Endoscopic and Clinicopathological Features of a Colorectal Mucin-Rich Variant of Traditional Serrated Adenoma

In the new WHO Classification of Tumors of the Digestive System (5th edition) (2019) [1], colonic serrated lesions are classified into hyperplastic polyps (HPs), sessile serrated lesions (SSLs), SSLs with dysplasia, and traditional serrated adenomas (TSAs). Histologically, TSA is characterized by slit-like serration, tall columnar cells with eosinophilic cytoplasm and penicillate nuclei, and ectopic crypt formations in the villous projections of protuberant polyps. The molecular features of TSAs have been thoroughly analyzed in previous studies [2‒5]. As in other serrated polyps, BRAF and KRAS mutations, which lead to the activation of the mitogen-activated protein kinase pathway, are present in most TSAs [2‒4]. However, compared with SSLs [5], TSAs consistently retain MLH1 expression irrespective of the presence of advanced histological features, suggesting that TSAs can be precursor lesions of an aggressive BRAF mutant, microsatellite stable subtype of colorectal carcinoma [2].

Several morphological variants of TSA have been reported. N Kalimuthu et al. [6] highlighted mucin-rich/goblet cell-rich TSA as a distinct morphological variant, stating that mucin-rich TSAs (MR-TSAs), compared with conventional TSAs (C-TSAs), are characterized by the presence of >50% goblet cells and fewer ectopic crypt formations, but with preservation of archetypal luminal serrations. In our previous study [7], we reported that immunohistochemical and genetic characteristics, such as MUC5AC expression and a BRAF mutation, were more closely associated with MR-TSAs than with C-TSAs.

Endoscopically, TSAs usually present as reddish protruded polyps, having a pinecone-like or coral-shaped appearance, and predominantly localized in the sigmoid colon and rectum [8‒10] (shown in Fig. 1). However, the endoscopic features of MR-TSAs remain unknown. Hence, this study aimed to elucidate the endoscopic features of MR-TSAs, including image-enhanced endoscopy findings, and clinicopathological features.

The Institutional Review Board and the Ethical Committee of our hospital approved this study (registration no.: 2017166). We reviewed lesions pathologically diagnosed as “traditional serrated adenoma” or “serrated adenoma” resected endoscopically at Juntendo University Hospital (Tokyo, Japan) between 2011 and 2023.

Based on accepted and established morphological features in the literature [1‒3], TSAs were diagnosed according to the following criteria (shown in Fig. 2a, b): (1) exophytic architecture and characteristic flat-topped, slit-like, luminal serrations with brush borders, resembling a small-bowel phenotype; (2) glands lined by columnar epithelial cells with dense eosinophilic cytoplasm and pencillate nuclei; and (3) the presence of ectopic crypt formations, which are abortive crypts with a “maelstrom-like” swirling configuration that are not anchored to or oriented toward the muscularis mucosa. All three features had to be present in >50% of an individual polyp for it to be classified as a C-TSA. MR-TSAs were diagnosed if they met the aforementioned criteria for TSA and showed the presence of goblet/mucin-rich cells in ≥50% of the entire lesion with a goblet cell/eosinophilic absorptive cell ratio of at least 1:1 (shown as Fig. 2c, d) [6, 7]. All samples were independently reviewed by two experienced gastrointestinal pathologists (T.S. and T.Y.).

Colonoscopy was performed using a magnifying endoscope (PCF-Q240ZI, PCF-Q260AZI, CF-H290AZI, or PCF-H290ZI; Olympus, Tokyo, Japan, or EC-L600ZP7; Fujifilm, Tokyo, Japan) with standard video processors (EVIS LUCERA system; Olympus, Tokyo, Japan, or LASEREO; Fujifilm, Tokyo, Japan). Following conventional endoscopic observation with white-light, the endoscopist performed narrow-band imaging (NBI) or blue laser imaging (BLI), and, if necessary, chromoendoscopy with 0.4% indigo carmine. Two endoscopists (E.K. and T.M.) who had the experience of performing 2,000 colonoscopic procedures retrospectively evaluated the size, color, macroscopic type, morphological characteristics, NBI findings, and pit patterns of all endoscopic images of lesions diagnosed as TSA. They were blinded to both the histopathologic diagnoses and clinical information. Differences in opinion between the endoscopists were resolved by consensus.

