et al., found a diagnostic accuracy of 82% for
distinguishing between malignant and non-malignant lesions of the esophagus.47 Kumagai,
et al., found that the need for histologic biopsy could be obviated in up to 93.5% of cases.48
More recently, Kumagai reported a sensitivity and specificity of 100% and 80%,
respectively, for the detection of ESCC using endocytoscopy.49 Further research in this field
includes the application of higher magnification endoscopes, in which preliminary results
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appear promising.50, 51

However, while endocytoscopy may obviate the need for biopsies, this approach is more
operator dependent, requiring experience to interpret the imaging and initiate appropriate
treatment. No studies on ESCC screening have reported the interobserver variability of this
technique. Furthermore, in order to highlight abnormalities, Lugol’s staining or NBI may be

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required, again increasing the costs. Further study is required to elucidate the role of
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endocystoscopy in the detection of esophageal squamous dysplasia.

High Resolution Microendoscopy: High resolution microendoscopy (HRME) utilizes a

contact probe containing a fiber optic microendoscope to visualize the esophageal
epithelium (Figure 5). Before insertion of the probe through the instrument channel of an
endoscope, the mucosal area of interest (usually identified by Lugol’s or NBI) is sprayed
with proflavine dye, which enables visualization of the superficial mucosal nuclei.52 Image
analysis software is then used to interpret the images and identify dysplastic cellular
features. This approach has been shown to be feasible for detection of ESCC, with
sensitivities ranging from 84% to 93% and specificities ranging from 92% to 97%.53, 54

HRME systems cost considerably less than other advanced screening platforms, making this
modality more attractive for developing nations. A screening trial of 147 patients in the
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United States and China showed that the addition of HRME to Lugol’s significantly
improved the specificity (88% vs 48%, p<0.001) and positive predictive value (45% vs 22%,
p<0.0001) for detecting severe esophageal squamous dysplasia and ESCC, compared to
Lugol’s alone. Fifty-five patients would have been spared from biopsy with HRME.51
Markov models incorporating both average and high risk populations in China have shown
that HRME is more cost-effective compared to no screening, but results were mixed when
compared to traditional screening methods.15

Image analysis advances that highlight abnormal nuclei or automatically calculate a score
for the degree of dysplasia present based on an algorithm of microscopic features may
improve accuracy by decreasing interobserver variation.55 Furthermore, portable tablet
systems have now been developed, potentially allowing for the use of HRME in remote
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regions.56

Video Capsule Endoscopy: Video capsule endoscopy has emerged as a non-invasive

method to image the gastrointestinal tract. Accordingly, there has been interest in using this
as a screening method for ESCC. However, an initial study comparing the use of video
capsule endoscopy to Lugol’s chromoendoscopy in a high-risk population of patients with
head and neck cancer found poor sensitivity and specificity in detecting neoplastic lesions in
the esophagus.57

Non-endoscopic Screening Methods

The community-assignment study by Wei et al shows that endoscopic screening with
appropriate treatment of esophageal squamous dysplasia and early ESCC can reduce ESCC
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mortality in a very high-risk population in China.11 However, widespread endoscopic

screening is not possible or cost-effective in most high-risk areas in Asia and Africa. Table 3
shows the esophageal cancer incidence rates in 2012 from high-quality population-based
cancer registries in ESCC endemic areas along with proportions of esophageal squamous
dysplasia and early ESCC diagnosed on screening endoscopies of asymptomatic adults in
three of these sites. The Wei study was done in Cixian, China, where ESCC incidence was
~150cases/100,000 population/year, and found HGD in 11 of every 100 endoscopies.58 In
such a site, primary endoscopic screening of the population may well be indicated and cost-

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effective. But in most high-risk populations, with ESCC incidence rates of ~20–40/100,000/
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year, only 1%–3% prevalence of HGD, and less developed health infrastructures, primary
endoscopic screening of the population will not be possible or cost-effective. To reduce
ESCC mortality in these populations, simple, inexpensive, and minimally-invasive tests need
to be developed that are acceptable to unsedated asymptomatic adults and can specifically
triage those with moderate to HGD to endoscopy for definitive diagnosis and treatment.
Table 4 and Table 5 summarize the characteristics and current status of non-endoscopic
screening methods for ESCC.

