Esophageal baseline impedance levels allow the identification of esophageal involvement in patients with systemic sclerosis

Author’s Accepted Manuscript Esophageal Baseline Impedance Levels Allow the Identification of Esophageal Involement in Patients with Systemic SclerosisEsophageal mucosal integrity in SSc patients Patrizia Zentilin, Vincenzo Savarino, Elisa Marabotto, Giuseppe Murdaca, Alberto Sulli, Carmen Pizzorni, Francesco Puppo, Edoardo Savarino

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To appear in: Seminars in Arthritis and Rheumatism Cite this article as: Patrizia Zentilin, Vincenzo Savarino, Elisa Marabotto, Giuseppe Murdaca, Alberto Sulli, Carmen Pizzorni, Francesco Puppo and Edoardo Savarino, Esophageal Baseline Impedance Levels Allow the Identification of Esophageal Involement in Patients with Systemic SclerosisEsophageal mucosal integrity in SSc patients, Seminars in Arthritis and Rheumatism, http://dx.doi.org/10.1016/j.semarthrit.2017.08.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ESOPHAGEAL BASELINE IMPEDANCE LEVELS ALLOW THE IDENTIFICATION OF ESOPHAGEAL INVOLEMENT IN PATIENTS WITH SYSTEMIC SCLEROSIS Patrizia Zentilin1, Vincenzo Savarino1, Elisa Marabotto1, Giuseppe Murdaca2, Alberto Sulli3, Carmen Pizzorni3, Francesco Puppo2, Edoardo Savarino4. 1

Department of Internal Medicine, Gastroenterology Unit, Genova, 2Department of Internal Medicine, Internal Medicine Unit, Genova, 3Department of Internal Medicine, Rheumatology Unit, Genova, Italy, 4Departement of Surgery, Oncology and Gastroenterology, Gastroenterology Unit, Padova Authors contribution: - Patrizia Zentilin, MD, PhD: data collection and analysis, writing of the manuscript, approving final version - Vincenzo Savarino, MD: data analysis, writing of the manuscript, approving final version - Elisa Marabotto, MD, PhD: data collection and analysis, writing of the manuscript, approving final version - Giuseppe Murdaca, MD: data collection and analysis, approving final version - Alberto Sulli, MD: data collection and analysis, approving final version - Carmen Pizzorni, MD: data collection, approving final version - Francesco Puppo, MD: data collection, approving final version - Edoardo Savarino, MD: writing of the manuscript, approving final version

Running Title: Esophageal mucosal integrity in SSc patients

Keywords: gastro-esophageal reflux disease; non-erosive reflux disease; impedance-pH monitoring; mucosal integrity; systemic sclerosis

Financial support: none

Potential competing interests: none

Electronic Word Count (excluding abstract, ref, tables, figures): 3214

Corresponding Author: Edoardo Savarino Division of Gastroenterology, Department of Surgery, Oncology and Gastroenterology 1

University of Padua Via Giustiniani 2 35128 Padova Italy Telephone: +390498217749; Fax: +390498760820; e-mail: [email protected]

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Abstract

Introduction: Distal esophageal baseline impedance (BI) levels reflect the esophageal mucosal integrity in reflux disease. Systemic sclerosis (SSc) could potentially affect the integrity of esophageal mucosa and consequently impair distal and proximal BI levels, but data in this regard are lacking.

Aim & Methods: We aimed to prospectively investigate and compare BI levels among non-erosive reflux disease (NERD), SSc patients and healthy controls (HCs). Consecutive patients with reflux symptoms and well defined diagnosis of SSc underwent upper endoscopy and, in case of no lesions encountered, manometry and impedance-pH testing off-therapy. In addition to traditional impedance-pH parameters, BI values at 3, 5, 7 and 17 cm above the lower esophageal sphincter were calculated.

