International Journal of Molecular Sciences Article Alterations in p53, Microsatellite Stability and Lack of MUC5AC Expression as Molecular Features of Colorectal Carcinoma Associated with Inflammatory Bowel Disease Míriam Gené 1,2,† , Míriam Cuatrecasas 3,4,† , Irene Amat 5 , Jesús Alberto Veiga 6 , María Jesús Fernández Aceñero 7 , Victòria Fusté Chimisana 8 , Jordi Tarragona 9 , Ismael Jurado 10 , Rebeca Fernández-Victoria 11 , Carolina Martínez Ciarpaglini 12 , Cristina Alenda González 13 , Carlos Zac 14 , Pilar Ortega de la Obra 15 , María Teresa Fernández-Figueras 16,17 , Manel Esteller 18,19,20,21 and Eva Musulen 16,18, * 1 Pathology Department, Hospital Universitari Joan XXIII, 43005 Tarragona, Spain; miriamgehi@hotmail.com 2 Surgery Department, Programme of Surgery and Morphological Sciences, Universitat Autònoma de Barcelona (UAB), 08193 Barcelona, Spain 3 Pathology Department, Hospital Clínic de Barcelona, Universitat de Barcelona (UB), 08007 Barcelona, Spain; mcuatrec@clinic.cat 4 School of Medicine, Campus Clínic, Universitat de Barcelona (UB), 08036 Barcelona, Spain 5 Pathology Department, Complejo Hospitalario de Navarra, 31008 Navarra, Spain; irene.amat.villegas@navarra.es 6 Pathology Department, Complejo Hospitalario Universitario de Ferrol, 15405 Ferrol, Spain; jesus.alberto.veiga.barreiro@sergas.es 7 Pathology Department, Hospital Clínico San Carlos, 28040 Madrid, Spain; mgg10167@gmail.com 8 Pathology Department, Hospital de la Santa Creu i Sant Pau, 08025 Barcelona, Spain; vfuste@santpau.cat 9 Pathology Department, Hospital Universitari Arnau de Vilanova, 25198 Lleida, Spain; jtarragona@gss.cat 10 Pathology Department, Consorci Sanitari de Terrassa, 08227 Terrassa, Spain; ijurado@cst.cat Citation: Gené, M.; Cuatrecasas, M.; 11 Pathology Department, Hospital Álvaro Cunqueiro, 36312 Vigo, Spain; rebeca.fernandez.victoria@sergas.es Amat, I.; Veiga, J.A.; Fernández 12 Pathology Department, Hospital Clínico Universitario de Valencia, Valencia INCLIVA-Instituto de Aceñero, M.J.; Fusté Chimisana, V.; Investigación Sanitaria, Universidad de Valencia, 46010 Valencia, Spain; carolaciarpaglini@gmail.com Tarragona, J.; Jurado, I.; 13 Pathology Department, Hospital General Universitario Dr. Balmis, Instituto de Investigación Sanitaria y Fernández-Victoria, R.; Martínez Biomédica de Alicante (ISABIAL), 03010 Alicante, Spain; alenda.cris@gmail.com 14 Pathology Department, Hospital Universitari i Politècnic La Fe, 46026 Valencia, Spain; Ciarpaglini, C.; et al. Alterations in p53, Microsatellite Stability and Lack carloszacromero@gmail.com 15 Pathology Department, Hospital General de Segovia, 40002 Segovia, Spain; mportegao@saludcastillayleon.es of MUC5AC Expression as Molecular 16 Pathology Department, Hospital Universitari General de Catalunya-Grupo QuironSalud, Sant Cugat del Features of Colorectal Carcinoma Vallès, 08195 Barcelona, Spain; maite.ffigueras@quironsalud.es Associated with Inflammatory Bowel 17 School of Medicine, Campus Sant Cugat del Vallès, Universitat Internacional de Catalunya (UIC), Sant Cugat Disease. Int. J. Mol. Sci. 2023, 24, 8655. del Vallès, 08017 Barcelona, Spain https://doi.org/10.3390/ 18 Institut de Recerca contra la Leucèmia Josep Carreras (IJC), Badalona, 08916 Barcelona, Spain; ijms24108655 mesteller@carrerasresearch.org 19 Institució Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain Academic Editor: Carmine Stolfi 20 Faculty of Medicine and Health Sciences, Department of Physiological Sciences, Universitat