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Histology of Symptomatic GastroesophagealReflux Disease
Is It Predictive of Response to Proton Pump Inhibitors?
Hiroto Miwa, Kaiyo Takubo, Tomohiko Shimatani, Takahisa Furuta, Tadayuki Oshima, Junji
Tanaka, Junko Aida, Masanori Ito, Susumu Kurosawa, Takashi Joh, Tsuneya Wada, Yasuki
Habu, Yusuke Watanabe, Michio Hongo, Tsutomu Chiba, Yoshikazu Kinoshita
J Gastroenterol Hepatol. 2013;28(3):479-487.
Abstract and Introduction
Abstract
Background and Aim: To examine the differences in esophageal histopathology between
non-erosive reflux disease (NERD) and reflux esophagitis (RE), and to investigate whether
baseline esophageal histopathology can predict the therapeutic response to proton pump
inhibitors (PPIs).
Method: The subjects comprised 94 patients with NERD (n = 71) or mild RE (n = 23). Tissuewas biopsied from 5 cm above the squamo-columnar junction (SCJ), and the degree or
presence of nine histopathological markers was assessed. The patients were treated with
rabeprazole (RPZ) 10 mg once daily for 4 weeks. If complete heartburn relief was not
achieved, RPZ was increased to 10 mg twice daily for another 2 weeks, and then to 20 mg
twice daily for another 2 weeks if heartburn remained.
Results: Features of esophageal histopathology 5 cm above the SCJ differed between NERD
and RE patients. The esophageal histopathology in patients unresponsive to RPZ was
characterized by Protein Gene Product (PGP) 9.5 negativity in those with NERD, and
intraepithelial bleeding in those with RE. In addition, the combination of dilated intercellular
spaces (DIS) (+)/PGP 9.5 () was indicative of strong resistance to PPI therapy in NERDpatients.
Conclusion: The therapeutic efficacy of PPI can be predicted from the features of biopsied
esophageal tissue. Factors predictive of resistance to treatment with PPI are negativity for
PGP 9.5 in NERD patients and intraepithelial bleeding in RE patients.
Introduction
Patients with gastroesophageal reflux disease (GERD) have been increasing in number in
recent years.[1] GERD is commonly treated with proton pump inhibitors (PPIs), but a high
proportion of patients show no response.[2,3] Cases of GERD in which the symptoms cannotbe relieved by PPI therapy are an important medical concern. Because about 20% of patients
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with reflux esophagitis (RE) and about 50% of patients with non-erosive reflux disease
(NERD) do not respond to PPI therapy,[4] it is desirable to obtain information on whether PPI
therapy would be effective or ineffective in individual patients before the start of therapy,
especially for those with NERD. In other words, it would be desirable to identify non-
responders to PPI before starting the therapy. Nocturnal gastric acid breakthrough (NAB),[5]
gene polymorphism of CYP2C19 (extensive metabolizer),[6]
esophageal reflux of duodenalfluid,[2] female gender, and poor treatment compliance have been noted as factors associated
with resistance to PPI therapy. However, no previous study has investigated whether
histopathological findings in the esophagus are predictive of resistance to PPI treatment.
It has been reported that GERD patients show changes in various esophageal
histopathological parameters,[79] and in recent years, consensus guidelines for histological
recognition of microscopically evident esophagitis in patients with GERD have been
formulated.[10] These parameters include an increased eosinophil count, intrapapillary blood
vessel dilatation, intraepithelial bleeding, papillary extension, basal cell hyperplasia
(thickening), dilated intercellular spaces (DIS), enhanced cellular proliferation, appearance of
Langerhans cells, and hyperplasia of nerve fibers. However, no study has compareddifferences in esophageal histopathological findings between patients with NERD and those
with RE. We therefore examined differences in esophageal histopathology between these
patient groups, and investigated whether the pathological features of esophageal biopsy
samples obtained before the start of treatment could predict the therapeutic efficacy of PPI.
This investigation was conducted as part of the TORNADO (Treatment with high dose Of
Rabeprazole for NERD patients conducted by AciD-related symptOm research group)
study.[11]
Methods
Patient Enrollment
This was a multi-center, cooperative open study in Japan belonging to the Acid-Related
Symptom (ARS) Research Group. The study was started after approval from the Ethics
Committee of each medical institution, and conducted in compliance with the Declaration of
Helsinki.
Outpatients with GERD who satisfied the following conditions were included in this study:
those who were 20 years of age or older, those who gave written informed consent to
participate in this study of their own free will, those who agreed to undergo esophagealbiopsy, and those who had suffered heartburn repeatedly at least once a week during a period
of one month before the study. Heartburn was defined as a "burning sensation" welling up
from the stomach or lower thoracic region that tended to occur after meals, or when in a bent-
over position or upon abdominal compression. The Modified Los Angeles Classification[12,13]
was used for diagnosis by esophageal endoscopic examination. Only patients with grade N or
M NERD and only patients with grade A or B RE were included in the study.