Results: Fifty-two patients with NERD, 50 with SSc and 50 HCs were enrolled. Nineteen (38%) SSc patients and 22 (42%) NERD patients had abnormal acid exposure. In patients with SSc, median BI values were significantly lower than in NERD patients and HCs (p<0.0001) at 3, 5 and 7 cm; there was no difference between HCs and NERD patients at 17 cm in the proximal esophagus, whereas a significant difference was observed at 17 cm between SSc and NERD as well as HCs (p<0.01).

Conclusion: Distal and proximal BI values in SSc patients were lower than in NERD and HCs, thus we speculated that these findings may be related to the deposition of collagen in the connective tissue. Measurement of BI may be used as an indirect, but simple and accurate marker of esophageal involvement in patients with SSc. 3

Key Messages: -

Systemic sclerosis frequently impairs the esophagus and the early identification of its involvement may permit to prevent serious complications

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In this study we showed that SSc patients with esophageal involvement present lower distal and proximal baseline impedance values compared to patients with reflux diseases or heathy controls, suggesting a severe impairment of esophageal mucosal integrity in them

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These data provide a rationale for the use of novel, poorly invasive, and easy to perform methods for the assessment of esophageal mucosal impedance that, potentially, will modify our diagnostic approach to SSc patients with suspect esophageal involvement

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INTRODUCTION Systemic sclerosis (SSc) is a clinically heterogeneous and generalized disease affecting the connective tissue of the skin and internal organs, such as the gastrointestinal tract, lungs, kidneys, and heart. SSc is characterized by deposition of collagen and other extracellular matrix proteins in the connective tissues, imbalance of the immune system, and microvascular abnormalities (1). Esophageal manifestations with dysphagia, heartburn and regurgitation are the most frequent gastrointestinal complaints, and serious complications can occur in 50% of SSc patients (2-6), as demonstrated by the increased incidence of esophageal adenocarcinoma and reflux-related pulmonary involvement, although in the latter case causality between reflux occurrence and interstitial lung disease has not been proven yet (7-8). Thus, the identification of esophageal involvement plays a crucial role in order to minimize these complications and improving survival in these patients (8). However, to date, this diagnostic workup is mainly based on the use of invasive tests such as manometry and reflux monitoring which provide unspecific diagnoses of ineffective esophageal motility or absent peristalsis and reflux disease, without the possibility to conclude that these alterations are due to scleroderma rather than concomitant gastro-esophageal reflux disease (GERD) (6). Further, their limited tolerability reduces their widespread application. Combined esophageal multichannel intraluminal impedance-pH monitoring (MII-pH) is currently used for the assessment of GERD by measuring changes in electrical impedance caused by fluid and/or gas reflux (9, 10). In the absence of reflux episodes and swallows, the esophageal lumen is collapsed and the metallic rings are in close contact with the esophageal mucosa; thus the resulting baseline impedance (BI) level is determined by the intrinsic electrical conductivity of the surrounding esophageal wall. In vitro and in vivo studies demonstrated that BI levels may reflect the integrity of esophageal mucosa and this finding has been further supported by several recent studies correlating BI values with histological abnormalities in GERD patients (11-16). In particular, distal esophageal BI levels were correlated with pathological acid exposure in GERD patients, whereas proximal BI levels were found not different between GERD and healthy controls 5

likely due to the limited proximal extension of the refluxate (17). Further, BI levels have been found lower in patients with physiological acid exposure but proven GERD, based on positive symptom association analysis or positive response to PPI therapy, than in patients with functional heartburn not responding to PPIs and in healthy volunteers (18). The deposition of collagen and other extracellular matrix proteins in the connective tissue of the gastrointestinal tract, typical of SSc patients with esophageal involvement, could potentially affect the conductivity of the esophageal wall and consequently BI levels, both in the distal and proximal esophagus, and therefore this new impedance parameter may be potentially adopted to clearly define an esophageal involvement in scleroderma patients. However, data in this regard are lacking. So far, we aimed to prospectively investigate and compare BI levels among non-erosive reflux disease (NERD), SSc patients and heathy controls (HCs).

