Alterations in p53, Microsatellite Stability and Lack of MUC5AC Expression as Molecular Features of Colorectal Carcinoma Associated with Inflammatory Bowel Disease

Colitis-associated colorectal carcinoma (CAC) occurs in inflammatory bowel disease (IBD) because of the “chronic inflammation-dysplasia-cancer” carcinogenesis pathway characterized by p53 alterations in the early stages. Recently, gastric metaplasia (GM) has been described as the initial event of the serrated colorectal cancer (CRC) process, resulting from chronic stress on the colon mucosa. The aim of the study is to characterize CAC analyzing p53 alterations and microsatellite instability (MSI) to explore their relationship with GM using a series of CRC and the adjacent intestinal mucosa. Immunohistochemistry was performed to assess p53 alterations, MSI and MUC5AC expression as a surrogate for GM. The p53 mut-pattern was found in more than half of the CAC, most frequently stable (MSS) and MUC5AC negative. Only six tumors were unstable (MSI-H), being with p53 wt-pattern (p = 0.010) and MUC5AC positive (p = 0.005). MUC5AC staining was more frequently observed in intestinal mucosa, inflamed or with chronic changes, than in CAC, especially in those with p53 wt-pattern and MSS. Based on our results, we conclude that, as in the serrated pathway of CRC, in IBD GM occurs in inflamed mucosa, persists in those with chronic changes and disappears with the acquisition of p53 mutations.


Introduction
Crohn's disease (CD) and ulcerative colitis (UC) are pathologies with a two-to threefold increased risk of colorectal cancer (CRC) [1,2]. Likewise, colitis-associated colorectal cancer (CAC) is the leading cause of death among long-standing inflammatory bowel disease (IBD) patients [3]. The continuous inflammatory stress of the colonic mucosa, with constant epithelial lesion repair and alterations of the microbiota, damage the colonic epithelia leading to dysplasia. As a result, CAC appears following the same pattern of distribution as inflammation in the different IBD subtypes. In CD, neoplasms are most often located in the small bowel or on the right side of the colon, whereas in UC tumors are usually located in the rectum and left colon. CACs are more frequently poorly differentiated, mucinous or signet ring cell carcinomas [4]. Recently, similar characteristics between CAC and CRC with microsatellite instability high (MSI-H), whether sporadic MSI-H or Lynch syndrome (LS)-related, have been described [5][6][7].
The sequence "chronic inflammation-dysplasia-cancer" should determine the driving genes that guide this carcinogenesis pathway of CRC, which is different from the canonical and serrated pathways [8]. In this "inflammatory" context the Wnt-signaling pathway is not involved, and TP53 alterations appear early and with different hotspot mutations as opposed to the canonical pathway where TP53 plays a role at later stages [9][10][11][12]. In addition, copy number alterations are frequently found involving genes such as c-MYC, IRS2 and HER2 as well as mutations in BRAF and IDH1 and the activation of transforming growth factor β related to the epithelial-mesenchymal transition [10,13,14]. In LS, the mutation of genes of the mismatch repair (MMR) system (MLH1, MSH2, MSH6 and PMS2) are the hallmark of this hereditary cancer predisposition syndrome [15]. In the serrated pathway, the mutation of genes in the MAP kinases cascade, especially BRAF, and the methylation of CpG islands are the main effectors [16].
In CD the presence of metaplastic changes such as pyloric gland metaplasia or gastric foveolar metaplasia have been described in the small bowel mucosa or in pouchitis in long-standing disease [17][18][19][20][21][22]. Recently, gastric metaplasia (GM) has been identified as a substrate for the initiation of the serrated pathway of CRC, due to the continuous lesion of the colonic epithelium of the right colon [23]. Hence, considering that both the serrated and IBD-associated pathways share the sequence "inflammation-dysplasia-carcinoma", we hypothesize that they could have a common origin in GM, as a mechanism of adaptation to colonic mucosal stress.
The aims of the study are to characterize CAC analyzing p53 alterations and microsatellite instability (MSI) and to explore their relation to GM.
According to location, CAC in patients with CD were more frequently located in the ileum (7/19, 37%) and the right colon (6/19, 31.5%), whereas CAC in patients with UC were more frequent in the left colon (15/38, 39.5%). This distribution was statistically significant (p = 0.003). All eight (21%) synchronous tumors were found in UC which was statistically significant (p = 0.042). In most cases (29/53, 55%) the colectomy mucosa showed active inflammation. Regarding histology, the majority of CRC (47/57, 82.5%) were adenocarcinomas in both CD and UC. Intestinal mucosa adjacent to the tumor was identified in 35 (61.4%) cases mostly with chronic changes, especially in UC. According to staging, stage III was more frequent in CD (10/19, 52.6%) while in UC the most prevalent was stage II (17/38, 44.7%) ( Table 1). The p53-mut pattern (defined in Section 4) was found in more than half (29/57, 51%) of the adenocarcinomas (22 with overexpression and 7 with null pattern). The differences in the distribution of both expression patterns between CD and UC were not statistically significant ( Table 2). When MSI was analyzed, we identified only six (11%) tumors that were unstable (MSI-H), all with a lack of expression for MLH1/PMS2, with the same distribution in both IBD subtypes (Table 2).
MUC5AC staining was identified in 44% of CACs, 9 adenocarcinomas with positivity in more than 50% of the tumor (extensive expression) and 16 with focal expression. The differences in the distribution of both expression patterns between CD and UC were not statistically significant (Table 2).

