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Notch3 signaling is associated with MUC5AC expression and favorable prognosis in patients with small intestinal adenocarcinomas
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Original Article
Notch3 signaling is associated with MUC5AC expression and favorable prognosis in patients with small intestinal adenocarcinomas Dae-Woon Eom a,∗ , Seung-Mo Hong b , Jihun Kim b , Gwangil Kim c , Young Kyung Bae d , Kee-Taek Jang e , Eunsil Yu b a
Department of Pathology, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung, Republic of Korea Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea c Department of Pathology, Bundang CHA Medical Center, CHA University, Seongnam, Republic of Korea d Department of Pathology, Yeungnam University College of Medicine, Daegu, Republic of Korea e Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 25 November 2013 Received in revised form 11 February 2014 Accepted 1 April 2014 Keywords: Small intestine Adenocarcinoma Prognosis Notch3 MUC5AC
a b s t r a c t Background: Notch signaling plays diverse roles not only in physiologic processes, including development and differentiation but also in tumorigenesis, either as a tumor promoter or suppressor depending on the cellular context, level of expression and cross-talk with other signaling pathways. In this study we investigated the expression of Notch3 and MUC proteins and their clinicopathological significance in small intestinal adenocarcinoma (SIAC). Methods: Surgically resected 191 SIACs and their clinical data were collected. Immunohistochemistry for Notch3, MUC2, MUC5AC, and MUC6 using tissue microarrays from formalin-fixed paraffin-embedded normal and matched tumor tissues was performed. Results: Notch3 expression was found in 52 (29.9%) cases of the tumors. MUC2, MUC5AC, and MUC6 were expressed in 52 (27.5%), 51 (31.9%), and 42 (22.0%) cases of the tumor, respectively. Notch3 expression was correlated with the absence of lymphovascular invasion (p = 0.009), lower T stage (p = 0.038), and histological subtype of tubular adenocarcinoma (p = 0.01), respectively. MUC2 was correlated with large tumor size (p = 0.013) and mucinous and signet ring cell adenocarcinomas (p = 0.01). MUC5A was correlated with proximal tumor location (p < 0.0001) and tumor differentiation (p = 0.027). MUC6 was correlated with proximal tumor location (p < 0.0001) and lower pT stage (p = 0.009), and absence of lymphovascular invasion, respectively. A significant correlation was noted between Notch3 and MUC5AC expression (p = 0.019). Notch3 expression was a relatively favorable prognostic factor in SIACs by univariate (p = 0.05) and multivariate analysis (p = 0.08, Cox Hazard ratio 0.841). Conclusion: Our findings indicate that Notch3 signaling, associated with MUC5AC expression, could be a more favorable prognostic factor in SIACs. © 2014 Elsevier GmbH. All rights reserved.
Introduction Primary small intestinal adenocarcinoma (SIAC) is a rare tumor although the small intestine occupies the largest absorptive surface areas (about 90%) of the gastrointestinal tract. Despite the recent development of endoscopic modalities and imaging techniques for the small intestine, the early detection of SIACs has been quite
∗ Corresponding author at: Department of Pathology, University of Ulsan College of Medicine, Gangneung Asan Hospital, 415, Bangdong-ri, Sacheon-myeon, Gangneung-si, Gangwon-do 210-711, Republic of Korea. Tel.: +82 033 610 3428; fax: +82 033 610 3430. E-mail address: [email protected] (D.-W. Eom).
difficult so far and the 5-year survival rate of SIACs diagnosed at an advanced disease state is 41.2% [1]. To improve the survival rate of patients with SIACs, it is necessary to understand more clearly their molecular pathogenesis. However, the molecular mechanism of SIAC has not been fully understood due to its rare incidence. MUC proteins are high-molecular-weight glycoproteins or mucopolysaccharides that are widely expressed in epithelial cells and show organ specificity [2,3]. To date, twenty distinct epithelial mucin genes, which can be broadly classified into two groups as secreted and membrane bound mucins, have been identified [2,4]. Among them, secreted gel-forming mucins (MUC2, MUC5A, and MUC6) are the major secreted glycoproteins of the gastrointestinal tract and play not only a role in normal physiological processes but also have a function in neoplastic progression. MUC2 is primarily
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expressed in goblet cells of the native intestinal epithelium in the small bowel and colon [5]. MUC5AC and MUC6 are markers of the gastric epithelial cell phenotype [6]. MUC5AC expression is localized in the gastric foveolar epithelium, whereas MUC6 expression is detected in the cytoplasm of the pyloric gland and mucous neck cells in the stomach [3,7]. The Notch signaling plays diverse roles not only in normal tissue development and differentiation but also in tumorigenesis [8–10]. The Notch signaling molecules are essential for controlling the fate of stem cells and expressed at different levels with varying spatial and temporal distribution in the developing small intestine [8]. Notch signaling regulates the proliferation and differentiation of intestinal stem and progenitor cells in crypt base to maintain epithelial cell homeostasis in the small intestine [11]. In malignant tumors, it has complex and controversial biological functions of either a tumor promoter or suppressor depending on the cellular context, level of expression and cross-talk with other signaling pathways [10,12,13]. The Notch signaling pathway associated proteins are composed of four Notch receptors (Notch1–4) and five Notch ligands in mammals. Notch1 and Notch2 have the highest homology with each other, while Notch3 and Notch4 are structurally and functionally diverged from Notch1 and Notch2 in both the extracellular and intracellular domains [14]. Notch-1 was first found to associate with hematological diseases, and its expression level increased in multiple myeloma, Hodgkin’s lymphoma, anaplastic large cell lymphoma and acute myeloid leukemia [16,17]. Notch2 has the highest homology with Notch1, but unlike Notch1, it can present a tumorsuppressive action in breast cancer [18,19]. It has recently been reported that Notch2 is a novel target for -catenin-dependent Wnt signaling [20]. Although the biological significance of the Notch4 has not been determined, activated it disrupts normal epithelial morphogenesis and induces an invasive phenotype in mammary epithelial cells [21]. Notch3 was initially reported as being expressed in the proliferating neuroepithelium and the underlying cause of cerebral arteriopathy with subcortical infarcts and leukoencephalopathy [15,22]. Although Notch3 activation was reported to be oncogenic in many various organs, such as the ovary, lung, and colon [23–25], it was also recently found to have the opposite effect for tumorigenesis or prognosis in some malignant tumors [26–28]. It is well known that Notch signaling regulates the terminal differentiation of goblet cells in normal intestinal mucosa [29,30], but the relationship between Notch3 signaling and MUC proteins has not been described in SIACs. In the present study, in order to investigate the role and clinical significance of Notch3 in small intestinal carcinogenesis, we evaluated the clinicopathologic significance of Notch3 expression and examined the association between the expression of Notch3 and those of MUC2, MUC5AC, and MUC6 proteins in patients with SIACs.