Esophageal cytology samples—Non-endoscopic esophageal sampling devices have

been studied extensively as inexpensive, tools for ESCC screening. The unsedated patient
swallows a brush, a deflated balloon, or a sponge in a gelatin capsule attached to a cannula
or string that is tethered outside the mouth. After it reaches the stomach, the balloon is
inflated or the gelatin capsule dissolves and the sponge expands, and then the cell collection
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device is pulled up the esophagus and out, collecting superficial squamous cells from the
esophagus. Traditionally, the recovered material is smeared on slides for Papanicolaou-
stained cytologic examination. Figure 6 illustrates some of the common balloons and
sponges used in China and Japan.

These methods are attractive as they can be performed without endoscopy or sedation, and
can be applied at scale in limited resource settings.59–61 Unfortunately, the results have been
less than optimal. A study of more than 700 patients comparing balloon cytology to
endoscopic biopsy reported sensitivities close to 40%.62 Similarly, cytology obtained with
sponges from a study of 439 Chinese patients also produced a disappointing sensitivity of
24% for esophageal squamous dysplasia and ESCC.63
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Esophageal cytology specimens combined with biomarkers—Combining

molecular biomarkers to esophageal cytology obtained non-endoscopically may improve the
sensitivity of the test. In a study of 147 Chinese patients with esophageal biopsies ranging
from normal to severe esophageal squamous dysplasia,89 a 4-marker panel of differentially
methylated promoter regions in AHRR, p16INK4a, MT1G, and CLDN3, assayed from cells
collected with an inflatable balloon, was able to detect HGD with a sensitivity and
specificity of 50% and 68%, respectively.

In another study, 301 Iranian adults were examined by a swallowed sponge (Cytosponge,
Medtronic, [Minneapolis, USA]), followed by Lugol’s chromoendoscopy. Combined
analyses of cytology and p53 staining showed a sensitivity of 100% at 97% specificity for
detecting four patients with HGD, although sensitivity was much lower for any grade of
dysplasia (64% by morphology, 11% by p53).64 Despite the small number of cases, this
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study shows promise for the Cytosponge as a safe and feasible tool for diagnosing
esophageal squamous dysplasia, and should be evaluated with other biomarkers in larger
studies.

Breath markers
Volatile organic compounds—The measurement of volatile organic compounds (VOCs)
from breath is completely non-invasive. Human breath is a complex biological sample

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containing more than 250 VOCs.65 The potential role of VOCs in detecting cancer was
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creatively demonstrated by Sonoda et al., who showed that canine olfaction of VOCs found
in exhaled breath and stool samples was accurate in discriminating patients with colorectal
cancer from normal controls (sensitivity 0.91 and specificity 0.99).66 Since this study, many
breath mass spectrometry devices have been developed to replace the canine nose.67
Although no studies specific to ESCC have been conducted, a pilot study from China on 29
patients with esophageal cancer and 57 healthy relatives reported an AUC of 0.943 with 7
ions analyzed on a home-made breath mass spectrometer—Ion Sniffer 2020Q (Hefei,
China).68 Since the study population was from China, where ESCC comprises over 90% of
esophageal cancer cases, one could assume that most of the esophageal cancer cases were
ESCC. However, these results should be interpreted with caution given the small sample size
and lack of blinding.

Blood Markers
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Autoantibodies—Auto-antibodies to tumor associated antigens (TAAs) have been

proposed as biomarkers for cancer due to their stability in serum. The most comprehensively
studied TAA is P53, in which a mutation results in a non-functioning protein that has a
longer half-life than the native protein, and the subsequent anti-P53 can be detected non-
invasively in serum. However, a meta-analysis of 15 studies reported a disappointing
sensitivity range of 15%–60% at specificity 91–100% for anti-P53 as a single marker.69 A 6-
marker panel of auto-antibodies in 237 late-stage ESCCs versus 134 normal did not do much
better, with a sensitivity of 45% at a specificity of 95%.70 Evidence is even more limited for
esophageal squamous dysplasia. Therefore, autoantibodies for screening purposes of
esophageal cancer are still far from clinical application.

