|Year : 2022 | Volume
| Issue : 3 | Page : 212-220
Histomorphological Evaluation of Chronic Gastritis and an Attempt to Prognosticate Gastric Intestinal Metaplasia with Selective Mucin Gene Expressions in Indian Population
Pooja E Moorthy, Vijayashree Raghavan, Rajesh Kanna
Department of Pathology, Chettinad Hospital and Research Institute, Chettinad Academy of Research and Education, Rajiv Gandhi Salai, Kelambakkam, Tamil Nadu, India
|Date of Submission||24-Mar-2022|
|Date of Decision||11-Jul-2022|
|Date of Acceptance||15-Jul-2022|
|Date of Web Publication||3-Oct-2022|
Professor, Department of Pathology, D Block, Chettinad Hospital and Research Institute, Kelambakkam, Kanchipuram, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Introduction Intestinal metaplasia (IM) is a well-established precancerous lesion. MUC2 and MUC5AC are mucin genes that have been studied in gastric IM to classify and determine which type of metaplasia will progress to malignancy. H.pylori, an important causative organism for gastric carcinoma, can be detected using anti-H. pylori antibodies (AHA) in case of suspicion when routine histochemical stains are negative.
Aim This study aims to determine the expression of MUC2 and MUC5AC in gastric IM in chronic gastritis to differentiate complete and incomplete IM and to predict and prognosticate its role in the possible evolution of malignancy. This study also aims to determine the expression of AHA in Giemsa negative cases with mild to severe activity.
Materials & Method A total of 345 gastric biopsy cases were included in the study. About 35 cases with IM on histology were subjected to Alcian Blue (AB) at pH 2.5 and markers MUC2 and MUC5AC to classify IM. To further subclassify the type II and type III IM, AB with periodic acid–Schiff at pH 1.5 was used. About 45 out of 171 Giemsa negative cases with activity were subjected to AHA.
Results 51.43% cases had complete IM and 48.57% had incomplete IM. And, 25.7% were type II and 22.8% were type III IM. About 20 out of 45 Giemsa negative cases showed AHA positivity, indicating sensitivity as high as 70.15%.
Conclusion MUC2 and MUC5AC expression are good parameters in the progression of IM togastric cancer (GC) and can be used as a good prognostic marker for GC. Detection and eradication of H.Pylori using AHA with appropriate treatment will reduce the risk of developing gastric carcinoma.
Keywords: Alcian Blue (AB), anti-H. pylori antibody (AHA), gastric cancer (GC), intestinal metaplasia (IM), MUC2, MUC5AC
|How to cite this article:|
Moorthy PE, Raghavan V, Kanna R. Histomorphological Evaluation of Chronic Gastritis and an Attempt to Prognosticate Gastric Intestinal Metaplasia with Selective Mucin Gene Expressions in Indian Population. Int J Nutr Pharmacol Neurol Dis 2022;12:212-20
|How to cite this URL:|
Moorthy PE, Raghavan V, Kanna R. Histomorphological Evaluation of Chronic Gastritis and an Attempt to Prognosticate Gastric Intestinal Metaplasia with Selective Mucin Gene Expressions in Indian Population. Int J Nutr Pharmacol Neurol Dis [serial online] 2022 [cited 2022 Nov 29];12:212-20. Available from: https://www.ijnpnd.com/text.asp?2022/12/3/212/357220
| Introduction|| |
Intestinal metaplasia (IM) is the partial replacement by metaplastic goblet cells of intestinal morphology of the antral or fundic mucosa. IM is a precancerous lesion and has the potential to evolve into gastric carcinoma. It can be identified by the routine stain hematoxylin and eosin (H&E), special stains like Alcian Blue (AB), high-iron diamine, and periodic acid–Schiff PAS/AB immunohistochemical staining markers like MUC1, MUC2, MUC5AC, and MUC6. Mucins are glycosylated glycoproteins which are the component of the mucous viscous gel which covers the surface of the epithelial tissue. Mucins play an important role in the pathogenesis of benign and malignant disease of the secretory epithelial cells. According the structure and function of the mucin, it can be divided into transmembrane and secreted mucins. MUC5AC belongs to secreted mucin. The genes for this mucin are found in chromosome 11p15.5. Its synthesis is regulated by biologically active molecules. The normal gastric mucosa shows the expression for MUC1, MUC5AC, and MUC6, where MUC1 and MUC5AC show expression in the superficial epithelium. MUC6 is seen in the deeper glands mainly in the gastric mucosa. Normal gastric mucosa produces mainly neutral mucin, except for the mucin secreting cells present in the neck glands of the stomach producing acid mucin. The human stomach expresses MUC1, MUC5AC, and MUC6. Small intestine expresses MUC2 in the goblet cells. In case of carcinoma, loss of expression of MUC5AC is noticed. Like in carcinoma, complete metaplasia and dysplasia also exhibit a loss of expression of MUC5AC. The possible reason could be due to glycosylation changes or increased mucin heterogeneity. These findings of mucin alteration can be regarded as molecular markers for malignant transformation of the gastric mucosa. Changes in the expression levels and/or distribution profile of MUC5AC lead to cancer of different organs like lungs, gastrointestinal tract, pancreas, hepatobiliary system, and male and female reproductive system. MUC2 is generally expressed in the duodenum and it is normally not expressed in normal gastric mucosa.
