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Abstract
Background: Epithelial-mesenchymal transition (EMT) is the heart of invasion. EMT associated with cancer progression and metastasis is known as type III EMT. Beta-catenin, E-cadherin, and MMP9 markers of EMT are routinely employed for diagnostic purposes. Aims: We employed these markers to study EMT by immunohistochemistry (IHC) in gall bladder cancer (GBC) with respect to depth of tumor invasion, clinical outcome, and disease-free survival. Settings and Design: This was a prospective case-control study. Material and Methods: Seventy gall bladders were included (50 GBC and 20 CC). After detailed histology, immunoexpression was studied in terms of percentage and strength of expression. Statistics Analysis Used: Expression was compared between CC and GBC by Student t test and analysis of variance. Kaplan–Meier was used for survival analysis, and the extent of agreement (“Kappa”) was calculated. Results and Conclusions: The age of incidence of GBC was 49.40 (+11.6) years with female predominance (F:M = 4:1). In 88% (44/50) of GBC, the fundus was involved. Moderately differentiated adenocarcinoma was most frequent [54%; 27/50]. Significant downregulation of E-cadherin (P = 0.022) and beta-catenin (P < 0.001) and upregulation in MMP9 (P < 0.001) were seen in GBC with respect to CC with significant association among them. MMP9 expression was significantly associated with higher tumor stage but with chemotherapeutic response. Our results display that epithelial-mesenchymal transition type III plays a role in GBC invasion. MMP9 overexpression and loss of membranous beta-catenin may be considered a marker for poor clinical outcomes and advanced disease.
Keywords: Beta-catenin, E-cadherin, epithelial-mesenchymal transition (EMT), gall bladder cancer, MMP9
How to cite this article: Introduction 
Gall bladder cancer (GBC) is often a terminal malignancy as the common presenting clinical symptoms such as abdominal pain, jaundice, and vomiting occur only when the cancer spreads to other organs. Transabdominal ultrasound, CT scan, endoscopic ultrasound, MRI, and MR cholangiopancreatography (MRCP) can be used for diagnosis. However, a biopsy is the only certain way to tell whether the growth is malignant or not.
Radical excision is currently the cornerstone of management of GBC and is considered as the only potentially curative modality for patients with GBC. However, more than 70% of cases are unresectable owing to local invasion and metastasis.
Metastatic cascade is divided into two phases: 1) invasion of the extracellular matrix (ECM) and 2) vascular dissemination, homing of tumor cells, and colonization. Invasion of the ECM initiates the metastatic cascade and is an active process that can be resolved into several steps, which are a) changes (“loosening up”) of tumor cell–cell interactions, b) degradation of ECM, c) attachment to novel ECM components, and d) migration of tumor cells.[1],[2]
An epithelial-mesenchymal transition (EMT) is a biologic process by which an epithelial cell undergoes multiple biochemical changes that enable it to assume a mesenchymal cell phenotype. These changes provide the cell with enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and greatly increased production of extracellular matrix (ECM) components. The completion of an EMT is signaled by the degradation of the underlying basement membrane and the formation of a mesenchymal cell that can migrate away from the epithelial layer in which it originated.
EMTs are encountered in three distinct biological settings that carry very different functional consequences and are accordingly classified into three types: 1) Type 1 EMT: EMT during implantation, embryogenesis, and organ development; 2) Type 2 EMT: EMT associated with tissue regeneration and organ fibrosis; 3) Type 3 EMT: EMT associated with cancer progression and metastasis.[3],[4]
Multiple molecular pathways have been implicated in EMT with complex mechanisms. Among them, some recognized markers are E-cadherin and beta-catenin.