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PATIENTS AND METHODS Throughout the entire 2014 year, we prospectively enrolled consecutive SSc patients, fulfilling the American College of Rheumatology (ACR) classification criteria (19), with esophageal involvement and patients with typical reflux symptoms (i.e., heartburn with/without regurgitation), presenting to the outpatient esophageal pathophysiology center at the University Hospital of Genoa. All of them underwent upper gastrointestinal endoscopy to assess the presence or absence of erosive esophagitis and Barrett’s esophagus, and in case of mucosal injuries, they have been excluded from further analysis in order to obtain data on mucosal integrity not influenced by macroscopic inflammation (20). Further, esophageal manometry was performed in all of them. Patients with reflux symptoms were identified as NERD and then included into the study if they had the following inclusion criteria (21,22): negative endoscopy, presence of heartburn with/without regurgitation at least twice in a week for 6 months in the previous year, pathological acid exposure or positive symptom association analysis at impedance-pH monitoring. The exclusion criteria were as follows: age lower than 18 years, pregnancy (excluded by urine analysis) and/or breast feeding; eating disorders; history of thoracic, esophageal, or gastric surgery; underlying psychiatric illness; peptic ulcer at a previous endoscopy. Fifty healthy subjects studied in our motility laboratory under the same conditions were used as controls (23,24). The study protocol was approved by our institutional review board. The study was conducted in accordance with the research protocol and in compliance with the Declaration of Helsinki, Good Clinical Practice guidelines, and local laws and regulations. All patients provided written informed consent to participate in the study.

Study Protocol Patients were asked to stop PPI treatment, if any, at least 15 days before the study enrolment and were only allowed to take alginates, on as-needed basis, as rescue therapy for controlling heartburn (25,26). Patients were asked to discontinue any medication that would influence 7

esophageal motor function. All subjects underwent stationary esophageal manometry and 24-hour MII-pH esophageal monitoring off therapy, that were performed after an overnight fast. A solid-state manometry 5 solid-state sensors spaced at 5-cm intervals (O.D. 4.2 mm) was used (Sandhill Scientific Instruments Inc., Highlands Ranch, CO, USA). Studies were done in supine position after at least a 6-h fast. The manometrical assembly was passed transnasally and positioned to record from the hypopharynx to the stomach with about 5 intragastric sensors. The catheter was fixed in place by taping it to the nose. The manometric protocol included a 5-min period to assess basal sphincter pressure and ten 5-mL water swallows to evaluate esophago-gastric junction function and esophageal body motility (27). Combined esophageal multichannel intraluminal impedance and pH monitoring (MII-pH) was performed using a polyvinyl catheter (diameter: 2.3 mm), equipped with an antimony pH electrode and several cylindrical electrodes, with a length of 4 mm, placed at intervals of about 2 cm (Sandhill Scientific Inc., Highland Ranch, CO, USA). Each pair of adjacent electrodes represented an impedance measuring segment corresponding to one recording channel. The singleuse MII-pH catheter was positioned with the pH electrode 5 cm above the lower esophageal sphincter (LES) and the six impedance recording channels at 3, 5, 7, 9, 15, and 17 cm above the LES. All patients consumed foods and beverages exclusively during three standard meals (lunch at 1.00 pm, dinner at 8.00 pm, and breakfast at 8.00 am of the next day) on the basis of a Mediterranean diet, without alcohol and coffee, to reduce variability due to alimentary habits (24). The patients were also requested to remain in upright position during the day and to indicate the recumbent period during nighttime (max 8 h). Each patient was instructed to press the ‘event marker’ button, on the pH datalogger, whenever they experienced reflux symptoms during the recording period. At the end of the examination, MII-pH tracings were reviewed manually by two investigators (EM, PZ) blinded for the conditions of the patients in order to ensure accurate detection and classification of reflux episodes and BI values. Meal periods were excluded from the analysis. Impedance and pH data were used to determine the number and type of reflux episodes 8

and acid exposure time (AET) (reflux percent time) in each patient. In particular, distal esophageal AET was defined as the total time with pH below 4.0 units, divided by the total time of monitoring. A percent time lower than 4.2% with pH < 4.0, over 24 h, was considered normal (25). Acid, weakly acidic, and weakly alkaline refluxes were defined according to medical literature (28). Proximal reflux extent was defined as a drop in impedance recorded at 15 cm from LES. Moreover, mean acid and median bolus clearance times were measured (28). Correlation between symptom and reflux events with symptom index and symptom association probability (SI and SAP, respectively) was evaluated for each patient, as previously described (29). Finally, BI levels were assessed from the distal channels (3 cm, 5 cm, 7 cm above the LES) and from the proximal channel (17 cm above the LES) during the overnight rest, excluding reflux episodes, swallows, and pH drops, as previously described (12).

