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Introduction:
Colorectal carcinoma (CRC) is the most common and one of the main causes of mortality and mor- bidity globally among the many gastrointestinal tract tumors 1. Generally accepted that CRC is a heterogeneous disease with a wide range of clinical and pathological behaviors, as well as predictions and treatment responses that can vary even among individuals with the same tumor, node, and metas- tasis (TNM) stage 2. This might be as a result of the different molecular events linked to the colon tu- morigenesis 3. A benign polyp is the first step in the multistage pathogenesis of colorectal cancer, which eventually progresses to an adenoma and a carcinoma. The slow polyp-cancer progression se- quence seen in the general population offers an op- portunity to detect and remove the polyps before they undergo malignant transformation 4. The de- mand for molecular markers to direct clinical deci- sion-makers on how to categorize patients into the most suitable therapy regiment has never been greater. Earlier in the 1990s, it was determined that mutations in the adenomatous polyposis coli gene (APC) were directly related to the hereditary can- cer condition familial adenomatous polyposis (FAP) 5,6. It rapidly became apparent that the Wnt/ β-catenin signaling pathway plays an initiating and a rate-limiting role in colorectal tumorgenesis as APC mutations were discovered at high frequen- cies in colorectal adenomas and carcinomas two years after it was first shown that APC and β- catenin interact closely 7. Wnt/ β-catenin signaling is disrupted in more than 90% of all CRCs, accord- ing to recent large-scale exome-sequencing 8.

In brief, when stromal cells and Paneth cells in the intestinal crypt release Wnt proteins, these proteins bind to heterodimeric receptor complexes on the surface of intestinal stem cells (Frizzled/Lrp6) and their effectors, initiating canonical Wnt signaling (i.e., Wnt/ β-catenin signaling) 9. After that, a signal is sent along a signaling cascade, effectively inhib- iting the degradation of cytoplasmic β-catenin 10. A number of genes involved in proliferation and dif- ferentiation are expressed as a result of β-catenin\\\\\\\'s quick translocation into the nucleus and interaction with DNA-bound TCF/Lef transcription factors 11. Mutations in APC, β-catenin in cancer cells result in constitutive activation of this signaling pathway, which promotes excessive proliferation and pre- vents stem cell progenitors from differentiating 12. Noticeably, β-catenin links E-cadherin to the cyto- skeleton in adherens junctions, which is a crucial cellular function 13. Over the past ten years, numer- ous datasets at the genetic and protein levels have evaluated the prognostic potential of various Wnt/ β-catenin pathway components in CRC. In particu- lar, deregulation of APC, β-catenin, and E-cadherin has drawn considerable attention 14,15. In fact, only a few studies have shown that this biomarker has a significant clinical impact. Saldanha et al. indicated that, at low levels of E-cadherin in the cells, E-cad- herin sequesters β-catenin at the cell membrane, leading to an increase in MYC and cyclin D1 ex- pression and changing the rate of tumor prolifera- tion 16. Therefore, this study aims to evaluate the association between the immunohistochemistry (IHC) expression of E-cadherin, and β-catenin. with the histopathological grade, stage of CRC in a subset of patients with primary CR. The results of this research could help in making decision- for newer targeted therapies for CRC.

Materials and Methods
The material of the present study was retrospec- tively collected from the archives of the Depart- ment of Pathology. All surgically resected CRC from 2021 to 2023 were included in the study. Par- affin blocks containing formalin-fixed primary tu- mors and hematoxylin and eosin-stained slides. The pathology reports of 82 patients of pre-malig- nant of different types of adenomas (n = 48) (tubu- lar, villous, and tubulovillous), and colorectal ade- nocarcinoma CRC (n = 34) were collected and the following data such as the histopathological diag- nosis, grade, and staging of the tumors were ob- tained. While clinical information was obtained from medical records and pathology reports. IHC staining was performed for all the cases using E- cadherin and β-catenin antibodies