Helicobacter pylori (H. pylori) is the microorganism responsible for the persistent bacterial infection of the stomach worldwide. Significant numbers of cases are affected by H. pylori. In developing countries, the prevalence of infection is as high as 90%, whereas in developed countries 50% of the population is affected. Idiosyncratic methods to detect H. pylori infection are many, and the choice of one method depends upon several factors, such as the availability of diagnostic tests, the need to perform an endoscopy, cost, accessibility, advantages and disadvantages of each method, and age of patients. H. pylori are associated with peptic ulcer disease, gastric ulcers, mucosa-associated lymphoid tissue lymphoma, and gastric cancer. Extermination of H. pylori is required for the treatment and prevention of the infection and malignancy. There are multiple methods that are currently available to detect the presence of H. pylori, each with its own advantages, disadvantages, and limitations. It is crucial to know the presence of H. pylori in gastric biopsy for giving timely and appropriate patient care. Though different methods have been described for identification of the gram negative H. pylori (invasive and noninvasive methods), however, there is no definite method to prove H. pylori. Thus, histological detection of H. pylori in gastric biopsies still remains the most common and the most sensitive test. A classic way to categorize the methods for diagnosing gastritis is according to the severity of the disease, whether or not an endoscopy is required. Invasive tests include histomorphological evaluation (biopsy), immunohistochemical staining (IHC), culture, polymerase chain reaction, and the rapid urease test, all of which are performed on tissue obtained during endoscopy. Alternatively, the urease breath test, serology, saliva antigen test, and stool antigen test can be performed as noninvasive procedures.
H&E, Giemsa, and IHC staining techniques have helped in the identification of H. pylori. H&E stain can directly identify H. pylori in high magnification, but it becomes difficult to identify H. pylori when it is present in low density and when atrophic mucosal changes are present. Giemsa stain is the preferred method over H&E in many laboratories, but it gives false-negative results when the organisms are few or in patients with prior incomplete treatment. IHC stains have advantages when patients are partially treated for H. pylori gastritis or have a low density of organism or atypical forms (coccoid) of H. pylori. IHC has high specificity as it can exclude other similar-shaped organisms.
In this study, we aim to determine the expression of MUC2 and MUC5AC in chronic gastritis cases, to classify IM to determine which type of metaplasia will progress to malignancy. We also aim to study the expression of anti-H. pylori antibody (AHA) in cases with mild to severe activity, which were reported negative for H. pylori with the aid of Giemsa stain.
| Materials and Methods|| |
Approval for the observational study was obtained from the Institutional Human Ethics Committee (IHEC).
Tissue sample collection
A total of 345 cases of gastric biopsies were taken on which demographic analysis was done. Histology-proven cases of IM in gastric biopsy and active gastritis with negative H. pylori cases between January 2019 and June 2020 were retrieved in the form of formalin-fixed, paraffin-embedded tissue blocks from the archives of the Department of Pathology, Chettinad Hospital and Research Institute. Of these, 35 cases of IM and 45 cases of active gastritis negative for H. pylori were identified. Four cases were not included in the study due to the unavailability of paraffin blocks.