Statistical analysis Statistical analysis was performed by comparing the differences among the three populations using the Kruskall-Wallis rank sum test. The Mann-Whitney "U" test was also used, when indicated. The estimate p-values were adjusted for multiple comparisons using Bonferroni correction method, and the differences with an adjusted p-value less than 0.05 were selected as significant in all the comparisons. The data are presented as median and 95% confidence intervals (CI). Data were acquired and analyzed in R v3.0.1 software environment (30).

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RESULTS We have globally studied 70 consecutive NERD patients (44 females and 26 males, median age 53 years), 50 patients with SSc (44 females and 6 males, median age 54 yrs), and 50 HCs (37 females and 13 males, median age 49 years). After manometric and 24-hour MII-pH esophageal monitoring, only 52 patients (33 females and 19 males, median age 51 years) were identified as NERD and were included in the final analysis; eighteen patients resulted affected by functional heartburn (21) and were excluded. The demographic and voluptuary characteristics of NERD, SSc patients and controls are showed in Table I, while the main clinical features of SSc patients are reported in Table II. All patients and controls were studied ‘off ’ PPI treatment. The results of manometric studies are displayed in Table III. We found that lower esophageal sphincter pressure (LESP) was significantly higher in HCs than in NERD and SSc patients (both p<0.0001). There was no difference in LESP values between NERD and SSc patients (p=0.3). The distal peristaltic wave amplitude was significantly higher in HCs than in NERD and SSc patients (p=0.0002 and p< 0.0001, respectively) and differed between the last two populations, as it was lower in SSc patients (p<0.0001). We found that 24-hour AET was significantly lower in HCs than in NERD and SSc patients during total recording time (both p<0.0001), in upright position (both p<0.0001) and in recumbent position (p<0.0001 and p=0.0002, respectively). However, no difference was observed between NERD and SSc patients in upright position (p=0.07), in recumbent position (p=0.62) and during total recording time (p=0.33), as shown in Table IV (a). The median reflux number was significantly lower in HCs than in NERD and SSc patients (both p<0.0001), while no statistical difference was found between NERD and SSc patients (p=0.58); the median number of proximal refluxes was significantly lower in HCs than in NERD and SSc patients (both p<0.0001) and NERD patients had a significantly higher number of proximal refluxes in comparison to SSc patients (p=0.005), as shown in Table IV (b).

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Median bolus clearing time (MBCT) was significantly lower in HCs than in NERD and SSc patients during total recording time, in upright and recumbent position (both p < 0.0001); no statistical difference was found between NERD and SSc patients (p = 0.84, p = 0.82, p = 0.31, respectively), as shown in Table IV(c). As to BI values, as shown in Table V, we found that at 3 cm above the LES they were significantly higher in HCs than in NERD and SSc patients (all p<0.0001). At 5 cm, a significant difference was found between median BI values of HCs and NERD patients (p=0.01), and they were significantly lower in SSc patients than in HCs and in NERD (both p<0.0001). At 7 cm above the LES, no significant difference was found between median BI values of HCs and NERD patients (p=0.1), while they were significantly lower in SSc patients than in HCs and NERD patients (both p< 0.0001). At 17 cm above LES, no statistical difference was found between median BI values of HCs and NERD patients (p = 0.92), while they were significantly lower in SSc than in the other two groups (both p<0.01).