Circulating tumor cells—Circulating tumor cells (CTCs) assayed from blood have been
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investigated for their prognostic use in esophageal cancer. The presence of CTCs has been
found to correlate strongly with nodal metastases, and poor progression-free survival in
patients with esophageal cancer.71–73 Since the presence of CTCs portends advanced
disease, CTCs would not be an appropriate screening test for early cancer.

Circulating miRNA—MicroRNAs (miRNAs) are small noncoding RNAs that bind to

target messenger RNAs (mRNAs) resulting in RNA degradation and/or post-translational
inhibition. MiRNAs are stable, abundantly expressed, and can be consistently detected in
blood. In a meta-analysis of 27 studies, the overall sensitivity and specificity of miRNAs for
detecting ESCC were 79.9% (95% CI: 76.2%–83.1%) and81.3% (95% CI: 75.7–85.9),
respectively, with an AUC of 0.87 (95% CI: 0.84–0.90).74 However, only four of the 19
miRNAs were studied in more than one of the publications. Other meta-analyses were
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limited by small numbers of studies and lack of validation.75 Nevertheless, the concept of
miRNAs does hold potential as a non-invasive screening tool for ESCC, and further studies
are underway.76–78

Methylated DNA markers

Oncogenesis by way of modifications in DNA methylation occurs via two fundamental
changes: (1) hypermethylation of CpG islands in gene promoters, which can silence tumor

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suppressor genes; and (2) hypomethylation of repetitive genetic elements, which may lead to
genomic instability or oncogene activation.79 Methylated DNA markers (MDMs) have been
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shown to be broadly informative markers of neoplasia82 and are a critical component of a

multi-target stool DNA test, FDA-approved for average risk colorectal cancer screening.83

MDMs have been utilized to develop minimally invasive diagnostic tools for BE and BE
related dysplasia. The identification, validation and pilot testing of MDMs for BE diagnosis
has been recently described. Following agnostic discovery by reduced representation
bisulfite sampling (RRBS) and validation in independent biopsy and whole esophageal
brushing cohorts84, the utility of these markers was confirmed in a pilot randomized trial. In
19 BE cases and 20 controls, esophageal cytology specimens obtained via a 25 mm capsule
sponge device (Sponge On a String (SOS) (EsophaCap) (Capnostics, New Jersey, USA),
coupled with a 2 marker MDM panel detected all 19 cases of BE (100% sensitivity at 100%
specificity).85 In a subsequent ongoing validation study (SOS 2 trial) including 90 BE and
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58 controls, a two-marker MDM panel assayed from SOS samples accurately distinguished
BE cases from controls with an AUC of 0.97 (95% CI 0.94–0.99).86 A three marker panel of
MDMs for the detection of BE related dysplasia has also been previously described.87
MDMs have also been applied to the Cytosponge as mentioned previously, with AUCs as
high as 0.877 (95% CI 0.84–0.91) in a pilot study with 20 BE cases and 10 normal controls,
and a validation cohort with 149 BE cases and 129 normal controls.88 In samples from non-
endoscopic balloons from 86 individuals, tests of CCNA1 and VIM DNA methylation
detected BE metaplasia with 90.3% sensitivity and 91.7% specificity in another recently
published study.84