Cell–cell interactions are mediated by the cadherin family of transmembrane glycoproteins. E-cadherins mediate homotypic adhesions in epithelial tissue, thus serving to keep the epithelial cells together and to relay signals between the cells. Intracellularly, the E-cadherins are connected to beta-catenin and the actin cytoskeleton. In several epithelial tumors, there is a downregulation of E-cadherin expression. Presumably, this downregulation reduces the ability of cells to adhere to each other and facilitates their detachment from the primary tumor and their advance into the surrounding tissues.[5],[6]
The second step in invasion is local degradation of the basement membrane and interstitial connective tissue. Tumor cells may either secrete proteolytic enzymes themselves or induce stromal cells (e.g., fibroblasts and inflammatory cells) to elaborate proteases. Many different families of proteases, such as matrix metalloproteinases (MMPs), cathepsin D, and urokinase plasminogen activator, have been implicated in tumor cell invasion. MMPs regulate tumor invasion not only by remodeling insoluble components of the basement membrane and interstitial matrix but also by releasing ECM-sequestered growth factors. Indeed, cleavage products of collagen and proteoglycans also have chemotactic, angiogenic, and growth-promoting effects; for example, MMP-9 is a gelatinase that cleaves type IV collagen of the epithelial and vascular basement membrane and stimulates the release of VEGF from ECM sequestered pools.[7],[8]
The third step in invasion involves changes in the attachment of tumor cells to ECM proteins. Normal epithelial cells have receptors, such as integrins, for basement membrane laminin and collagens that are polarized at their basal surface. These receptors help to maintain the cells in a resting, differentiated state. Loss of adhesion in normal cells leads to induction of apoptosis; not surprisingly, tumor cells are resistant to this form of cell death. In addition, the matrix itself is modified in ways that promote invasion and metastasis.
The final step of invasion involves propelling tumor cells through the degraded basement membranes and zones of matrix proteolysis.
Thus, with the above understanding, the question arises that 'Will immunoexpression alterations of markers such as E-cadherin, beta-catenin, and MMP-9 be seen in GBC with respect to chronic cholecystitis (CC) and is there any clinical significance of their up or downregulation?'.
We hypothesized that downregulation of E-cadherin and beta-catenin and upregulation of MMP-9 will be seen in GBC with respect to CC and associated with poor survival higher tumor stage.
Thus, the present study was carried out with the following primary objectives: 1) To see the role of IHC markers—E-cadherin, beta-catenin, and MMP-9 alterations—in GBC; and 2) To correlate the IHC expression with the depth of invasion, clinical outcome, and disease-free survival.
The results are expected to provide a better comprehension of the early events related to GBC metastasis and may help us interfere early and thus reduce mortality in further received cases of GBC.
Material and Methods A prospective case-control study was carried out after approval from the institutional ethical committee approval via letter number 213/Ethics/R. cell-2018. A total of 70 cases of gall bladder disease were studied, which included 50 cases of malignant lesions (adenocarcinomas) and 20 cases of chronic cholecystitis serving as hospital controls received from September 2016 to 2018. Cases and controls were age- and sex-matched.
The clinical history and demographic profile of the patients were recorded along with the radiological details. Detailed histomorphological evaluation, pathological tumor staging, and immunohistochemistry for E-cadherin, beta-catenin, and MMP-9 were done after taking written consent from patients/patient's attendants. Two major study groups were formed: cases and controls. GBC cases were further divided into three subgroups: 1) carcinoma limited to gall bladder, 2) locally advanced gall bladder carcinoma, and 3) nodal and distant metastasis.
For GBC cases, tissue samples from patients were included in the study when a definite resection specimen was received with a clinical diagnosis of gall bladder carcinoma confirmed histologically and when adequate tissue for further IHC was present in the tissue block.
For CC cases tissue samples from patients were included in the study when a definite resection specimen was deposited with a clinical diagnosis of chronic cholecystitis and there was no evidence of intestinal or antral metaplasia, dysplasia, or malignancy in any of the sections. Adequate tissue for further IHC was available. Cases with inadequate biopsy samples, the patient unwilling to participate, and sections that floated during the IHC procedure even after repeat were excluded from the study.
Hematoxylin and Eosin (H&E)-stained slides of all these cases were evaluated in detail and all the morphological parameters under College of American Pathologists (CAP) protocol, which included the diagnostic information such as specimen identification, pro-cedure, laterality, lymph node sampling, site and size of the tumor, histological type and tumor grading were record-ed along with evaluation of prognostic histopathological param-eters. The prognostic parameters studied were tumor necrosis, lymph vascular and perineu-ral invasion, nodal metastasis etc. After which IHC was performed.