Materials and methods Patients and specimens 191 cases of surgically resected SIACs were collected from 22 institutions in Korea1 [1]. Representative H&E slides and paraffin tissue blocks were obtained with the appropriate human protection approval from the institutional review board from each institution that participated in the Korean Small Intestinal Cancer Study Group. Primary carcinomas arising in the duodenum, jejunum, and ileum were included in this study. Malignant tumors from other gastrointestinal tract organs, such as the stomach, ampulla of Vater, pancreas or appendix with extension into the small intestine were excluded. Data were collected after reviewing the medical records
of the patients including the patients’ sex, age, accompanying tumors, and recent follow-up date for survival status. The pathologic characteristics that were evaluated included tumor location, size, histological subtype, differentiation, growth pattern, pT stage, retroperitoneal seeding, other intestinal loop invasion, metastasis to lymph node, and presence of perineural and lymphovascular invasion. Tissue microarray To construct tissue microarrays, tissue cores of 1 mm diameter were taken from morphologically representative areas of the formalin-fixed paraffin-embedded blocks. The tissue array blocks consisted of 2 or 3 cores of tumor tissue and one core of matched normal mucosa far from the tumor, not adjacent area. Immunohistochemistry and interpretation Immunohistochemical staining for Notch3 (rabbit polyclonal, Abcam, Cambridge, UK, 1:100), MUC2 (mouse monoclonal, Novocastra, Newcastle, UK, 1:200), MUC5AC (mouse monoclonal, Novocastra, Newcastle, UK, 1:200), and MUC6 (mouse monoclonal, Novocastra, Newcastle, UK, 1:200) was performed on the arrayed blocks. All immunostaining was performed with the Bond-Max automatic immunostaining device (Leica Biosystem, Newcastle, UK) using a bond polymer intensity detection kit (Leica Biosystem) for formalin-fixed, paraffin-embedded tissue sections. Four-micrometer-thick sections were obtained by microtome, transferred onto adhesive slides, and dried at 62 ◦ C for 30 min. Antigen retrieval was carried out in all cases. Slides were counterstained with Harris hematoxylin. Immunoreactivity was interpreted by light microscopic examination and independently evaluated by pathologists who authored this study (D-WE and S-MH) and who were blind to the clinicopathological information. Immunostained slides were scored for unequivocally positive epithelial cells and 10 representative fields of each tumor and normal tissue cores were analyzed at ×200 for determining expression levels. Immunohistochemical staining was successfully performed on the tissue microarrays for 174, 189, 191, and 191 tumors for Notch3, MUC2, MUC5AC, and MUC6 respectively, due to loss of several tissue cores during sectioning or the staining process. for Notch3, both the percentage of positive tumor cells and the intensity of positive staining were graded. The percentage of staining was graded as follows: 0, staining in <5% of tumor cells; 1, staining in 6–25% of tumor cells; 2, staining in 26–50% of tumor cells; 3, staining in 51–75% of tumor cells; and 4, staining in 76–100% of tumor cells [31]. The intensity of staining was graded as follows: 0, no or weak staining; 1, moderate staining; and 2, strong staining. The mean value of multiplication of the extent and intensity of Notch3 was 2.99. Therefore, when the product of the multiplication of the extent and intensity was ≥3, the staining was interpreted as positive by modified previously reported methods [31,32]. Because the staining intensity of MUC proteins was relatively even, only the percentage of unequivocally positive epithelial cells was evaluated and sections demonstrating >5% positivity were considered positive, as previously described [32]. Statistical analysis In order to compare associations between immunohistochemical markers and clinicopathologic parameters, chi-square and Fisher’s exact tests were performed. Overall patients’ survival was defined as the time from the surgical resection of the SIACs to the death of the patient or the last follow-up of the patient. The survival rate was calculated by the Kaplan–Meier method. A comparison of the survival rate with regard to various clinicopathologic
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Table 1 Notch3 and MUCs expressions and a clinicopathologic characteristics in small intestinal adenocarcinoma patients. Variable
Characteristics
All Case No.a
Notch3+ (%)
p-Value
MUC2+ (%)
p-Value
MUC5AC+ (%)
p-Value
MUC6+ (%)
p-Value
Age (y)
<60 ≥60 Male Female <4.0 cm ≥4.0 cm Proximal (duodenal) Distal (jejunal/ileal) Polypoid Nodular Infiltrative Tubular adenocarcinoma b Other type than tubular adenocarcinoma Well differentiated Moderately differentiated Poorly differentiated Undifferentiated pT1-2 pT3-4 Present Absent Present Absent Present Absent Present Absent Present Absent
93 98 120 71 92 97 102 89 31 12 140 173
26 (30.0) 26 (29.2) 32 (29.4) 20 (30.8) 28 (35.0) 24 (26.1) 27 (95%) 68 (71.6%) 8 (28.6) 2 (18.2) 41 (32.3) 52 (32.5)
0.843
28 (30.4) 24 (24.7) 33 (28.0) 19 (26.8) 18 (19.6) 34 (35.8) 22 (22.0) 30 (33.1) 10 (32.3) 4 (33.3) 34 (24.6) 42 (24.6)
0.381
31 (33.3) 30 (30.6) 40 (33.3) 21 (29.6) 35 (38.0) 26 (26.8) 44 (43.1) 17 (19.1) 8 (25.8) 4 (33.2) 46 (32.9) 58 (33.5)
0.687
18 (19.4) 24 (24.5) 33 (27.5) 9 (12.7) 24 (26.1) 18 (18.6) 34 (33.3) 8 (9.0) 9 (29.0) 2 (16.7) 27 (19.3) 40 (23.1)
0.392
Sex Tumor size Location Growth pattern
Histologic subtype
Differentiaton
pT classification Lymph node metastasis Other loop invasion Retroperitoneal invasion Perineural invasion Lymphovascular invasion a b
18
0.844 0.204 0.644 0.601
0.011
0 (0)
39 105
11 (31.4) 31 (32.6)
42 5 7 9 90 84 5 186 14 177 63 128 51 140
10 (25.0) 0 (0) 8 (53.3) 44 (27.9) 21 (25.9) 26 (33.3) 1 (25.0) 51 (30.0) 3 (25.0) 49 (30.2) 13 (22.0) 39 (33.9) 19 (21.1) 33 (39.3)
0.857 0.013 0.072 0.588
0.01
10 (55.6)
0.468
0.038 0.306 1 1 0.105 0.009
12 (30.8) 30 (28.8) 10 (24.4) 0 (0) 2 (12.5) 50 (28.9) 22 (24.7) 26 (31.3) 1 (20.0) 51 (27.7) 5 (35.7) 47 (26.9) 28 (44.4) 46 (35.9) 23 (23.5) 29 (31.9)
0.591 0.099 <0.001 0.741
0.188
3 (16.7)
0.494
0.242 0.334 1 0.536 0.257 0.196
16 (41.0) 38 (36.2) 7 (16.7) 0 (0) 88 (50.0) 53 (30.3) 29 (32.2) 30 (35.7) 1 (20.0) 60 (32.3) 4 (28.6) 57 (32.2) 17 (27.0) 35 (27.8) 27 (27.3) 34 (37.0)
0.019 0.213 <0.001 0.45
0.371
2 (11.1)
0.027
0.105 0.627 1 1 0.908 0.151
10 (25.6) 26 (24.8) 6 (14.3) 0 (0) 8 (50.0) 34 (19.4) 18 (20.0) 22 (26.2) 1 (20.0) 41 (22.0) 4 (28.6) 38 (21.5) 10 (15.9) 32 (25.0) 13 (13.1) 29 (31.5)
0.304
0.009 0.332 1 0.513 0.152 0.002
Total patient numbers in this study. Immunohistochemical staining was performed in 174, 189, 191, and 191 cases for Notch3, MUC2, MUC5AC, and MUC6 respectively. The other histological types include 9 mucionous carcinomas, 4 signet ring cell carcinomas, and 5 undifferentiated carcinomas.
