AIM: To clarify the correlation with phenotypic expression, clinicopathological features, genetic alteration and microsatellite-instability status in small intestinal adenocarcinoma (SIA).

METHODS: The cases of 47 patients diagnosed with primary SIAs that were surgically resected at our institution in 1975-2005 were studied. We reviewed clinicopathological findings (age, gender, tumor size, gross appearance, histological morphologic type, invasion depth, lymphatic permeation, venous invasion, and lymph node metastasis), and the immunohistochemical expression of MUC5AC, MUC6, MUC2, CD10, and mismatch-repair (MMR) proteins (MLH1 and MSH2). We analyzed KRAS and BRAF gene mutations, and the microsatellite instability (MSI) status. The immunohistochemical staining of CD10, MUC2, MUC5AC and MUC6 was considered positive when distinct staining in > 5% of the adenocarcinoma cells was recorded. To evaluate of MMR protein expression, we used adjacent normal tissue including lymphoid follicles, inflammatory cells, and stromal cells as an internal positive control. Sections without nuclear staining in the tumor cells were considered to have lost the expression of the respective MMR protein.

RESULTS: There were 29 males and 18 females patients (mean age 59.9 years, range: 23-87 years). Tumors were located in the duodenum in 14 cases (30%), the jejunum in 21 cases (45%), and the ileum in 12 cases (25%). A phenotypic expression analysis revealed 20 MUC2-positive tumors (42.6%), 11 MUC5AC-positive (23.4%), 4 MUC6-positive (8.5%), and 7 CD10-positive (14.9%). The tumor sizes of the MUC2(+) tumors were significantly larger than those of the MUC2(-) tumors (mean, 5.7 ± 1.4 cm vs 4.7 ± 2.1 cm, P < 0.05). All three tumors with adenomatous component were positive for MUC2 (P < 0.05). Polypoid appearance was seen significantly more frequently in the CD10(+) group than in the CD10(-) group (P < 0.05). The tumor size was significantly larger in the CD10 (+) group than in the CD10(-) group (mean, 5.9 ± 1.4 cm vs 5.0 ± 2.1 cm, P < 0.05). Of 34 SIAs with successfully obtained MSI data, 4 were MSI-high. Of the 4 SIAs positive for both MUC5AC and MUC2, 3 showed MSI-H (75%) and 3 were mucinous adenocarcinoma (75%). KRAS mutations were detected in 4 SIAs. SIAs had KRAS mutation expressed only MUC2, but were negative for MUC5AC, MUC6 and CD10.

CONCLUSION: These findings suggest that the phenotypic expression of SIAs is correlated with their biological behavior, genetic alteration, and MSI status.

Primary small intestinal adenocarcinomas (SIAs) are rare, accounting for only about 0.8% of all malignant gastrointestinal tumors, although the small intestine represents 75% of the length and 90% of the mucosal surface of the gastrointestinal tract[1,2]. Despite major advances in imaging and therapeutic techniques, the prognosis of SIAs remains dismal, with an overall 5-year survival rate of 25%-30%[3,4].

Mucins are a family of high-molecular-weight, heavily glycosylated proteins that are widely produced by epithelial tissue. In the alimentary canal, MUC2 is produced by goblet cells of the small and large intestinal mucosa, and MUC5AC is produced by gastric foveolar epithelium. MUC6 is produced by the gastric pyloric gland and duodenal Brunner’s glands. Compared to normal mucosa, adenocarcinomas occasionally produce different types of mucin. Some researchers have reported that in gastric carcinoma (GC) or colorectal carcinoma (CRC), the mucin phenotype was related to the tumor growth pattern, appearance, aggressiveness, prognosis, genetic alternation and microsatellite instability (MSI) status. For example, it was reported that MUC2-positive CRCs have a relatively favorable prognosis[5], whereas loss of MUC2 was an adverse prognostic factor in CRCs[6]. MUC5AC was often found to be expressed in CRCs with MSI-high (MSI-H) and/or mucinous adenocarcinomas, and intriguingly, this type of CRCs often express MUC2 as well[7,8].

The “serrated pathway” is an evolving pathway in the carcinogenesis of CRC, characterized by BRAF gene mutations and MSI-H[9]. Sessile serrated adenomas, considered precursors of MSI-H CRCs, often express both MUC2 and MUC5AC, the same as MSI-H CRCs do[10].

It has been suggested that not only mucin expression but also CD10 expression, which is usually seen in the brush border of the small intestinal villi, are correlated with aggressive biological behavior of CRCs. CD10-positive CRCs in particular were suggested to be prone to the development of metastasis to the liver[11,12]. Analyses of the mucin core proteins and CD10 expression in adenocarcinomas of the alimentary canal are thus important to determine these tumors’ biological behavior and carcinogenesis.

In SIAs, however, the phenotypic expressions of mucin core proteins and CD10 have not been established, and the clinicopathological features and pathogenesis of SIAs are less known than those of CRCs.

The purposes of the present study were to evaluate the mucin core proteins and CD10 expression of primary SIAs and to clarify their correlation with the phenotypic expression, clinicopathological features, biological behavior, genetic alterations and MSI status.

CD10 is a membrane-bound, zinc-containing metalloendopeptidase, that is expressed in a wide variety of normal and neoplastic cells including pre-cursor B and T cells, follicle center B cells in the hematopoietic system, myoepithelial cells of the breast and salivary glands, basaloid cells of the prostatic gland, glomerular cells of the kidney, and stromal cells in the endometrium[18,19]. In the alimentary canal, normal small intestine shows immunoreactivity for CD10 in the brush border. On the other hand, except for intestinal metaplasia of the stomach, the normal gastric foveolae and the colorectal epithelium show no CD10 expression.