Immunohistochemical evaluation using the streptavidin-biotin immunoperoxidase method was done. Primary antibodies used were 1) Anti-MMP-9 antibody: AbcamTM (Rabbit Polyclonal to MMP-9, dilut-ed in PBS, in a dilution of 1:100), Clone ab38898) 2) Anti-Human Beta-Catenin: Ready to use (Dako TM, Denmark, auto-stainer), Mouse monoclonal antibody, Clone: Beta-Catenin- and 3) Anti-Human E-Cadherin: Ready to use (Dako TM, Denmark, autostainer). Second-ary antibody used was DacoEnVision-FlexTM high pH.
For antigen retrieval, deparaffinized sections were microwaved in TRIS-EDTA (TRIS: 1.21 gm, EDTA: 0.37 gm, Tween: 20–500 μL) buffer (pH: 9.0)/citrate buffer (pH: 6.0) at 98°C for 15 min and cooled to room temperature. Concentrated diaminobenzene solution (DAB) was diluted with substrate buffer (500 μL of substrate buffer + 2 drops of DAB) was used as chromogen with 10% hematoxylin as counter stain.
The slides were examined at 400 Χ magnification for immunohistochemical expression. For comparison of immunoexpression between GBC and CC, survival and association between IHC markers the percentage expression was taken as continuous variable. For comparison of immunoexpression with clinic-pathological parameters, they were taken as dichotomous variables (negative and positive).
Membranous expression of E-cadherin and beta-catenin was seen as a continuous variable.[9],[10],[11],[12] Faint expression was considered negative. Moderate to strong membranous expression was considered positive. The percentage area of complete and incomplete membranous expression was recorded separately along with cytoplasmic expression if seen in any of the cases or controls. Cytoplasmic expression of MMP9 was seen. Faint expression was considered negative. The intensity of expression was recorded as mild, moderate, and strong.[13],[14]
E-cadherin and beta-catenin were also interpreted as dichotomous variables, that is, positive and negative. Cytoplasmic and no immunoexpression was considered negative. Moderate to strong membranous expression (complete or incomplete) > 10% of tumor cells was considered positive. For MMP-9, Allred score guidelines were followed. Immunoreactivity scores were calculated by adding the number representing the percentage of immunoreactive cells by the number representing staining intensity; a total score of 0–3 was taken as negative and 4–8 was considered as positive.
Statistical software IBM SPSS® platform 24.0 was used to analyze the data, and statistical tools used were student t test, analysis of variance (ANOVA), Kaplan–Meier (for survival analysis), and extent of agreement (Kappa).
Results In the present study, we examined 70 cases of gall bladder disease, among which 50 (71.4%) were of GBC and 20 (28.5%) were of CC (hospital control). Among CC, 19 had gall stones and one had xanthogranulomatous cholecystitis. Out of the 50 GBC cases, three cases were associated with xanthogranulomatous cholecystitis, and 13 cases were associated with cholelithiasis.
Predominant population was of females with a M: F ratio of 1:4. The age of GBC presentation ranged from 26 to 70 years with a mean and standard deviation of 49.4 ± 11.6, and the age of CC ranged from 26 to 65 years with a mean and standard deviation of 46.5 ± 10.1. The GBC and CC groups were age and sex-matched.
All 50 cases of GBC were adenocarcinoma (not otherwise specified (NOS)) type. [Table 1] summarizes the morphological parameters of GBC cases.
The clinical follow-up of all malignant cases was sought. Adequate follow-up was found in 78% (39/50) cases. Eleven patients (22%) could not be contacted either by phone or hospital retrieval system. Twenty-six out of 50 patients were alive, of which 22 (44%) patients had received chemotherapy and four (8%) patients had not received chemotherapy. Eight out of 50 patients died, of which seven (14%) patients died due to disease and one (2%) died due to other causes (myocardial infarction). Five (10%) patients were alive with a debilitating condition.