characteristics was performed by the log rank. Statistical analyses were performed using IBM SPSS 12.0 software (SPSS, IL, USA). A p-value less than 0.05 was considered statistically significant. Results Expression of Notch3 and MUC proteins (MUC2, MUC5A, and MUC6) and their correlation with clinicopathologic characteristics in SIACs Table 1 summarizes the correlation between the expression of Notch3 and MUC proteins and clinicopathologic characteristics. In the normal small intestinal mucosa, focal expressions of Notch3 were found in the cytoplasm and nuclei of some epithelial cells of the crypt bottoms (Fig. 1A). In 52 (29.9%) of the tumors, Notch3 was aberrantly expressed in the cell membrane and/or cytoplasm with an occasional nuclear stain (Fig. 1B). The cytoplasm of endothelial cells and some stromal cells were also positive for Notch3. MUC2 expression was universally present in the majority of non-neoplastic small intestinal mucosa (119, 78.8%) and was predominantly seen in goblet cells (Fig. 1C). In tumor cells, MUC2 expression was noted in the cytoplasm in 52 (27.5%) cases (Fig. 1D). In non-neoplastic small intestinal epithelium, MUC5AC was expressed in 8 (5.1%) cases in the cytoplasm of some columnar cells (Fig. 1E), but 61 (31.9%) SIACs showed MUC5AC overexpression with a cytoplasmic staining pattern (Fig. 1F). The expression profile of MUC6 was similar to that of MUC5AC. That is, in 19 (12.4%) cases, the cytoplasm of the non-neoplastic epithelium stained positive (Fig. 1G), but 42 cases (22.0%) showed diffuse cytoplasmic expression for MUC6 (Fig. 1H). Notch3 expression was correlated with the absence of lymphovascular invasion (p = 0.009), lower
pT stage (p = 0.038), and the tubular adenocarcinoma histological subtype (p = 0.011), respectively. MUC2 was correlated with a large tumor size (p = 0.013), distal tumor location (p = 0.072), and other histological subtypes (9 mucinous, 4 signet ring cell, and 5 undifferentiated carcinomas) (p = 0.01), respectively. MUC5AC was correlated with proximal tumor location (p < 0.001), and tumor differentiation (p = 0.027), respectively. MUC6 was correlated with a proximal tumor location (p < 0.001), lower pT stage (p = 0.009), and the absence of lymphovascular invasion (p = 0.002), respectively.
Correlation between expression of Notch3 and MUC proteins in SIACs Table 2 summarizes the correlation between expressions of Notch3 and MUC proteins. Notch3 was frequently expressed in patients with MUC5AC expression (42.3%) compared with patients with MUC5AC negative expression (24.6%) (p = 0.019; Fig. 1C and D). There was no correlation between Notch3 and MUC2 or MUC6 expression.
Table 2 Correlation between Notch3 and MUC protein expression. Variable
Characteristics
Notch3+ (%)
p-Value
MUC2
Positive Negative Positive Negative Positive Negative
15 (31.3) 37 (29.8) 22 (42.3) 30 (24.6) 13 (35.1) 39 (28.5)
0.857
MUC5A MUC6
0.019 0.432
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Fig. 1. (A) In the normal small intestinal mucosa, Notch3 are expressed in the cytoplasm and nuclei of some epithelial cells of the crypt bottoms. (B and D) The tumor cells show strong cytoplasmic immunoreactivity for both NOTCH3 and MUC2. (C) MUC2 is predominantly expressed in the goblet cells in the normal mucosa. (E and G) In the normal mucosa, MUC5AC and MUC6 are expressed in the cytoplasm of some columnar cells. (F and H) MUC5AC and MUC6 are diffusely positive in the cytoplasm in the tumor cells.
Survival analyses with clinicopathologic factors for patients with SIACs Table 3 summarizes the correlation between the clinicopathologic factors including the expressions of Notch3 and MUC proteins and survival data. Notch3 expression was significantly associated with more favorable patient survival (median survival time: 74.10 months vs. 36.20 months, p = 0.05; Fig. 2) according to the
univariate analysis. MUC2 showed a worse survival when it was expressed, but there was no statistical significance (median survival time: 48.50 months vs. 36.20 months, p = 0.7710). Expressions of MUC5AC and MUC6 also were not significantly correlated with patient survival although high expressions were associated with somewhat favorable outcomes (MUC5AC: median survival time, 41.70 vs. 36.20 months, p = 0.8985; MUC6 median survival time, 49.90 vs. 30.70 months, p = 0.4253).