Relation of the Three IHC Markers in Adenocarcinoma
Analyzing p53 expression with MSI, we observed that the six unstable tumors were p53 wt-pattern (100%), and most of the stable tumors presented a p53 mut-pattern (29/51, 57%) establishing a statistically significant relation (p = 0.010) ( Table 3). When we analyzed p53 according to MUC5AC expression, we found that 60% of CACs with MUC5AC expression had a p53 wt-pattern. In contrast, 59% of CACs with p53 mut-pattern were MUC5AC negative. Nevertheless, this distribution was not statistically significant (Table 4 and Figure 1). When we analyzed p53 according to MUC5AC expression, we found that 60% of CACs with MUC5AC expression had a p53 wt-pattern. In contrast, 59% of CACs with p53 mut-pattern were MUC5AC negative. Nevertheless, this distribution was not statistically significant (Table 4 and Figure 1).  Analyzing the relation between MUC5AC expression and MSI, a statistically signification was found, with all MSI-H CACs being positive for MUC5AC and all MSS CACs being MUC5AC negative (Table 5).  Analyzing the relation between MUC5AC expression and MSI, a statistically signification was found, with all MSI-H CACs being positive for MUC5AC and all MSS CACs being MUC5AC negative (Table 5). The clinicopathological characteristics and IHC expression of MSI-H CAC are shown in Figure 2 and Table 6. Of notice, these adenocarcinomas were in the ileum or right colon and showed histological features characteristic of sporadic MSI-H or LS-associated CRC-i.e., medullary, mucinous or signet ring cell carcinomas. Only one patient with MSH-H CAC was affected by LS, and another had two synchronous sporadic tumors harboring the V600E BRAF mutation. The remaining unstable CRCs were BRAF wild type, and the hypermethylation of MLH1 was not performed.    Only one patient with MSH-H CAC was affected by LS, and another had two synchronous sporadic tumors harboring the V600E BRAF mutation. The remaining unstable CRCs were BRAF wild type, and the hypermethylation of MLH1 was not performed.

p53 and MUC5AC Expression in the Mucosa Adjacent to Adenocarcinomas
To determine whether p53 and MUC5AC staining were present in the non-tumoral intestinal mucosa, we explored the mucosa adjacent to the adenocarcinomas, which was available in 35 cases, 1 from the small intestine and 34 from the colon.
p53 immunostaining showed focal positivity in the nuclei of the glands surrounding the adenocarcinomas in all cases, corresponding to wt-pattern.
MUC5AC immune-expression was found in 77% (27/35) of the mucosa adjacent to CAC, being in the ileum (100%, 1/1), in 70% (7/10) of the mucosa located in the right colon, in 80% in the left colon (12/15) and in 100% (4/4) of the rectal mucosa. Regarding the type of lesion, MUC5AC was positive in all cases with active inflammation, in 79% (15/19) of the mucosa with chronic changes and in 67% (8/12) of the mucosa without morphological lesions, although these differences were not statistically significant (Figure 3).

R PEER REVIEW
10 of 18  The relation between MUC5AC expression in adenocarcinoma and adjacent mucosa was not statistically significant. MUC5AC-positive non-tumoral mucosa was adjacent to negative adenocarcinoma in more than 60% (16/27), and MUC5AC-negative mucosa was adjacent to positive or negative adenocarcinoma in the same proportion (50%, 4/8) (Table  7 and Figure 4). The relation between MUC5AC expression in adenocarcinoma and adjacent mucosa was not statistically significant. MUC5AC-positive non-tumoral mucosa was adjacent to negative adenocarcinoma in more than 60% (16/27), and MUC5AC-negative mucosa was adjacent to positive or negative adenocarcinoma in the same proportion (50%, 4/8) (Table 7 and Figure 4).

