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ORIGINAL ARTICLE  
Year : 2022  |  Volume : 65  |  Issue : 4  |  Page : 781-785
Spectrum of histopathological changes and its quantification using a scoring system in patients with gastroesophageal reflux disease


1 Department of Pathology, Tel Aviv Sourasky Medical Center, Israel
2 Department of Pathology, M.L.N. Medical College, Prayagraj, Uttar Pradesh, India
3 Department of Gastroenterology, M.L.N. Medical College, Prayagraj, Uttar Pradesh, India

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Date of Submission02-Jan-2021
Date of Decision05-Jan-2022
Date of Acceptance06-Jan-2022
Date of Web Publication02-Jun-2022
 

   Abstract 


Aim: The aim of this study was to evaluate the role of histopathological and histomorphometric features in oesophageal biopsy of patients presenting with symptoms of Gastroesophageal Reflux Disease (GERD). Material and Methods: Present study included 42 patients and 12 controls. Complete clinical evaluation followed by endoscopic examination of the patients was done and multipleoesophageal biopsies were taken. Biopsies were processed routinely and stained with Hematoxylin and Eosin and examined for any changes related to GERD. Morphometric assessment was done by using Leitz optical micrometer. The histological scoring was done based on the parameters: basal cell hyperplasia, stromal papillae elongation, cells with irregular nuclear contour (CINC), eosinophilic infiltrate, gastric and intestinal metaplasia. A numerical score was assigned to each parameter and sum of these scores represented the total score. Statistics: The statistical analysis was done using graph pad prism, Medcalc software and Windows MS office. P value and mean standard deviation (SD) was calculated. Results: The endoscopic findings of all the controls and 83.33% of patients were normal. Only 16.67% of patients had reflux associated changes of varying grades on endoscopy. Oesophageal biopsy of all patients had changes related to GERD on histology. Immunohistochemistry confirmed that cells with irregular nuclear contour were T- lymphocytes. The mean (SD) histological scoring of control and patients were 1.75 (0.62) and 5.66 (1.31) respectively. The difference was considered to be statistically significant (P < 0.001). Thus, it was suggested that a cut-off of histological score > 3 can be used to indicate GERD. Conclusion: Patients with gastroesophageal reflux symptoms can have normal endoscopic findings but can be diagnosed on the basis of histological changes in the squamous epithelium. Scoring of the histopathological parameters along with the cut-off value can give a definitive diagnosis of GERD.

Keywords: Cells with irregular nuclear contour, endoscopy, gastroesophageal reflux disease, histomorphometry

How to cite this article:
Thakur K, Varma K, Bhargava M, Singh T, Misra V, Misra SP, Dwivedi M. Spectrum of histopathological changes and its quantification using a scoring system in patients with gastroesophageal reflux disease. Indian J Pathol Microbiol 2022;65:781-5

How to cite this URL:
Thakur K, Varma K, Bhargava M, Singh T, Misra V, Misra SP, Dwivedi M. Spectrum of histopathological changes and its quantification using a scoring system in patients with gastroesophageal reflux disease. Indian J Pathol Microbiol [serial online] 2022 [cited 2022 Nov 30];65:781-5. Available from: https://www.ijpmonline.org/text.asp?2022/65/4/781/346514





   Introduction Top


Gastroesophageal Reflux Disease (GERD), gastric reflux disease or acid reflux disease is a chronic symptom of mucosal damage caused by stomach acid coming up into the esophagus.[1] There is increase in prevalence of symptom-based GERD and endoscopic reflux esophagitis in Asia.[2] Prevalence of GERD in an urban adult population from northern India is 16.2% which is similar to other industrialized countries.[3]

Reflux of gastric juice is central to the development of mucosal injury in GERD. Conditions that decrease lower oesophageal sphincter tone or increases abdominal pressure contribute to GERD. Tobacco, alcohol, obesity, and pregnancy are important etiological factors. GERD can present with various clinical symptoms like heartburn, epigastric pain, regurgitation and dysphagia. If left untreated it can lead to complication such as reflux esophagitis, oesophageal strictures, Barrett's esophagus and oesophageal adenocarcinoma.[4]

No definitive criteria have been established for diagnosis of GERD and it was diagnosed based on a history of classic symptoms and favorable response to antisecretory medical therapy. Latest guidelines have included oesophageal reflux and impedance monitoring studies for its diagnosis. However, if results are inconclusive by above techniques or patient fails to respond to appropriate antisecretory medical therapy or show clinical signs suggestive of complicated GERD, oesophageal gastro duodenoscopy and biopsy is advised.[5]

Endoscopically GERD can be classified into erosive and non-erosive subtypes; however, most of these patients may be normal on endoscopy and may show only histological evidence of GERD.[6] Histologically features of GERD are edema, basal cell hyperplasia, elongation of the papillae, lymphocytic inflammation, thinning of the squamous cell layer, and gastric or intestinal metaplasia.