In our evaluation of the distribution of CD10, we observed CD10 expression in the superficial area of the SIAs. The expression diminished with greater depth and was not detected in the deepest area of almost all of the CD10(+) SIAs. Yao et al[13] reported that the CD10 expression of CRCs was more frequently observed in the center or deep area of the tumors than their periphery. The distribution of CD10-positive areas seemed to be opposite in SIAs and CRCs.

Regarding the CD10 expression of advanced CRCs, the significant correlations have been reported between CD10 expression and a high incidence of liver metastasis[11,12,20], and between CD10 expression and venous invasion[13]. Carl-McGrath et al[21] reported that CD10 was significantly up-regulated in GCs and lymph node metastases. Although the concrete function of CD10 in carcinogenesis remains unclear, the acquisition of CD10 expression correlates with the malignant potential in CRCs and GCs. In the present study, the incidence of lymphatic permeation tended to be lower in the CD10(+) SIAs than in the CD10(-) SIAs, regardless of the tendency for larger tumor sizes in the CD10(+) SIAs. The CD10-positive adenocarcinoma glands showed lower atypia than the CD10-negative adenocarcinoma glands did in the same SIAs. These results suggest that CD10(+) SIAs are less aggressive than CD10(-) SIAs, and that the loss of CD10 expression in SIAs may be correlated with the phenotypic dedifferentiation and malignant phenotype.

The relationship between mucin phenotype and clinicopathological or biological features has been investigated in various types of GC and CRC[7,8,11-14,22-25]. It is well established that MUC2 expression is related to the progression or prognosis of CRC[5,6,25], an association that suggests that the expression of MUC2 is also related to the biological behavior of SIA. Although in the present study the tumor sizes of the MUC2(+) SIAs were significantly larger than those of the MUC2(-) SIAs, the frequency of lymphatic permeation tended to be lower in the MUC2(+) SIAs than in the MUC2(-) SIAs. Poorly differentiated adenocarcinomas were prevalent in the MUC2(-) SIA group. From these findings, we infer that MUC2(-) SIAs are more aggressive than MUC2(+) SIAs. Loss of MUC2 expression in SIAs may also be correlated with malignant potential.

The serrated neoplastic pathway of CRCs is characterized by BRAF mutation, abnormal DNA methylation, and MSI-H status, and some authors suggested that serrated polyps were the precursor of MSI-H CRCs[9,26]. The MUC5AC+/ MUC2+ mucinous phenotype is common in serrated polyps (including hyperplastic polyps and serrated adenomas) and MSI-H CRCs[10,27,28]. Arai et al[28] also demonstrated a higher proportion of MSI-H in mucinous adenocarcinoma. In the present study, we observed the 4 SIAs that expressed both MUC5AC and MUC2, and these 4 SIAs contained 3 MSI-H tumors and 3 mucinous adenocarcinomas. The data suggested that the co-expression of MUC5AC and MUC2 in SIAs was also correlated with MSI-H tumors and mucinous adenocarcinomas as in CRCs.

We did not detect any serrated polyps or any mutations in codon 600 of BRAF gene. Fu et al[29] investigated 99 duodenal adenocarcinomas and detected no BRAF mutation among them, and therefore they suggested that BRAF mutations are not involved in MSI-H duodenal adenocarcinomas. In other investigations, the BRAF mutations were rare in SIAs[30,31]. In the carcinogenesis of SIAs, it is still unclear whether there is a serrated neoplastic pathway as same as in CRCs.

The present study revealed that 4 of 34 (12%). SIAs had microsatellite instability. This result is in line with reports by other investigators, who described the frequency of MSI-H of SIAs of 5% to 30%[32-35]. We also found that 2 of 4 MSI-H tumors showed the immunohistochemical loss of MMR protein expression, which affected MLH1 in 1 case and MSH2 in 1 case. Mangold et al[36] reported that 32% of CRCs from MLH1 mutation-carrier patients showed partly positive or weak staining of MLH1. In the present study, we considered only the completely negative staining of MLH1 as the loss of MLH1 protein, and thus some of the SIAs we assessed as positive for MLH1 protein could be false-positive. Planck et al[35] investigated a large population-based series of 89 SIAs, and eight of 15 MSI-H tumors expressed both MLH1 and MSH2. We suggest that the immunohistochemical staining of MLH1 and MSH2 proteins is generally useful to detect MSI-H SIAs, but a molecular analysis is necessary to detect some MSI-H SIAs.

Previous studies detected KRAS mutations in 14%-83% of SIA cases[32,34,37-39]. We detected mutations in KRAS codon 12 and codon 13 in 4 of 37 SIAs. The frequency and the location were in line with those of the previous studies. Three of the 4 present SIAs with KRAS mutations were positive for only MUC2 (75%). We found a tendency for the SIAs with KRAS mutations to have the colonic phenotype.

In conclusion, we suggest that the mucinous phenotype and CD10 expression of SIAs also correlates with the tumors’ biological behavior, genetic alteration, and MSI status. In particular, the preservation of CD10 expression, correlated with larger tumor size, polypoid appearance and less lymphatic permeation, features that are suggestive of less aggressive biological behavior. The co-expression of MUC5AC and MUC2 of the SIAs was correlated with the MSI-H status and mucinous adenocarcinoma, as in CRCs.

We are grateful to Dr. Shimokawa M, National Hospital Organization Kyushu Cancer Center, for reviewing the statistical method of this study.