A total of 345 cases were included in the study. From the 345 cases, 35 cases had morphology of IM and were subjected to AB at pH 2.5. These were also subjected for IHC marker MUC2 and MUC5AC to classify as complete or incomplete IM. The 35 IM cases were subjected for AB/PAS at pH 1.5 to further subclassify as type II or type III incomplete IM.
All the 345 cases had Giemsa staining performed for the detection of H. Pylori, out of which, 174 cases showed positivity. About 171 cases were negative for H. pylori on Giemsa. Forty-five cases which were reported as negative for H. pylori with the aid of Giemsa stain were subjected to the IHC marker AHA with mild to severe activity. Normal intestinal biopsy and chronic active H. pylori gastritis were taken as the control for the cases to compare with the IHC markers. The tissue blocks were procured and sections were cut at 3.5 μ. AB staining was done on the selected 35 cases. Then, appropriate areas for performing immunohistochemistry were marked. The pathological characteristics using Updated Sydney classification were obtained from the retrieved slides from the Department of Pathology.
MUC2, MUC5AC, and AHA
IHC was performed on the selected blocks for MUC2, MUC5AC, and AHA. About 3.5 μ thick sections were cut in a semiautomated microtome (Leica Biosystems, RM2245) using disposable blades. For IM (35 cases), two sections − one for MUC2 and one for MUC5AC − were taken. Positive control for MUC2 and MUC5AC from the normal intestine was used. One section was taken for AHA (45 cases), and one positive control from H. pylori gastritis was used. Positively charged hydrophobic slides were used. The staining was done using MUC2 (PR194-6ml RTU, PathnSitu, Chennai, India; Clone: EP187), MUC5AC primary antibody (PM233-6ml RTU, PathnSitu; Clone: CLH2), and AHA (PR145-6ml RTU, PathnSitu; Clone: Polyclonal).
Assessment of MUC2, MUC5AC, and AHA
All slides were evaluated for both the intensity of the staining and the percentage of positive cells. MUC2 shows expression in cytoplasm and membranes of goblet cells. MUC5AC shows expression in membranes of the columnar epithelium of gastric mucosa. The intensity of the staining was classified [Table 1]. AHA shows expression in the H. pylori organism. The scoring was classified based on staining intensity and background was assessed for nonspecific staining of extracellular mucin and nonbacterial particles [Table 2].
The frequencies and percentage analysis of various parameters was done using SPSS software version 23. Data visualization was done using SPSS software version 25.
| Results|| |
A total of 345 cases of gastric biopsies were studied between January 2019 and June 2020. Out of these 345 cases, 67 cases were subjected to IHC markers MUC2, MUC5AC, and AHA. About 35 cases were histomorphologically proven cases of IM, 51.43% cases (n = 18) had complete IM and 48.57% cases (n = 17) had incomplete IM. The incomplete IM were subclassified using AB/PAS at pH 1.5. About 25.7% cases (n = 9) were type II and 22.8% cases (n = 8) were type III IM [Figure 1], [Table 2]. All 345 cases had Giemsa staining. About 171 cases were negative for H. pylori on Giemsa. Forty-five cases which were reported as negative for H. pylori with the aid of Giemsa stain were subjected to the IHC marker AHA (n = 45). Out of 45 cases, 20 (44.4%) of them were positive for AHA, indicating sensitivity as high as 70.15% [Figure 2], [Table 3] and [Table 4].