Since the following conditions may affect evaluation of the therapeutic effect of PPIs, patients
to whom these conditions applied were excluded: those who had been definitively diagnosed
as having RE by endoscopic examination in the past, those who had used a PPI within one
month previously, those who had a history of upper digestive tract surgery, those who had anactive gastric ulcer or duodenal ulcer, those who had Barrett's esophagus (long-segment
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Barrett's esophagus: LSBE), those who had a history or presence of angina pectoris, those
who had malignant disease, serious hepatic disease, or serious renal disease, and other
patients judged inappropriate for the study by the investigators.
Study Protocol
After acquisition of informed consent, biopsy samples were taken from the esophageal
mucosa using an endoscope. In the "Treatment Period" at the beginning of the study, a 10-mg
rabeprazole (RPZ) tablet was administered orally once daily for 4 weeks. Changes in the
symptoms of heartburn over time were recorded using the following four grades in a self-
administered patient diary every day. No symptoms, bothersome but tolerable (mild),
bothersome (moderate), very bothersome (severe).
Patients having complete heartburn relief (defined as no heartburn during the previous 7 days)
were defined as "Responders to 4-week treatment," and completed the study drug
administration at Week 4. Patients not having complete heartburn relief at the end of the
Treatment Period were defined as "Non responders to 4-week treatment" and the dose of RPZwas increased. Such patients received oral administration of a RPZ 10-mg tablet twice daily
for 2 weeks in "Continuous Treatment Period I." Patients who had complete heartburn relief
at the end of Continuous Treatment Period I (after successive 6-week administration)
completed the study drug administration. Patients who did not have complete heartburn relief
at the end of Continuous Treatment Period I received a further increased dose of RPZ orally
with a RPZ 20-mg tablet twice daily for another 2 weeks (successive 8-week administration)
in "Continuous Treatment Period II." Patients who were completely relieved of heartburn
after the 4-week, 6-week, or 8-week treatment were defined as "Cumulative responders to 8-
week treatment," while patients who were not completely relieved after the 8-week treatment
were defined as "Cumulative non-responders to 8-week treatment."
During the period of RPZ administration, concomitant use of the following drugs and therapy
was prohibited: other PPIs, H2 receptor antagonists, M3 receptor antagonists, prokinetics, anti-
cholinergic agents, antacids, anti-gastrin agents, prostaglandin preparations, mucosal
protective agents, sodium alginate, andHelicobacter pylori eradication therapy.
In investigation 1, one endoscopic biopsy specimen was obtained from each patient before
treatment. The features of nine histopathological and immunohistopathologically stained
sections () from each NERD patient were compared with those from RE patients. PGP 9.5 is a
protein belonging to the ubiquitin C-terminal hydrolase proteins and highly expressed in
vertebral neurons and nerve fibers.
[14]
Free ending nerves have been described in interepithelial spaces and probably mediated esophageal pain and dysphagia in patients GERD.[15]
To estimate increased number of nerve fibers in patients with GERD, we stained
immunohistochemical biopsy specimens with antibody for PGP 9.5. In Investigation 2, the
therapeutic effects of RPZ (rate of complete heartburn relief after the 4-week treatment and
rate of cumulative relief after the 8-week treatment) in NERD patients and RE patients were
calculated and aggregated. Then, the aggregated data and findings for the nine different
histopathological and immunohistopathological sections were compared between
"Responders to 4-week treatment" and "Non-responders to 4-week treatment," and
"Cumulative responders to 8-week treatment" and "Cumulative non-responders to 8-week
treatment."
Table 1. Histopathological parameters investigated
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Item Evaluation
EosinophilsTotal number of intraepithelial
eosinophils in biopsy sample
Intrapapillary vessel dilatation
Presence of intrapapillary vessel
dilatation
Intraepithelial bleeding
Presence of red blood cell
diapedesis from intrapapillary
vessels into epithelium
Dilated intercellular spaces (DIS) Positive (+) or negative ()
Papillary extension(Length of papilla)/(thickness of
whole epithelium) 100 = %
Basal cell hyperplasia
(Thickness of basal cell
layer)/(thickness of whole
epithelium) 100 = %
Ki-67 immunoreactivity (MIB-1; anti-human
monoclonal antibody, Dako Cytomation, Glostrup,
Denmark)
Number of cell layers positive for
Ki-67 (cellular marker of
proliferation = marker of DNA
synthesis)
Langerhans cells (positivity on CD1a immunostaining;
CD1a101, Dako Cytomation, Glostrup, Denmark)
Number of positive cells (from
most concentrated area, cell nuclei
must be recognized in a 400
view)
PGP 9.5 immunoreactivity (a soluble protein isolatedfrom brain, used as a general marker for neuronal and
neuroendocrine tissue;14 anti-PGP 9.5 polyclonal
antibody, Dako Cytomation, Glostrup, Denmark)
Number of positively stained nerve
fiber sections per squamous
epithelial area (2 3 mm)
It has been reported that, in gastroesophageal reflux disease (GERD) patients, the basal cell
layer is thickened (basal cell hyperplasia) to 1550% of the thickness of stratified squamousepithelium and that the thickness of the lamina propria mucosal papilla exceeds 1/22/3 of the
thickness of the epithelium (papillary extension).16 In addition, it has been reported that if Ki-
67 (MIB-1) immunoreactivity is positive in three or more layers of esophageal epithelium
cells, some type of abnormality consistent with GERD is present.