de Barcelona (UB), 08007 Barcelona, Spain Received: 29 March 2023 21 Centro de Investigación Biomédica en Red Cáncer (CIBERONC), 28029 Madrid, Spain Revised: 7 May 2023 * Correspondence: eva.musulen@quironsalud.es or emusulen@carrerasresearch.org Accepted: 8 May 2023 † These authors contributed equally to this work. Published: 12 May 2023 Abstract: Colitis-associated colorectal carcinoma (CAC) occurs in inflammatory bowel disease (IBD) because of the “chronic inflammation-dysplasia-cancer” carcinogenesis pathway characterized by p53 Copyright: © 2023 by the authors. alterations in the early stages. Recently, gastric metaplasia (GM) has been described as the initial event Licensee MDPI, Basel, Switzerland. of the serrated colorectal cancer (CRC) process, resulting from chronic stress on the colon mucosa. This article is an open access article The aim of the study is to characterize CAC analyzing p53 alterations and microsatellite instability distributed under the terms and (MSI) to explore their relationship with GM using a series of CRC and the adjacent intestinal mucosa. conditions of the Creative Commons Immunohistochemistry was performed to assess p53 alterations, MSI and MUC5AC expression as a Attribution (CC BY) license (https:// surrogate for GM. The p53 mut-pattern was found in more than half of the CAC, most frequently creativecommons.org/licenses/by/ stable (MSS) and MUC5AC negative. Only six tumors were unstable (MSI-H), being with p53 4.0/). Int. J. Mol. Sci. 2023, 24, 8655. https://doi.org/10.3390/ijms24108655 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2023, 24, 8655 2 of 17 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. Keywords: intestinal bowel disease; colorectal cancer; MUC5AC expression; gastric metaplasia; DNA-damage response 1. Introduction Crohn’s disease (CD) and ulcerative colitis (UC) are pathologies with a two- to three- fold 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 [5–7]. 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 [9–12]. 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 [17–22]. 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 mi- crosatellite instability (MSI) and to explore their relation to GM. 2. Results 2.1. Patients and Specimens A total of 57 CACs were studied from 38 (72%) men and 15 (28%) women, all Caucasian, aged between 34 and 90 years (mean 62.6), 34 (64%) with UC and 19 (36%) with CD. 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 Int. J. Mol. Sci. 2023, 24, 8655 3 of 17 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). Table 1. Clinicopathological features of IBD patients and adenocarcinomas. Crohn’s Disease (%) Ulcerative Colitis (%) Total (%) p Value Patients 19 (36) 34 (64) 53 (100) Age Mean years [range] 58.4 [35–88] 64.8 [34–90] 62.6 [34–90] Gender 1.000 Male 14 (74) 24 (71) 38 (72) Female 5 (26) 10 (29) 15 (28) IBD duration 0.244 Mean years [range] 13.2 [1–43] Concominant * 5 (26) 2 (6) 7 (13.2) ≤8 years 4 (21) 13 (38) 17 (32.1) 9–15 years 3 (16) 7 (20.6) 10 (18.9) 16–20 years 1 (5) 5 (14.7) 6 (11.3) >21 years 5 (26) 6 (17.6) 11 (20.8) Unknown 1 (5) 1 (3) 2 (3.8) IBD activity 0.780 Active 11 (58) 18 (53) 29 (55) Quiescent 8 (42) 16 (47) 24 (45) Synchronous CRC (n = 57) 0.042 Non synchronous CRC 19 (100) 30 (79) 49 (86) 2 synchronous CRC 0 8 (21) 8 (14) Adenocarcinoma location (n = 57) 0.003 Ileum 7 (37) 0 7 (12.3) Right colon 6 (31.5) 11 (28.9) 17 (29.8) Left colon 3 (16) 15 (39.5) 18 (31.6) Rectum 1 (5) 6 (15.8) 7 (12.3) Colon 