Gastric Mucin Phenotype Indicated Aggressive Biological Behavior in Early Differentiated Gastric Adenocarcinomas by Endoscopic Treatment

Background: The distribution of mucin phenotypes and its relationship with clinicopathological features in early differentiated gastric adenocarcinomas among the Chinese cohort is unknow. We aim to investigate the mucin phenotypes and analyze the relationship between mucin phenotypes and clinicopathological features, especially the biological behavior, in early differentiated gastric adenocarcinomas from endoscopic specimens in a Chinese cohort. Methods: Immunohistochemical staining of CD10, MUC2, MUC5AC, MUC6 was performed in 257 patients with early differentiated gastric adenocarcinomas. Tumor location, gross type, tumor size, histological type, depth of invasion, lymphovascular invasion, mucosal background and other clinicopathological parameters were evaluated. Analyzed the relationship between mucin phenotypes and clinicopathological features through chi-square test.


Background
Gastric cancer (GC), one of the most common human cancers worldwide, is a sort of disease with multiple pathogenic factors, various prognoses and different responses to treatments. Thus, distinguishing the worse prognose one from the better one properly seems signi cantly important. There are four different morphological-based classi cation systems, the World Health Organization (WHO/2019) [1], the Japanese Gastric Cancer Association (JGCA/2017)(1), Laurén(2) and Nakamura (3).
According to the WHO classi cation, GCs are subclassi ed into papillary, tubular, poorly cohesive, mucinous and mixed types. In JGCA classi cation, the subtypes are papillary(pap), tubular 1, tubular 2, poorly 1 (solid type), signet-ring cell, poorly 2 (non-solid type), mucinous, which are similar to those in WHO. GCs are diveded into intestinal and diffuse type using the Laurén's classi cation, or as differentiated and undifferentiated type based on Nakamura's classi cation (2)(3)(4). Differentiated type contains pap, tub1, tub2 in JGCA classi cation and papillary, well/moderately differentiated adenocarcinoma in WHO classi cation. These different histological types exhibit distinct biological behaviors.
The mucus produced by cancers is one of the factors determining the nature of biological behavior. The main component is a high molecular weight glycoprotein called mucin (5). As the cancer progresses, the nature of mucus changes, which is related to the degree of biological malignancy. In the 1990s, with the progress of structural analysis of mucin and the widespread use of monoclonal antibodies to the core protein of mucin, mucin phenotype subclassi cation emerged. Mucin phenotype subclassi cation entirely based on the mucin expression mode, independent of histological features. Thus GCs are classi ed into gastric, intestinal, gastrointestinal and null mucin phenotype(4-6). Previous researches have reported the gastric-phenotype determined a higher potential of invasion and metastasis than the intestinal type, which resulted in a worse prognosis of GCs (7)(8)(9)(10)(11). But the researches were mostly focused on advanced gastric caners, the early gastric cancers were rarely involved.
The early gastric cancer (EGC) is de ned as the tumor invasion is con ned to mucosa and submucosa, irrespective of the regional lymph nodes metastasis (12). Endoscopic mucosal resection(EMR) and endoscopic submucosal dissection(ESD) are used as the treatment of some intramucosal carcinoma and submucosal lesions, which have a very low possibility of lymph node metastasis (13,14).
To our knowlege, there is no research discussed mucin phenotypes in EGCs by EMR/ESD from China investigators. And there is little information on the effects of the mucin phenotypes on the clinicopathological features of EGCs among the Chinese cohort. To this end, we examined the mucin expressions and mucin phenotypes, and explored mucin phenotypes' clinicopathological characteristcs and biological behavior.

Patients and tissue specimens
Our series consisted of 257 consecutive patients who underwent EMR/ESD for differentiated EGCs between January 2012 and June 2018 at The Second A liated Hospital of Zhejiang University, China. The group comprised 182 men and 75 women with an age ranged from 29 to 87 (mean 64) years old.
The location of lesion was classi ed in terms of upper (27 cases), middle (46 cases) and lower (184 cases) third of the stomach. The size of the lesion was measured by the maximum diameter, it ranged from 0.1 to 6.5 (mean 1.5) centimeters. The category of protruding (52 cases) included type 0-I and 0-IIa, depressed (102 cases) category contained type 0-IIc and III, and all the others went to the category of protruding and depressed (103 cases). Based on the WHO and JGCA classi cation, the EGCs were subclaasi ed into well differentiated tubular adenocarcinoma (tubular, well-diff / tub 1, 198 cases), moderately differentiated tubular adenocarcinoma (tubular, mod-diff / tub 2, 37 cases), papillary adenocarcinoma (3 cases) and mixed (tub-pap / sig / por, 19 cases). In the mixed cases, the undifferentiated components (sig / por) were less than 50%.