17
Histopathological Features of the Esophageal Mucosa
One biopsy specimen was taken from 5 cm above the squamo-columnar junction (SCJ) using
an endoscope. The specimen was taken from part of the anterior wall of the esophagus and at
the top of the esophageal mucosal plicae; any erosion or ulcer was excluded from biopsy. The
biopsy specimen was fixed in 10% formalin solution, embedded in paraffin, subjected to
hematoxylin-eosin staining or immunostaining, and histopathologically assessed at the Tokyo
Metropolitan Institute of Gerontology using light microscopy. Two pathologists (KT, JA)
blind to the patient backgrounds assessed the degree or presence of nine items[79] listed in .
Table 1. Histopathological parameters investigated
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Item Evaluation
EosinophilsTotal number of intraepithelial
eosinophils in biopsy sample
Intrapapillary vessel dilatation
Presence of intrapapillary vessel
dilatation
Intraepithelial bleeding
Presence of red blood cell
diapedesis from intrapapillary
vessels into epithelium
Dilated intercellular spaces (DIS) Positive (+) ornegative ()
Papillary extension(Length of papilla)/(thickness of
whole epithelium) 100 = %
Basal cell hyperplasia
(Thickness of basal cell
layer)/(thickness of whole
epithelium) 100 = %
Ki-67 immunoreactivity (MIB-1; anti-human
monoclonal antibody, Dako Cytomation, Glostrup,
Denmark)
Number of cell layers positive for
Ki-67 (cellular marker of
proliferation = marker of DNA
synthesis)
Langerhans cells (positivity on CD1a immunostaining;
CD1a101, Dako Cytomation, Glostrup, Denmark)
Number of positive cells (from
most concentrated area, cell nuclei
must be recognized in a 400
view)
PGP 9.5 immunoreactivity (a soluble protein isolatedfrom brain, used as a general marker for neuronal and
neuroendocrine tissue;14 anti-PGP 9.5 polyclonal
antibody, Dako Cytomation, Glostrup, Denmark)
Number of positively stained nerve
fiber sections per squamous
epithelial area (2 3 mm)
It has been reported that, in gastroesophageal reflux disease (GERD) patients, the basal cell
layer is thickened (basal cell hyperplasia) to 1550% of the thickness of stratified squamousepithelium and that the thickness of the lamina propria mucosal papilla exceeds 1/22/3 of the
thickness of the epithelium (papillary extension).16 In addition, it has been reported that if Ki-
67 (MIB-1) immunoreactivity is positive in three or more layers of esophageal epithelium
cells, some type of abnormality consistent with GERD is present.
17
Immunostaining with the two antibodies was carried out on 3-m-thick tissue sections using
ChemMate Envision/HRP (Polymer-Immuno Complex method, Dako Cytomation, Glostrup,
Denmark). Micrographs of intraepithelial bleeding, dilated intercellular spaces (DIS), Ki-67
immunoreactivity, and PGP 9.5 immunoreactivity are shown in Figure 1.
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Figure 1.
Histopathological findings. (a) Intraepithelial bleeding in a 41-year-old man with m-LA grade
M, who achieved complete heartburn relief after 4 weeks; (b) Dilated intercellular spaces
(DIS) circled (dotted line) in a 54-year-old woman with m-LA grade M, who achieved
complete heartburn relief after 4 weeks; (c) Ki-67 immunoreactivity (34 cell layers) in a 55-
year-old man with m-LA grade B, who achieved complete heartburn relief after 4 weeks; (d)
Protein Gene Product (PGP) 9.5 immunoreactivity revealed as dots or dotted lines (arrows) in
intercellular spaces of the epithelium in a 23-year-old woman with m-LA grade N, who
achieved complete heartburn relief after 6 weeks.
m-LA, modified Los Angeles Classification.
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Statistical Analysis
Data distributions were determined by summary statistics for continuous variables and by
frequency tables for classified variables. The significance level for statistical tests was set at
5% on both sides. Intergroup comparisons of demographic data for the study subjects and
esophageal histopathological findings were performed between NERD and RE patients usingthe two-sample Wilcoxon test or 2 test. In addition, intragroup comparisons of each
esophageal histopathological feature were also performed between the two patient groups
using the two-sample Wilcoxon test, 2 test, or Fisher's exact test.
For pathologic parameters deemed likely to exhibit significant differences between responders
and non-responders, subgroup analyses were performed for complete heartburn relief rates
after the 4-week treatment, and for cumulative complete heartburn relief rates after the 8-
week treatment.
Results
Subject Disposition and Demographics
Ninety-four (94) subjects who gave consent to participate in the study, and for whom
esophageal histopathological findings were evaluable, were included in the analyses. These
subjects comprised 71 patients with NERD and 23 patients with RE. Disease severity graded
by the modified Los Angeles Classification was Grade N in 24 subjects, Grade M in 47
subjects, Grade A in 17 subjects, and Grade B in six subjects. Demographics of the subjects
are shown for NERD and for RE separately in .