2 (10.5) 5 (13.2) 7 (12.3) Ileal reservory 0 1 (2.6) 1 (1.8) Histology (n = 57) 0.504 Adenocarcinoma NOS 15 (78.9) 32 (84.2) 47 (82.5) Mucinous 3 (15.8) 4 (10.5) 7 (12.3) Mucinous and signet ring cell 1 (5.3) 0 1 (1.8) Medullary 0 1 (2.6) 1 (1.8) Undifferentiated 0 1 (2.6) 1 (1.8) Histologic grade (n = 57) 1.000 Low-grade 15 (79) 31 (82) 46 (81) High-grade 4 (21) 7 (18) 11 (19) Adjacent mucosa to adenocarcinoma (n 0.077 = 57) Absent 10 (53) 12 (32) 22 (39) Present 9 (47) 26 (68) 35 (61) Active inflammation 0 4 (15) 4 (11.4) Chronic changes 4 (21) 15 (58) 19 (54.3) Normal 5 (26) 7 (27) 12 (34.3) Stage (8th ed) (n = 57) 0.305 I 3 (15.8) 9 (23.7) 12 (21) II 6 (31.6) 17 (44.7) 23 (40) III 10 (52.6) 12 (31.6) 22 (39) Abbreviations: IBD, intestinal bowel disease; NOS, not otherwise specified. Concomitant*, in this case the diagnosis of IBD was made at colectomy. Int. J. Mol. Sci. 2023, 24, 8655 4 of 17 2.2. Immunohistochemistry 2.2.1. p53, MUC5AC and MSI in Adenocarcinoma 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). Table 2. Expression of p53, MSI and MUC5AC in CAC according to IBD subtypes. Crohn’s Disease (%) Ulcerative Colitis (%) Total (%) p Value p53 pattern 0.914 mut-pattern Overexpression 8 (42) 14 (37) 22 (39) Null pattern 2 (11) 5 (13) 7 (12) Focal nuclear wt-pattern 9 (47) 19 (50) 28 (49) staining Microsatellite 0.389 instability MSI-H 3 (16) 3 (8) 6 (11) MSS 16 (84) 35 (92) 51 (89) MUC5AC 0.511 Negative 10 (52.6) 22 (57.9) 32 (56.1) Positive Extensive 2 (10.5) 7 (18.4) 9 (15.8) Focal 7 (36.8) 9 (23.7) 16 (28.1) Abbreviation: mut, mutation; wt, wild type; MSI-H, microsatellite instability high grade; MSS, microsatellite stable. 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). 2.2.2. 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). Table 3. Relation between p53 immunohistochemical patterns and MSI. Microsatellite Instability MSI-H (%) MSS (%) Total (%) p Value p53 pattern 0.010 mut-pattern 0 29 (57) 29 (51) wt-pattern 6 (100) 22 (43) 28 (49) Abbreviation: MSI-H, microsatellite instability high-grade; MSS, microsatellite stable; mut, mutation; wt, wild type. 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). p53 pattern 0.010 mut-pattern 0 29 (57) 29 (51) wt-pattern 6 (100) 22 (43) 28 (49) Abbreviation: MSI-H, microsatellite instability high-grade; MSS, microsatellite stable; mut, muta- tion; wt, wild type. Int. J. Mol. Sci. 2023, 24, 8655 5 of 17 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 Table 4.significant Relation (Table of p534and andMUC5AC Figure 1). expression in CAC. Table 4. Relation of p53 and MUC5AC expression in CAC. MUC5AC Negative (%)MUC5AC Positive (%) Total (%) p Value Negative (%) Positive (%) Total (%) p Value p53 pattern p53 pattern 0.186 0.186 mut-pattern mut-pattern 19 (59) 19 (59) 10 10 (40) (40) 29 (51) 29 (51) wt- pattern wt- pattern 13 (41) 13 (41) 15 15 (60) (60) 28 (49) (49) 28 Abbreviation: mut, mutation; Abbreviation: wt, wild mut, mutation; wt,type. wild type. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 6 of 18 (a) (b) Figure 1. Two CACs with different expression of p53 and MUC5AC. (a) p53 shows a wt-pattern Figure 1. Two CACs with different expression of p53 and MUC5AC. (a) p53 shows a wt-pattern with with focal nuclear staining in tumor cells. In the same tumor, MUC5AC expression is diffuse (20×, focal nuclear staining scale bar 50 µm). in(b)tumor cells.tumor, In this other In thenuclear same overexpression tumor, MUC5AC expression is observed of mut-(20×, scale is diffuse in the form pattern bar 50 µm). staining (b) of other In this p53 while MUC5AC tumor, is negative. nuclear Note the MUC5AC overexpression positivity is observed in in non-dysplastic the form of mut-pattern glands on the left side (10×, scale bar 100 µm). staining of p53 while MUC5AC is negative. Note the MUC5AC positivity in non-dysplastic glands on the left side (10×, scale Analyzing bar 100between the relation µm). MUC5AC expression and MSI, a statistically signifi- cation was found, with all MSI-H CACs being positive for MUC5AC and all MSS CACs being MUC5AC negative (Table 5). Table 5. Relation between MUC5AC expression and MSI. MUC5AC Negative (%) Positive (%) Total (%) p Value Int. J. Mol. Sci. 2023, 24, 8655 6 of 17 Analyzing the relation between MUC5AC expression and MSI, a statistically signifi- cation was found, with all MSI-H CACs being positive for MUC5AC and all MSS CACs being MUC5AC negative (Table 5). Table 5. Relation between MUC5AC expression and MSI. MUC5AC Negative (%) Positive (%) Total (%) p Value Microsatellite 0.005 instability MSI-H 0 6 (24) 6 (11) MSS 32 (100) 19 (76) 51 (89) Abbreviation: MSI-H, microsatellite instability high-grade; MSS, microsatellite stable. The clinicopathological characteristics and IHC expression of MSI-H CAC are shown in Figure Int. J. Mol. Sci. 2023, 24, x FOR PEER 2 and REVIEWTable 6. Of notice, these adenocarcinomas were in the ileum or right 7 of 18 colon and showed histological features characteristic of sporadic MSI-H or LS-associated CRC—i.e., medullary, mucinous or signet ring cell carcinomas. (a) Figure 2. Cont. Int. J. Mol. Sci. 2023, 24, x FOR PEER REVIEW 8 of 18 Int. J. Mol. Sci. 2023, 24, 8655 7 of 17 (b) Figure Figure 2. 2. TwoTwo examples examples of of MSI-H MSI-H CAC. CAC. (a)(a) medullary medullary andand (b)(b) mucinous. mucinous. In In both both adenocarcinomas adenocarcinomas p53 showed scattered nuclear positivity corresponding to a wt-pattern, MUC5AC expression was p53 showed scattered nuclear positivity corresponding to a wt-pattern, MUC5AC expression was focal positive, and MLH1 showed an absence of nuclear staining in tumor cells. Note in both MLH1 focal positive, and MLH1 showed an absence of nuclear staining in tumor cells. Note in both MLH1 staining the positive internal control in lymphocytes (20×, scale bar 50 µm). staining the positive internal control in lymphocytes (20×, scale bar 50 µm). Only one patient with MSH-H CAC was affected by LS, and another had two syn- Table 6. Clinicopathological chronous sporadic tumorsandharboring IHC features theofV600E unstable CRC.mutation. The remaining unstable BRAF CRCs were BRAF wild type, and the hypermethylation of MLH1 was not performed. IBD Age p53 BRAF MLH1 Predisposition Patient Gender IBD Duration Location Histology MUC5AC MLH1 (Years) Pattern Status Methylation to Cancer (Years) Table 6. Clinicopathological and IHC features of unstable CRC. Right Focal Sporadic vs. 8 73 Male UC 12 Medullary wt Negative V600E NP IBD colon positive IBD-associated Age p53 BRAF MLH1 Meth- Predisposition Patient Gender IBD Duration Location Histology NOS with MUC5AC MLH1 (Years) Right mucinous Pattern Focal Status ylation to Cancer Sporadic vs. 8 (Years) colon wt Negative V600E NP compo- positive IBD-associated (transvers) Right co- Focal Sporadic vs. 8 73 Male UC 12 nent Medullary wt Negative V600E NP lon positive IBD-associated Right Extensive No Lynch 14 52 Male UC 4 Right co- NOS wt Negative wt colon NOS with positive