To reduce the variation in size assessment between assessors, the size of polyps was estimated based on the review of endoscopic images conducted by two endoscopists (E.K. and T.M.). The macroscopic type was classified according to the Paris classification [11]. Polyp color was classified into three categories (shown in Fig. 3): reddish (defined as showing significant redness in the entire lesion compared to the surrounding mucosa), weak reddish (defined as showing a slight redness locally or entirely compared to the surrounding mucosa), and pale (defined as showing a significant pallor in the entire lesion or the same color compared to the surrounding mucosa). Additionally, we evaluated the following endoscopic findings on white-light images: a pinecone-like/coral-shaped appearance (defined as an appearance of clustered spherical or branched protrusions, visually resembling a pinecone or coral, a macroscopic feature of C-TSAs) [8‒10]; mucous cap (defined as a focal collection of mucus on the mucosal surface that can be removed with irrigation); and a flat elevation at the base (exhibiting a mucosal structure at the base of the lesion that resembles a HP; shown in Fig. 4).

Magnification endoscopy with NBI or BLI was performed to evaluate expanded crypt openings (indicating crypt dilatations, which are characteristics of a histological feature of SSLs). Furthermore, the Japan NBI Expert Team (JNET) system [12] was used to evaluate magnifying NBI or BLI endoscopic findings. The pit pattern of each lesion was evaluated by chromoendoscopy. The pit pattern was classified using Kudo’s pit patterns [13], with two additional variant pit patterns (types III_H and IV_H), which are characteristics of serrated lesions [14, 15]. Type III_H, also known to have a fern-like appearance, is characterized by elongated and/or branched pits with serrated features. Type IV_H is defined as an enlarged villous structure with serrations.

The following immunohistochemical parameters were assessed similarly as in our previous study [7]: cytokeratin (CK) 7, CK20, MUC2, MUC5AC, MUC6, CD10, β-catenin, MLH1, and p53. Appropriate positive and negative controls were used for each antibody. Additionally, we used the same evaluation method as that reported in our previous study [7]. In short, all markers, including CK7, CK20, MUC2, MUC5AC, MUC6, and CD10, were assessed for both intensity and extent of staining as follows. Intensity was scored as 0–3 (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining), and extent was scored as 0–4 (0, no staining; 1, 1–10% of cells; 2, 11–50% of cells; 3, 51–90% of cells; 4, >90% of cells). A final score was determined by multiplying the intensity and extent scores, and positive immunostaining required cytoplasmic staining with a score of ≥3. Nuclear β-catenin expression in ≥10% of tumor cells was considered to be nuclear-accumulation positive. Loss of MLH1 expression was noted when at least one cluster of tumor cells or all tumor cells showed no nuclear staining. Expression of p53 was recorded as positive if distinct and strong nuclear staining was observed in >10% of tumor cells. Immunohistochemical staining was assessed by two independent authors (E.K. and T.M.).

Genetic analysis was performed using the same method as that of our previous study [7]. Briefly, genomic DNA samples separated with a modified microdissection approach were extracted. The resulting DNA samples were subjected to polymerase chain reaction, followed by direct sequencing. The sequences of primers used, corresponding exons (codons), annealing temperatures, and product sizes were as previously described [7]. Mutations were considered to be legitimate when the height of the mutated peak reached 20% of the height of the normal peak. All mutations were verified by sequencing of sense and antisense strands.

All statistical analyses were performed using STATVIEW for Windows (v5.0; SAS Institute, Cary, NC, USA). Continuous data were compared using the Mann-Whitney U-test. Categorical analysis of variables was performed using either the chi-square test (with Yates’ correction) or Fisher’s exact test, as appropriate. A p value of <0.05 was considered to be statistically significant.

Among 344 lesions diagnosed as TSAs or serrated adenomas, 36 lesions that did not meet the criteria for a diagnosis of C-TSA or MR-TSA were excluded. Additionally, 23 cases were excluded due to the unavailability of evaluable endoscopic images. Finally, the lesions of interest in this study were obtained from 49 MR-TSAs and 236 C-TSAs, each representing a different patient. Regarding MR-TSA, 31 cases were used from our previous study [7]. The clinicopathological characteristics of the studied lesions are summarized in Table 1. The MR-TSA and C-TSA groups had similar median ages, with no significant difference between the two groups. In both groups, men were higher in number than the women. Many lesions in both groups were predominantly located in the sigmoid colon or rectum.