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Table 3 Univariate analysis of clinicopathologic factors. Variable
Notch3 expression MUC2 expression MUC5A expression MUC6 expression Tumor size Location Growth pattern
Histologic subtype Differentiaton
pT classification Lymph node metastasis Other loop invasion Retroperitoneal seeding Perineural invasion Lympovascular invasion
Characteristics
Median survival (mo)
Positve Negative Positve Negative Present Absent Positve Negative <4.0 cm ≥4.0 cm Proximal (duodenal) Distal (jejunal/ileal) Polypoid Nodular Infiltrative Tubular adenocarcinoma Other types Well Moderately Poorly Undifferentiated pT1-2 pT3-4 Present Absent Present Absent Present Absent Present Absent Present Absent
95% Confidence interval
74.1 36.2 48.5 36.2 41.7 36.2 49.9 30.7 49.9 28.2 74.1 24.6 48.5 36.2 38.5 39.9 20.1 49.9 28.2 30 7.9 – 30.7 22.6 80.8 5.1 39.9 11.1 48.5 26.3 49.9 22.6 74.1
Next, the Notch3+ expression group was examined with multivariate statistical analysis, with other significant prognostic valuables examined by univariate analysis. T stage, tumor location, lymph node metastasis, lymphovascular invasion, perineural invasion, other loop invasion, and retroperitoneal seeding were included as cofactors. As shown in Table 4, together with retroperitoneal seeding and lymph node metastasis, Notch3+ expression was an independent favorable prognostic factor, with marginal statistical significance (p = 0.08, Cox hazard ratio 0.841), for patient survival. Discussion In this study, we demonstrate that Notch3 expression was associated with favorable prognosis in patients with SIACs, a result which was in contrast with the results of previous reports on colorectal cancers, in which Notch signaling was associated with an aggressive tumor prognosis [25,32]. However, it has been known that Notch signal pathway plays diverse and complex roles as not only a tumor promoter but also a suppressor in malignant tumors depending on the cellular context, level of expression, and kinds of organ. In fact, Kang et al. recently reported that the expression of Notch3 was associated with a favorable prognosis with intestinal or glandular differentiation in gastric cancer [26]. Kim et al. also reported that Notch1 signaling
Lower
Upper
– 21.84 2.5 21.35 17.22 16.08 29.19 17.15 33.06 20.22 36.34 15.98 0 – 18.88 22.68, 0
– 50.56 94.5 51.5 66.18 56.12 70.61 44.25 66.74 36.18 111.86 33.22 100.64 – 58.12 57.12 44.19
3.83 10.57 0 – 18.16 15.73 – 0 22.37 0.98 15.59 15.59 14.89 14.32 39.22
52.57 49.43 20.35 – 43.24 29.47 – 19.02 57.43 21.22 81.41 37.01 84.91 30.88 108.98
p-Value
0.05 0.771 0.8985 0.4253 0.3486 0.0169 0.9311
0.2171 0.6619
0.0774 0.0006 0.0801 0.0002 0.1508 0.0042
retained the capability of suppressing the expression of WNT target genes in colorectal cancers and the activation of Notch1 even converted high-grade adenoma into low-grade adenoma in a mouse colon cancer model [13]. They suggested that Notch signaling promotes the onset of intestinal tumors, and after tumor onset, Notch signaling inhibits tumor progression by repression of the WNT target gene through an epigenetic chromatin modification. Although a contrasting result has been reported [25], it is also well known that Notch signaling is strongly expressed in the adenoma of the colon, suggesting the role of Notch signaling as an essential initial event triggering colorectal cancer [33]. These findings suggest that the role of Notch signaling in intestinal tumors is more complex; that is, Notch signaling is a strong promoter in intestinal tumor initiation, and it might also have a suppressive role in established tumors. We also found MUC5AC expression was closely related with the expression of Notch3 (p = 0.02). MUC5AC was expressed diffusely in 8 cases (5.1%) in the cytoplasm of columnar cells or focal goblet cells in non-neoplastic small-intestinal epithelium, but 61 SIAC cases (31.9%) showed MUC5AC overexpression with a mixed apicoluminal and cytoplasmic staining pattern. There are a few studies on the relationship between MUC5AC expression and tumor progression in the colorectum. Kocer B et al. reported that in colorectal cancer, decreased MUC5AC expression can be a prognostic factor indicating the existence of a more aggressive form of colorectal
Table 4 Multivariate analysis of prognosis. Variable
p-Value
Relative risk
95% confidence interval
Notch3 expression Retroperitoneal seeding Metastasis to lymph node
0.08 0.002 0.0002
0.841 3.61 2.667
0.609–1.02 1.590–8.195 1.577–4.510
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of tumor progression and enhancement of tumor initiation. Further studies are required to reveal more detailed mechanisms of the influence of Notch signaling on tumor progression in the small intestine. Acknowledgements
Fig. 2. Patients with Notch3+ expression show significantly better overall survival rates (p = 0.05) than patients with Notch3-expression by Kaplan–Meier survival analysis.
cancer [34]. MUC5AC mRNA and protein were present more frequently in colorectal polyps which were intermediate in size, and in polyps which had villous histology and low or moderate dysplasia [6]. MUC5AC protein expression was higher in hyperplastic polyps, and in polyps with low and high grade dysplasia, and in mucinous carcinoma groups than in normal mucosa and conventional adenocarcinoma groups [35]. On the other hand, Kang JH et al. recently described the possible involvement of Notch signaling in the expression of MUC5AC, a defining characteristic of the goblet cell phenotype which manifests as insults to the respiratory airway epithelial cells [12]. They suggested that Notch signaling not only involves epidermal growth factor induced MUC5AC expression, but that it is also able to induce MUC5AC expression itself [12]. Considering that the role of Notch signaling for tumorigenesis depends on the cellular context and cross-talk with other signaling pathway molecules, MUC5AC expression might partially contribute to the favorable prognosis of patients with Notch-3 expression in SIACs. On the contrary, Ou-Yang HF et al. recently reported that Notch signaling could directly downregulate MUC5AC promoter activity through Hes1-dependent mechanisms in the airway epithelium in asthma. Therefore, more studies are needed to determine the exact molecular mechanisms of how Notch signaling regulates MUC5AC expression [36]. To the best of our knowledge, this is the first description of the prognostic significance of Notch3 expression and its relationship with the expression of MUC proteins in SIACs. The limitations of this study include the retrospective nature of the study design and the fundamental problems of an immunohistochemical study that uses a TMA block, including a subjective scoring system and the intrinsic heterogeneity of the immunoreactivity in the tumor. In summary, the present study revealed that Notch3 was expressed in 52 (29.9%) of the tumors and significantly correlated with the absence of lymphovascular invasion, lower T stage, and the histological subtype of tubular adenocarcinoma, and MUV5AC expression. Furthermore, Notch3 expression was a favorable prognostic factor in patients with SIACs. These findings suggest dual roles of Notch signaling in SICAs, including both the suppression
