Abstract
Background/Aim: Nearly 70 years ago, Slaughter launched the hypothesis of field cancerization for oral carcinomas; that hypothesis was subsequently also claimed for carcinomas in other organs. We previously found in the colon mucosa adjacent to nonpolypoid adenomas, branching crypts lined by normal epithelium (BCNE). Here, we explored whether BCNE could also be found in the colon mucosa adjacent to sporadic polypoid tubular adenomas (TA), the most prevalent of all colon adenomas. Patients and Methods: Nondysplastic mucosa adjacent to TA was found in 103 out of 131 TA. All BCNE adjacent to TA were recorded. Results: In 98 (95.1%) out of 103 TA having nondysplastic adjacent mucosa, 645 BCNE were registered: 82.6% were in asymmetric branching and 17.4% in symmetric branching. Thus, BCNE in asymmetric branching predominated. The frequency of BCNE adjacent to TA was influenced by the adenoma size and degree of dysplasia severity. Contrarywise, the frequency of BCNE adjacent to TA was neither influenced by the age or sex of the patients, nor by the colon localization of TA. Conclusion: BCNE often occur in the normal mucosa adjacent to TA. BCNE emerge as integral components of TA. The majority of the BCNE were in asymmetric branching, considered as aberrations of cryptogenesis. We propose that the accretion of asymmetric BCNE adjacent to TA supports Slaughter’s hypothesis of field cancerization.
Based on the study of large numbers of histological sections from cases with recurrent oral squamous cell carcinomas, Slaughter et al. proposed the notion of field cancerization (1). The authors found that oral squamous carcinoma developed from coalescent areas with multifocal precancerous changes (1). The Slaughter’s field cancerization hypothesis was subsequently extended to carcinomas in other organs, such as the oropharynx (2), larynx (3), lung (4), esophagus (5), vulva uterine cervix (6), breast (7), bladder (8), skin (9), and colon (10).
Years ago, Filipe (11) found increased sialomucins, decreased or absent sulphomucins, and large goblet cells in the “transitional” mucosa adjacent to colon carcinomas. At the TEM level, large goblet cells displayed 0.15- 0.3~m electron-dense bodies and enlarged Golgi zone with increased secretory activity. Filipe suggested that the changes found in the “transitional” mucosa were due to a failure of cell differentiation in the crypts (11). More recent studies, such as peripheral nuclear heterochromatin loss (12), deficient expression of DNA repair enzymes (13), increased folate concentrations (14), abnormal gene expression (15), and mutations of the K-ras codon 12 (10), have contributed to support the notion of field cancerization hypothesis in the colon.
The expeditious turnover rate of the gastrointestinal mucosa is orchestrated by cell proliferation, differentiation, migration, and apoptosis (16). Quantitative studies indicate that the frequency of branching crypts in the small intestine increases during infancy and childhood (17). A similar age-dependent, quantitative study of branching crypts in the human colorectal mucosa has not been published. However, Mirjialili et al. recently assessed the length of the large bowel in 112 children using computed tomography scan (18). The length of the large bowel increased from a mean of 52 cm in children aged <2 years to 73 cm at 4-6 years and 95 cm at 9-11 years (18). The increased length of the colon mucosa in children must had been evoked by an increased frequency of branching crypts. Paradoxically, branching crypts are rarely found in adults (19-21), despite that the total area of the large bowel increases to approximately 2 m2 (22). This phenomenon is substantiated by the recent studies of Mandir et al. in rats (23) showing that crypt fission declined rapidly in the first 9 weeks (from 67.6 to 23.1) and then continued to decline, although at a lower rate. The crypt fission index in 48 weeks old rats was 9.8±1.0 (23). Inversely, crypt branching was frequently found in the colon mucosa of adult patients having either infectious colitis (24), ulcerative colitis (25), or Crohn’s colitis (26). Moreover, crypt branching was also frequent in the nondysplastic colon mucosa adjacent to nonpolypoid adenomas (27). The increase in crypt branching in these instances implies that not only inflammatory insults, but also proximity to mutated neoplastic tissue of nonpolypoid adenomas can unchain crypt replication in the colon mucosa.