The endoscopic findings of the studied lesions are summarized in Table 2. No significant differences in lesion size were observed between MR-TSA and C-TSA. Macroscopically, approximately half of MR-TSAs (49%) were of the 0-Is type, while 66% of C-TSA were of the 0-Ip type, with only 18% showing the 0-Is type (p < 0.001). Regarding color, more than half of MR-TSAs (55%) were weak reddish, whereas 52% of C-TSAs showed a reddish color (p < 0.001). A pinecone-like/coral-shaped appearance was found in 57% of MR-TSAs, which was lower than that of C-TSAs (85%, p < 0.001). Most cases of MR-TSAs (82%) had a mucous cap, whereas only 30% of C-TSA cases had a mucous cap (p < 0.001). A flat elevation at the base were identified in 39% and 44% of MR-TSAs and C-TSAs, respectively, with no significant difference. Figure 5 shows several representative endoscopic images of MR-TSA.

NBI/BLI endoscopic images were available for 30 MR-TSAs and 177 C-TSAs. In the JNET classification, cases showing Type 2A (shown in Fig. 6) were observed in 26 cases (87%) of MR-TSA and 151 cases (85%) of C-TSA, with no significant difference between them. Most MR-TSAs (80%) had expanded crypt openings, whereas only 23% of C-TSAs had expanded crypt openings (p < 0.001).

Pit patterns were available for 26 MR-TSAs and 149 C-TSAs. The numbers of cases showing type III_H and type IV_H patterns were 5 and 21 for MR-TSA, and 35 and 114 for C-TSA, respectively (shown in Fig. 7). Figure 8 shows magnifying endoscopic images of a representative case of MR-TSA.

We randomly selected and immunostained 49 cases of MR-TSA, including 31 cases from our previous study [7], and the same number of C-TSA cases. The immunohistochemical characteristics of the studied lesions are summarized in Table 3. Regarding the immunohistochemical expression of CKs, all studied cases were positive for CK20, whereas few cases were positive for CK7, with no significant differences between MR-TSAs and C-TSAs. Furthermore, regarding mucin-phenotypic expression, all studied cases were positive for MUC2 but not for MUC6 or CD10. MUC5AC positivity was found more frequently in MR-TSAs than in C-TSAs (p = 0.004). Nuclear β-catenin expression in MR-TSAs was less frequent than that in C-TSAs (p = 0.003). MLH1 nuclear staining was retained in all cases. Strong and abnormal nuclear immunostaining of p53 was detected in only one case of C-TSA. The immunohistochemical staining patterns in a representative MR-TSA case are illustrated in Figure 9.

We screened for mutations in BRAF (exon 15) and KRAS (exon 2) in each of the 49 MR-TSA and 49 C-TSA tissues evaluated in this study (Table 3). On one hand, 76% of MR-TSAs harbored a BRAF mutation; this frequency was higher than that for C-TSA (37%, p < 0.001). All BRAF mutations were V600E (c.1799 T>A). On the other hand, only 12% of MR-TSAs exhibited KRAS mutations, which was lower in frequency than those of C-TSA (31%, p = 0.047). KRAS mutations in MR-TSA were G12V (c.35 G>T), G12D (c.35 G>A), and G13d (c.38 G>A), whereas in 15 cases of C-TSA, five cases were G12V, five cases were G12D, four cases were G13D, and one case was G12C (c.34 G>T), The BRAF and KRAS mutations were mutually exclusive.

In the chapter on colorectal serrated lesions and polyps in the WHO Classification of Tumors of the Digestive System (5th edition) [1], one sentence was dedicated to mucin/goblet cell-rich TSAs without much details. In this study, clinical and molecular evaluations of MR-TSAs showed that MR-TSAs had several features. Like C-TSA lesions, MR-TSA lesions were more common in men and predominantly localized in the sigmoid colon. More than 75% of lesions exhibited BRAF mutations but not KRAS mutations, and this finding is consistent with that of our previous study [7]. Endoscopically, MR-TSAs showed a range of morphological variations, although they mostly shared several endoscopic findings, including a type 0-Is nodular morphology, weak reddish color, and mucous cap. Furthermore, MR-TSAs less frequently had a pinecone-like/coral-shaped appearance, which is characteristic of C-TSAs. Based on these features, we expect that MR-TSAs can be endoscopically distinguished from C-TSAs.