Processing Procedures:
For each case, a representative paraffin-embedded tissue was chosen. The paraffin wax sections were cut at 4 microns and stained by hematoxylin and eosin stain for routine histopathological examina- tion and immunohistochemical staining by B- catenin and E-cadherin monoclonal antibodies. Each section was obtained from the blocks was placed on positive charge slides, dewaxed in xy- lene, rehydrated in consecutive descending concen- trations of ethanol (100%, 90%, 80%, and 70%), and rinsed in distilled water. For antigen retrieval, slides were placed in a plastic container filled with sufficient citrate buffer pH 6 and heated in a micro- wave oven at 100ºC for three successive times, five minutes each. The amount of fluid in the container was checked and added if necessary to prevent slides from drying out. The slides were immersed in 3% hydrogen peroxide for 10 minutes to block endogenous peroxidase.

Immunohistochemical analysis:
Sections were incubated at room temperature with the following Dako Monoclonal Mouse Anti-Hu- man β-Catenin (β-CateninC-1) antibodies (Santa Clara, CA, USA; Catalog number 610154), recog- nizing a C-terminal epitope between residue 571 and residue 781 of β-catenin. Mouse monoclonal anti-E-cadherin (Clone 36) antibodies were ob- tained from BD Biosciences (catalog number 610181), recognizing a C-terminal epitope be- tween residue 735 and residue 883 of E-cadherin. Chromogen application by using DAB (3,3-dia- minobenzidine tetrahydrochloride). The counter- staining of the sections was done with Mayer\\\\\\\'s He- matoxylin. Positive and negative controls have been simultaneously run to verify the accuracy of the technique. Slides were scanned by light micros- copy and representative fields were selected for analysis. Evaluation: Slides were mounted for light microscope (Olympus BX45 manual microscope, Germany) evaluation of immunoreactivity by a pathologist. The staining of β-catenin and E-cad- herin was scored according to the proportion and intensity categories proposed by Allred et al. The intensity score represents their average staining in- tensity (0 = negative, 1 = weak, 2 = intermediate, and 3 = strong). Staining was evaluated and scored separately for membranous, cytoplasmic, and nu- clear staining patterns. Staining score 0 and 1 is considered low expression while staining score 2 and 3 is considered high expression
 
Statistical Analysis:
The data entry was done using Microsoft Excel (Microsoft Corporation, Redmond, WA). The col- lected clinical data and histopathological character- istics were analyzed using Microsoft Excel soft- ware. Categorical variables were summarized us- ing frequencies and proportions and all results were presented in tabular form and graphs. The groups were tested for statistical significance using the chi-square test and Fisher\\\\\\\'s exact test, and a p-value less than 0.05 was considered statistically signifi- cant

Results:
Clinico-pathological Features:
A total of 82 cases were taken in this study the cases are different types of adenomas (n = 48) (tub- ular, villous, and tubulo villous), and colorectal ad- enocarcinoma CRC (n = 34). Age ranging from 30 years to 80 years with a mean age of 52.9 (SD±15.8). The mean age for patients with tubular adenoma (N=18) 47.6 (SD ± 14.6), villous ade- noma (N=16) 50.4 (SD ± 15.4), and tubulo-villous adenoma (N=14) 50.8 (SD± 17.9). The maximum number of adenoma cases were observed between the age of 30– 40 years. The mean age for Non- mucinous CRC (N=21) 56.2 (SD ± 16.1), and mu- cinous CRC (N=13) 60.4 (SD ± 13). The peak in- cidence was in the 61-70 years for both types of CRC as shown in (Table 1). Most patients were males (62.2%) and (37.8%) were females, with a male-to-female ratio of 1.6:1 (Figure 1). 

Table 1: Distribution of adenomas and colorectal carcinoma according to age group.