methylated syndrome lon Focal Sporadic vs. 8 mucinous wt Negative V600E NP (trans- positive IBD-associated component vers) Int. J. Mol. Sci. 2023, 24, 8655 8 of 17 Table 6. Cont. IBD Age p53 BRAF MLH1 Predisposition Patient Gender IBD Duration Location Histology MUC5AC MLH1 (Years) Pattern Status Methylation to Cancer (Years) NOS with mucinous Extensive 20 50 Male CD Concomitant Ileum wt Negative wt NP Unknown compo- positive nent Focal 24 54 Male CD 1 Ileum NOS wt Negative wt* NP Unknown positive Mucinous Right and Extensive 53 52 Female CD 2 wt Negative wt NP Unknown colon signet positive ring cell Abbreviations: IBD, Intestinal bowel disease; UC, ulcerative colitis; CD, Crohn’s disease; wt, wild type; IHC, immunohistochemistry; NP, not performed. wt*, in this case a G13D mutation in KRAS was identified. Only one patient with MSH-H CAC was affected by LS, and another had two syn- chronous sporadic tumors harboring the V600E BRAF mutation. The remaining unstable CRCs were BRAF wild type, and the hypermethylation of MLH1 was not performed. 2.2.3. 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, OR PEER REVIEW 10 of 18 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). Figure 3. Cont. Int. J. Mol. Sci. 2023, 24, 8655 9 of 17 Figure 3. Colon mucosa 3. Colon to adjacent Figure mucosa a CAC.adjacent to a CAC. Severe Severe distortion distortion of colonic of colonic epitheliawith epithelia with chronic chronic in- inflammation, glandular atrophy and granulation tissue (H&E, 10×, scale bar 100 µm). Strong flammation, glandular atrophy and granulation tissue (H&E, 10×, scale bar 100 µm). Strong expres- expression of MUC5AC was seen in the epithelium re-epithelizing the granulation tissue and in part sion of MUC5AC wasofseen in the epithelium the irregular re-epithelizing glands (10×, scale bar 100 µm). p53-wtthepattern granulation showed fewtissue and positive in part nuclei in theof the irregular glands (10×,surface scaleepithelium bar 100 and µm). p53-wt in the pattern glands (10 showed ×, scale bar 100 µm; few positive 40 in magnification, nuclei ×). in the surface epithelium and in the glands (10×, scale bar 100 µm; in magnification, 40×). The relation between MUC5AC expression in adenocarcinoma and adjacent mucosa was not statistically significant. MUC5AC-positive non-tumoral mucosa was adjacent to The relation between MUC5AC expression negative adenocarcinoma in adenocarcinoma in more than 60% and adjacent (16/27), and MUC5AC-negative mucosa mucosa was adjacent to positive or negative adenocarcinoma in the same proportion (50%, 4/8) (Table 7 was not statisticallyand significant. Figure 4). 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). MUC5AC in adenocarcinoma Negative 4 (50) 16 (59) 20 (57) Int. J. Mol. Sci. 2023, 24, 8655 10 of 17 Positive 4 (50) 11 (41) 15 (43) J. Mol. Sci. 2023, 24, x FOR PEER REVIEW (a) (b) Figure Figure4.4.Colonic mucosa Colonic mucosa withwith chronic chronic changeschanges adjacent toadjacent a CAC. (a)to a staining p53 CAC. (a) p53astaining showed wt- sh pattern patternwith fewpositive with few positive nucleinuclei (in magnification) (in magnification) in the in the colon glands colon adjacent glands to an adjacent to an adenocarcinoma with p53 nuclei overexpression (p53 mut-pattern) (10 × , scale noma with p53 nuclei overexpression (p53 mut-pattern) (10×, scale bar bar 100 µm). (b) MUC5AC strongly 100 µm). (b positive in the colon mucosa adjacent to a negative neoplasm (5×, scale bar 200 µm). strongly positive in the colon mucosa adjacent to a negative neoplasm (5×, scale bar 20 3. 