Table 2. Patient demographics (non-erosive reflux disease [NERD]vs
reflux esophagitis[RE])
Items NERD (n= 71) RE (n= 23) P-value
Gender (male/female) 32/39 17/6 0.016*a
Age, years (mean SD) 44.6 17.8 41.7 18.5 0.428
BMI, kg/m (mean SD) 22.5 3.4 23.9 5.9 0.402
Frequency of heartburn, days/week (mean SD) 4.3 2.4 3.7 2.1 0.308
Severity of heartburn (%)
Mild 59.2 43.5 0.351a
Moderate 35.2 52.2
Severe 5.6 4.3
Hiatal hernia + (%) 40 52.2 0.306a
Atrophy: Kimura-Takemoto Classification (%)
C-O 42.9 52.2 0.738a
C-I-III 41.4 34.8
O-I-III 15.7 13.0
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Helicobacter pylori + (%) 31 17.4 0.205a
CYP2C19 genotype (%)
homo EM 38.2 34.8 0.679a
hetero EM 44.1 39.1PM 17.6 26.1
Smoking+: sometimes +every day (%) 21.4 43.5 0.103a
Alcohol+: sometimes + every day (%) 47.1 56.5 0.220a
Caffeine +: sometimes +every day (%) 71.6 69.6 0.864a
*P< 0.05,a)2 test, b)Wilcoxon two-sample test, SD = standard deviation.n = 70.n = 68.n
= 67.
Comparison of Esophageal Histopathological Findings Between NERD and RE
Nine (9) esophageal histopathological markers in biopsy specimens taken before treatment
were compared between NERD and RE, and the results are shown in . There was no
statistically significant difference in any marker between NERD and RE. Though there was
no significant difference in the proportions of patients showing Ki-67 immunoreactivity in 3
cell layers, the proportion in the RE group (61.9%) was higher than that in the NERD group
(39.4%) (P= 0.071). In addition, the mean number of nerve fibers showing staining for PGP
9.5 (per squamous epithelial area) was higher in the NERD group than in the RE group (4.4 vs
1.9 respectively;P= 0.078).
Table 3. Comparison of baseline histopathological parameters in the non-erosive reflux
disease (NERD) vsreflux esophagitis (RE) groups
Items NERD (n) RE (n)P-
value
Number of eosinophils (mean SD)0.1 0.3
(68)
0.0 0.2
(23)0.990b)
Proportion of patients with intrapapillary vessel
dilatation (+)30.3% (66) 36.4% (22) 0.597a)
Proportion of patients with intraepithelial bleeding (+) 47.8% (67) 52.2% (23) 0.715a
Proportion of patients with dilated intercellular spaces
(DIS) (+)70.4% (71) 73.9% (23) 0.448a)
Papillary extension % (mean SD)47.2 17.2
(49)
52.8 12.6
(17)0.192b)
Basal cell hyperplasia % (mean SD)10.6 4.8
(55)
11.0 7.3
(17)0.651b)
Proportion of patients with Ki-67 (+) immunoreactivity
3 cell layers
39.4% (66) 61.9% (21) 0.071a)
Number of Langerhans cells (+) (mean SD) 6.8 2.6 6.9 3.4 0.557
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(66) (23)
Number of PGP 9.5 (+) nerve fibers (/unit area) (mean
SD)
4.4 5.1
(51)
1.9 2.5
(15)0.078b)
*P< 0.05,a)
2
test,b)
Wilcoxon two-sample test. Note: The number of patients is differentaccording to the histopathological parameters because of presence of biopsy specimens unable
to assess by each parameter.
Comparison of Esophageal Histopathology Between Responders and Non-responders in
the NERD Group
Histopathological features of the esophagus in NERD patients were compared between
responders and non-responders to RPZ for the 4-week treatment and between responders and
non-responders (cumulative) for the 8-week treatment, and the results are shown in .
Table 4. Comparison of histopathological parameters in the esophagus after 4 or 8weeks of rabeprazole (RPZ) treatment between responders and non-responders
Items Time NERD group RE group
Responde
rs (n)
Non-
responde
rs (n)
P-
value
Responde
rs (n)
Non-
responde
rs (n)
P-
value
Number of
eosinophils
(mean SD)
4 weeks0.0 0.0
(27)
0.1 0.4
(34)0.121b)
0.0 0.0
(17)
0.2 0.4
(6)0.113b)
8 weeks(Cumulativ
e)
0.1 0.4
(37)
0.0 0.2
(24)0.828b)
0.0 0.0
(19)
0.3 0.5
(4)
0.039*b)
Proportion of
patients with
intrapapillary
vessel
dilatation (+)
4 weeks23.1%
(26)
33.3%
(33)0.388a)
25.0%
(16)66.7% (6) 0.070a)
8 weeks
(Cumulativ
e)
25.7%
(35)
33.3%
(24)0.526a)
33.3%
(18)50.0% (4) 0.531a)
Proportion of
patients withintraepithelial
