Study on the Relationship Between Different Pathological Features of Achalasia and Prognosis of Per-oral Endoscopic Myotomy
Conditions: Esophageal Achalasia
Sponsor: Fandong Meng

Study Protocol and Statistical
Analysis Plan
Study on the Relationship Between Pathological
Features of Achalasia and Prognosis of Per-oral
Endoscopic Myotomy
Main research institute: Beijing Friendship Hospital,
Capital Medical University
Study Director: Fandong Meng,
Date: April 1st, 2021

Directory
1. Background ……………………………………………3
2. Research objectives ……………………………………4
3. Research content ………………………………………4
4. Research proposal ……………………………………4
5. Data processing and analysis ………………………9
6. Quality control …………………………………………9
7. References ………………………………………………9

Background
Achalasia is the most common motility disorder of esophagus,
characterized by disorders of the lower esophageal sphincter (LES).
Normal peristalsis of the esophagus is eliminated and replaced by
synchronous
or
ineffective
contraction.
Based
on
high-resolution
manometry (HRM), the patients with achalasia were categorized into 3
subtypes, type I: achalasia with minimum esophageal pressurization,
type II: achalasia with esophageal compression and type III: achalasia
with spasm.
Previous studies have found that the pathological features of the
esophageal muscular layers in patients with achalasia are degeneration
of nerve plexus, reduction of interstitial cells of Cajal (ICCs), muscular
atrophy, fibrosis and infiltration of different inflammatory cells. Different
subtypes
of
achalasia
have
different
clinical
characteristics
and
esophageal motility. Studies have found that ganglion cells in type I
patients are significantly reduced compared with type II patients, but the
pattern is similar, which may indicate that type I achalasia represents the
progression of type II achalasia. Studies have shown that ICCs are
preserved in type III achalasia compared with type I and II achalasia. In
addition, Achalasia is related to viral infection and autoimmune disease.
Nowadays, per-oral endoscopic myotomy (POEM) is a main therapy for
patients with achalasia. Most studies have focused on the relationship

between pathological features and motility characteristics of achalasia,
but there are few studies on the relationship between pathological
features and therapeutic effect of POEM.
This study will evaluate the pathological characteristics of full-thickness
esophageal muscle in patients with achalasia in order to discover the
relationship
between
different
pathological
characteristics
and
prognosis of POEM.
Research objective
To determine whether there is difference in prognosis of POEM
among patients with achalasia of different pathological characteristics.
Research content
This study prospectively collects the information of 50 participants
undergoing POEM for achalasia in Beijing Friendship Hospital, Capital
Medical University. During POEM, all patients are required to obtain
esophageal muscle biopsy for pathological examination. Participants will
be followed up by telephone at 1, 3, 6, and 12 months after POEM for
improvement in clinical symptoms.
Research proposal
1. Research design
This is a prospective study based on Beijing Friendship Hospital
affiliated
to
Capital
Medical
University.
The
data
of
50
patients
undergoing
POEM
due
to
achalasia
were
prospectively
collected,

including demographic data, drug and surgical treatment data during
hospitalization.At POEM, all patients are required to obtain esophageal
muscle
biopsy
and
mucosal
tissue
samples
for
pathological
examination.The patients were followed up by telephone at 1, 3, 6, and
12 months for improvement in clinical symptoms.
2. The research object
2.1 Study Population
The subjects of this study were patients undergoing concurrent
POEM surgery for achalasia in Beijing Friendship Hospital affiliated to
Capital Medical University.
2.2
Number of planned enrollment cases
50 cases.
2.3
Into the discharge standard
Inclusion criteria:
1) Patients with achalasia treated in Beijing Friendship Hospital, Capital
Medical University
2) Age 18-80, no gender limitation
3) Fit and agree to receive POEM therapy
4) Signed informed consent
Exclusion criteria:
1)
the
previously
received
treatment
of
achalasia,
Barrett's
esophagus
lesions,
esophageal
stricture,
liver
cirrhosis,
and/or