|Figure 1 Positivity of different tests in IM (IM- Intestinal Metaplasia).|
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|Figure 2 Relationship between Giemsa and Anti H pylori antibody testing.|
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| Discussion|| |
Expression of MUC2 and MUC5AC in IM
Total of 35 cases which exhibited IM in H&E stain from the total of 345 cases were stained with AB at pH 2.5 and were positive for IM [Figure 3]. These 35 cases were subjected to IHC MUC2 and MUC5AC. About 18 cases (51.43%) showed absence or weak expression of MUC5AC in the gastric columnar epithelium and goblet cells with de novo expression of MUC2 in the goblet cells (vacuolar staining). These 18 cases with the IHC profile we concluded as complete IM or type I IM [Figure 4] and [Figure 5]. Out of these 18 cases, two were positive for malignancy − gastric adenocarcinoma (Lauren diffuse and WHO 2019–poorly cohesive-signet-ring cell phenotype), and another case of mucin secreting adenocarcinoma with signet-ring cells according to WHO 2019, they showed complete IM with faint expression of MUC2 and MUC5AC. About 17 cases (48.57%) retained the expression of MUC5AC in the gastric columnar epithelium and in the goblet cells (cytoplasm and supranuclear pattern of staining). They also showed de novo expression of MUC2 in the goblet cells (vacuolar staining). So, these 17 cases were concluded as incomplete IM [Figure 3]. There were two cases which showed moderate to strong expression of MUC5AC in the gastric columnar epithelium and the goblet cells which gradually became weak suggesting that these two patients are going for complete IM or type I IM [Figure 6]. But these cases were taken as incomplete IM as predominant pattern was taken into account. To further differentiate between type II and type III IM, a combined histochemical stain of AB/PAS at pH 1.5 was done. Nine cases (25.7%) belonged to type II and eight cases (22.8%) belonged to type III IM. Of these 35 cases, 21 cases were Giemsa proven H. pylori positive cases which were subjected only to the mucin stains (AB, MUC2, MUC5AC, and AB/PAS). The 13 cases which had mild to moderate activity, which are negative for H. pylori (Giemsa stain) were subjected to AHA along with MUC5AC and MUC2. Of these 13 cases, three showed positivity for H. pylori, one of which was positive for malignancy (poorly differentiated gastric adenocarcinoma) [Figure 7]. These three cases had complete IM with weak to no expression of MUC5AC and goblet cell positivity by MUC2
|Figure 3 Alcian blue positivity in the goblet cells (a- 10X) (b) Type III IM which has taken Alcian blue stain and PAS is negative at 40X (c) & (d) Alcian blue/PAS (pH 1.5) positivity in the goblet cells, the goblet cells have taken a purplish tone - Type II IM or Incomplete IM (Arrow head red & black denotes the goblet cells with purple colour) (c-40X and d-20X) (IM- Intestinal Metaplasia, PAS - Periodic Acid Sciff).|
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|Figure 4 IHC for MUC2 and MUC5AC in complete IM (a) and (b) shows dense positivity on goblet cells by MUC2 (20X & 40X). (c) shows no expression of MUC5AC on the goblet cells (40X) (IM- Intestinal Metaplasia).|
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|Figure 5 (a) MUC2 showing strong positivity in goblet cells (IHC at 20X) (b & c) MUC5AC showing strong positivity in goblet cells and columnar epithelium (IHC at 40X), (d) AB/PAS at pH 1.5 showing AB positive on goblet cells (40X)- Type III IM (AB -Alcian Blue, PAS - Periodic Acid Schiff, IM- Intestinal metaplasia).|
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|Figure 6 IHC for MUC2 and MUC5AC in a case going to complete IM from incomplete IM (a) and (b) shows dense positivity on goblet cells by MUC2 (20X & 40X). (c) An area with strong expression of MUC5AC in the columnar cells and goblet cell cytoplasm (red arrow) (d & e) show no expression of MUC5AC on the goblet cells (arrow black and red) with some retains the expression of MUC5AC (20X & 40X) (IHC - Immunohistochemistry, IM- Intestinal Metaplasia).|
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|Figure 7 IHC for MUC2, MUC5AC and anti H pylori antibody in a case of poorly differentiated adenocarcinoma. (a) Poorly differentiated adenocarcinoma (H&E 20X). (b) Mucinous adenocarcinoma (20X). (c) Alcian blue showing focal mucinous area long with the tumour cells. (d) Focal weak staining for MUC2 in the goblet cells and few tumour cells (20X). (e) Focal weak staining for MU5AC in the tumour cells (20X). (f) Anti H pylori antibody show positivity for H pylori (blackarrow) (100X).|
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According to the study done by Celso et al., the overall mucin expression in type I or complete IM is characterized by absence or weak expression of MUC1, MUC5AC, and MUC6 expression in the gastric mucosa with de novo expression of MUC2 in the goblet cells alone. In type IIA IM, the overall mucin expression of MUC1, MUC5AC, and MUC6 is maintained in the gastric epithelium along with de novo expression of MUC2 in the goblet cells. The immunodetection of mucin in type IIB or type III of incomplete IM is similar to that of type IIA IM.