Discussion
Colitis-associated colorectal cancer (CAC) is the result of the alteration o volved in the sequence of "chronic inflammation-dysplasia-cancer" which dra ent landscape from the other colorectal cancer pathways.
It is well known that p53 alterations appear in early stages even in the co without dysplastic changes, contributing to establish a field cancerization [9

Discussion
Colitis-associated colorectal cancer (CAC) is the result of the alteration of genes involved in the sequence of "chronic inflammation-dysplasia-cancer" which draw a different landscape from the other colorectal cancer pathways.
It is well known that p53 alterations appear in early stages even in the colon mucosa without dysplastic changes, contributing to establish a field cancerization [9,14]. In line with published data, in our series a pattern of p53 mutation was present in more than half of the CAC, independently of the IBD subtype [9][10][11][12][13][14]. However, not any of the p53 mut-patterns were found in any adjacent intestinal mucosa.
MSI-H in IBD, especially in UC, has been reported between 3 and 18% [5][6][7][24][25][26][27][28][29][30]. In our study, MSI-H was found in 10.5%, within the described range and with the same incidence in CD and UC. Furthermore, we found a statistical signification between p53 and MSI, such as that all p53 mut-pattern were stable tumors and all MSI-H CAC showed a p53 wt-pattern. Therefore, our results support a role for MSI in a subset of CAC. In addition, our unstable tumors had other clinical and pathological features of MSI-H CRC, such as the right location in the colon and histology of mucinous, medullary or signet ring cell carcinomas, in line with previous observations of other authors [5][6][7]. In our study, LS was excluded in one patient older than 70 years with UC and two synchronous tumors, since the molecular characteristics of both CRC and family history did not support LS diagnosis. Multifocal neoplasms are present in 38% of UC patients, and these synchronous neoplasms are usually heretogeneous [14]. In contrast, our patient had two MSI-H CACs which shared the molecular alterations and histological features probably due to both neoplasms appearing in the same side of the colon.
The other four patients with MSI-H CRC were in their 50s. One was a LS with UC of 4-year duration. The CRC was probably the result of the summatory risk of the two conditions. The presence of IBD and LS in the same patient is a rare situation given the scarce literature reporting these cases [31][32][33][34][35][36][37] and the low rate of germline mutations found in IBD patients [12]. Nevertheless, it is important to exclude LS in those MSI-H CACs to define the outcome in patients with early onset CRC, which is worse in the IBD-related group, intermediate in the sporadic group and best in the hereditary group [35]. In the remaining three patients with MSI-H CRC, short course IBD and tumors with wt-BRAF, LS cannot be ruled out.
MUC5AC staining, characteristic of gastric foveolar epithelium [38], was identified in less than half (44%) of the CACs but was retained in 77% of the 35 available adjacent intestinal mucosa. The MUC5AC-positive CACs were more frequently those with a p53 wt-pattern and MSS, although MUC5AC expression was present in all cases with MSI-H. Furthermore, considering that most of the tumors with p53 mut-pattern lack MUC5AC expression, it can be deduced that the appearance of gastric epithelial change occurs in the inflamed mucosa, persists in those with chronic-regenerative changes and is maintained in CAC without p53 mutation, to finally disappear in parallel to the acquisition of p53 mutations. Our results agree with the recently published data by Chen et al. who identified GM as a substrate for the initiation of the serrated pathway of CRC, due to continuous injury of the colonic epithelium of the right colon [23]. Therefore, we could argue that GM is the change shared by CAC and serrated CRC in which the sequence "chronic inflammationdysplasia-cancer" is initiated. Considering all this, we cannot rule out that the group of MSI-H CRC that appear in IBD, with characteristics like MSI-H sporadic tumors with MLH1 hypermethylation, are nothing more than serrated pathway CRCs that appear in a context of continuous colon injury.
Further studies are needed to explore the expression of MUC5AC in dysplastic epithelia IBD-associated, an intermediate step between colon mucosa with chronic changes and the onset of neoplastic events of CAC.

Materials and Methods
We retrospectively reviewed a series of 53 IBD colectomies with CAC from 14 hospitals. Specimens were classified as CD or UC based on histology, radiological studies and clinical information collected from patient histories. Intestinal mucosa adjacent to the tumor was evaluated for the presence of inflammatory activity or chronic changes.
For the evaluation of p53 immunostaining, we used the 3-level scores described by Köbel et al. that correlated with TP53 alterations: the overexpression pattern, when strong and intense p53 staining was observed in all nuclei, and the complete absence (null pattern), when a total absence of nuclear staining was observed, were considered as surrogates for TP53 mutation (p53-mut pattern) [39]. The wild-type pattern, corresponding to focal nuclear positivity of varying intensity, was considered normal (p53-wt pattern), even knowing that it could be a false negative for a mutated TP53 [39] (Figure 5). An external positive control was included on each slide. Positive staining for the MMR antibodies were in the nucleus of the neoplastic cells. We considered the tumor stable if nuclear staining was retained in tumor cells for all four MMR antibodies and unstable if one or more of the four markers lacked nuclear expression in tumor cells. Nuclear immunoreaction in lymphocytes, normal colonic mucosa or stromal cells served as the internal anti-MMR positive control. Loss of MMR staining was considered when the nuclei of all neoplastic cells were negative.
MUC5AC in adenocarcinomas was considered positive extensively if more than 50% of the tumor glands showed positive staining in the cytoplasm, focally positive if less than 50% of the glands had expression in the cytoplasm and negative if 100% of the tumor glands lacked expression. MUC5AC in the mucosa adjacent to the CRC was consider positive if more than 50% of the gland showed cytoplasmic staining. An external positive control was included on each slide.
Microscopy images were acquired with a digital slide scanner (Ultra Fast Scanner 1.8, Philips, Amsterdam, The Netherlands).