Figure 1: Histopathological pattern of adenoma subtypes and both types of colorectal adenocarcinoma distribution by gender

β-catenin expression
Immunohistochemical examination for determina- tion of β-catenin was accomplished to define sub- cellular localization and score the all-colorectal ne- oplasms types included in the study as shows in (Figure 2 first raw). On examination of IHC results and their analysis, the following were distinguished that highest β-catenin membranous expression in benign adenomas; namely tubular adenoma, vil- lous adenoma and tubulo-villous adenoma. There were highest cytoplasmic β--catenin scores noted in low grade colorectal adenocarcinomas and high- est β-catenin nuclear score noted in high grade col- orectal adenocarcinomas. By scoring the intensity, there was high expression in 27 cases (79.4%) out of 34 malignant adenocarcinoma and low expres- sion in 7 cases (20.6%). While intensity score in 48 cases of adenomas was showing high expression in 12 cases (25%) low expression in 36 cases (75%) as indicated in (Figure 2 second raw). Univariate analysis revealed significant differences between β-catenin expression (high versus low) in malignant adenocarcinomas and tumor grade, tu- mor stage and lymph node metastasis (Table 2). And there are significant differences between β- catenin expression (high versus low) and various types of adenomas (Table 3).

Table 2: Correlation between β-catenin and clinicopathologic characteristics of patients with colorectal

E-cadherin expression
In contrast to immunohistochemical examination for determination of E-cadherin, that highest mem- branous and cytoplasmic E-cadherin expression in benign adenomas. There was decrease in E-cad- herin expression in high grade colorectal adenocar- cinomas. By scoring the intensity, there was high expression in 14 cases (41.2%) out of 34 malignant adenocarcinoma and low expression in 20 cases (58.8%). While intensity score in 48 cases of adenomas was showing high expression in 39 cases (81.3%) low expression in 9 cases (18.7%) as shown in (Figure 2 third raw). Univariate analysis revealed significant differences between E-cadherin expression (high versus low) in malignant adenocarcinomas and tumor grade, tu- mor stage and lymph node metastasis (Table 4). And there are significant differences between E- cadherin expression (high versus low) and various types of adenomas (Table 5).

Table 4: Correlation between E-cadherin and clinicopathologic characteristics of patients with colorectal carcinoma.

Discussion:
In this work, using immunohistochemical analysis, we aimed at determining whether Wnt signaling- associated proteins, β-catenin, and E-cadherin were related to progression of colorectal cancer and ver- ify whether it could be used as a prognostic indica- tor. One of the fundamental goals of translational research in colonic adenoma is to distinguish the small number of individuals who progress to CRC from the majority who do not. Currently, periodic colonoscopic biopsies with histological assessment of adenomas are used to assess the risk of progres- sion to CRC in patients with adenomas 17. A multi- functional protein known as β-catenin is essential for the Wnt/β-catenin signaling pathway and in cel- lular adherens. Increased expression of nuclear β- catenin has been suggested to be a sign of an aber- rant activation of the β-catenin signaling pathway and could play a role in tumor progression 18. Since colorectal tumorigenesis occurring as a result of mutation in the APC gene (85% cases) follows a gradual multistep sequence, β-catenin expression by immunohistochemistry (nuclear positivity) can be used to determine the malignant potential of col- orectal polyps and adenomas 19. In a recent study, Mårtensson A et al. revealed a correlation between elevated nuclear β-catenin levels and a poor prog- nosis in CRC 20.

In the present study, the mean age was 52 years with peak incidence was in the 61-70 years for both types of adenocarcinoma, which was similar to findings from other studies 21,22. In the current study, 62.2% were of the male gender, while 37.8% were female. Consistent with the studies by Iseki et al.22, Gomaa et al.23, and Melincovici et al.24 had a male predominance. Conversely, the studies by Choi et al.21 and Tunuguntla et al.25 show female preponderance. In the present study, 52.9% of ma- lignant cases were considered T3 stage, and similar findings were observed by Gomaa et al.23, Me- lincovici et al.24 and Tunuguntla et al.25, whereas the T4 stage was the major type in the study by Iseki et al.22.

Histological subgroupings of adenomas are associ- ated with different capabilities to develop malig- nancy. The three main histological subtypes (tubu- lar, villous, and tubulovillous) of adenomas, were included to provide insights into the expression pattern of β-catenin, and E-cadherin. Our data re- vealed most of benign adenomas had high expres- sion of E-cadherin, while most of malignant adenocarcinomas had low E-cadherin expression. There are significant correlation with tumor grade, tumor stage and lymph node metastases. This result is correspond- ing to Choi et al21. and Melincovici et al 24 found a significant association between the loss of expression of E-cadherin and cancer grading. Tunuguntla et al.25 shows significant correlation with tumor staging and lymph node metastases. Similarly, in another study, loss of E-cadherin be- ing indispensable in transformation from adenoma to carcinoma sequence in CRC 26. Lugli et al., demonstrate that nuclear β-catenin expression and loss of E-cadherin membranous expression could be adverse independent prognostic markers in co- lon carcinoma 27.