Though studies have shown that histology has limited value in diagnosis of GERD, if systematically and objectively applied, it may render definitive diagnostic criteria especially in cases that appear normal on endoscopy. Hence, the aim of this study was to evaluate the spectrum of histopathological changes in esophagus of patients presenting with GERD in accordance with scoring system of Esposito et al.[7] to establish definitive criteria for diagnosis of GERD along with its endoscopic correlation.[8]


   Material and Methods Top


This prospective study was done in the Departments of Pathology and Gastroenterology. It included 42 patients presenting to Gastroenterology OPD with clinical features of gastroesophageal reflux disease over a period of one year. Symptoms included typical and atypical features of GERD as detailed below. Body mass index of all patients was noted and obesity was defined BMI of 30 kg/m2 or more.

Controls included 12 patients. These were subjects who did not present with symptoms of GERD and underwent UGI endoscopy for some indication other than GERD including malabsorption, weight loss, porto-caval gastropathy etc.

After informed consent history and clinical features were noted and UGI endoscopy was performed. Reflux-associated changes were graded according to the Modified Los Angeles Classification of GERD.[9] Biopsy was taken from four quadrants of oesophagus2 cm above the squamo-columnar junction, fixed in 10% buffered formalin solution, and processed routinely. Hematoxylin and Eosin stained sections were examined for histopathological details. Only sections which were properly oriented, adequately stained and showed the presence of lamina propria were included in the study. Reflux-related changes such as basal cell hyperplasia, elongation of papillae and intraepithelial lymphocytes, gastric or intestinal metaplasia was noted as defined in the criteria as Esposito et al.[7] The intraepithelial lymphocytes showing irregular nuclear contour (CINC) were examined in five HPF in areas that were showing maximum density and their average number per high power field was calculated manually. Immunohistochemical (IHC) staining of formalin-fixed paraffin-embedded sections was performed using anti-CD3 monoclonal antibody to confirm the nature of CINC. The subepithelial zone was thoroughly examined to look any neutrophils, eosinophils, lymphocytes and fibrosis. Morphometric assessment was done using Leitz optical micrometer. The total thickness of squamous epithelium and basal cell thickness and elongation of stromal papilla was noted in five well-oriented areas. Averages of thickness of squamous epithelium, basal cell thickness and distance of stromal papilla in squamous epithelium were calculated. Percentage of basal cell thickness and distance transversed by stromal papillae in squamous epithelium was also calculated. A modified histological scoring was done based on scoring system of Esposito et al.[7] Basal cell hyperplasia, stromal papillae elongation, intraepithelial lymphocytes (CINC), eosinophils, neutrophils and gastric and intestinal metaplasia were noted in all oesophageal biopsies of control and patients. A significant increase in CINC was found in our study population in contrast to eosinophil and neutrophils. A numerical score was assigned to all the above parameters except neutrophilic scores. The sum of these scores represented the total score for each biopsy specimen [Table 1].
Table 1: Scoring scale of Histological parameters

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Ethics

The study was approved by the ethical committee of our institute. Written informed consent was taken from each patient before the procedure (Date of approval : 12-09-2018).

Statistical analysis

The statistical analysis was done using graph pad prism, Medcalc and Windows MS office, mean and standard deviation (SD) were calculated. A value of P ≤ 0.05 was taken as critical level of significance


   Results Top


The mean (±SD) age of presentation of patients in this study was 47.19 ± 13.8 years with male preponderance and male: female ratio of 2.5:1. Maximum numbers of patients were in the age group of 31 – 50 years. The most common presentation was found to be heartburn (90.47%), followed by regurgitation (80.95%), epigastric pain (59.52%), upper abdominal discomfort (42.85%) and vomiting in 4.76% of patients. Obesity was present in 47.61% of patients. Out of 42 patients' endoscopic findings of GERD was present in only 7 (16.67%) patients. Endoscopy was normal in 83.33% of patients. 16.67% of patients had reflux associated changes of varying grades (Modified Los Angeles Classification A through D) on endoscopy including erythema, erosions and ulcerations.