In our previous study [7], patients with MR-TSAs were described to have an average age of 65 years, a male predominance, and a higher incidence in the sigmoid colon. Similar findings were observed in this study. These clinicopathological features of MR-TSAs are considered to be similar to those of C-TSAs. Endoscopically, C-TSAs were morphologically dominated by the 0-Ip (pedunculated) type, whereas in MR-TSAs, the 0-Is (sessile) type were common. Furthermore, a pinecone-like/coral-shaped appearance was less frequently observed in MR-TSAs. Regarding color, C-TSAs often had a reddish tone, whereas MR-TSAs commonly had a weak reddish tone. The exact reason for the increased frequency of the weak reddish tone in MR-TSAs is unclear. However, according to one hypothesis drawn from histopathological observations, the abundance of proliferative goblet cells in layers reduces the intensity of the red color. Interestingly, a mucous cap was observed in most cases of MR-TSA (82%). This finding seems to corroborate the mucin-rich/goblet cell-rich histological characteristics of MR-TSA. In other words, the most important morphological features of MR-TSAs may be their nodular and (hemi)spherical structure and mucin-rich layer without a pinecone-like/coral-shaped appearance.

Generally, SSLs and HPs are known as precursor lesions of TSAs, and their coexistence is often recognized as a flat elevation at the base [16]. Previous studies have reported that HP and SSL pathologically coexist with and are present at the base of TSAs. Seventy-eight of the 149 TSAs showed evidence of another polyp (52%): Of these, 32 were low-grade tubular/tubulovillous adenomas (TAs/TVAs; 41%), 28 were HPs (36%) and 18 were SSLs (23%) [17]. In this study, no difference in frequency of a coexisting flat elevation at the base was observed between MR-TSAs and C-TSAs. Therefore, MR-TSA, like C-TSA, may have HP or SSL as precursor lesions. In this regard, the number of cases showing a flat elevation at the base in MR-TSA was lower than those in previous reports. This might be due to the retrospective observation of endoscopic images, which included some cases in which the base could not be adequately evaluated.

Regarding magnifying endoscopy using NBI/BLI, most cases in this analysis showed JNET classification Type 2A. According to the JNET classification, Type 2A reflects adenoma, suggesting that this classification can also be applied to MR-TSAs. Furthermore, expanded crypt openings, which are generally characteristic of SSLs [18], were relatively frequently found in MR-TSAs. The reason for this was thought to be the accumulation of a large amount of mucus within the gland ducts, causing their expansion. This is similar to the one in which the gland duct openings dilate in SSL (shown in Fig. 8f).

Magnifying chromoendoscopy is a valuable tool for distinguishing serrated lesions, such as TSAs, from other polyps. In this study, MR-TSAs, similar to C-TSAs, exhibited type III_H and IV_H pit patterns; this has been suggested as helpful for diagnosing TSAs, including MR-TSAs. When encountering lesions with a villous structure, differentiating villous tumors (such as tubulovillous adenomas) from TSAs can be challenging. Under magnifying chromoendoscopy with indigo carmine, lesions displaying a type IV_H pit pattern with serration are more likely to be TSAs (including MR-TSAs), whereas those showing a type IV pit pattern without serration are more indicative of villous tumors. Furthermore, when only using conventional colonoscopy with white-light imaging, differentiating between MR-TSAs and conventional tubular adenomas with regard to type 0-Is lesions with a weak reddish color – especially those lacking a pinecone-like or coral-shaped appearance—can be difficult. Typically, conventional tubular adenomas are differentiated from TSAs because conventional tubular adenomas exhibit a type III_L pit pattern on magnifying chromoendoscopy [13]. Thus, type III_H and IV_H pit patterns, as identified by magnifying chromoendoscopy, are critical for the accurate diagnosis of TSAs, including MR-TSAs.