Increased expression of β-catenin showed a strong association with increasing grading and staging of the tumor with lymph node metastases, which was also seen in the studies by Choi et al. 21 and Tunu- guntla et al. 25 and decreased expression in adeno- mas. However, no such association was found in other studies by Gomaa et al.23 Our result showed the gradual intracellular translo- cation of β-catenin from a membranous to a nuclear expression in a stepwise manner, following the polyp-adenoma-carcinoma sequence, which is in concordance with the findings of Wong et al.28 In this study a statistically significant changes in the subcellular localization and increase in β-catenin expression was noted between benign and malig- nant neoplasms, with the subsequent presence of a higher nuclear β-catenin score in the malignant ne- oplasms. In the studies by Bhattacharya et al.29, Wong et al.28 and Kovacs et al 30, a significant positive correla- tion was accomplished between β-catenin subcellu- lar localization and their corresponding membra- nous, cytoplasmic, and nuclear score. However, the study by Gomaa et al.23 revealed that loss of β- catenin expression was significantly related with aggressive behavior, high stage, and distant metas- tases, and aggressive CRC is associated with a de- creased expression of β-catenin.

The present study indicates that both β-catenin, and E-cadherin exhibited different expression pattern among adenomas and CRC cases, could provide a useful marker for disease progression, risk stratification, or monitoring of treatment response. It is however now necessary to validate this result using larger panel of pre-clinical and clinical pa- tients in a multicentre setting in order to investigate large cohorts study. Heterogeneity in patient popu- lations and sampling bias are always a challenge in histopathology.

Limitations of the study
Our study has some limitations. First, a small sam- ple size was used to identify the value of β-catenin, and E-cadherin expression in benign and malignant colorectal neoplasms because of the short study pe- riod. Second, there is no follow-up of the patients because of the lack of patient registrations.

Future work
To overcome the subjectivity in interpretation of visual scoring, alternative approaches using auto- mated IHC measurements (IHC profiler) which are precise and produce continuous data are being de- veloped. Additionally, automated IHC profilers considerably improves both intra- and inter-ob- server agreement. Also, the digital methods for IHC quantifications are ideal for large samples size

Conclusion
From the results of the present study, we can con- clude that, the type of histological differentiation of colorectal adenomas and adenocarcinoma is di- rectly correlated with the intensity of expression of β-catenin. While E-cadherin expression showed an inversely correlation. β-catenin and E-cadherin could be used as useful prognostic markers for col- orectal adenocarcinoma. A long-term follow-up study will be necessary to identify the clinical value of β-catenin and E-cadherin expression in colorec- tal adenomas and adenocarcinoma.

Funding: No funding sources

Conflict of interest: No conflict of interest rele- vant to this article

References:

1 Xi Y, Xu P. Global colorectal cancer burden in 2020 and projections to 2040.Translational Oncofogy.2021;14(10):101174. doi:10.1016/.tranon.2021.101174. 2. Grizzi F, Basso G, Borroni EM, et al. Evolving notions on immune response in colorectal can-cer and their implications for Biomarker De-velopment. Inflammation Research. 12. 2018;67(5):375-389. doi:10.1007/s00011-017- 1128-1.

3. Scarps M, Ruffolo C, Canal F, et al. Sporadic mismatch repair genes defects are associated to HICD80+ lamina propria mononuclear cells 13. infiltration in Colo-rectal cancer. European Journal of Surgical Oncology (EJSO). 2015;41(1). doi:10.1016/j.ejso.2014.10.022.