Immunohistochemistry
All the specimens were xed with 10% buffered formalin, embedded in para n and cut into 4μm thick sections, in addition to hematoxylin and eosin (HE) staining. MUC2 was detected by mAb Ccp58 (Zsbio, 1:100), MUC5AC by mAb MRQ-19 (Zsbio, 1:100), MUC6 by mAb MRQ-20 (Zsbio, 1:100), and CD10 (Zsbio, Classi cation of mucin phenotype Based on MUC5AC, MUC6, MUC2 and CD10, the EGCs could be classi ed into gastric-phenotype (G-type), gastrointestinal-phenotype (GI-type), intestinal-phenotype (I-type) and null-phenotype (N-type)(4,6,15-17). The following criteria were used for the classi cation of mucin phenotypes: G-type presents positive to at least one of the MUC5AC and MUC6, while CD10 and MUC2 were both negative; I-type presents positive to CD10 and/or MUC2, while MUC5AC and MUC6 were both negative; GI-type presents positive to marker CD10 and/or MUC2 associated to one of the markers MUC5AC and MUC6 positive; None of the four markers was positive, the phenotype was classi ed as N-type.
In addition, in GI-type, we labelled the one expressed MUC5AC and/or MUC6 more than MUC2 and CD10 as gastric-predominant GI phenotype (GI-G type), otherwise, labelled as intestinal-predominant phenotype (GI-I type).

Statistical analysis
Associations between mucin expression pro les and clinicopathologic parameters were examined by chisquare test or Fisher's exact test. Statistical signi cance was used, and was established to be P<0.05. Statistical calculations were performed with the IBM SPSS Statistics (version 23.0).

Relationship between mucin phenotype and clinicopathological features
The relationship between mucin phenotype and clinicopathological features were summarized in table 1.

Relationship between mucin phenotypes and background mucosa
Intestinal metaplasia (IM) of background mucosa was observed in 199 of 249 (79.9%) cases (G-,GI-and GI-type), including 38 cases of incomplete IM and 161 cases of complete IM. IM did not signi cantly differ among mucin phenotypes (P 0.05). But incomplete and complete IM were signi cantly differed in different mucin phenotypes (P=0.004, P=0.018). The expression of incomplete IM in GI-type was higher than that of G-type and I-type GC (21.5%vs. 9.3% vs. 3.9%). On the contrary, there were 77.8% (42/54) Gtype cases and 70.6% (36/51) I-type GCs appeared complete IM, which was higher than GI-type 57.6% (83/114). The IM status of background mucosa and the relationship with mucin phenotypes were showed in Table 2.

Biological behavior of mucin phenotypes
In addition to tubular adenocarcinoma components, 22 of the 257 cases also contained components of papillary adenocarcinoma, or poorly differentiated carcinoma, or signet ring cell carcinoma. 15 cases (68.18%) contained por/sig components, and the other 7 (31.82%) contained pap components. And 11 cases showed G-type, 10 cases showed GI-type, only one case showed N-type, none of them showed Itype. Among the 10 GI-type cases, 9 cases showed GI-G type, one showed GI-I type . Almost all the 22 cases showed G-and GI-G type, which was signi cantly higher than I-type (P=0.011). In addition, the 22 cases got a higher proportion of in ltration into the submucosa layer (P 0.001). (Table 3, Fig. 3, Fig. 4).

Follow-up
Six patients received additional gastrectomy, and there was no residual tumor or lymph node metastasis. All cases were under close follow-up, neither recurrence nor metastasis were detected.