The purpose of the present work was to explore whether branching crypts could also be found in the nondysplastic mucosa adjacent to sporadic polypoid colon adenomas. Tubular adenomas (TA) were selected because they are the most prevalent histologic phenotype of all colon adenomas.
Patients and Methods
The material consists of 131 consecutive sporadic TA, retrieved from the electronic archive (DC Pathos database), Institute of Pathology, Klinikum Bayreuth GmbH (DC Systeme, Heiligenhaus, Germany). Cases were diagnosed at the Department of Pathology, Klinikum Bayreuth, Friedrich-Alexander-University Erlangen-Nuremberg, Bayreuth, Germany, on hematoxylin and eosin (H&E)-stained slides (4 mm sections). Sections were subsequently scanned and digitalized with a Hamamatsu NanoZoomer Digital Pathology S360 (NDP, Hamamatsu, Herrsching am Ammersee, Germany). Images were made available online.
The size of TA was recorded using the digital ruler of the NPD View 2 of the NanoZoomer Digital Pathology S360. Small TA were those measuring <5 mm in diameter and larger TA were those measuring ≥5 mm in diameter. The number of branching crypts lined by normal epithelium (BCNE) found in the nondysplastic mucosa adjacent to TA was recorded. BCNE included those in symmetric branching and those in asymmetric branching.
The following independent confounders that could influence the frequency of BCNE in the mucosa adjacent to TA were investigated: i) age, ii) sex, iii) location (right colon or left colon), and iv) degree of TA dysplasia.
Statistical analysis. The non-parametric Mann–Whitney U two-tailed test was applied to compare differences between groups. The Pearson’s correlation coefficient was used to evaluate the possible linear association between two variables. Statistical significance was defined as p<0.05.
Ethical approval. Ethical approval was obtained from the Ethics Committee of Friedrich-Alexander University, Erlangen-Nuremberg, Germany, for adenomas (No. 21-475-Br).
Results
Out of the 131 TA, 103 (78.6%) had adjacent normal (nondysplastic) mucosa; the remaining 28 (21.4%) showed no normal adjacent mucosa. BCNE adjacent to TA were found in 98 of the 103 cases. A total of 645 BCNE adjacent to these 98 TA were found: 533 were BCNE in asymmetric branching (82.6%) and the remaining 112, BCNE in symmetric branching (17.4%). Thus, the vast majority of the BCNE were in asymmetric branching (Figure 1). The following independent variables that could possibly influence the frequency of NDBC, such as age, sex, TA localization, TA size, and degree of TA dysplasia, were investigated.
Nondysplastic colon mucosa adjacent to polypoid tubular adenomas. (a-i) Colon mucosa adjacent to polypoid tubular adenomas showing crypt in asymmetric branching [at arrows; hematoxylin and eosin (H&E), a-f original ×10, g, h, i, original ×20].
Age. The frequency of BCNE was analyzed for its correlation with the age of the patients (mean 64.8 years, range=22-87 years) in the 98 TA. No correlation was found (Pearson correlation coefficient R=0.0434; the results were not significant at p<0.05; p=0.09894).
Sex. The material included 34 females and 64 males. In females, a total of 250 BCNE were registered (mean 7.3, range=0-23). Of these, 209 were BCNE in asymmetric branching (mean 5.2, range=0-21), and 41 BCNE in symmetric branching (mean 1.0, range=0-13). In males, a total of 401 BCNE were logged (mean 6.3, range=0-50). Of these, 330 were BCNE in asymmetric branching (mean 5.2, range=0-31), and 71 BCNE in symmetric branching (mean 1.0, range=0-19). The difference between the frequency of BCNE in females and in males was not significant (Pearson correlation coefficient R=0.22, p-value=0.172548).