4. Grady WM, Markowitz SD. The molecular pathogenesis of colorectal cancer and its poten-tial application to colorectal cancer screen- 14. ing. Digestive Diseases and Sciences. 2014;60(3):762-772. doi:10.1007/s10620-014- 3444-4.

5. lchii S, llorii A, Nakatsuru S, Furuyarna Utsunomiya J. Nakamura Y. Inactivation of both apc alleles in an early stage of colon ade- 15. nomas in a patient with familial adenomatous polyposis (FAP). Human Molecular Genetics. 1992;1(6):387-390. doi:10.1093/lang/1.6.387.

6. Cottrell S, Bodmer WF, Bicknell D, Kaklam-anis L. Molecular analysis of APC mutations in familial adenomatous polyposis and spo- 16. radic colon carcinomas. The Lancet. 1992;340(8820):626-630. dot 10.1016/0140- 6736(92)92169-g.

7. Aghabozorgi AS, Bahreyni A, Soleimani A, et S. Role of adenomatous polyposis coli (APC) gene mutations in the pathogenesis of colorec-tal cancer; current status and Perspectives. Bi- 17. ochimie. 2019;157:64-71. doi:10.1016/j.bio-chi.2018.11.003.

8. Grasso CS, Giannakis M. Genomic mecha-nisms of immune evasion in colorectal cancer. From discovery to clinical practice. Oncotar-get 2018;9(73):33743-33744. dot 10.18637Joncotarget.26105.

9. San Roman AK, Tovaglieri A, Bneault DT, 18. Shivdasani RA. Distinct processes and tran-scriptional targets underlie CDX2 require-ments in intestinal stem cells and differentiated villus cells. Stem Cell Reports. 2015;5(5):673-681. doi:10.1016/j.stemcr.2015.09.006 19.

10. Li VSW, Ng SS, Boerserna PJ, et al. Wnt sig-naling through inhibition of p-catenin degradation in an intact Axin I Complex. Cell. 2012;149(6):1245-1256. doi:10.1016/j.ce11.2012.05.002. Sederholm S, Cantu C. The witt/p-catenin de-pendent transcription: A tissue-specific busi-ness. WIRES Mechanisms of Disease. 2020;13(3). doi:10.1002Avsbm.15

11. Söderholm S, Cantù C. The wnt/β‐catenin de- pendent  transcription:  A  tissue‐specific  busi- ness. WIREs Mechanisms of Disease. 2020;13(3). doi:10.1002/wsbm.1511.
12. Deitrick J, Pruitt WM. Wnt/β catenin-mediated signaling commonly altered in colorectal can- cer. Progress in Molecular Biology and Trans- lational Science. Published online 2016:49-68. doi:10.1016/bs.pmbts.2016.09.010.
13. Hartsock A, Nelson WJ. Adherens and tight junctions: Structure, function and connections to the actin cytoskeleton. Biochimica et Bio- physica    Acta    (BBA) - Biomembranes. 2008;1778(3):660-669. doi:10.1016/j.bbamem.2007.07.012.
14. Albuquerque C, Pebre Pereira L. Wnt signal- ling-targeted therapy in the CMS2 tumour sub- type: A new paradigm in CRC treatment? Tar- geted Therapy of Colorectal Cancer Subtypes. Published online 2018:75-100. doi:10.1007/978-3-030-02771-1_6.
15. Subramanian S, Posey T, Jacob J, Carmon KS. Development of a novel combination therapy targeting met and LGR5 to overcome colorec- tal cancer resistance. Leading Edge of Cancer Research Symposium: Poster Session. Pub- lished online 2022. doi:10.52519/00086.
16. Saldanha G, Ghura V, Potter L, Fletcher A. Nuclear β-catenin in basal cell carcinoma cor- relates with increased proliferation. British Journal of Dermatology. 2004;151(1):157- 164. doi:10.1111/j.1365-2133.2004.06048.x.