Genetically, most MR-TSAs harbored BRAF mutations in this study, and the frequency was remarkably higher than that of C-TSAs. Contrastingly, KRAS mutations in MR-TSAs were fewer than those in C-TSAs. These observations, consistent with those in our previous report [7], suggest that BRAF mutations might be one of the distinctive genetic characteristics of MR-TSAs. TSAs can show either BRAF or KRAS mutations, although the rate of BRAF mutation varied among previous studies, ranging from 10% to 67% [19‒23]. The varying BRAF mutation rates in TSAs might depend on the proportion of MR-TSA among the TSAs studied. Furthermore, TSAs are thought to be initiated by either BRAF or KRAS, with subsequent activation of the WNT/β-catenin signaling pathway, which is associated with RNF43 mutations [2, 24]. Regarding TSA progression, TSAs show infrequent nuclear β-catenin expression, whereas advanced areas of TSAs show a highly significant increase in nuclear β-catenin staining, indicating that WNT/β-catenin signaling pathway activation is an important step in malignant progression [2]. In this study, aberrant nuclear β-catenin expression was less frequently detected in MR-TSAs than in C-TSAs, suggesting that WNT/β-catenin signaling activation might not be associated with the early stage of MR-TSA progression. Additionally, our immunohistochemical results showed that MLH1 expression was retained in all MR-TSAs. BRAF mutants in TSA are considered to be associated with an aggressive, microsatellite-stable subtype (MSS) of colorectal carcinoma [2], with these subtypes often showing mucinous differentiation [25]. Considering the higher frequency of BRAF mutations in MR-TSA, our findings suggest that MR-TSAs might represent precursor lesions for the aggressive BRAF mutated microsatellite-stable subtype of colorectal carcinoma. MSS colorectal carcinomas with BRAF mutations generally have a poor prognosis. Therefore, it is important to differentiate MR-TSAs, which are more likely to progress to aggressive MSS-type colorectal carcinoma, from C-TSAs. On the other hand, among serrated lesions, SSL and SSL with dysplasia also follow a carcinogenic pathway involving BRAF mutations, as with MR-TSA. Many such lesions are characterized by a high-microsatellite instability subtype of colorectal carcinomas associated with MLH1 deficiency, while certain SSLs progress to MSS colorectal carcinomas via TSA [26]. Based on these findings, it is suggested that the carcinogenic progression of MR-TSA might partially overlap with a pathway leading to a BRAF-mutated MSS-type colorectal cancer through SSL.

Regarding the mucin phenotypes, MUC5AC is immunohistochemically expressed by gastric foveolar cells but not by normal colonic epithelium. However, expression of MUC5AC is reportedly elevated in colorectal adenomas showing villous histology or polypoid growth [27, 28]. In this study, phenotypic expression of MUC5AC was significantly more frequent in MR-TSAs than in C-TSAs, which was consistent with findings from our previous study [7]. MUC5AC expression is thought to be one of the distinctive immunohistochemical features of MR-TSAs, as in gastric foveolar cells.

This study had several limitations. First, this study was retrospective in nature based on the histological review of endoscopically resected colorectal lesions and the methods used for case selection. Second, due to the retrospective study design, some cases had low-quality images, which made precise evaluation of all endoscopic features challenging. Third, a certain number of cases had inadequate magnification of endoscopic findings. Fourth, the endoscopic equipment used in this analysis was not consistent. Finally, interobserver and intra-observer variability in the interpretation of endoscopic findings was not analyzed. Since the endoscopic findings relied on the evaluators’ interpretation, there was potential for bias. This study focuses on identifying characteristic endoscopic findings and does not evaluate their validity. Therefore, assessing the validity of these findings remains a task for future research. Further investigation is needed to elucidate the endoscopic characteristics of MR-TSA in detail, and confirm their clinical usefulness since the number of MR-TSA cases was limited in this study.

This study outlined the endoscopic and clinicopathological characteristics of MR-TSAs, which were distinct from those of C-TSAs. Considering the predilection for the sigmoid colon and rectum, characteristic endoscopic findings, such as a weak reddish color, sessile morphology without pinecone-like/coral-shaped appearance, and the presence of a mucous cap, would assist the endoscopic diagnosis of MR-TSA. However, this study did not include any cases of cancerization in MR-TSA and, therefore, could not assess the difference in cancer incidence or malignancy between MR-TSA and C-TSA. Consequently, further accumulation of MR-TSA cases is necessary to determine the appropriate clinical management of MR-TSA.

This study protocol was reviewed and approved by the Institutional Review Board and the Ethics Committee of Juntendo University Hospital, Approval No. 2017166. The study was performed in accordance with the principles of the Declaration of Helsinki. The need for informed consent was waived by the Institutional Review Board and the Ethics Committee of Juntendo University School of Medicine.

The authors have no conflicts of interest to declare.

This study was not supported by any sponsor or funder.

Eiji Kamba and Takashi Murakami contributed significantly to the conception and design of the study. Naoki Tsugawa, Yudai Otsuki, Kei Nomura, Hirofumi Fukushima, and Tomoyoshi Shibuya contributed to the acquisition of the clinical data. Yuichiro Kadomatsu, Tsuyoshi Saito, and Takashi Yao pathologically reviewed the cases. Eiji Kamba, Takashi Murakami, and Naoki Tsugawa analyzed the data. Eiji Kamba interpreted the data and drafted the manuscript. Takashi Murakami and Akihito Nagahara contributed to the critical revision of the manuscript. All the authors have read and approved the final version of the manuscript.

The data that support the findings of this study are not publicly available due to their containing information that could compromise the privacy of research participants but are available from the corresponding author, T.M., upon reasonable request.