This research was supported by the Gangneung Asan Hospital Biomedical Research Center Promotion Fund & Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0004807). We thank to members of the Korean small intestinal cancer study groups: Dr. Hee-Kyung Chang, Kosin University College of Medicine, Pusan; Dr. Eun Sun Jung, The Catholic University of Korea College of Medicine, Seoul; Drs. Ghil Suk Yoon and Han-Ik Bae, Kyungpook National University, Dague; Dr. Joon Mee Kim, Inha University College of Medicine, Incheon; Dr. YoungHa Oh, Hanyang University College of Medicine, Seoul; Dr. Soo Jin Jung, Inje University College of Medicine, Busan; Dr. Mi Jin Gu, Fatima Hospital, Daegu; Dr. Jung Yeon Kim, Inje University Sanggye Paik Hospital, Seoul; Dr. Kyu Yun Jang, Chonbuk National University Medical School, Jeonju; Dr. Kye Won Kwon, Bundang Jesaeng General hospital, Seongnam; Dr. Gyeong Hoon Kang, Seoul National University College of Medicine, Seoul; Dr. Jae Bok Park, Catholic University of Daegu, Daegu; Dr. SoonWon Hong, Yonsei University College of Medicine, Seoul; Dr. Sun-Young Jun, Inchoen Saint Mary Hospital, The Catholic University of Korea College of Medicine, Incheon; and Dr. Ji Shin Lee, Chonnam National University Medical School, Gwangju, South Korea. References [1] H.K. Chang, E. Yu, J. Kim, et al., Adenocarcinoma of the small intestine: a multiinstitutional study of 197 surgically resected cases, Hum. Pathol. 41 (2010) 1087–1096. [2] J.C. Byrd, R.S. Bresalier, Mucins and mucin binding proteins in colorectal cancer, Cancer Metastasis Rev. 23 (2004) 77–99. [3] J.A. Gibson, H.P. Hahn, A. Shahsafaei, R.D. Odze, MUC expression in hyperplastic and serrated colonic polyps: lack of specificity of MUC6, Am. J. Surg. Pathol. 35 (2011) 742–749. [4] S.J. Gendler, A.P. Spicer, Epithelial mucin genes, Annu. Rev. Physiol. 57 (1995) 607–634. [5] S.K. Chang, A.F. Dohrman, C.B. Basbaum, et al., Localization of mucin (MUC2 and MUC3) messenger RNA and peptide expression in human normal intestine and colon cancer, Gastroenterology 107 (1994) 28–36. [6] A.E. Bartman, S.J. Sanderson, S.L. Ewing, et al., Aberrant expression of MUC5AC and MUC6 gastric mucin genes in colorectal polyps, Int. J. Cancer 80 (1999) 210–218. [7] G.R. Van den Brink, K.M. Tytgat, R.W. Van der Hulst, et al., H pylori colocalises with MUC5AC in the human stomach, Gut 46 (2000) 601–607. [8] N. Schroder, A. Gossler, Expression of Notch pathway components in fetal and adult mouse small intestine, Gene Expr. Patterns 2 (2002) 247–250. [9] M. Katoh, Notch signaling in gastrointestinal tract (review), Int. J. Oncol. 30 (2007) 247–251. [10] X.Y. Ding, J. Ding, K. Wu, et al., Cross-talk between endothelial cells and tumor via delta-like ligand 4/Notch/PTEN signaling inhibits lung cancer growth, Oncogene 31 (2012) 2899–2906. [11] K.L. VanDussen, A.J. Carulli, T.M. Keeley, et al., Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells, Development 139 (2012) 488–497. [12] J.H. Kang, E.H. Lee, S.W. Park, I.Y. Chung, MUC5AC expression through bidirectional communication of Notch and epidermal growth factor receptor pathways, J. Immunol. 187 (2011) 222–229. [13] H.A. Kim, B.K. Koo, J.H. Cho, et al., Notch1 counteracts WNT/-catenin signaling through chromatin modification in colorectal cancer, J. Clin. Invest. 122 (2012) 3248–3259. [14] D. Bellavia, S. Checquolo, A.F. Campese, M.P. Felli, A. Gulino, I. Screpanti, Notch3: from subtle structural differences to functional diversity, Oncogene 27 (2008) 5092–5098. [15] M. Lardelli, J. Dahlstrand, U. Lendahl, The novel Notch homologue mouse Notch 3 lacks specific epidermal growth factor-repeats and is expressed in proliferating neuroepithelium, Mech. Dev. 46 (1994) 123–136. [16] F. Jundt, O. Acikgoz, S.H. Kwon, et al., Aberrant expression of Notch1 interferes with the B-lymphoid phenotype of neoplastic B cells in classical Hodgkin lymphoma, Leukemia 22 (2008) 1587–1594.
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Original Article
Notch3 signaling is associated with MUC5AC expression and favorable prognosis in patients with small intestinal adenocarcinomas Dae-Woon Eom a,∗ , Seung-Mo Hong b , Jihun Kim b , Gwangil Kim c , Young Kyung Bae d , Kee-Taek Jang e , Eunsil Yu b a
Department of Pathology, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung, Republic of Korea Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea c Department of Pathology, Bundang CHA Medical Center, CHA University, Seongnam, Republic of Korea d Department of Pathology, Yeungnam University College of Medicine, Daegu, Republic of Korea e Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 25 November 2013 Received in revised form 11 February 2014 Accepted 1 April 2014 Keywords: Small intestine Adenocarcinoma Prognosis Notch3 MUC5AC
a b s t r a c t Background: Notch signaling plays diverse roles not only in physiologic processes, including development and differentiation but also in tumorigenesis, either as a tumor promoter or suppressor depending on the cellular context, level of expression and cross-talk with other signaling pathways. In this study we investigated the expression of Notch3 and MUC proteins and their clinicopathological significance in small intestinal adenocarcinoma (SIAC). Methods: Surgically resected 191 SIACs and their clinical data were collected. Immunohistochemistry for Notch3, MUC2, MUC5AC, and MUC6 using tissue microarrays from formalin-fixed paraffin-embedded normal and matched tumor tissues was performed. Results: Notch3 expression was found in 52 (29.9%) cases of the tumors. MUC2, MUC5AC, and MUC6 were expressed in 52 (27.5%), 51 (31.9%), and 42 (22.0%) cases of the tumor, respectively. Notch3 expression was correlated with the absence of lymphovascular invasion (p = 0.009), lower T stage (p = 0.038), and histological subtype of tubular adenocarcinoma (p = 0.01), respectively. MUC2 was correlated with large tumor size (p = 0.013) and mucinous and signet ring cell adenocarcinomas (p = 0.01). MUC5A was correlated with proximal tumor location (p < 0.0001) and tumor differentiation (p = 0.027). MUC6 was correlated with proximal tumor location (p < 0.0001) and lower pT stage (p = 0.009), and absence of lymphovascular invasion, respectively. A significant correlation was noted between Notch3 and MUC5AC expression (p = 0.019). Notch3 expression was a relatively favorable prognostic factor in SIACs by univariate (p = 0.05) and multivariate analysis (p = 0.08, Cox Hazard ratio 0.841). Conclusion: Our findings indicate that Notch3 signaling, associated with MUC5AC expression, could be a more favorable prognostic factor in SIACs. © 2014 Elsevier GmbH. All rights reserved.
Introduction Primary small intestinal adenocarcinoma (SIAC) is a rare tumor although the small intestine occupies the largest absorptive surface areas (about 90%) of the gastrointestinal tract. Despite the recent development of endoscopic modalities and imaging techniques for the small intestine, the early detection of SIACs has been quite
∗ Corresponding author at: Department of Pathology, University of Ulsan College of Medicine, Gangneung Asan Hospital, 415, Bangdong-ri, Sacheon-myeon, Gangneung-si, Gangwon-do 210-711, Republic of Korea. Tel.: +82 033 610 3428; fax: +82 033 610 3430. E-mail address: [email protected] (D.-W. Eom).