bleeding (+)
4 weeks57.7%
(26)
44.1%
(34)0.297a)
41.2%
(17)83.3% (6) 0.076a)
8 weeks
(Cumulativ
e)
52.8%
(36)
45.8%
(24)0.598a)
42.1%
(19)100% (4)
0.035*a)
Proportion of
patients with
dilated
intercellular
spaces (DIS)
(+)
4 weeks56.7%
(30)
79.4%
(34)0.050a)
76.5%
(17)66.7% (6) 0.638a)
8 weeks
(Cumulativ
e)
62.5%
(40)
79.2%
(24)0.164a)
73.7%
(19)75.0% (4) 0.957a)
Papillary 4 weeks 43.8 49.2 0.273 53.3 51.6 9.9 0.597
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extension %
(mean SD)
18.8 (18) 17.0 (25) 13.9 (12) (5)
8 weeks
(Cumulativ
e)
46.6
18.4 (26)
47.6
17.2 (17)0.931b)
53.8
13.5 (13)
49.5
10.1 (4)0.427b)
Basal cellhyperplasia %
(mean SD)
4 weeks 10.8 5.6(22)
10.7 4.4(27)
0.739b) 11.4 8.4(12)
10.0 4.3(5)
0.958b)
8 weeks
(Cumulativ
e)
10.9 5.5
(30)
10.6 4.1
(19)0.829b)
10.8 8.3
(13)
11.5 3.8
(4)0.393b)
Proportion of
patients with
Ki-67 (+)
immunoreactiv
ity 3 cell
layers
4 weeks42.3%
(26)
42.4%
(33)0.993a)
73.3%
(15)33.3% (6) 0.088a)
8 weeks
(Cumulativ
e)
40.0%
(35)
45.8%
(24)
0.656a)64.7%
(17)
50.0% (4) 0.586a)
Number of
Langerhans
cells (+) (mean
SD)
4 weeks6.7 2.9
(26)
6.9 2.4
(33)0.859b)
6.3 3.0
(17)
8.5 4.1
(6)0.256b)
8 weeks
(Cumulativ
e)
6.7 2.8
(35)
6.9 2.3
(24)0.779b)
6.6 3.4
(19)
8.3 3.3
(4)0.285b)
Ratio of
patients with
PGP 9.5 (+)
immunoreactivity
4 weeks88.2%
(17)
60.7%
(28)
0.048*a)
50.0%
(10)60.0% (5) 0.714a)
8 weeks
(Cumulativ
e)
84.6%(26)
52.6%(19)
0.019*a)
50.0%(12)
66.7% (3) 0.605a)
*P< 0.05,a)2 test,b)Wilcoxon two-sample test. Note: Responder or non-responder status was
defined as achieving or failing to exhibit lack of heartburn onset during the 7-day period
preceding evaluation, respectively. Patients who were completely relieved of heartburn after
the 4-week, 6-week, or 8-week treatment were defined as "Cumulative responders to 8-week
treatment." The number of patients is different according to the histopathological parameters
because of presence of biopsy specimens unable to assess by each parameter.
The proportion of patients whose biopsy samples showed staining for PGP 9.5 wassignificantly higher in responders to the 4-week treatment and in cumulative responders after
the 8-week treatment (responders vs non-responders: 88.2% [15/17] vs 60.7% [17/28],P=
0.048, for 4-week treatment and 84.6% [22/26] vs 52.6% [10/19],P= 0.019, for cumulative
8-week treatment). (See Fig. 2a "Rate of complete heartburn relief in the NERD group in
relation to presence or absence of PGP 9.5 immunoreactivity").
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Figure 2.
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Rate of complete heartburn relief in the non-erosive reflux disease (NERD) group in relation
to presence or absence of Protein Gene Product (PGP) 9.5 immunoreactivity (a) and dilated
intercellular spaces (DIS) (b). Note: Complete heartburn relief was defined as no heartburn
onset during the 7-day period preceding evaluation.
The proportion of patients whose biopsy samples showed DIS tended to be higher in non-responders to the 4-week treatment and in cumulative responders to the 8-week treatment
(responders vs non-responders: 56.7% [17/30] vs 79.4% [27/34],P= 0.050, for 4-week
treatment and 62.5% [25/40] vs 79.2% [19/24],P= 0.164, for 8-week treatment), although
this difference was not significant. (See Fig. 2b "Rate of complete heartburn relief in relation
to presence or absence of DIS in the NERD group").
There were no significant differences in the other pathological parameters between responders
and non-responders for either the 4-week treatment or the 8-week treatment.
Comparison of Esophageal Histopathology Between Responders and Non-responders in
the RE Group
Histopathological features of the esophagus in RE patients were compared between
responders and non-responders to RPZ for the 4-week treatment and between responders and
non-responders (cumulative) to the 8-week treatment, and the results are shown in .