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 Int. J. Mol. Sci. 2023, 24, 8655 11 of 17 Table 7. Expression of MUC5AC in mucosa adjacent to adenocarcinomas. MUC5AC Negative (%) Positive (%) Total (%) p Value Adjacent mucosa 1.000 (n = 35) Location Colon 8 (100) 26 (96) 34 (97) Small bowel 0 1 (4) 1 (3) Specific location 0.614 Ileum 0 1 (4) 1 (3) Right 3 (37.5) 7 (26) 10 (29) Left 3 (37.5) 12 (44) 15 (43) Rectum 0 4 (15) 4 (11) Colon 2 (25) 3 (11) 5 (14) Status 0.374 Active 0 4 (14.8) 4 (11.4) inflammation Chronic changes 4 (50) 15 (55.6) 19 (54.3) Normal 4 (50) 8 (29.6) 12 (34.3) MUC5AC in 0.700 adenocarcinoma Negative 4 (50) 16 (59) 20 (57) Positive 4 (50) 11 (41) 15 (43) 3. Discussion Colitis-associated colorectal cancer (CAC) is the result of the alteration of genes in- volved 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 [9–14]. 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% [5–7,24–30]. 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 [5–7]. 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 [31–37] 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 Int. J. Mol. Sci. 2023, 24, 8655 12 of 17 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 inflammation- dysplasia-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 epithe- lia IBD-associated, an intermediate step between colon mucosa with chronic changes and the onset of neoplastic events of CAC. 4. 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. 4.1. Immunohistochemistry Formalin-fixed, paraffin-embedded tissue sections were analyzed using standard IHC techniques. The primary antibodies used were anti-p53 (clone DO-7, Ventana Med- ical Systems, Inc., 1910 E. Innovation Park Drive, Tucson, AZ, USA), anti-MUC5AC (clone MRQ-19, Ventana Medical Systems), anti-MLH1 (clone ES05, Leica Biosystems, Newcastle Upon Tyne, UK), anti-MSH2 (clone 79H11, Leica Biosystems), anti-MSH6 (clone EP49, Leica Biosystems) and anti-PMS2 (clone EP51, Leica Biosystems, Newcastle Upon Tyne, UK). Immunostaining was performed automatically using a Ventana BenchMark ULTRA machine (Roche, Basel, Switzerland) for p53 and MUC5AC and a Bond-III machine (Leica Biosystems, Newcastle Upon Tyne, UK) for the MMR antibodies. Immunostaining was independently evaluated by two pathologists (M.G. and E.M.). 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. Int.J. J.Mol. Int. Mol.Sci. Sci.2023, 24,x 8655 2023,24, FOR PEER REVIEW 14 13 ofof1817 (a) (b) (c) Figure Figure5.5.Three Threepatterns patternsofofp53 p53staining. staining.(a) (a)Overexpression Overexpressionpattern, pattern,allalltumor tumornuclei nucleiare arestrongly strongly positive positive(scale (scalebar bar10 10 µm). µm). (b) (b) Null pattern, no Null pattern, no staining stainingisisobserved observedinintumor tumorcell cell nuclei nuclei inin thethe pres- presence ence of a positive internal control in stromal cells (scale bar 10 µm). (c) Wild-type pattern, focal of a positive internal control in stromal cells (scale bar 10 µm). (c) Wild-type pattern, focal nuclear nuclear positivity of varying intensity in tumor cell nuclei (scale bar 50 µm). positivity of varying intensity in tumor cell nuclei (scale bar 50 µm). Positive Positivestaining stainingforforthe theMMR MMRantibodies antibodieswerewereininthethenucleus nucleusofofthe theneoplastic neoplasticcells. cells. We considered the tumor stable if nuclear staining was