difficult so far and the 5-year survival rate of SIACs diagnosed at an advanced disease state is 41.2% [1]. To improve the survival rate of patients with SIACs, it is necessary to understand more clearly their molecular pathogenesis. However, the molecular mechanism of SIAC has not been fully understood due to its rare incidence. MUC proteins are high-molecular-weight glycoproteins or mucopolysaccharides that are widely expressed in epithelial cells and show organ specificity [2,3]. To date, twenty distinct epithelial mucin genes, which can be broadly classified into two groups as secreted and membrane bound mucins, have been identified [2,4]. Among them, secreted gel-forming mucins (MUC2, MUC5A, and MUC6) are the major secreted glycoproteins of the gastrointestinal tract and play not only a role in normal physiological processes but also have a function in neoplastic progression. MUC2 is primarily
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expressed in goblet cells of the native intestinal epithelium in the small bowel and colon [5]. MUC5AC and MUC6 are markers of the gastric epithelial cell phenotype [6]. MUC5AC expression is localized in the gastric foveolar epithelium, whereas MUC6 expression is detected in the cytoplasm of the pyloric gland and mucous neck cells in the stomach [3,7]. The Notch signaling plays diverse roles not only in normal tissue development and differentiation but also in tumorigenesis [8–10]. The Notch signaling molecules are essential for controlling the fate of stem cells and expressed at different levels with varying spatial and temporal distribution in the developing small intestine [8]. Notch signaling regulates the proliferation and differentiation of intestinal stem and progenitor cells in crypt base to maintain epithelial cell homeostasis in the small intestine [11]. In malignant tumors, it has complex and controversial biological functions of either a tumor promoter or suppressor depending on the cellular context, level of expression and cross-talk with other signaling pathways [10,12,13]. The Notch signaling pathway associated proteins are composed of four Notch receptors (Notch1–4) and five Notch ligands in mammals. Notch1 and Notch2 have the highest homology with each other, while Notch3 and Notch4 are structurally and functionally diverged from Notch1 and Notch2 in both the extracellular and intracellular domains [14]. Notch-1 was first found to associate with hematological diseases, and its expression level increased in multiple myeloma, Hodgkin’s lymphoma, anaplastic large cell lymphoma and acute myeloid leukemia [16,17]. Notch2 has the highest homology with Notch1, but unlike Notch1, it can present a tumorsuppressive action in breast cancer [18,19]. It has recently been reported that Notch2 is a novel target for -catenin-dependent Wnt signaling [20]. Although the biological significance of the Notch4 has not been determined, activated it disrupts normal epithelial morphogenesis and induces an invasive phenotype in mammary epithelial cells [21]. Notch3 was initially reported as being expressed in the proliferating neuroepithelium and the underlying cause of cerebral arteriopathy with subcortical infarcts and leukoencephalopathy [15,22]. Although Notch3 activation was reported to be oncogenic in many various organs, such as the ovary, lung, and colon [23–25], it was also recently found to have the opposite effect for tumorigenesis or prognosis in some malignant tumors [26–28]. It is well known that Notch signaling regulates the terminal differentiation of goblet cells in normal intestinal mucosa [29,30], but the relationship between Notch3 signaling and MUC proteins has not been described in SIACs. In the present study, in order to investigate the role and clinical significance of Notch3 in small intestinal carcinogenesis, we evaluated the clinicopathologic significance of Notch3 expression and examined the association between the expression of Notch3 and those of MUC2, MUC5AC, and MUC6 proteins in patients with SIACs.
Materials and methods Patients and specimens 191 cases of surgically resected SIACs were collected from 22 institutions in Korea1 [1]. Representative H&E slides and paraffin tissue blocks were obtained with the appropriate human protection approval from the institutional review board from each institution that participated in the Korean Small Intestinal Cancer Study Group. Primary carcinomas arising in the duodenum, jejunum, and ileum were included in this study. Malignant tumors from other gastrointestinal tract organs, such as the stomach, ampulla of Vater, pancreas or appendix with extension into the small intestine were excluded. Data were collected after reviewing the medical records
of the patients including the patients’ sex, age, accompanying tumors, and recent follow-up date for survival status. The pathologic characteristics that were evaluated included tumor location, size, histological subtype, differentiation, growth pattern, pT stage, retroperitoneal seeding, other intestinal loop invasion, metastasis to lymph node, and presence of perineural and lymphovascular invasion. Tissue microarray To construct tissue microarrays, tissue cores of 1 mm diameter were taken from morphologically representative areas of the formalin-fixed paraffin-embedded blocks. The tissue array blocks consisted of 2 or 3 cores of tumor tissue and one core of matched normal mucosa far from the tumor, not adjacent area. Immunohistochemistry and interpretation Immunohistochemical staining for Notch3 (rabbit polyclonal, Abcam, Cambridge, UK, 1:100), MUC2 (mouse monoclonal, Novocastra, Newcastle, UK, 1:200), MUC5AC (mouse monoclonal, Novocastra, Newcastle, UK, 1:200), and MUC6 (mouse monoclonal, Novocastra, Newcastle, UK, 1:200) was performed on the arrayed blocks. All immunostaining was performed with the Bond-Max automatic immunostaining device (Leica Biosystem, Newcastle, UK) using a bond polymer intensity detection kit (Leica Biosystem) for formalin-fixed, paraffin-embedded tissue sections. Four-micrometer-thick sections were obtained by microtome, transferred onto adhesive slides, and dried at 62 ◦ C for 30 min. Antigen retrieval was carried out in all cases. Slides were counterstained with Harris hematoxylin. Immunoreactivity was interpreted by light microscopic examination and independently evaluated by pathologists who authored this study (D-WE and S-MH) and who were blind to the clinicopathological information. Immunostained slides were scored for unequivocally positive epithelial cells and 10 representative fields of each tumor and normal tissue cores were analyzed at ×200 for determining expression levels. Immunohistochemical staining was successfully performed on the tissue microarrays for 174, 189, 191, and 191 tumors for Notch3, MUC2, MUC5AC, and MUC6 respectively, due to loss of several tissue cores during sectioning or the staining process. for Notch3, both the percentage of positive tumor cells and the intensity of positive staining were graded. The percentage of staining was graded as follows: 0, staining in <5% of tumor cells; 1, staining in 6–25% of tumor cells; 2, staining in 26–50% of tumor cells; 3, staining in 51–75% of tumor cells; and 