[Table 2] shows the immunoexpression of E-cadherin, beta-catenin, and MMP9 in GBC cases and Controls (CC). The distribution of morphological variables was also evaluated. The mean age of presentation was 49.4 years with a standard deviation of 11.68. Tumor thickness was between 0.3 and 4.3 cm with a mean of 1.9 + 1.5 cm. Almost one-third of patients with GBC displayed immunohistochemical expression of E-cadherin (32.6%), beta-catenin (37%), and MMP9 (36.4%). The expression was seen in the tumor cells of the GBC cases. The mean follow-up was of 13.66 months with patients succumbing to the disease in a minimum of 5 months after surgery. Only beta-catenin downregulation was significantly associated with the pT stage of GBC cases (P = 0.005). No significant association was found between E-cadherin and MMP-9 immunoexpression and the pT stage, and even for tumor thickness, tumor grade and nodal metastasis. However, there was reduced expression of complete membranous E-cadherin in pT3 and pT4 tumors as compared to pT1 and pT2.
Table 2: Immunoexpression of E-cadherin, beta-catenin, and MMP9 in GBC cases and controls (CC)Further, when the study subgroups as detailed in material and methods were evaluated for immunoexpression, it was observed that IHC expression of E-cadherin in the epithelium lining was high in CC cases [Figure 1] (with a mean of 52 ± 22.6) as compared to GBC cases. In malignant subgroups, cases limited to gall bladder had higher expression of E-cadherin in tumor cells (35.8 ± 29.8) than locally advanced GBC cases (24.5 ± 23.3) and nodal and distant metastatic cases (34.0 ± 23.2) with a significant P value (P = 0.022) [Table 3].
Figure 1: Microscopic image of chronic cholecystitis with immunohistochemical results displaying the expression of beta-catenin and E-cadherin and the absence of MMP9. Images E–H display well-differentiated adenocarcinoma limited to gall bladder with complete membranous expression of E-cadherin in 90% of the tumor area, complete membranous expression of beta-catenin in 90% of the tumor area, and mild expression of MMP-9 in 30% of the tumor area
Table 3: Correlation between the immunoexpression of E-cadherin, beta-catenin, and MMP-9 among study subgroupsIHC expression of beta-catenin in the epithelium lining was also high in CC cases [[Figure 1]a, [Figure 1]b, [Figure 1]c, [Figure 1]d (with a mean of 73.5 ± 19.8) as compared to GBC cases. In malignant subgroups, cases with nodal and distant metastasis had higher expression of beta-catenin in tumor cells (40.4 ± 30.7) than cases limited to gall bladder (38.8 ± 27.8) [Figure 1]e, [Figure 1]f, [Figure 1]g, [Figure 1]h and [Figure 2]a, [Figure 2]b, [Figure 2]c, [Figure 2]d, [Figure 2]e, [Figure 2]f, [Figure 2]g, [Figure 2]h locally advanced GBC cases (27.2 ± 18.4) with a very significant P value (P < 0.001) [Table 3].
Figure 2: Microscopic images A–D show pT2 tumor with incomplete membra-nous expression of E-cadherin in 30% of the tumor area, incomplete membranous expression of beta-catenin in 50% of the tumor area, and moderate cytoplasmic expression of MMP-9 in 80% of the tumor area. Images E–H display tumor cells infiltrating into adjacent liver parenchyma (pT3) with complete membranous expression of E-cadherin in 80% of the tumor area, incomplete membranous expression of beta-catenin in 50% of the tumor area, and strong cytoplasmic expression of MMP-9 in 90% of the tumor area.IHC expression of MMP-9 in the epithelial lining was low in CC cases (with a mean of 4.5 ± 14.6) as compared to GBC cases. In malignant subgroups, cases limited to gall bladder had higher expression of MMP-9 in tumor cells (40.5 ± 25.1) than cases with nodal and distant metastasis (37.7 ± 28.9) and locally advanced GBC cases (27.27 ± 24.5) with a very significant P value (P < 0.001) [Figure 1] and [Figure 2]d, [Figure 2]h and [Table 3].
Two cases had no immunoexpression of either E-cadherin, beta-catenin, or MMP-9. One of these cases was pT3 with liver involvement and the other was pT1. None of them had nodal metastasis.
The above results show significant downregulation of E-cadherin and beta-catenin along with upregulation of MMP-9 in GBC as compared to CC [[Figure 3]; Scatter plots].