Discussion
Mucin phenotype is a classi cation based on the mucin markers expression mode. After the year of 2000, the mucin phenotypes of gastric and intestinal were analyzed by IHC (15).The mucin markers of MUC5AC, MUC6, MUC2 and CD10 are considered necessary, though there is no consensus on the amount of markers which should be used to de ne a mucin phenotype, or the percentage of tumor cells must be stained (6-8,11,15,18). MUC5AC is a secreted mucin expressed in the surface mucous epithelium of normal gastric mucosa. High expression of MUC6 is observed in fundic neck mucous cells and pyloric glands of gastric mucosa. CD10 is a marker for the brush border on the luminal surface of small intestinal. In the normal adult intestine, MUC2 expression is observed in the perinuclear areas of goblet cells.
The relationship between mucin phenotypes and clinicopathological features was investigated. We found the histology classi cation (both the JGCA and WHO classi cation) were closely related with mucin phenotype. The I-type had a higher proportion than G-, GI-and N-type (100.0% vs.79.7%, 93.1%, 87.5%, P = 0.027). The G-type was correlated signi cantly with the mixed type (with poorly differentiated/papillary carcinoma) histologically. Our data showed the proportion of G-type turned greater during the transition from solely differentiated type to mixed type carcinoma. Mixed type carcinoma in early stage more frequently expressed G-type mucin, and the G-type tumors were associated with a higher rate of undifferentiated-type as compared with the I-type tumors (7,22).
While there were no signi cant differences between mucin phenotypes and other parameters, including gender, age, margin, color, tumor size, gross type, depth of invasion, lymphovascular invasion (P 0.05). These results perfectly shared the same viewpoint with some researchers (22,25,29), there was no clear correlation between phenotypes and clinicopathologic characteristics, including sex, age, tumor size, location, macroscopic features, lymphatic or venous invasion, or lymph node metastases in the case of the differentiated type (22,25,29). While Koseki et al. (7) and Oya et al. (30) have reported the incidence of lymphatic invasion, venous invasion and lymph node metastasis in gastric phenotype carcinomas was signi cantly more frequent than that in intestinal phenotype carcinomas. In addition, G-type GCs had a correlation with some distinguished macroscopic features: the smaller tumor diameter(28), the discolored surface and non-wavy tumor margins (31,32). G-type differentiated adenocarcinomas showed depressed type, indistinct margins and monotonous color tone across the mucosal layer, whereas I-type had an elevated, distinct margin and a red mucosa (3,33,34). The discrepancy of these results may have been due to heterogeneous components which contained poorly differentiated adenocarcinoma (29).
Intestinal metaplasia has been frequently observed surrounding the GC, especially differentiated adenocarcinomas. IM had a malignant potential and has been regareded as a precursor of gastric neoplasms. According to Laurén, The intestinal type adenocarcinoma preceded by metaplastic changes, while the diffuse type adenocarcinoma arised in non-IM gastric mucosa (2). In the current study, the backgroud mucosa IM was observed in 79.9% cases (G-,GI-and I-type) and 87.0% in G-type. 25.5% cases of I-type arised from the normal mucosa without IM. IM did not signi cantly differ among mucin phenotypes (P 0.05). But incomplete and complete IM were signi cantly differed in different mucin phenotypes (P = 0.004, P = 0,018). There were 77.78% (42/54) G-type and 70.6% (36/51) I-type appeared complete IM, which was higher than GI-type (83/114,57.6%). The expression of incomplete IM in GI-type was higher than that of G-and I-type (21.5%vs. 9.3% vs. 3.9%). Our results demonstrated a remarkable difference between mucin phenotypes and the background mucosa. Similar results have been repoted by Kabashima et al.(36)and Matsuoka (37). The mucin phenotype of the carcinoma was independent of mucin phenotypic changes in the surrounding mucosa, they might perform individual intestinalization. Gtype might imitate the surrounding mucosa, the carcinomas and the background mucosa had an unstable status, then commonly possess the hybrid phenotype of the stomach and the small intestine(36,37).
Mucin phenotypes can indicate biological behavior in GCs. G-type GCs got increased potential for invasion and metastasis: in ltrating deeper layers or more surrounding structures, higher rate of lymph node metastasis, and poorer prognosis (3,12,18,21). Even, the differentiated adenocarcinoma with G-type had a similar outcome, focused on the prognosis, with undifferentiated adenocarcinoma(7-10). In our research, six patients received additional gastrectomy, and there was no residual tumor or lymph node metastasis. All cases were under close follow-up, neither recurrence nor metastasis were detected. The mixed type (mixed with poorly differentiated or papillary adenocarcinoma) were mainly of G-type, which was signi cantly higher than that of purely differentiated tubular adenocarcinoma (P < 0.05), and the depth of in ltration was deeper (P < 0.05). G-type had the highest portion (11/54,20.37%) with poorly differentiated/undifferentiated components, and almost all of them (19/22,86.36%) expressed G-and GI-G type. The mixed type may represent a progressive loss of glandular structure during its progression from mucosal to advanced stage, and those with submucosal invasive was a risk factor for lymph node metastatsis. (7,22,38) Differentiated GC with G-type often changed histologically into signet ring-cell carcinoma or poorly differentiated adenocarcinoma. Those imply the fact of more aggressive biological behavior and poorer prognosis.

Conclusion
Our study rstly reported the expression of mucin markers (MUC5AC, MUC6, MUC2 and CD10) and mucin phenotypes in differentiated EGC samples from the treatment of ESD/EMR among the Chinese population. Mucin phenotypes of early differentiated gastric cancer is of clinical signi cance, and G-type have aggressive biological behavior in early differentiated gastric cancers, especially in those mixed with poorly differentiated adenocarcinoma or papillary adenocarcinoma component.    Figure 1 Page 16/18