4, staining in 76–100% of tumor cells [31]. The intensity of staining was graded as follows: 0, no or weak staining; 1, moderate staining; and 2, strong staining. The mean value of multiplication of the extent and intensity of Notch3 was 2.99. Therefore, when the product of the multiplication of the extent and intensity was ≥3, the staining was interpreted as positive by modified previously reported methods [31,32]. Because the staining intensity of MUC proteins was relatively even, only the percentage of unequivocally positive epithelial cells was evaluated and sections demonstrating >5% positivity were considered positive, as previously described [32]. Statistical analysis In order to compare associations between immunohistochemical markers and clinicopathologic parameters, chi-square and Fisher’s exact tests were performed. Overall patients’ survival was defined as the time from the surgical resection of the SIACs to the death of the patient or the last follow-up of the patient. The survival rate was calculated by the Kaplan–Meier method. A comparison of the survival rate with regard to various clinicopathologic
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Table 1 Notch3 and MUCs expressions and a clinicopathologic characteristics in small intestinal adenocarcinoma patients. Variable
Characteristics
All Case No.a
Notch3+ (%)
p-Value
MUC2+ (%)
p-Value
MUC5AC+ (%)
p-Value
MUC6+ (%)
p-Value
Age (y)
<60 ≥60 Male Female <4.0 cm ≥4.0 cm Proximal (duodenal) Distal (jejunal/ileal) Polypoid Nodular Infiltrative Tubular adenocarcinoma b Other type than tubular adenocarcinoma Well differentiated Moderately differentiated Poorly differentiated Undifferentiated pT1-2 pT3-4 Present Absent Present Absent Present Absent Present Absent Present Absent
93 98 120 71 92 97 102 89 31 12 140 173
26 (30.0) 26 (29.2) 32 (29.4) 20 (30.8) 28 (35.0) 24 (26.1) 27 (95%) 68 (71.6%) 8 (28.6) 2 (18.2) 41 (32.3) 52 (32.5)
0.843
28 (30.4) 24 (24.7) 33 (28.0) 19 (26.8) 18 (19.6) 34 (35.8) 22 (22.0) 30 (33.1) 10 (32.3) 4 (33.3) 34 (24.6) 42 (24.6)
0.381
31 (33.3) 30 (30.6) 40 (33.3) 21 (29.6) 35 (38.0) 26 (26.8) 44 (43.1) 17 (19.1) 8 (25.8) 4 (33.2) 46 (32.9) 58 (33.5)
0.687
18 (19.4) 24 (24.5) 33 (27.5) 9 (12.7) 24 (26.1) 18 (18.6) 34 (33.3) 8 (9.0) 9 (29.0) 2 (16.7) 27 (19.3) 40 (23.1)
0.392
Sex Tumor size Location Growth pattern
Histologic subtype
Differentiaton
pT classification Lymph node metastasis Other loop invasion Retroperitoneal invasion Perineural invasion Lymphovascular invasion a b
18
0.844 0.204 0.644 0.601
0.011
0 (0)
39 105
11 (31.4) 31 (32.6)
42 5 7 9 90 84 5 186 14 177 63 128 51 140
10 (25.0) 0 (0) 8 (53.3) 44 (27.9) 21 (25.9) 26 (33.3) 1 (25.0) 51 (30.0) 3 (25.0) 49 (30.2) 13 (22.0) 39 (33.9) 19 (21.1) 33 (39.3)
0.857 0.013 0.072 0.588
0.01
10 (55.6)
0.468
0.038 0.306 1 1 0.105 0.009
12 (30.8) 30 (28.8) 10 (24.4) 0 (0) 2 (12.5) 50 (28.9) 22 (24.7) 26 (31.3) 1 (20.0) 51 (27.7) 5 (35.7) 47 (26.9) 28 (44.4) 46 (35.9) 23 (23.5) 29 (31.9)
0.591 0.099 <0.001 0.741
0.188
3 (16.7)
0.494
0.242 0.334 1 0.536 0.257 0.196
16 (41.0) 38 (36.2) 7 (16.7) 0 (0) 88 (50.0) 53 (30.3) 29 (32.2) 30 (35.7) 1 (20.0) 60 (32.3) 4 (28.6) 57 (32.2) 17 (27.0) 35 (27.8) 27 (27.3) 34 (37.0)
0.019 0.213 <0.001 0.45
0.371
2 (11.1)
0.027
0.105 0.627 1 1 0.908 0.151
10 (25.6) 26 (24.8) 6 (14.3) 0 (0) 8 (50.0) 34 (19.4) 18 (20.0) 22 (26.2) 1 (20.0) 41 (22.0) 4 (28.6) 38 (21.5) 10 (15.9) 32 (25.0) 13 (13.1) 29 (31.5)
0.304
0.009 0.332 1 0.513 0.152 0.002
Total patient numbers in this study. Immunohistochemical staining was performed in 174, 189, 191, and 191 cases for Notch3, MUC2, MUC5AC, and MUC6 respectively. The other histological types include 9 mucionous carcinomas, 4 signet ring cell carcinomas, and 5 undifferentiated carcinomas.
characteristics was performed by the log rank. Statistical analyses were performed using IBM SPSS 12.0 software (SPSS, IL, USA). A p-value less than 0.05 was considered statistically significant. Results Expression of Notch3 and MUC proteins (MUC2, MUC5A, and MUC6) and their correlation with clinicopathologic characteristics in SIACs Table 1 summarizes the correlation between the expression of Notch3 and MUC proteins and clinicopathologic characteristics. In the normal small intestinal mucosa, focal expressions of Notch3 were found in the cytoplasm and nuclei of some epithelial cells of the crypt bottoms (Fig. 1A). In 52 (29.9%) of the tumors, Notch3 was aberrantly expressed in the cell membrane and/or cytoplasm with an occasional nuclear stain (Fig. 1B). The cytoplasm of endothelial cells and some stromal cells were also positive for Notch3. MUC2 expression was universally present in the majority of non-neoplastic small intestinal mucosa (119, 78.8%) and was predominantly seen in goblet cells (Fig. 1C). In tumor cells, MUC2 expression was noted in the cytoplasm in 52 (27.5%) cases (Fig. 1D). In non-neoplastic small intestinal epithelium, MUC5AC was expressed in 8 (5.1%) cases in the cytoplasm of some columnar cells (Fig. 1E), but 61 (31.9%) SIACs showed MUC5AC overexpression with a cytoplasmic staining pattern (Fig. 1F). The expression profile of MUC6 was similar to that of MUC5AC. That is, in 19 (12.4%) cases, the cytoplasm of the non-neoplastic epithelium stained positive (Fig. 1G), but 42 cases (22.0%) showed diffuse cytoplasmic expression for MUC6 (Fig. 1H). Notch3 expression was correlated with the absence of lymphovascular invasion (p = 0.009), lower
pT stage (p = 0.038), and the tubular adenocarcinoma histological subtype (p = 0.011), respectively. MUC2 was correlated with a large tumor size (p = 0.013), distal tumor location (p = 0.072), and other histological subtypes (9 mucinous, 4 signet ring cell, and 5 undifferentiated carcinomas) (p = 0.01), respectively. MUC5AC was correlated with proximal tumor location (p < 0.001), and tumor differentiation (p = 0.027), respectively. MUC6 was correlated with a proximal tumor location (p < 0.001), lower pT stage (p = 0.009), and the absence of lymphovascular invasion (p = 0.002), respectively.
Correlation between expression of Notch3 and MUC proteins in SIACs Table 2 summarizes the correlation between expressions of Notch3 and MUC proteins. Notch3 was frequently expressed in patients with MUC5AC expression (42.3%) compared with patients with MUC5AC negative expression (24.6%) (p = 0.019; Fig. 1C and D). There was no correlation between Notch3 and MUC2 or MUC6 expression.
Table 2 Correlation between Notch3 and MUC protein expression. Variable
Characteristics
Notch3+ (%)
p-Value
MUC2
Positive Negative Positive Negative Positive Negative
15 (31.3) 37 (29.8) 22 (42.3) 30 (24.6) 13 (35.1) 39 (28.5)
0.857
MUC5A MUC6
0.019 0.432
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Fig. 1. (A) In the normal small intestinal mucosa, Notch3 are expressed in the cytoplasm and nuclei of some epithelial cells of the crypt bottoms. (B and D) The tumor cells show strong cytoplasmic immunoreactivity for both NOTCH3 and MUC2. (C) MUC2 is predominantly expressed in the goblet cells in the normal mucosa. (E and G) In the normal mucosa, MUC5AC and MUC6 are expressed in the cytoplasm of some columnar cells. (F and H) MUC5AC and MUC6 are diffusely positive in the cytoplasm in the tumor cells.