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DISCUSSION. Esophageal baseline impedance has been recognized as an accurate marker of mucosal integrity in several recent pathophysiological investigations using 24-hour impedance-pH testing (11-16) and, therefore, this parameter has been proposed to achieve an indirect, but simple and accurate measurement of esophageal damage. Based on this concept, we decided to explore the value of this finding in a large population of patients with SSc. To the best of our knowledge. this is the first study in which esophageal BI levels were studied in SSc patients and were compared to those obtained in a population of patients with reflux disease and healthy controls. We found that BI values were significantly lower in SSc patients than in NERD and HCs and this occurred both in the distal esophagus, that is at 3, 5, and 7 cm above the LES and in the proximal esophagus at 17 cm above the LES. This suggests that in SSc patients the deposition of collagen and other extracellular matrix proteins in the connective tissue of the esophagus may impair the epithelial integrity of both distal and proximal esophagus, whereas in GERD only the distal esophagus is involved. Therefore, this finding could be used as a confirmatory evidence of esophageal involvement in SSc patients and could represent a potential target for novel therapies targeted towards molecular and cellular pathways (31). Recent studies underlined the correlation between BI levels, detected during impedance-pH monitoring, and mucosal integrity, thus validating this measurement as an indirect but accurate method to assess the alterations at esophageal mucosa level (11-18). It is relevant to note that there is no international agreement on how and when measuring BI levels during reflux monitoring, since in the above mentioned studies different methods have been adopted (11,12,14,15,17). In this study, BI levels have been assessed during the night, as in this time period impedance registration is less affected by swallows and refluxes, which occur rarely during the nocturnal period. Furthermore, Kessing et al (17) analyzed the 24-hour measurement of BI levels in GERD patients and did not find a marked diurnal variation in such values, despite AET decreased in all patients during the

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night. So, they confirmed the low variability over time of this parameter and emphasized its reliability. Although it is well known that the prevalence of reflux esophagitis is highly variable and can range from 33% to 63% of patients with SSc (32-34), we selected only cases without any esophageal mucosal lesion visible at endoscopy in order to match better them with our group of reflux patients not bearing any macroscopic damage at the esophagus, as occurs in case of NERD. We opted for this decision because of different reasons. First, NERD represents the majority of patients with typical reflux symptoms and they are generally studied with impedance-pH testing because of reduced response to medical treatment or before proposing them for surgery (21,35). Second, in the PPI era, it is quite uncommon to observe erosive esophagitis because of the high rate of mucosal healing induced by these drugs (36). Third, we preferred to exclude patients with esophageal mucosal injuries in order to obtain data on epithelial integrity not influenced by macroscopic inflammation and therefore more reflecting the potential role of the underlying disease on mucosal status (37,38). In our study there was no difference in acid esophageal exposure between the two populations of SSc and NERD patients, thus confirming that they were totally comparable in terms of pathophysiological parameters. Indeed, the proportions of cases with excess of acid in the esophagus were 38% and 42%, respectively. The percentage of 42% is in agreement with previous data already published in NERD (23,29), while 38% represents a lower rate than that reported in other studies on patients with SSc, which varied from 54% to 86% (39-41). However, it must be considered that these previous investigations were performed in SSc patients with erosive esophagitis and Barrett’s esophagus. In contrast, we enrolled only patients without any esophageal mucosal lesions detected at endoscopy and therefore with likely mild or moderate reflux disease. Nevertheless, despite the occurrence of a similar acid exposure between SSc and NERD populations, the former presented significantly lower BI values than the latter one at both distal and proximal esophagus. Our findings seem to indicate that the alterations in BI levels documented 13