Bhat et al. in their study came up with the evidence like from other studies that expression of MUC5AC was highly expressed in the gastric mucosa, also it was retained in both goblet and columnar cells in case of incomplete IM. But MUC5AC expression was weak or absent in complete IM and in gastric carcinoma like in our studies.
Subramani et al. studied the expression of mucins on normal gastric epithelium, IM, dysplasia, and carcinoma. In normal gastric epithelium, MUC5AC was diffusely and strongly expressed in the superficial foveolar epithelium, MUC6 was expressed in the antral glands, whereas, MUC2 was not detected in the normal gastric mucosa. MUC2 was expressed in most of the specimens with presence of goblet cells (cytoplasmic pattern of staining); MUC5AC and MUC6 showed decreased expression in both the goblet cells and columnar cells with variation of stain from case to case in IM. The expression of MUC5AC and MUC6 was decreased in dysplastic areas. Early carcinoma showed expression of MUC5AC and MUC6, whereas in advanced stages faint or no expression of MUC5AC and MUC6 was seen. They explained that the possible expression of MUC5AC and MUC6 in early gastric carcinomas could be due to the retained gastric phenotype cells before the complete neoplastic development.,
Expression of AHA
Total of 345 cases from the study were subjected to Giemsa stain for H. pylori, 174 (50.7%) cases were positive and 171 (49.3%) were negative. The positive cases were between the ages of 35 and 50 years with mean age of 42.4 years. Males were affected more than females. From the 171 negative cases, 45 cases which had mild to severe activity in H&E stain were subjected to AHA. So, these cases were doubtful for the presence of H. pylori. Out of 45 cases, 20 cases (44.44%) were positive for AHA and 25 were negative for both AHA and Giemsa. Out of the 20 positive cases, two were in coccoid form. The grading for AHA according to Ali et al. was done. Out of the 20 positive cases for AHA, 17 of them had mild activity, three had moderate activity, and one has severe activity. IHC is the gold standard for detecting H. pylori organism. It should be considered in case of doubt when Giemsa shows negativity; it can detect possible low density or coccoid forms of organism. Posttreatment, some of the H. pylori, instead of the classical spiral or curvilinear form, can exhibit atypical forms which can be picked up in IHC. Our study had two cases with coccoid form of H. pylori [Figure 8].
|Figure 8 IHC for anti H pylori antibody in 4 different cases showing positivity for H pylori (a) the arrow mark with double head and red arrow shows the H pylori organism, (b, c and d) coccoid forms of H pylori taken up the stain (Arrow black and red) (40X and 100X). (IHC- Immunohistochemistry).|
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In a study done by Sunita et al., 20.57% cases of H. pylori were identified in both Giemsa and IHC in mild chronic gastritis whereas, 81% and 87.34% in moderate chronic gastritis, 92.8% and 95.71% in severe chronic gastritis in Giemsa followed by IHC. In a study by Ali et al., a comparison between conventional histochemical stain and IHC marker was done. It was suggested that IHC was highly sensitive but to employ it as a routine stain like Giemsa would be difficult in terms of cost and time. But should be considered when histochemical stains are negative or posttreatment to check on the bacterial load.,,,,
The important limitation in our study was the small number of cases which were included in the study and the lack of follow-up of details in the cases. If the studies were done on a larger group of population then it would be useful to implicate for prognosis and treatment modality with good follow-up details. These markers can be routinely used for future prognostication and categorization in the high-risk group for follow-up.
| Conclusion|| |
MUC2 and MUC5AC expression are good parameters in the progression of IM to gastric cancer (GC) and can be used as a good prognostic marker for GC. Detection and eradication of H.Pylori using anti H pylori antibody marker with severe activity and negative for Giemsa stain with appropriate treatment will reduce the risk of developing gastric carcinoma.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]
[Table 1], [Table 2], [Table 3], [Table 4]