Statistical Analysis
Analysis was carried out using SSPS software version 26.0 (SPSS, Chicago, IL, USA). The χ2 test was used to analyze the association between qualitative variables, whereas the Fisher's exact test and the Student's t-test or the Mann-Whitney test were used for quantitative variables. A p < 0.05 was considered statistically significant.

Conclusions
Our results reflect that CAC shows a molecular profile characterized by p53 alterations and an absence of both MSI and MUC5AC staining, which differentiate it from CRC of the canonical and serrated carcinogenesis pathways. In relation to the expression of MUC5AC in CAC and surrounding intestinal mucosa, we could hypothesize that the appearance of GM occurs in inflamed mucosa, persists in those with chronic-regenerative changes and disappears with the acquisition of p53 mutations ( Figure 6). Positive staining for the MMR antibodies were in the nucleus of the neoplastic cells. We considered the tumor stable if nuclear staining was retained in tumor cells for all four MMR antibodies and unstable if one or more of the four markers lacked nuclear expression in tumor cells. Nuclear immunoreaction in lymphocytes, normal colonic mucosa or stromal cells served as the internal anti-MMR positive control. Loss of MMR staining was considered when the nuclei of all neoplastic cells were negative.
MUC5AC in adenocarcinomas was considered positive extensively if more than 50% of the tumor glands showed positive staining in the cytoplasm, focally positive if less than 50% of the glands had expression in the cytoplasm and negative if 100% of the tumor glands lacked expression. MUC5AC in the mucosa adjacent to the CRC was consider positive if more than 50% of the gland showed cytoplasmic staining. An external positive control was included on each slide.
Microscopy images were acquired with a digital slide scanner (Ultra Fast Scanner 1.8, Philips, Amsterdam, The Netherlands).

Statistical Analysis
Analysis was carried out using SSPS software version 26.0 (SPSS, Chicago, IL, USA). The χ2 test was used to analyze the association between qualitative variables, whereas the Fisher's exact test and the Student's t-test or the Mann-Whitney test were used for quantitative variables. A p < 0.05 was considered statistically significant.

Conclusions
Our results reflect that CAC shows a molecular profile characterized by p53 alterations and an absence of both MSI and MUC5AC staining, which differentiate it from CRC of the canonical and serrated carcinogenesis pathways. In relation to the expression of MUC5AC in CAC and surrounding intestinal mucosa, we could hypothesize that the appearance of GM occurs in inflamed mucosa, persists in those with chronic-regenerative changes and disappears with the acquisition of p53 mutations ( Figure 6). Funding: This research was funded by Roche through a collaboration agreement for research in Oncology (activity code: SP210830002).

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of the Hospital Universitari General de Catalunya (2020/139-APA-HUGC-HUSC-HQV and 10 November 2020).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Acknowledgments:
We want to particularly acknowledge the patients and the technical staff of the Hospital Universitari General de Catalunya-Grupo Quironsalud and Patologia Integrada, S.L. Work supported by the Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d'Oncologia de Catalunya (XBTC).

Conflicts of Interest:
The authors declare no conflict of interest.

CAC
colitis-associated colorectal carcinoma IBD inflammatory bowel disease Figure 6. Pathways of CRC carcinogenesis in IBD. GM appears in the inflamed colic mucosa as an adaptive mechanism to continued stress, persists in the mucosa with chronic changes and disappears with the acquisition of p53 mutations. CACs are characterized by p53 alterations, a lack of MUC5AC expression and MSS, whereas those CRCs with MSI-H, MUC5AC expression and p53 wt-pattern could represent sporadic CRC of the serrated pathway that appear in a context of "chronic inflammationdysplasia-cancer" from a gastric metaplastic epithelium. Funding: This research was funded by Roche through a collaboration agreement for research in Oncology (activity code: SP210830002).

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of the Hospital Universitari General de Catalunya (2020/139-APA-HUGC-HUSC-HQV and 10 November 2020).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data that support the findings of this study are available from the corresponding author upon reasonable request.