Oesophageal biopsy of controls showed normal histological findings whereas biopsy of all patients had changes related to GERD [Table 2]. Basal cell thickness was increased in biopsies from all the patients and was >25% in 90.47% of patients [Figure 1]a. Stromal papillae showed marked elongation in most of the biopsies. It was >66% in 88.09% of patients [Figure 1]b. The epithelium showed marked infiltration by lymphocytes (IEL). IEL/HPF was >10 in 61.90% of patients [Figure 2]a. Most of these intraepithelial lymphocytes were seen as CINC. IHC staining using anti-CD-3 monoclonal antibody confirmed them to be T- lymphocytes [Figure 2]b. Increased eosinophils/HPF >10 was seen in only one patient and none of the controls. None of the cases or controls showed a significant increase in neutrophils hence it was not included in the scoring system. Gastric metaplasia was present in 9.52% and intestinal metaplasia was present in 7.14% of patients. None of the cases showing metaplasia showed clinical or endoscopic evidence of Barretts hence was not classified as such. The subepithelial zone was seen only in 14.28% of patients.
Figure 1: (a) Showing expansion of the basal cells leading to basal cell hyperplasia (H and E, x400). (b) Showing stromal papillae elongation (H and E, x400)

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Figure 2: (a) Showing increased intraepithelial lymphocytes (black arrows) seen as cells with irregular contour (H and E, x400). (b) Immunohistochemical staining using anti CD-3 monoclonal antibody confirmed the cells with irregular nuclear contour (red arrows) to be T-lymphocytes (IHC × 400)

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Table 2: Comparative table showing Histomorphometric findings of oesophageal biopsies in Controls and Patients

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Histological scoring showed that most of controls had a histological score of 1 and 2 (58.3% and 33.3% respectively) whereas only 8.3% had a score of 3. In contrast, patients showed a higher score i.e., 6 (35.7%), 5 (33.3%), 4 (16.6%), 8 (9.5%) and 9 (4.76%). When mean (SD) score of patient and control was compared, the difference was found to be statistically significant (P < 0.001). Mean + 2 SD of control was calculated. It was found to be 2.99 [Figure 3].
Figure 3: Bar diagram showing mean histological score

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   Discussion Top


The prevalence of GERD has increased over the years ranging from 2.5% to 25%. Accurate diagnosis is important as even mild disease can progress to severe forms in 1–22% cases and 13% can develop Barrett's esophagus. It is also a major risk factor for development of oesophageal adenocarcinoma.[10] Our study shows that GERD is more prevalent in males with mean (±SD) age of presentation being 47.19 ± 13.84 years which is in accordance with the results of a meta-analysis revealed that the male to female ratio was 1.57 in the prevalence of reflux esophagitis Obesity was present in 47.61% of patients and thus appeared as important risk factor of GERD as has been reported in earlier studies. Tobacco consumption was present in 28.57% of patients. These findings are in accordance with previous studies.[3],[11]

Clinical definition of GERD according to Montreal consensus is based on troublesome symptoms due to reflux of stomach contents. The most common presentation was found to be heartburn (90.47%), followed by regurgitation (80.95%), epigastric pain (59.52%), upper abdominal discomfort (42.85%) and vomiting in 4.76% of patients. Simadibrata et al.[12] also found heartburn whereas as Zuberi et al.[13] reported epigastric pain to be most common presentation in patients with GERD. But diagnosis based on purely symptoms has low sensitivity (38%).[12],[13]

The revised ROME IV criteria recommend upper GI endoscopy with or without biopsy in cases of refractory GERD. We used the Los Angeles classification system for endoscopic assessment.[14] In present study, out of 42 patients' endoscopic findings of GERD were present in only 7 (16.67%) of patients. Endoscopy was normal in 83.33% of patients. Thus, we found that non-erosive subtype to be more prevalent which is in concordance with a study done by Zuberi et al.[13] On the other hand, histopathological changes associated with reflux were present in all the 42 patients included in this study. Studies have shown sensitivity of endoscopy to be low 30-50% as was seen in our study. But it does play a role in grading the severity of esophagitis, predicting its progression and taking a targeted biopsy. Thus, we found histology to be sensitive enough to diagnose GERD even when endoscopic findings were normal in patients with clinical diagnosis of GERD. Previous study also found that histological examination of oesophageal mucosa to be more sensitive in detecting subtle changes in mucosa and diagnosing non-erosive reflux disease (NERD).[15],[16]