Along these same lines, MDMs for ESCC and squamous dysplasia, partnered with a device
such as SOS, can lead to the development of a cost-effective, minimally-invasive, and highly
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accurate way to screen for this deadly disease. In a prior discovery study, we reported
MDMs with high discrimination for the detection of ESCC.89 The 15 best-performing
MDMs were then chosen for a multi-national validation study with tissue from 86 ESCC
cases from the US, 114 from Iran, and 133 from China, with similar numbers of country-
specific normal controls. Although the subjects came from three distinct populations with
varying risks of ESCC, our candidate MDMs performed well at discriminating ESCC tissue
from normal esophageal tissue across all sites. Using a marker panel of 2–4 MDMs, the
AUCs were 0.997 (95% CI 0.99–1.0), 0.99 (95% CI 0.98–1.0), and 0.97 (95% CI 0.93–
0.99), for US, Iran, and China respectively, corresponding to sensitivities and specificities
>95%.90 These markers need to be tested on esophageal cytology specimens obtained via a
capsule sponge sampling device. A report from China applied MDMs to esophageal balloon
cytology samples from 147 patients with endoscopic biopsy diagnoses ranging from normal
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to severe squamous dysplasia in an endemic area, and a 4-marker panel (AHRR, p16INK4a,
MT1G, and CLDN3) was able to detect severe squamous dysplasia with a sensitivity and
specificity of 50% and 68%.58

Not only can ESCC-specific MDMs be detected in esophageal tissue, we have also
demonstrated its feasibility as a plasma-based biomarker for esophageal cancer in a recent
pilot study (ref DDW abstract).91 Using MDMs discovered through the aforementioned
whole methylome discovery effort, 12 candidate MDMs were chosen for a Phase I study

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with 85 cases (76 EAC and 9 ESCC) and 98 controls. At a specificity of 91%, a five-marker
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panel assayed from plasma detected 74% of esophageal cancer overall (74% of EAC, and
78% of ESCC), with an overall AUC of 0.93 (95% CI 0.89–0.96). The test was more
sensitive for higher-stage disease, but was still able to detect Stage I and Stage II disease at
sensitivities of 56% and 83%, respectively. Although only 9 cases of ESCC were included in
this study, this preliminary research has established a potential for using MDMs for
detection of ESCC in an entirely non-invasive way. With ongoing larger-studies and
optimized assays, active research in this area may transform the future of screening for
ESCC especially in resource-poor endemic areas.

Future Directions
ESCC is a lethal disease largely due to its usually advanced stage at diagnosis. Screening
programs targeting high-risk populations are needed to detect curable dysplasia and early
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stage ESCC. Critical advances have been made in high quality endoscopic screening
modalities. However, the majority of patients afflicted by ESCC are found in resource-poor
regions. For screening to occur successfully at a population level in these regions,
minimally-invasive tools that are accurate and cost effective need to be developed.

The use of molecular biomarkers in esophageal cell samples acquired non-endoscopically

(via a sampling device such as a sponge) or in blood holds great potential in the future for
screening in populations at high risk for ESCC. However, the performance of such
biomarkers is yet to be optimized, and results need to be validated in large studies.
Additionally, further risk-stratification of individuals needs to be achieved in areas with
lower incidence of ESCC and esophageal squamous dysplasia. Such risk-stratification
models combined with a minimally-invasive test could transform screening and reduce
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mortality from ESCC.

Acknowledgments
Funding: None

Acronyms:
ESCC esophageal squamous cell carcinoma

HGD high grade dysplasia

EAC esophageal adenocarcinoma

BE Barrett’s esophagus
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NBI narrow band imaging

TNE transnasal endoscopy

WLE white light endoscopy

FICE Fuji Intelligent Chromoendoscopy

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BLI Blue Laser Imaging

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HRME High Resolution Microendoscopy