Figure 3: Scatter plots of Immunohistochemistry results show CC controls have 60-80% expres-sion of Beta Catenin with significant down regulation in GBC; expression ranging from 25-40% in tumor cells. While for E- Cadherin more than 35% (30- 70%) of tumor cells display expression in CC (Mild downregulation of E-Cadherin are seen in inflamed mucosa) however around 30% tumor cells also express E- Cadherin in GBC. Most of the CC cases have no immuno-expression of MMP-9 with significant up-regulation of MMP-9 in GBC (average expression being 30-50% tumor cells)Immunoexpression of E-cadherin, beta-catenin, and MMP-9 studied in GBC was then subjected to c2 tests to see whether the immunoexpression if these IHC markers correlated with each other. We found that between E-cadherin and beta-catenin, c2 = 79.150, P < 0.001; between E-cadherin and MMP-9: c2 = 11.188, P = 0.011; and between beta-catenin and MMP-9: c2 = 14.406, P = 0.002.
The above results show a significant association between immunoexpression of E-cadherin, beta-catenin, and MMP-9 in GBC cases, supporting the hypothesis that they are linked to each other directly or by some other molecular pathway.
Kaplan–Meier plots for overall survival in 39 patients with gall bladder adenocarcinoma in relation to IHC expression, tumor stage, grade, and tumor metastasis were observed. Better clinical outcome was seen in GBC that had lower tumor stage, grade, and without tumor metastasis [Figure 4]. The overall survival time was more in cases with complete membranous immunoexpression of E-cadherin as compared to incomplete membranous and negative immunoexpression of E-cadherin (P = 0.13). Though not statically significant, better survival was seen in cases with complete membranous expression as compared to incomplete membranous and no expression of E-cadherin. We found significantly better survival in GBC with complete membranous immunoexpression of beta-catenin as compared to incomplete membranous and negative immunoexpression of beta-catenin (P = 0.004) [Figure 4] There was no significant difference in survival of cases with MMP-9 immunoexpression as compared to cases with no expression of MMP-9. However, when the response to chemotherapy in this group was analyzed, that is, MMP-9 expression with respect to the response of chemotherapy, the mortality in patients with MMP-9 IHC expression who received chemotherapy was lower as (75%) compared to that in those who did not receive chemotherapy (55%).
Figure 4: Survival graphs of the study subjects with respect to grade o tumor where better sur-vival is seen in well differentiated tumor as compared to poorly differentiated and significantly better survival in GBC with complete membranous immuno-expression of Beta-Catenin as com-pared to incomplete membranous and negative immuno-expression of Beta-Catenin. (p=0.004)The above results confer that as far as IHC expression was considered complete membranous immunoexpression of beta-catenin confer better outcome as compared to incomplete membranous expression or downregulation. As far as MMP-9 is considered, it confers better response to chemotherapy in GBC, leading to better survival in positive cases.
Discussion The expression of E-cadherin, beta-catenin, and MMP-9 were evaluated in GBC and chronic cholecystitis by immunohistochemistry. Fifty patients who underwent radical procedures for GBC were taken as cases and 20 patients with symptomatic benign gall bladder disease who had undergone cholecystectomy served as hospital controls in our study.
The mean age of cases of GBC was 49.4 years (SD: 11.6 years), almost two decades younger than reported western literature; however, it was supported by data presented in Indian studies.[9],[14],[15],[16],[17] Food, dietary habits, and geographical distribution can be some of many possible reasons for GBC presenting almost two decades earlier in our country as compared to the west. GBC is also known to have female predominance, which has been linked to female hormones. Females formed the majority part of our dataset with a female: male ratio of 4:1.[18]
On collating the clinic-pathological parameters, we found that out of 50 GBC cases, 44 cases were seen involving body and/or fundus with or without the involvement of adjacent neck [Table 1], which is in concordance with the previous studies reporting that approximately 90% of GBC are seen involving body and fundus.[18] We also found that majority of these cases were moderately differentiated (54%), followed by well-differentiated (32%) and poorly differentiated tumor grade (14%). Published studies from north India, such as Ghosh M et al.[12] (2012) and Chandrawati et al.[15] (2018), also noted that moderately differentiated tumors are more frequent in the North Indian population. Like their observation, we also found that pT2, that is, peri-muscular fat involvement, is more frequent in our population [Table 1]. Reasons for moderate differentiation and pT2 being more common may be that the patients remain undiagnosed in the early stages of carcinoma due to subtle symptoms. Moreover, there is a lack of awareness, lack of targeted approach, and cancer control programs for GBC.