17. Orsetti B;Selves J;Bascoul-Mollevi C;Lasorsa L;Gordien K;Bibeau F;Massemin B;Paraf F;Soubeyran I;Hostein I;Dapremont V;Guimbaud R;Cazaux C;Longy M;Theillet C; Impact of chromosomal instability on colo- rectal cancer progression and outcome. BMC cancer. Accessed June 18, 2023. https://pub- med.ncbi.nlm.nih.gov/24559140/.
18. Jung Y-S, Jun S, Lee SH, Sharma A, Park J-I. WNT2 complements Wnt/β-catenin signaling in colorectal cancer. Oncotarget. 2015;6(35):37257-37268. doi:10.18632/onco- target.6133
19. Zhang M, Yang D, Gold B. The adenomatous polyposis coli (APC) mutation spectra in dif- ferent anatomical regions of the large intestine
in colorectal cancer. Mutation Research/Fun- damental and Molecular Mechanisms of Muta- genesis. 2018;810:1-5. doi:10.1016/j.mrfmmm.2018.04.003
20. Mårtensson A, öberg åke, Jung A, Cederquist K, Stenling R, Palmqvist R. β-catenin expres- sion in relation to genetic instability and prog- nosis in colorectal cancer. Oncology Reports. Published online 2007. doi:10.3892/or.17.2.447
21. Choi JE, Bae JS, Kang MJ, et al. Expression of epithelial-mesenchymal transition and cancer stem cell markers in colorectal adenocarci- noma: Clinicopathological Significance. On- cology Reports. 2017;38(3):1695-1705. doi:10.3892/or.2017.5790.
22. Iseki Y, Shibutani M, Maeda K, Nagahara H, Ikeya T, Hirakawa K. Significance of E-cad- herin and CD44 expression in patients with un- resectable metastatic colorectal cancer. Oncol- ogy Letters. 2017;14(1):1025-1034. doi:10.3892/ol.2017.6269.
23. Gomaa W, Al-Maghrabi H, Al-Maghrabi J. The prognostic significance of immunostain- ing of Wnt signaling pathway molecules, E- cadherin and β-catenin in colorectal carci- noma. Arab Journal of Gastroenterology. 2021;22(2):137-145. doi:10.1016/j.ajg.2021.05.00.
24. Melincovici CS, Boşca AB, Şuşman S, et al. Assessment of mismatch repair deficiency, CDX2, β-catenin and E-cadherin expression in colon cancer: Molecular characteristics and impact on prognosis and survival – an im- munohistochemical study. Romanian journal of Morphology and Embryology. 2021;61(3):715-727. doi:10.47162/rjme.61.3.10.
25. Tunuguntla A, Suresh TN, PN S. Association between the immunohistochemistry expression of E-cadherin, β-catenin, and CD44 in colorec- tal adenocarcinoma. Cureus. Published online 2023. doi:10.7759/cureus.35686.
26. Herzig M, Savarese F, Novatchkova M, Semb H, Christofori G. Tumor progression induced by the loss of E-cadherin independent of β- catenin/TCF-mediated wnt signaling. Onco- gene. 2006;26(16):2290-2298. doi:10.1038/sj.onc.1210029
27. Lugli A, Zlobec I, Minoo P, et al. Prognostic significance of the Wnt Signalling Pathway Molecules  APC, ?-catenin  and  E-cadherin  in colorectal cancer?a tissue microarray-based analysis. Histopathology. 2007;50(4):453-464. doi:10.1111/j.1365-2559.2007.02620.x
28. Wong SC, Lo ES, Lee KC, Chan JK, Hsiao WL. Prognostic and diagnostic significance of β-catenin nuclear immunostaining in colorectal cancer. Clinical Cancer Research. 2004;10(4):1401-1408.    doi:10.1158/1078- 0432.ccr-0157-03.
29. Bhattacharya I, Barman N, Maiti M, Sarkar R. Assessment of β-catenin expression by im- munohistochemistry in colorectal neoplasms and its role as an additional prognostic marker in colorectal adenocarcinoma. Medicine and Pharmacy Reports. Published online 2019. doi:10.15386/mpr-1218
30. Kovacs Z. Loss of E-cadherin and B-catenin nuclear translocation in the invasion front is the central phenomenon of epithelial mesen- chymal transition in colorectal cancer. Pub- lished online 2017. doi:10.26226/morress- ier.596dfd59d462b8029238754.