esophageal
varices,
the
digestive
tract
tumor,
active
esophagitis,
connective tissue disease, allergic disease, blood or blood coagulation
patients and hiatal hernia.
2) Use of nonsteroidal anti-inflammatory drugs, corticosteroids, or
other immunosuppressive drugs 6 months prior to examination.
3. Research methods
3.1 Diagnosis and classification of achalasia
According
to
clinical
manifestations,
endoscopy
and
high-resolution esophageal manometry, achalasia was diagnosed.
3.1.1 Clinical symptom score
The Eckardt score was used to score clinical symptoms based on
preoperative dysphagia, regurgitation, retrosternal pain, and reduced
body mass. Doctors need to collect information about the patient's
history of viral infections and autoimmunity, such as herpes simplex virus
(HSV),
varicella
zoster,
measles,
and
human
papillomavirus
(HPV).
Autoimmune diseases include urticaria, eczema, ankylosing spondylitis,
Hashimoto's thyroiditis, rheumatoid arthritis and sjogren's syndrome.
3.1.2 Endoscopy
All patients underwent endoscopy to exclude tumors, inflammatory
reactions or large ulcers in the esophagus and stomach, especially in the
lower esophagus or the gastroesophageal junction.
3.1.3 High-resolution manometry

The pressure measurement included resting pressure data, 10
times of swallowing with 5ml water in supine position, and fast
swallowing data. The data includes UES, LES, and esophageal body
pressure measurement data. Achalasia is divided into three subtypes.
3.2 POEM and muscular pathology
Patients
received
routine
POEM
surgery,
and
full-thickness
esophageal muscle biopsy was performed from LES, distal esophagus
(about 5cm above EGJ) and middle esophagus (about 10-20cm above
EGJ) during the operation. Intraoperative biopsy to take pathological
tissue depends on the patient's condition, and the collection method is
to use biopsy forceps to take tissue, and take 1 piece at one place.
3.3 Pathological staining and evaluation
Tissue samples were fixed with formalin and embedded in paraffin
before sectioning and staining.
3.3.1 Specific dyeing items
1) HE staining is used to evaluate the atrophy degree of smooth
muscle and the number of inflammatory cells.
2) Azan-Mallory staining was used to assess the degree of fibrosis.
3) C-KIT immunohistochemistry: C-KIT was positive, and trypsin-like
IHC staining was negative to evaluate ICC cells.
4) S100: Evaluate the degree of ganglion loss
5) The number of eosinophil cells was evaluated by HE staining, MBP

and EDN
6) Mast cells: C-kit positive, trypsin-like IHC staining positive.
7) Lymphocyte inflammation.
3.4 Follow-up contents
Follow-up data were collected, including symptom relief (Eckardt
score), reflux symptom score (GerdQ score), gastroscopic manifestations,
reflux
esophagitis,
esophagography
and
esophageal
manometry.
Follow-up collection time is 1 month, 3 months, 6 months and 12
months after POEM. Patients' symptom improvement and postoperative
reflux will be inquired.
4. Clinical outcomes
4.1 Main clinical outcomes
The primary end point event of patients is Eckardt score in 1 month,
3 months, 6 months and 12 months after POEM. Any Eckardt score >3
points are defined as poor prognosis after POEM.
4.2 Secondary clinical outcomes
Secondary end points were the presence of reflux symptoms (GerdQ
score >7) or evidence of reflux esophagitis under endoscopy during
follow-up. In addition, we will evaluate pathological differences between
different sites to provide evidence for selecting the best biopsy site.
Data processing and analysis
Measurement data are expressed as x±s or median, as appropriate,

and count data rate and percentage are expressed. The chi-square test or
Fisher's exact test was used to analyze the count data, and the Student's
T test or Mann-Whitney test was used to analyze the qualitative and
quantitative differences among the subgroups. All P values were bilateral,
and P <0.05 was considered as statistically significant difference.
Quality control
In this study, two doctors will participate in patient enrollment, input
of baseline data and follow-up data, and jointly check the accuracy and
authenticity of data.
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