TA localization. Out of the 98 TA, 32 were found in the right colon (including the transverse colon), and the remaining 66, in the left colon. A total of 201 BCNE (mean 6.3, range=0-31) were found in the right colon, and 438 BCNE (mean 6.6, range=0-50) in the left colon. The difference between the frequency of BCNE in the right and left colon was not significant (p=0.62414, Mann–Whitney U two-tailed test).
TA size. The total digital size in the 98 TA was 558.8 mm (mean 5.7, range=1.4-16.6 mm). The frequency of BCNE was significantly higher in the 46 larger TA than in the 52 small TA (p<0.05; p-value=0.00104; Mann–Whitney U two-tailed test).
Degree of TA dysplasia. A total of 336 BCNE were recorded in the 67 TA with LGD (mean 5.0, range=0-24), and 308 BCNE in 31 TA with HGD (mean 9.9, range=0-50). The difference between the frequency of BCNE in TA with LGD and TA with HGD was significant at p<0.05 (p-value=0.00328; Mann–Whitney U two-tailed test).
Discussion
In the present survey we found that the colonic mucosa adjacent to TA displayed an unprecedent accretion of BCNE (predominantly in asymmetric branching and less frequently, in symmetric branching). The occurrence of this phenomenon within a few mm in the colon mucosa contrasts with the infrequency of isolated crypts in symmetric branching, and with the absence of asymmetric branching crypts in the entire normal colon mucosa in adults (19-21). This survey also showed, as expected, that the frequency of BCNE near TA increased with the increasing TA size, as the adjacent mucosa in larger TA was larger than that in small TA. The frequency of BCNE was also higher in TA with HGD than in that with LGD. Importantly, the frequency of BCNE adjacent to TA was neither influenced by the age or the sex of the patients, nor by the localization of TA in the colon.
It is generally accepted that the entire colon mucosa is constantly battered by a series of oncogenic factors able to generate mucosal mutations leading to colonic adenomas (28). Therefore, it remains enigmatic why mutated TA and the associated BCNE occupies barely a few mm of the colonic mucosa. Knowing that TA are triggered by somatic mutations (29), the pertinent question is: do BCNE also evolve as a result of somatic mutations in the same area? While studying nondysplastic crypts underneath the neoplastic canopy of nonpolypoid and polypoid sporadic colon adenomas (30), we noticed that some of the crypts in the pedicle exhibited haphazardly distributed Ki67-labeled cells and p53-upregulated cells, reaching the luminal aspect of nondysplastic crypts (30). It was suggested that the relocation of the normal Ki67 domains in the cells of the crypts of the stalk and the occurrence of p53-up-regulated cells in the same crypts evolved as a result of somatic mutations (29). This suggestion was validated in a recent mega-analysis of colorectal transcriptomes of histologically normal mucosa adjacent to colorectal carcinomas (CRC) (31). These authors found that in the mucosa adjacent to CRC, the gene expression was at an intermediate state between healthy mucosa expression and CRC expression (31).
In conclusion, BCNE often occur in the normal mucosa adjacent to TA. BCNE emerge as integral components of TA. The majority of the BCNE were in asymmetric branching, considered as aberrations of cryptogenesis. We propose that the accretion of asymmetric BCNE adjacent to TA supports Slaughter’s hypothesis of field cancerization (1).
Footnotes
Authors’ Contributions
CAR is responsible for conceptualization, conducting the project, visualization, writing the original draft, data curation, formal analysis, investigation, and methodology; MV and CL-S for the scanning of sections and the reviewing of the original draft. The final draft was approved by all Authors.
Conflicts of Interest
The Authors have no conflicts of interest to declare regarding this study.
- Received October 20, 2022.
- Revision received October 25, 2022.
- Accepted October 26, 2022.
- Copyright © 2022 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