Earlier studies have reported histology to have a limited role in diagnosis of GERD as the changes seen were non-specific. However, a multicenter study in 2010 developed subjective and standardized criteria for GERD diagnosis which was validated in 2014 (histoGERD trial). Using their criteria, the diagnostic histological findings of patients with GERD seen in our cases were basal cell hyperplasia > 15%, elongation of papillae > 60%, lymphocytic infiltration, neutrophilic inflammation and columnar and intestinal metaplasia.[17] We also found a predominant lymphocytic infiltration in our study in comparison to other inflammatory cells i.e., eosinophils and neutrophils. Mild eosinophilia in biopsy samples as a marker of GERD was the prevailing notion in the 1970s and1980s but this concept has been refuted by many studies. Also the role of adhesion molecules, particularly eotaxin-3, has been identified as a highly expressed biomarker of Eosinophilic Esophagitis and not GERD. Hence dense oesophageal eosinophilia was distinctly uncommon in patients with GERD and similarly our study population did not show a significant increase in eosinophils.[18]

A significant increase in Intraepithelial lymphocytes was found in our study. These lymphocytes were seen as cells with irregular nuclear contour which have been reported in previous studies and named as “squiggle cells”.[18] In GERD, lymphocytes may be most significant inflammatory cells present, however, their number is relatively low and if > 10-50 IELs/HPF, a diagnosis of lymphocytic esophagitis may be considered.[19],[20] Immunohistochemically profiling of these cells showed them to be T- lymphocytes. Recent studies have also shown a high prevalence of specialized intestinal metaplasia at the gastroesophageal junction in patients without endoscopic evidence of columnar lining of the esophagus. We found an incidence of 7.1% unsuspected specialized intestinal metaplasia in our cases which was similar to previous studies.[19]

We found that the above spectrum of histological changes were present in all patients presenting with GERD. These histological changes were well documented in oesophageal biopsies in patient presenting with GERD.[16] Takubo et al.[21] found that a set of histologic changes that are invariably reflux associated were not found in the small set of biopsy specimens examined and these histologic changes may exist, but these changes were not specific to patients with GERD. They found that changes were seen individually in 23% to 71% of the GERD group, but no single change was seen in 100% of patients. They suggested that to enable pathologists to find such a set of histologic changes, endoscopists should take targeted biopsies from multiple sites that have different endoscopic findings.[21]

The histological scoring done in the present study was based on the scoring done by Esposito et al.[7] They evaluated oesophageal biopsies of children with suspected esophagitis and concluded that histological score derived by evaluating and grading all classical parameters had a satisfactory diagnostic sensitivity and a good specificity when biopsy specimen was adequate, well oriented and the lamina propria was present, whereas increased CINC number provided a higher sensitivity in a superficial biopsy where all parameters were not assessable. They found that cut-offs of 6 for score and of 4 for CINC density provided the best sensitivity and specificity.[7]

Histological scoring done in patients was in range of 4 -9 whereas in controls it was between 1-3. The mean (SD) histological scoring of control was 1.75 (0.62). The mean (SD) histological scoring of patients was 5.66 (1.31) and P value calculated was found to be statistically significant. Mean + 2 SD of control was found to be 2.99. Thus, it was suggested that a cut off of histological score > 3 can be used to indicate GERD. Hence, this study proposes that subjective criteria and cut-off score using the Esposito score can be applied to cases with typical symptoms of GERD even in absence of endoscopic findings and can help in establishing the diagnosis.

To conclude, patients with GERD may have normal endoscopic findings but can be diagnosed on histology i.e., non-erosive GERD is more prevalent. Histological changes in squamous epithelium such as basal cell thickness >15%, elongation of papilla >60% and intraepithelial T lymphocytes >5/HPF along with their scoring with a cut off value can provide the diagnosis of GERD even in superficial biopsy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Kachnar Varma
Department of Pathology, M.L.N. Medical College, Prayagraj, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijpm.ijpm_5_21

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