retained in tumor We considered the tumor stable if nuclear staining was retained in tumor cells for all cells for all four MMR antibodies four MMR and unstable antibodies if one or and unstable if more one orofmore the four markers of the lacked nuclear four markers lackedexpres- nuclear sion in tumor cells. Nuclear immunoreaction in lymphocytes, expression in tumor cells. Nuclear immunoreaction in lymphocytes, normal colonicnormal colonic mucosa mucosaor stromal cells served as the internal anti-MMR positive control. Loss of or stromal cells served as the internal anti-MMR positive control. Loss of MMR staining MMR staining was considered was consideredwhenwhenthe nuclei of all of the nuclei neoplastic cells were all neoplastic negative. cells were negative. MUC5AC MUC5ACininadenocarcinomas adenocarcinomaswas wasconsidered consideredpositive positiveextensively extensivelyififmore morethan than50%50% ofofthe thetumor tumorglands glandsshowed showedpositive positivestaining stainingininthe thecytoplasm, cytoplasm,focally focallypositive positiveififless lessthan than 50% 50%of of the glands glands had hadexpression expressioninin thethe cytoplasm cytoplasm andand negative negative if 100% if 100% of theoftumor the tumor glands glands lacked expression. MUC5AC in the mucosa adjacent to the lacked expression. MUC5AC in the mucosa adjacent to the CRC was consider positive CRC was consider pos-if itive moreifthan more50% than 50%gland of the of the gland cytoplasmic showed showed cytoplasmic staining. Anstaining. externalAnpositive external positive control was control included wasonincluded each slide.on each slide. Microscopy Microscopyimages imageswerewereacquired acquiredwith withaadigital digitalslide slidescanner scanner(Ultra (UltraFast FastScanner Scanner1.8,1.8, Philips, Philips,Amsterdam, Amsterdam,The TheNetherlands). Netherlands). 4.2.Statistical 4.2. StatisticalAnalysis Analysis Analysiswas Analysis wascarried carriedoutoutusing usingSSPS SSPSsoftware softwareversion version26.0 26.0(SPSS, (SPSS,Chicago, Chicago,IL,IL,USA). USA). The χ2 test was used to analyze the association between qualitative variables, The χ2 test was used to analyze the association between qualitative variables, whereas the whereas the Fisher’s Fisher’s exact exact testthe test and and the Student’s Student’s t-test t-test or the or the Mann–Whitney Mann–Whitney testused test were wereforused for quan- quantitative variables. A p < 0.05 was considered statistically titative variables. A p < 0.05 was considered statistically significant. significant. 5. Conclusions 5. Conclusions Our results reflect that CAC shows a molecular profile characterized by p53 alterations Our results reflect that CAC shows a molecular profile characterized by p53 altera- and an absence of both MSI and MUC5AC staining, which differentiate it from CRC of the tions and an absence of both MSI and MUC5AC staining, which differentiate it from CRC canonical and serrated carcinogenesis pathways. In relation to the expression of MUC5AC of the canonical and serrated carcinogenesis pathways. In relation to the expression of in CAC and surrounding intestinal mucosa, we could hypothesize that the appearance of MUC5AC in CAC and surrounding intestinal mucosa, we could hypothesize that the ap- GM occurs in inflamed mucosa, persists in those with chronic-regenerative changes and pearance of GM occurs in inflamed mucosa, persists in those with chronic-regenerative disappears with the acquisition of p53 