In the present study, we found a significant downregulation in the expression of E-cadherin and beta-catenin and upregulation in MMP-9 [[Figure 3]; Scatter Plots]. Choi et al. (2004), Puhalla et al. (2005), Priya et al. (2010), and Mukai et al. (2001) reported similar results with respect to E-cadherin.[11],[16],[19],[20],[21] With regards to beta-catenin, Choi et al. (2004), Puhalla et al. (2005), and Moon et al. (2005) found similar observations.[10],[11],[20] Though there is an increased MMP-9 expression reported in the literature in GBC as compared to CC, the percentage expression results are variable.[22] Significant variation in IHC expression of MMP-9 in GBC ranging from 37% to 100% has been reported. Karadag et al. (2008) reported 100% and Kirimlioğlu et al. (2009) reported 37% immunoexpression of MMP-9 in GBC.[13],[14]
Some Indian studies have reported that downregulation of membranous expression of beta-catenin is accompanied with weak-to-moderate nuclear expression in GBC (Ghosh[12] (2012) and Chandrawati et al.[15] (2018)), with an explanation that internalization of beta-catenin triggers the expression of EMT-inducing transcription factors.[23] Heterogenous expression of beta-catenin ranging from cytoplasmic to nuclear is seen. The results from multiple studies have been summarized in [Table 4]. Membranous positivity has been seen in CC. Mutations in beta-catenin may lead to cytoplasmic accumulation with subsequent nuclear translocations. Mutations in the N terminus of beta-catenin causes its phosphorylation and degradation, or if the phosphorylation is prevented with any further mutation, the increased stability of beta-catenin may lead to internalization and cellular proliferation. In our study, we saw the loss of membranous expression and cytoplasmic accumulation on IHC; however, we found that the expression of beta-catenin was seen up to the cytoplasmic accumulation stage only. The reason for this observation in our study group was extensively searched and could not be apprehended; the most plausible justification may be the make and clonality of the antibody used or that the mutation occurring in our study cohort was up to the terminus level only. Further blocking of phosphorylation might have not occurred, which would have led to increased stability of beta-catenin protein and eventual nuclear localization.[24],[25]
Table 4: Summary of published literature review of E-cadherin, beta-catenin, and MMP-9 in gall bladder cancerOn comparing the percent expression of E-cadherin, we found complete membranous expression of E-cadherin in 36% with incomplete membranous expression in 54% cases of GBC. Kohya et al. (2002) reported 48.6%, Priya et al. (2010) reported 67%, and Hirata et al. (2007) reported 61% expression of E-cadherin in GBC.[16],[26],[27] Though the overall percentage expression in our cases lies in between their observation, we could not find any published literature that has reported the percent expression of tumor cells as complete membranous and incomplete membranous for adequate comparison [Figure 1] and [Figure 2]; [Table 2].
Significant downregulation of E-cadherin and beta-catenin and upregulation of MMP-9 among study subgroups supports our hypothesis that loosening of cellular junctions due to loss of E-cadherin and beta-catenin with the increased dissolution of stroma due to increased MMP-9 occurs in metastatic and locally advanced GBC as compared to GBC limited to gall bladder, supporting the fact that EMT type III plays a role in GBC invasion and metastasis.[14]
Multiple EMT markers have been studied in GBC, such as 1) epithelial markers- E-cadherin, beta-catenin, Claudin-1, and occluding; 2) mesenchymal markers- N-cadherin, vimentin, fibronectin, and S100 A4; and 3) EMT-inducing transcription factors (EMT-TFs)- Snail, ZEB1, and Twist1. Immunoexpression of E-cadherin and beta-catenin has been linked to miRNA and IncRNA such as miR 20a, 33a, 29c-5p, IncRNA- ROR, KIAA0125, and Linc-ITGB1. The above observations make them relevant, providing a molecular backup and showing that a complex cellular talk occurs involving many of them at various levels. The use of a single marker to predict EMT in GBC or even any cancer may lead to erroneous observations. EMT is not only dependent on the cellular makeup of the stroma; other clinical, histological, and immunological factors also play an equal role in metastasis. The above confounding may sometimes lead to some altered results; however, the advantage of promising targeted therapy provided by their mere expression abundant for example HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) is seen to increase the expression of E-cadherin and inhibit GBC cell growth by exclusively targeting HDAC1/2.[29],[30]
Only beta-catenin immunoexpression was significantly associated with one of the morphological parameters, pT stage of GBC cases. We found reduced cytoplasmic immunoexpression of beta-catenin as the pT increased (64.1% cases; 25/32 of pT1 and pT2 compared to 35.9%; 14/18 cases of pT3 and pT4). Hirata et al.[27] (2006) reported increased nuclear beta-catenin immunoexpression in advanced GBC (83%) as compared to that in pT1 stage (63%).