Table 4. Comparison of histopathological parameters in the esophagus after 4 or 8
weeks of rabeprazole (RPZ) treatment between responders and non-responders
Items Time NERD group RE group
Responde
rs (n)
Non-responde
rs (n)
P-
value
Responde
rs (n)
Non-responde
rs (n)
P-
value
Number of
eosinophils
(mean SD)
4 weeks0.0 0.0
(27)
0.1 0.4
(34)0.121b)
0.0 0.0
(17)
0.2 0.4
(6)0.113b)
8 weeks
(Cumulativ
e)
0.1 0.4
(37)
0.0 0.2
(24)0.828b)
0.0 0.0
(19)
0.3 0.5
(4)
0.039*b)
Proportion of
patients withintrapapillary
vessel
dilatation (+)
4 weeks23.1%
(26)
33.3%
(33)0.388a)
25.0%
(16)66.7% (6) 0.070a)
8 weeks
(Cumulativ
e)
25.7%
(35)
33.3%
(24)0.526a)
33.3%
(18)50.0% (4) 0.531a)
Proportion of
patients with
intraepithelial
bleeding (+)
4 weeks57.7%
(26)
44.1%
(34)0.297a)
41.2%
(17)83.3% (6) 0.076a)
8 weeks
(Cumulativ
e)
52.8%
(36)
45.8%
(24)0.598a)
42.1%
(19)100% (4)
0.035*a)
Proportion ofpatients with
4 weeks 56.7%(30)
79.4%(34)
0.050a) 76.5%(17)
66.7% (6) 0.638a)
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dilated
intercellular
spaces (DIS)
(+)
8 weeks
(Cumulativ
e)
62.5%
(40)
79.2%
(24)0.164a)
73.7%
(19)75.0% (4) 0.957a)
Papillaryextension %
(mean SD)
4 weeks 43.8 18.8 (18) 49.2 17.0 (25) 0.273b) 53.3 13.9 (12) 51.6 9.9(5) 0.597
b)
8 weeks
(Cumulativ
e)
46.6
18.4 (26)
47.6
17.2 (17)0.931b)
53.8
13.5 (13)
49.5
10.1 (4)0.427b)
Basal cell
hyperplasia %
(mean SD)
4 weeks10.8 5.6
(22)
10.7 4.4
(27)0.739b)
11.4 8.4
(12)
10.0 4.3
(5)0.958b)
8 weeks
(Cumulativ
e)
10.9 5.5
(30)
10.6 4.1
(19)0.829b)
10.8 8.3
(13)
11.5 3.8
(4)0.393b)
Proportion of
patients with
Ki-67 (+)
immunoreactiv
ity 3 cell
layers
4 weeks42.3%
(26)
42.4%
(33)0.993a)
73.3%
(15)33.3% (6) 0.088a)
8 weeks
(Cumulativ
e)
40.0%
(35)
45.8%
(24)0.656a)
64.7%
(17)50.0% (4) 0.586a)
Number of
Langerhans
cells (+) (mean
SD)
4 weeks6.7 2.9
(26)
6.9 2.4
(33)0.859b)
6.3 3.0
(17)
8.5 4.1
(6)0.256b)
8 weeks
(Cumulative)
6.7 2.8
(35)
6.9 2.3
(24) 0.779b)6.6 3.4
(19)
8.3 3.3
(4) 0.285b)
Ratio of
patients with
PGP 9.5 (+)
immunoreactiv
ity
4 weeks88.2%
(17)
60.7%
(28)
0.048*a)
50.0%
(10)60.0% (5) 0.714a)
8 weeks
(Cumulativ
e)
84.6%
(26)
52.6%
(19)
0.019*a)
50.0%
(12)66.7% (3) 0.605a)
*P< 0.05,a)2 test,b)Wilcoxon two-sample test. Note: Responder or non-responder status was
defined as achieving or failing to exhibit lack of heartburn onset during the 7-day periodpreceding evaluation, respectively. Patients who were completely relieved of heartburn after
the 4-week, 6-week, or 8-week treatment were defined as "Cumulative responders to 8-week
treatment." The number of patients is different according to the histopathological parameters
because of presence of biopsy specimens unable to assess by each parameter.
The mean eosinophil count was 0 in cumulative responders to the 8-week treatment and 0.3 in
cumulative non-responders to the 8-week treatment, being significantly higher (P= 0.039) in
the latter. The proportion of patients with intraepithelial bleeding was 42.1% in cumulative
responders to the 8-week treatment and 100% in cumulative non-responders to the 8-week
treatment, being also significantly higher (P= 0.035) in non-responders.
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There were no significant differences in other pathological parameters between responders
and non-responders to the 4-week or 8-week treatment.
Relationship Between Presence or Absence of DIS and PGP 9.5 Immunoreactivity in
NERD and RE Patients The above findings suggested that both the presence or absence of
DIS or PGP 9.5 immunoreactivity might be related to the efficacy of RPZ in NERD patients.The efficacy of RPZ was therefore compared among patients classified into the following four
groups: DIS (+)/PGP 9.5 (+), DIS (+)/PGP 9.5 (), DIS ()/PGP 9.5 (+), and DIS ()/PGP 9.5
().
With regard to the efficacy of RPZ in 45 NERD patients, the rate of complete heartburn relief
after 4 weeks was 0% for DIS (+)/PGP 9.5 (), representing the lowest value, and there were
significant differences among the four histopathology groups (P= 0.046) ( ). Similarly, the
rate of cumulative complete heartburn relief after 8 weeks was lowest (22.2%) in the DIS
(+)/PGP 9.5 () group (P= 0.093), although the difference relative to the other groups was
not significant. In 15 RE patients, there were no significant differences in the rates of
complete heartburn relief among the four groups after the 4-week treatment, or in thecumulative rate after the 8-week treatment.