HGIN High Grade Intraepithelial Neoplasia

LGIN Low Grade Intraepithelial Neoplasia

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Figure 1:
The Asian and African esophageal cancer belts (in dark blue), where over 90% of cases are
esophageal squamous cell carcinoma.
(Reprinted with permission from Ferlay J SI, et al. GLOBOCAN 2012 v1.0, [Internet].
Lyon, France: International Agency for Research on Cancer, 2013.)
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 Codipilly et al. Page 18
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Figure 2:
Original intrapapillary capillary loop (IPCL) pattern classification.
The IPCL pattern classification includes two sets of diagnostic criteria. IPCL pattern
classification from IPCL type I to type V-1 is used for the tissue characterization of flat
lesions (red outline). IPCL pattern classification from IPCL type V-1 to type VN reflects
cancer infiltration depth (blue outline). IPCL type III corresponds to borderline lesions
which potentially include esophagitis or low-grade intraepithelial neoplasia. IPCL type III
should be considered for endoscopic follow up. In IPCL type IV, high-grade intraepithelial
neoplasia appears, and then further treatment with endoscopic mucosal resection (EMR) /
endoscopic submucosal dissection is recommended. EMR/esophageal squamous dysplasia
for IPCL types V-1 and V-2 should be also considered as they are definite M1 or M2 lesion
with no risk of lymph node metastasis. IPCL type V3 corresponds to an M3 lesion and
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diagnostic EMR/esophageal squamous dysplasia should be applied as a “complete biopsy”

to decide on a final treatment strategy. IPCL type VN corresponds to a “new tumor vessel”
often associated with sm2 invasion with significantly increased risk of lymph node
metastasis. Surgical treatment should be recommended.
(Reprinted with permission from Inoue H, et al. Ann Gastroenterol. 2015 Jan-Mar; 28(1):
41–48.)

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Figure 3:
Endoscopic images of the same lesion of esophageal squamous dysplasia, with WLE, NBI,
Magnifying NBI, and Lugol’s chromoendoscopy.
a. | Endoscopic finding with conventional white light imaging. A flat reddish lesion was seen
in the upper thoracic esophagus (between the arrows). At the edge of the lesion, the vascular
pattern in the surrounding mucosa was interrupted.
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b. | Endoscopic finding with narrow band imaging of the lesion from panel a. The lesion was
contrasted as darker area (arrow) and the boundary of the lesion is clearer than in white light
images.
c. | Endoscopic finding with magnifying narrow-band imaging. Dilated irregular
microvascular pattern was clearly visualized in the lesion. A distinct demarcation line
between the background mucosa and the lesion is visible (arrow). Note the color of the
epithelium in the lesion is browner than the surrounding mucosa. Accordingly, endoscopic
diagnosis of intramucosal squamous cell carcinoma or high-grade dysplasia can be made
with high confidence.
d. | Endoscopic finding using Lugol’s chromoendoscopy for the lesion depicted in panels a
and b. The lesion is clearly visualized as a lighter (yellow) unstained area within the brown
stained area. The boundary of the lesion can be clearly delineated with chromoendoscopy.
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(Reprinted with permission from Veitch AM, et al. Nat Rev Gastroenterol & Hepatol. 2015)
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Figure 4:
Microendoscopic imaging of squamous dysplasia.
a. | Endocytoscope probe which can be passed through the instrument channel of an
endoscope.
b. | The border between esophageal squamous cancer (upper) and normal squamous
epithelium (lower). The density and nucleus:cytoplasm ratio is much higher in cancer
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compared to normal epithelium.

(Reprinted with permission from Kumagai Y, et al. Endoscopy 2004;36:590–594.)

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Figure 5:
High-resolution microendoscope92
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Lugol’s unstained lesions (left) are imaged with HRME (“optical” biopsy) and compared
with corresponding tissue biopsy (histopathologic biopsy) (original magnification, 100x).
Only 1 of the 2 Lugol’s abnormal areas was neoplastic (upper panel) as determined by the
imaging software, based on mean nuclear area, nuclear-to cytoplasmic ratio, nearest inter-
nuclear distance, nuclear eccentricity, nuclear solidity, and the major axis of the ellipse best
approximating each nucleus.
(Reprinted with permission from Protano et al. Gastroenterology 2015; 149: 321–329.)
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Figure 6:
Balloons and sponges used in prior studies for screening of esophageal squamous cell
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carcinoma and/or dysplasia.