mutations (Figure 6). changes and disappears with the acquisition of p53 mutations (Figure 6). Int. Int.J.J.Mol. Mol.Sci. 2023,24, Sci.2023, 24,8655 x FOR PEER REVIEW 1514ofof18 17 Figure 6. Pathways of CRC carcinogenesis in IBD. GM appears in the inflamed colic mucosa as an Figure 6. Pathways adaptive mechanismofto CRC carcinogenesis continued in IBD.in stress, persists GMtheappears mucosainwith the inflamed colic mucosa chronic changes as an and disap- adaptive mechanism to continued stress, persists in the mucosa with chronic changes and pears with the acquisition of p53 mutations. CACs are characterized by p53 alterations, a lack ofdisappears with the acquisition MUC5AC expressionofand p53 MSS, mutations. whereasCACs areCRCs those characterized by p53 with MSI-H, alterations, MUC5AC a lack of expression andMUC5AC p53 wt- expression andrepresent pattern could MSS, whereas thoseCRC sporadic CRCs ofwith MSI-H, MUC5AC the serrated expression pathway that appearand in ap53 wt-pattern context could of “chronic inflammation-dysplasia-cancer” from a gastric metaplastic epithelium. Figure created with represent sporadic CRC of the serrated pathway that appear in a context of “chronic inflammation- BioRen- der.com. dysplasia-cancer” from a gastric metaplastic epithelium. Figure created with BioRender.com. Author Contributions: Conceptualization, M.G., M.C. and E.M; methodology, M.G., M.C. and E.M; formal analysis, Author M.G., M.C. Contributions: and E.M.; data curation, Conceptualization, M.G.,and M.G., M.C. M.C., E.M., E.M; I.A., J.A.V., M.J.F.A., methodology, M.G., M.C. V.F.C., and J.T., I.J., E.M; R.F.-V., formal C.M.C., analysis, C.A.G., M.G., C.Z. M.C. and and P.O.d.l.O.; E.M.; writing—original data curation, draft M.G., M.C., preparation, E.M., M.G.; I.A., J.A.V., writ- M.J.F.A., ing—review V.F.C., J.T., I.J.,and editing, R.F.-V., M.C.,C.A.G., C.M.C., E.M., M.T.F.-F. C.Z. andand M.E.; funding P.O.d.l.O.; acquisition,draft writing—original E.M.preparation, All authors have M.G.; read and agreed and writing—review to the published editing, version M.C., of the manuscript. E.M., M.T.F.-F. and M.E.; funding acquisition, E.M. All authors have read and Funding: Thisagreed researchto the waspublished funded byversion Rocheof the manuscript. through a collaboration agreement for research in Oncology This Funding: (activity code:was research SP210830002). 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 Declara- tion of Helsinki Institutional and approved Review by the ethics Board Statement: committee The study of the Hospital was conducted Universitari in accordance withGeneral de Ca- the Declaration talunya (2020/139-APA-HUGC-HUSC-HQV and 10 November 2020). of Helsinki and approved by the ethics committee of the Hospital Universitari General de Catalunya (2020/139-APA-HUGC-HUSC-HQV Informed Consent Statement: Informedand 10 November consent was 2020). obtained from all subjects involved in the study. 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 Data Availability Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request. corresponding author upon reasonable request. Acknowledgments: We want to particularly acknowledge the patients and the technical staff of the 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 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 supported by the Xarxa de Bancs de Tumors de Catalunya sponsored by Pla Director d’Oncologia de de Catalunya (XBTC). 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