Table 5. Response to rabeprazole (RPZ) therapy based on presence or absence of
dilated intercellular spaces (DIS) and Protein Gene Product (PGP) 9.5 immunoreactivity
in non-erosive reflux disease (NERD) and reflux esophagitis (RE) patients (%, rates of
complete heartburn relief)
Time
Presence or
absence
NERD (n = 45) RE (n = 15)
DIS (+) DIS ()
P-
value DIS (+) DIS ()
P-
value
After 4 weeksPGP 9.5 (+)
47.8%
(11/23)
44.4%
(4/9)0.046*
60.0%
(3/5)
66.7%
(2/3)1.000
PGP 9.5 () 0% (0/9)50.0%
(2/4)
80.0%
(4/5)
50.0%
(1/2)
After 8 weeks
(cumulative)PGP 9.5 (+)
69.6%
(16/23)
66.7%
(6/9)0.093
80.0%
(4/5)
66.7%
(2/3)1.000
PGP 9.5 ()22.2%
(2/9)
50.0%
(2/4)
80.0%
(4/5)
100%
(2/2)
*P< 0.05, Fisher's exact test.
Discussion
The present study is the first to have examined differences in esophageal histopathology
between NERD and RE. Although several reports have described histopathological findings
in the esophagus of patients with GERD,[79] it has been unclear whether they are
physiological characteristics of gastroesophageal reflux and not specific to GERD.[18] One
reason for this lack of clarity is probably the diverse nature of the sites from which biopsy
samples have been obtained.[9,19]
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In the present investigation, biopsy specimens were taken from 5 cm above the SCJ near the
site of an indwelling sensor used for pH monitoring to detect morbid gastric acid reflux. This
biopsy site was selected because it might enable clarification of the relationship between the
duration of exposure to gastric acid and tissue histopathology. Although there was no
significant correlation between any of the histopathological features of esophageal biopsy
specimens and pH parameters (% period during which pH < 4, frequency of reflux) (data notshown), one difference in pathology between NERD and RE was of interest: a greater
proportion of nerve fibers were immunopositive for PGP 9.5 in NERD patients than in RE
patients, though the difference was not statistically significant. This finding may explain the
greater sensory hypersensitivity of NERD patients.[20] On the other hand, a greater proportion
of RE patients had three or more Ki 67-positive cell layers, an indicator of cell proliferation,
than was the case in NERD patients, though the difference was also not statistically
significant. Enhancement of cellular proliferative potential may reflect inflammation due to
mucosal damage by gastric acid. This is consistent with the reported finding that the
expression of Ki 67 increases in proportion to worsening of esophageal histopathology.[21,22]
Although it has been speculated that NERD may be a mild form of RE, the present results
suggest that the spectra of these diseases may differ.
Here we also investigated the histopathological features of the esophagus that might predict
the therapeutic efficacy of PPIs in patients with GERD. In this study, we compared the
esophageal histopathology between responders and non-responders. The results indicated that
among patients with NERD, the proportion of responders to PPI was significantly higher in
those showing PGP 9.5 immunoreactivity in nerve fibers, and that the proportion of non-
responders to PPI was significantly higher in patients lacking such PGP 9.5 immunoreactivity
even after an increase in the PPI dose. As mentioned above, positive PGP 9.5
immunoreactivity in nerve fibers may be related to hypersensitivity of NERD patients.
Therefore, the patients with positive PGP9.5 will respond to the increased dose of PPI, which
somehow decreases the amount of acid or weakly acid reflux. On the other hand, the
negativity to PGP9.5 suggests that their symptoms are not related to acid reflux event.
Therefore such patients are unlikely to respond to the treatment with PPI even if its dosage is
increased. In fact, it is reported that the total esophageal acid exposure time correlates
significantly with density of PGP 9.5 immunoreactivity in NERD patients.[23] Thus, lack of
PGP 9.5 immunoreactivity appeared to be a useful predictive factor of resistance to treatment
with PPI.
Because NERD patients with DIS tended to be more resistant to standard therapy with PPI
than NERD patients without DIS (although not to a significant degree;P= 0.050), DIS was
also considered to be a potentially important factor predicting the efficacy of PPI. DIS hasbeen studied mainly in the context of GERD symptoms.[24] Since it has been reported that DIS
disappears with improvement of heartburn,[25] and that it is evident microscopically in 90% of
RE patients, 68% of NERD patients, and 8% of control subjects, being correlated with
mucosal damage to some extent,[26] it is not surprising that DIS might be a factor predictive of
the therapeutic effectiveness of PPI. In the present study, the difference in the symptom relief
rate between patients with and without DIS decreased (P= 0.164) after PPI dose escalation.
This suggested that strong and long-term suppression of gastric acid might ameliorate DIS.
With regard to the efficacy of PPI, as shown in , NERD patients who showed an especially
low response to the therapy were DIS (+)/PGP 9.5 (), and the difference in the efficacy rate
was significantly lower than in the other three groups. Since these patients continued to showa low efficacy rate even after the dose of RPZ had been increased, these two parameters
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appeared to be potentially predictive of therapeutic effectiveness of PPI. The precise
mechanism why NERD patients with DIS (+)/PGP 9.5 () is still unclear. However, it is not
surprising that the combination of these is likely to serve as a predictor for poor responder to
PPI because positive DIS is a marker of symptoms and negative PGP 9.5 suggests the
symptoms are less related to acid reflux.