along the entire esophagus cannot be only explained by reflux occurrence, but also by a deeper damage of the wall of the esophagus due to the deposition of collagen, which is the characteristic feature of scleroderma (1). This concept is further corroborated by the fact that we did not find any significant difference in proximal BI values between NERD patients and HCs. Beyond the poor integrity of the mucosa and the structural damage of the esophageal wall, the decrease in BI values could help to explain the high frequency of heartburn in SSc patients (42). In fact, the normal structure of esophageal epithelium prevents the passage of acid and other substances through the mucosa, whereas, when erosions are present, they are able to cross the epithelium and reach the sensory nerve endings, thus provoking heartburn. In patients with NERD, esophageal acid exposure is frequently normal and then visceral hypersensitivity appears to be an important pathogenic mechanism for the development of heartburn. An impaired mucosal integrity may underlie the occurrence of esophageal visceral hypersensitivity, suggesting that the status of the esophageal mucosa has a key role in heartburn perception in patients with GERD without any erosions (43-45). A limitation of the present study is that we did not take biopsies from the esophageal mucosa in order to search for the possible presence of dilation of intercellular spaces and microscopic esophagitis. In patients with NERD, as well as in those with erosive esophagitis, dilated intercellular spaces within the squamous epithelium are typically present and this microscopic abnormality can be readily identified with optical microscopy and is considered a histopathological marker of the presence of reflux disease. Another limitation is that we did not enroll a group of SSc patients without esophageal involvement as controls. However, given the invasiveness of the manometry and impedance-pH testing, we opted to study only symptomatic patients with scleroderma in order to justify the need of an invasive test. Finally, we did not assessed symptoms in our SSc patients in a structured manner. However, our experience and previous works on SSc patients support the reproducibility of our data and their clinical significance (3,5,6,8,46). 14

In conclusion, this is the first study evaluating BI levels in SSc without erosive esophagitis and these values were compared with those obtained in NERD patients and HCs. We showed that in patients with SSc, BI levels were lower than those in both NERD and HCs in both distal and proximal esophagus and this result is particularly interesting if we consider that SSc and reflux patients had similar exposure to acid. This supports the presence of a greater alteration of esophageal wall in SSc patients as a likely result of a deeper damage secondary not only to the increased reflux of gastric contents, but also to the deposition of collagen, which is the characteristic pathologic feature of this disease. Therefore, we suggest that the measurement of BI levels at both proximal and distal esophagus can be adopted as an indirect, but valid marker of the esophageal wall impairment in SSc patients. The advent of novel, poorly invasive, and easy to perform methods for the assessment of esophageal mucosal impedance (47) will provide the opportunity of performing this measurement without the need of probe insertion and, potentially, will modify our diagnostic approach to SSc patients with suspect esophageal involvement. However, further study are mandatory to prove the clinical utility of these data.

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Table I. Demographic and voluptuary characteristics of systemic sclerosis (SSc), non-erosive reflux disease (NERD) patients and healthy controls. P value

SSc

NERD

Healthy Controls

(n=50)

(n=52)

(n=50)

54 (18–83)

51 (22–78)

49 (24–73)

0.88

6 (12%)

19 (38%)

13 (26%)

0.03

Female n (%)

44 (88 %)

33 (62%)

37 (74%)

BMI, Kg/m2, median (range)

24 (17–31)

25 (17-36)

22 (18–33)

0.90

Coffee, n (%)

37 (74%)

35 (37%)

32 (64%)

0.83

Alcohol, n (%)

25 (50%)

26 (50%)

19 (38%)

0.50

Smokers, n (%)

8 (16%)

9 (17%)

5 (10%)

0.50

Median age (range), years Male n (%)

21

Table II. Clinical features of systemic sclerosis (SSc) patients. Clinical features

SSc (n=50)

Disease subtype Limited, n (%)

32 (64%)

Diffuse, n (%)

18(36%)

Disease duration Limited, median (95% CI), years

14 (0.5–32)

Diffuse, median (95% CI), years

2.5 (0.5–26)

Modified Rodnan skin score > 14

16/50

Active vascular ulcers

4/50

Pulmonary hypertension

5/50

ANA Pattern Homogeneous

9/50

Nucleolar

8/50

Spekled

8/50

Anti-centromere antibodies

27/50

Anti-Scl70

11/50

Patients with immunosuppressive therapy Cyclosporine

3/50

Hydroxychloroquine

2/50

Mycophenolate mofetil

2/50

Azathioprine

1/50

22

Table III. Median values and 95% CI of lower esophageal sphincter pressure (LESP) and distal waves amplitude in healthy controls, non-erosive reflux disease (NERD) and systemic sclerosis (SSc) patients. Manometry features Median LESP (CI 95%)