MMP-9 and E-cadherin were not seen to significantly correlate with morphological parameters studied in GBC. Mukai et al.[19] (2001) studied E-cadherin expression with respect to the grade of the tumor. They found that E-cadherin expression with membranous localization was universally found in the well-differentiated type of GBC. Our findings are supported by those of Karadag et al. (2008),[14] who reported that MMP-9 expression with morphological parameters did not correlate with the grade of differentiation, level of infiltration, and liver and lymph node involvement.
We found a significant association of immunoexpression of E-cadherin, beta-catenin, and MMP-9 in GBC, further supporting our hypothesis that they are linked to each other directly or by some other molecular pathway. To the best of our knowledge, we could not find any literature available in which association of all these three markers has been described [Table 4].
On doing the survival analysis on cases of GBC, we found that there was significantly better survival in cases that were well-differentiated, without nodal metastasis, and where pT was either pT1 or pT2 when compared to moderately and poorly differentiated with nodal metastasis and high pT. These findings support the well-known fact that tumor stage (pT and nodal status are part of which) and grade are one of the most important prognostic parameters in GBC [Figure 4].[18] On comparing the survival outcome with the immunoexpression of studied markers, we found that there was no significant correlation between survival with E-cadherin (P = 0.13) and MMP-9 expression (P = 0.3). However, complete membranous immunoexpression of beta-catenin was seen significantly seen in patients with better survival in GBC (P = 0.004).
It has been reported that cases of GBC with MMP-9 immunoexpression respond better to chemotherapy as compared to cases with no MMP-9 expression.[21] Our observation was in close concurrence with their findings. On follow-up, we found that in our study population, 58% (29/50) cases had MMP-9 expression, out of which 68.9% (20/29) cases received chemotherapy and 31.0% (9/29) cases did not receive chemotherapy. Better clinical outcome was seen in patients who received postoperative chemotherapy and had MMP-9 expression as compared to patients having MMP-9 expression who did not receive postoperative chemotherapy.
Multiple chemotherapeutic agents such as wogonin in hepatocellular carcinoma, doxycycline, antagonist peptide 9, and Lupeol in GBC have been found to be effective both in vitro and in vivo against MMP-9 expressing tumor cells.[21],[28],[31] Cytotoxic drugs such as lupeol, which induce apoptosis and inhibit invasion by suppression of EGFR/MMP-9 signaling pathway, have been seen to be effective in cases with MMP-9 expression as suggested by Liu et al. in 2016.[28] The increased MMP-9 expression in GBC cases suggests that this group of patients might benefit from targeted therapy such as lupeol, doxycyclin, antagonist peptide 9, and wogonin.
Our study showed that there was significant downregulation of E-cadherin and beta-catenin along with upregulation of MMP-9 in GBC as compared to CC with a strong association of these IHC markers with each other. Apart from lower stage and grade, complete membranous expression of beta-catenin showed better survival outcomes as compared to incomplete membranous expression or internalization of beta-catenin.
The study suggested that beta-catenin and MMP-9 can be used to predict clinical outcomes and response to chemotherapy, which has to be confirmed further. The limitations of the present study are that it is a single-center study with a small sample size; larger studies with more robust follow-up may reinforce our observations.
Acknowledgements
We are thankful to King George's Medical University for providing the infrastructure to perform our work. We are also thankful to our immunohistochemistry and histopathology laboratory staff for their support. The study has been approved by the Ethical Committee of KGMU.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ijpm.ijpm_876_21
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