Table 5. Response to rabeprazole (RPZ) therapy based on presence or absence of
dilated intercellular spaces (DIS) and Protein Gene Product (PGP) 9.5 immunoreactivity
in non-erosive reflux disease (NERD) and reflux esophagitis (RE) patients (%, rates of
complete heartburn relief)
Time
Presence or
absence
NERD (n = 45) RE (n = 15)
DIS (+) DIS ()P-
valueDIS (+) DIS ()
P-
value
After 4 weeksPGP 9.5 (+)
47.8%
(11/23)
44.4%
(4/9) 0.046*60.0%
(3/5)
66.7%
(2/3) 1.000
PGP 9.5 () 0% (0/9)50.0%
(2/4)
80.0%
(4/5)
50.0%
(1/2)
After 8 weeks
(cumulative)PGP 9.5 (+)
69.6%
(16/23)
66.7%
(6/9)0.093
80.0%
(4/5)
66.7%
(2/3)1.000
PGP 9.5 ()22.2%
(2/9)
50.0%
(2/4)
80.0%
(4/5)
100%
(2/2)
*P< 0.05, Fisher's exact test.
Interestingly, the present results suggested that the pathologic factors possibly reflecting the
efficacy of RPZ differed between NERD and RE groups. That is, DIS (+)/PGP 9.5 ()
indicated resistance to PPI therapy in NERD patients, while the presence of both eosinophil
infiltration and intraepithelial bleeding were predictive of resistance to PPI therapy in RE
patients. RE patients whose biopsy samples had eosinophil infiltration exhibited a poor
response to PPI therapy. Based on the reported finding that the eosinophil count reflects the
severity of gastric acid reflux,[19] it can be inferred that the greater the degree of eosinophil
infiltration, the longer the symptoms have continued, despite treatment with PPI. However,
since only one of the four patients in the non-responders of RE group showed eosinophil
positivity, no statistically firm conclusion can yet be reached. Similarly, patients who hadintraepithelial bleeding also exhibited a poor response to PPI therapy. Intraepithelial bleeding
may be caused by dilation or hyperplasia of intrapapillary blood vessels,[27,28] and since
bleeding is an erythrogenically severe condition clinically, it may prolong the symptoms.
The advantages of the study were that it was a multi-center study, biopsy specimens were
assessed objectively at a completely independent center, and that the site of biopsy was
carefully specified. However, since biopsy specimens were taken from only one site in each
subject at each study center, and assessed on this basis, it was unclear whether the data
obtained were actually representative of each subject. In addition, the number of subjects may
have been too small to allow detection of statistically significant differences among the
variables investigated. Although the sample size reflected the actual epidemiologic ratio ofNERD and RE patients (71 vs 23), it would have been better if the number of RE subjects had
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approximated that of NERD patients. A further study including healthy adults, patients with
Grade C and D reflux esophagitis, and patients with asymptomatic reflux esophagitis would
allow a more comprehensive assessment of the characteristics of biopsy specimens from
GERD patients and their clinical significance.
In conclusion, our study demonstrated the histological assessment of biopsied esophagealtissue is likely to predict therapeutic efficacy of PPI, and the current study suggests factors
predictive of resistance to treatment with PPI are negativity for PGP 9.5 in NERD patients
and intraepithelial bleeding in RE patients.
Appendix
Members of the ARS (Acid-Related Symptom) Research Group
Shuichi Ohara, Department of Gastroenterology, Tohoku Rosai Hospital, Sendai, Japan;
Tomoyuki Koike, Division of Gastroenterology, Tohoku University Graduate School of
Medicine, Sendai, Japan; Motoyasu Kusano and Yasuyuki Shimoyama, Department ofEndoscopy and Endoscopic Surgery, Gunma University Hospital, Maebashi, Japan; Osamu
Kawamura, Department of Medicine and Molecular Science, Gunma University Graduate
School of Medicine, Maebashi, Japan; Yoshio Hoshihara, Clinic of the Ministry of Economy,
Trade, and Industry, Tokyo, Japan; Kouji Yakabi, Department of Gastroenterology and
Hepatology, Saitama Medical Center, Saitama Medical University, Kawagoe, Japan;
Mitsushige Sugimoto and Masafumi Nishino, First Department of Medicine, Hamamatsu
University School of Medicine, Hamamatsu, Japan; Makoto Sasaki, Department of
Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical
Sciences, Nagoya, Japan; Yasuhiro Fujiwara, Department of Gastroenterology, Osaka City
University Graduate School of Medicine, Osaka, Japan; Kazuhide Higuchi, Second
Department of Internal Medicine, Osaka Medical College, Takatsuki, Japan; Ken Haruma,
Division of Gastroenterology, Department of Internal Medicine, Kawasaki Medical School,
Kurashiki, Japan; Noriaki Manabe, Division of Endoscopy and Ultrasonography, Kawasaki
Medical School, Kurashiki, Japan; Kyoichi Adachi, Department of Clinical Nursing, Shimane
University Faculty of Medicine, Izumo, Japan; Kenji Furuta, Department of Internal Medicine
II, Shimane University Faculty of Medicine, Izumo, Japan; Kazuma Fujimoto, Department of
Internal Medicine, Saga Medical School, Saga, Japan.
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Financial support
Unrestricted donation was offered by Eisai Co., Ltd. for this study.
J Gastroenterol Hepatol. 2013;28(3):479-487. 2013 Blackwell Publishing