Healthy Controls (n=50)

NERD (n=52)

SSc (n=50)

18.1

11.1

13.2

(10.1 – 13.1)

(10.4 – 15.40)

120.0

89.5

26.0

(110.6 – 144.7) °

(74.2 - 100.9) ¥

(4.0 - 32.0)

(14.6 – 24.0) Median distal peristaltic wave amplitude (CI 95%)

*,§

*

p<0.0001 HCs vs NERD patients; §HCs vs SSc patients; ° p=0.0001 HCs vs NERD patients;

p<0.0001 NERD vs SSc patients

23

¥

Table IV. Median values and 95% CI of acid exposure time (AET) in upright and recumbent positions and total AET (a), median number of total and proximal refluxes (b) and median bolus clearing time (MBCT) in upright and recumbent positions and total MBCT (c) in healthy controls, non-erosive reflux disease (NERD) and systemic sclerosis (SSc) patients. a) pH-metry features

Healthy Controls (n=50)

NERD (n=52)

SSc (n=50)

0.3*

4.6

2.0

(0.2 – 0.6)

(2.9 – 5.1)

(0.8 – 4.2)

0.0

0.7

0.1

(0.0 – 0.0)*

( 0.1 – 1.9)

(0.0 – 1,5)

0.2

3.5

2.8

( 2.5 – 4.8)

( 1.1 – 4.7)

Healthy Controls (n=50)

NERD (n=52)

SSc (n=50)

26

67

71

( 16 – 28)*

( 55 – 75)

( 58 – 85)

4.5

26

15

(3 – 7)*

( 20 – 33)¥

( 9 – 20)

Median %T pH < 4.0 up (CI 95%) Median %T pH < 4 rec (CI 95%) Median %T pH < 4 tot (CI 95%)

*

(0.2 – 0,5)

*p <0.0001 HCs vs NERD and SSc patients b) Impedance features Median number of refluxes (CI 95%) Median number of proximal refluxes (CI 95%)

*p<0.0001 HCs vs NERD and SSc patients; ¥ p=0.005 NERD vs SSc patients .

c) Impedance features

Healthy Controls (n=50)

NERD (n=52)

SSc (n=50)

10

14

16

(7 – 11)*

(13 – 16)

(10.76 – 20.23)

4.5

11

15

(0 – 8)*

(9 – 14)

(8.76 – 17)

10

13

15

(8 – 11)*

(13 – 16.37)

(11.76 – 19)

Median Bolus Clearance Time up (CI 95%) Median Bolus Clearance Time rec (CI 95%) Median Bolus Clearance Time tot (CI 95%)

*p < 0.0001 HCs vs NERD and SSc patients

24

Table V. Median Baseline Impedance (BI) values with 95% CI at 3, 5, 7 and 17 cm above LES in healthy controls, non-erosive reflux disease (NERD) and systemic sclerosis (SSc) patients.

Median BI values

Healthy Controls (n=50)

NERD (n=52)

SSc (n=50)

2786.7

1737.3

979.3

(2443.8–2959.7)*,§

(1500.7–2959.7) ¥

(656.5–1318.2)

2479.0

2091.1

1088.7

(1822.4–2439.2) ¥

(879.9–1288.6)

2834.9

2462.7

1486.4

(CI 95%)

(2530.8–3161.8)§

(2107.6–2964.5) ¥

(1224.8–1713.6)

at 17 cm

2918.7

3106.2

2145.4

(2698.0–3448.5) ¥

(1978.8–2697.7)

at 3 cm (CI 95%) at 5 cm (CI 95%) at 7 cm

(CI 95%)

*,§

(2133.8–2853.3)

§

(2732.1–3475.5)

*p < 0.01 HCs vs NERD patients; § p < 0.0001 HCs vs SSc patients; ¥ p < 0.01 NERD vs SSc patients

25