(Reprinted with permission from Pan QJ, et al. Acta Cytol 2008;52:14–23.)
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Table 1:

Recommended Screening Guidelines for High Risk Populations

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Level of
Risk factor Screening method and duration Outcome
evidence^20
Endoscopy with Lugol’s or NBI every 6 months to • Detects earlier stage disease II-III (moderate)
Head and neck
1 year after completion of therapy for HNSCC, for • Improved survival
cancer
10 years • No evidence for cost-effectiveness,

4 quadrant biopsies from proximal, middle, and • Effective for early diagnosis III-IV (low)
Tylosis distal esophagus starting at age 30; repeat every 1– • Only beneficial for Type A (late onset)
3 years Tylosis

• No evidence for cost effectiveness III (low)

Yearly EGD 10–15 years after disease onset +/−
Achalasia • Need to screen many patients to detect
Lugol’s solution
one cancer

Asian or African II-III (moderate)

One time Lugol’s chromoendoscopy beginning at • Screened groups have lower ESCC
high-risk
the age of 40 incidence and mortality rates
populations

History of caustic Endoscopy every 2–3 years 10–20 years following IV (low)
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• No evidence for effectiveness

esophageal injury the injury

^
Levels of evidence: Level I evidence: presence of at least one prospective, randomized, controlled trial, level II evidence: well-designed cohort or
case-controlled studies; level III evidence: case series or flawed clinical trials; level IV evidence: opinions of respected authorities or expert
committees; level V evidence: insufficient evidence to form any opinions
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Table 2:

Pros and Cons of Various Endoscopic Screening Methods

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PIVI criteria
Endoscopic screening for Squamous
Pros Cons
modalities Dysplasia
Reached?
• Uncomfortable if not done with sedation (which is •
frequently not available in resource poor areas)
All modalities •Visualizes the mucosa
• Requires operator training and experience
• Expensive equipment

Conventional white • Readily available in developed • No

• Lacks sensitivity for precursor lesions
light endoscopy areas

• Inexpensive • Yes
• Improves sensitivity for • Irritant/allergic reactions to iodine
Chromoendoscopy precursor lesions/dysplasia • Lower specificity for precursor lesions (before biopsy
• Short learning curve diagnosis)
• Clarity of lesion borders

• Improves sensitivity and • Yes

• Increased cost of equipment
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Endoscopy with specificity for precursor lesions/

• Longer learning curve
Narrow Band Imaging dysplasia
• Requires more operator expertise
• Does not require iodine

• Currently not standard of care, not widely available • Yes (only if

• Lack of trained operators combined with
• Cannot be combined with Lugol’s or NBI to detect FICE)
• No need for sedation precursor lesions.
Transnasal endoscopy
• Improves cost effectiveness • Not yet tested on precursor lesions
• Smaller biopsies
• No therapeutic capabilities
• Increased risk for patients with ENT pathology

• A contact probe technology • Yes

• Small field of view
• Cellular level resolution may • Can only be used with other lesion localization
Endocytoscopy obviate need for some or all technologies (Lugol’s or NBI)
biopsies • Increased cost of equipment
• Additional training needed
• Not yet tested on precursor lesions

• Inexpensive and portable • Yes

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• Cellular level resolution may • A contact probe technology

obviate need for some or all • Small field of view
biopsies • Can only be used with other lesion localization
Microendoscopy • Can be portable and added to technologies (Lugol’s or NBI)
standard endoscope • Additional training needed
• High sensitivity and specificity • Variable cost-effectiveness when compared to standard
for dysplasia and early stage endoscopic screening techniques
cancer.
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Table 3:

Esophageal Cancer Incidence and the Prevalence of High-Grade Dysplasia in Endoscopic Screening Studies
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from Selected Sites in Asia and Africa11, 28, 64, 93

EC incidence HGD Prevalence

Males Females

Cixian, China 193 109 11%

Yanting, China 101 68

Golestan, Iran 23 19 1%

Nairobi, Kenya 21 15 3%

Blantyre, Malawi 38 23

Eastern Cape Province, RSA 32 20

*
Esophageal cancer incidence/100,000 population/year from population-based cancer registries in Cancer Incidence in Five Continents, Vol X,
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IARC;
§
Wei et al11;
ɣ
Roshandel et al68;
Ʃ
Mwachiro et al.28
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Table 4:

Non-endoscopic screening methods and characteristics

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Screening Study Dysplasia and

Author (year) Assay method Sensitivity Specificity
Method Size (N) ESCC (N)
Direct Sampling Techniques

123 (28%)
47% esophageal 81% esophageal
esophageal
Inflatable Balloon Roth (1997)63 439
squamous dysplasia
Cytology only squamous squamous
dysplasia+ dysplasia+
16 (4%) ESCC

232 (32%)
46% esophageal 84% esophageal
esophageal
Inflatable Balloon Pan (2008) 62 725
squamous dysplasia
Cytology only squamous squamous
dysplasia+ dysplasia+
+

232 (32%)
39% esophageal 85% esophageal
Mechanical esophageal
Balloon Pan (2008)62 725
squamous dysplasia
Cytology only squamous squamous
dysplasia+ dysplasia+
+
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123 (28%)
24% esophageal 92% esophageal
esophageal
Sponge Roth (1997)63 439
squamous dysplasia
Cytology only squamous squamous
dysplasia+ dysplasia+
16 (4%) ESCC

72 esophageal
squamous dysplasia Cytology + Panel of
Adams Methylated DNA
Inflatable Balloon 147 (25 mild, 26 50% HGD+ 28% HGD+
(2008)58 moderate, 21 markers*
severe)

22% esophageal
Roshandel 18 esophageal
squamous
Sponge 301 squamous dysplasia, Cytology + p53 97% HGD+
(2014)64 (4 with HGD)
dysplasia+_
100% HGD+

Breath Markers
7 ions analyzed on a
home-made breath
29 esophageal 89.5% for
Volatile Organic mass spectrometer 86.2% for
Compounds Zou (2016)68 86 cancers (not
—PTR-MS Esophageal cancer
Esophageal
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specified) cancer
apparatus (Ion
Sniffer 2020Q).

Blood Markers
64% (for ESCC)
65 esophageal 38% for
Autoantibodies Zhou (2014)94 567 squamous dysplasia 6 marker panel^ esophageal 94% (for ESCC)
88 ESCC squamous
dysplasia+

Autoantibodies Xu (2014)71 513 237 ESCC 6 marker panel~ 45% for ESCC 95% for ESCC

miRNA Zhang 201095 430 290 ESCC 7 miRNA panel# 80% for ESCC 90% for ESCC

Survivin mRNA
Circulating tumor expressing
cells and mRNA Cao (2009)96 182 108 ESCC
circulating tumor
47.2% for ESCC 100% for ESCC
cells
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*
AHRR, p16INK4a, MT1G, and CLDN3
^
P53, IMP1, P16, cyclin B1, P62, and C-myc
~
p53, NY-ESO-1, MMP-7, Hsp70, Prx VI, and BMI-1

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 Codipilly et al. Page 27

#
miR-10a, miR-22, miR-100, miR-148b, miR-223, miR-133a, and miR-127–3p
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Table 5:

Pros and Cons of Non-endoscopic Screening Methods

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Pros Cons
• Minimally invasive direct tissue sampling
Esophageal mucosal sampling • Cytology-only yields low sensitivity
•Can be combined with any molecular test
devices (brushes, balloons, • Devices are hard to swallow and may lead to poor
• Low operating cost
sponges) compliance
• High specificity

• Non-invasive (serum)
Auto-antibodies (blood) • Low sensitivity
• Stable assay

• Non-invasive
• Low sensitivity
Circulating tumor cells (blood) • High specificity
• Not found in early disease
• Prognostic value

• Non-invasive (detected from plasma or serum)

Circulating miRNA (blood • Stable and consistently expressed •Limited number of studies
• High sensitivity and specificity

Methylated DNA (cell sample or • Can be non-invasively detected in plasma

• Limited number of studies
blood) • Stable and consistently expressed
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Volatile Organic Compounds

• Non-invasive • Limited number of studies
(breath)
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