Survival analyses with clinicopathologic factors for patients with SIACs Table 3 summarizes the correlation between the clinicopathologic factors including the expressions of Notch3 and MUC proteins and survival data. Notch3 expression was significantly associated with more favorable patient survival (median survival time: 74.10 months vs. 36.20 months, p = 0.05; Fig. 2) according to the
univariate analysis. MUC2 showed a worse survival when it was expressed, but there was no statistical significance (median survival time: 48.50 months vs. 36.20 months, p = 0.7710). Expressions of MUC5AC and MUC6 also were not significantly correlated with patient survival although high expressions were associated with somewhat favorable outcomes (MUC5AC: median survival time, 41.70 vs. 36.20 months, p = 0.8985; MUC6 median survival time, 49.90 vs. 30.70 months, p = 0.4253).
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Table 3 Univariate analysis of clinicopathologic factors. Variable
Notch3 expression MUC2 expression MUC5A expression MUC6 expression Tumor size Location Growth pattern
Histologic subtype Differentiaton
pT classification Lymph node metastasis Other loop invasion Retroperitoneal seeding Perineural invasion Lympovascular invasion
Characteristics
Median survival (mo)
Positve Negative Positve Negative Present Absent Positve Negative <4.0 cm ≥4.0 cm Proximal (duodenal) Distal (jejunal/ileal) Polypoid Nodular Infiltrative Tubular adenocarcinoma Other types Well Moderately Poorly Undifferentiated pT1-2 pT3-4 Present Absent Present Absent Present Absent Present Absent Present Absent
95% Confidence interval
74.1 36.2 48.5 36.2 41.7 36.2 49.9 30.7 49.9 28.2 74.1 24.6 48.5 36.2 38.5 39.9 20.1 49.9 28.2 30 7.9 – 30.7 22.6 80.8 5.1 39.9 11.1 48.5 26.3 49.9 22.6 74.1
Next, the Notch3+ expression group was examined with multivariate statistical analysis, with other significant prognostic valuables examined by univariate analysis. T stage, tumor location, lymph node metastasis, lymphovascular invasion, perineural invasion, other loop invasion, and retroperitoneal seeding were included as cofactors. As shown in Table 4, together with retroperitoneal seeding and lymph node metastasis, Notch3+ expression was an independent favorable prognostic factor, with marginal statistical significance (p = 0.08, Cox hazard ratio 0.841), for patient survival. Discussion In this study, we demonstrate that Notch3 expression was associated with favorable prognosis in patients with SIACs, a result which was in contrast with the results of previous reports on colorectal cancers, in which Notch signaling was associated with an aggressive tumor prognosis [25,32]. However, it has been known that Notch signal pathway plays diverse and complex roles as not only a tumor promoter but also a suppressor in malignant tumors depending on the cellular context, level of expression, and kinds of organ. In fact, Kang et al. recently reported that the expression of Notch3 was associated with a favorable prognosis with intestinal or glandular differentiation in gastric cancer [26]. Kim et al. also reported that Notch1 signaling
Lower
Upper
– 21.84 2.5 21.35 17.22 16.08 29.19 17.15 33.06 20.22 36.34 15.98 0 – 18.88 22.68, 0
– 50.56 94.5 51.5 66.18 56.12 70.61 44.25 66.74 36.18 111.86 33.22 100.64 – 58.12 57.12 44.19
3.83 10.57 0 – 18.16 15.73 – 0 22.37 0.98 15.59 15.59 14.89 14.32 39.22
52.57 49.43 20.35 – 43.24 29.47 – 19.02 57.43 21.22 81.41 37.01 84.91 30.88 108.98
p-Value
0.05 0.771 0.8985 0.4253 0.3486 0.0169 0.9311
0.2171 0.6619
0.0774 0.0006 0.0801 0.0002 0.1508 0.0042
retained the capability of suppressing the expression of WNT target genes in colorectal cancers and the activation of Notch1 even converted high-grade adenoma into low-grade adenoma in a mouse colon cancer model [13]. They suggested that Notch signaling promotes the onset of intestinal tumors, and after tumor onset, Notch signaling inhibits tumor progression by repression of the WNT target gene through an epigenetic chromatin modification. Although a contrasting result has been reported [25], it is also well known that Notch signaling is strongly expressed in the adenoma of the colon, suggesting the role of Notch signaling as an essential initial event triggering colorectal cancer [33]. These findings suggest that the role of Notch signaling in intestinal tumors is more complex; that is, Notch signaling is a strong promoter in intestinal tumor initiation, and it might also have a suppressive role in established tumors. We also found MUC5AC expression was closely related with the expression of Notch3 (p = 0.02). MUC5AC was expressed diffusely in 8 cases (5.1%) in the cytoplasm of columnar cells or focal goblet cells in non-neoplastic small-intestinal epithelium, but 61 SIAC cases (31.9%) showed MUC5AC overexpression with a mixed apicoluminal and cytoplasmic staining pattern. There are a few studies on the relationship between MUC5AC expression and tumor progression in the colorectum. Kocer B et al. reported that in colorectal cancer, decreased MUC5AC expression can be a prognostic factor indicating the existence of a more aggressive form of colorectal
Table 4 Multivariate analysis of prognosis. Variable
p-Value
Relative risk
95% confidence interval
Notch3 expression Retroperitoneal seeding Metastasis to lymph node
0.08 0.002 0.0002
0.841 3.61 2.667
0.609–1.02 1.590–8.195 1.577–4.510
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of tumor progression and enhancement of tumor initiation. Further studies are required to reveal more detailed mechanisms of the influence of Notch signaling on tumor progression in the small intestine. Acknowledgements
Fig. 2. Patients with Notch3+ expression show significantly better overall survival rates (p = 0.05) than patients with Notch3-expression by Kaplan–Meier survival analysis.
cancer [34]. MUC5AC mRNA and protein were present more frequently in colorectal polyps which were intermediate in size, and in polyps which had villous histology and low or moderate dysplasia [6]. MUC5AC protein expression was higher in hyperplastic polyps, and in polyps with low and high grade dysplasia, and in mucinous carcinoma groups than in normal mucosa and conventional adenocarcinoma groups [35]. On the other hand, Kang JH et al. recently described the possible involvement of Notch signaling in the expression of MUC5AC, a defining characteristic of the goblet cell phenotype which manifests as insults to the respiratory airway epithelial cells [12]. They suggested that Notch signaling not only involves epidermal growth factor induced MUC5AC expression, but that it is also able to induce MUC5AC expression itself [12]. Considering that the role of Notch signaling for tumorigenesis depends on the cellular context and cross-talk with other signaling pathway molecules, MUC5AC expression might partially contribute to the favorable prognosis of patients with Notch-3 expression in SIACs. On the contrary, Ou-Yang HF et al. recently reported that Notch signaling could directly downregulate MUC5AC promoter activity through Hes1-dependent mechanisms in the airway epithelium in asthma. Therefore, more studies are needed to determine the exact molecular mechanisms of how Notch signaling regulates MUC5AC expression [36]. To the best of our knowledge, this is the first description of the prognostic significance of Notch3 expression and its relationship with the expression of MUC proteins in SIACs. The limitations of this study include the retrospective nature of the study design and the fundamental problems of an immunohistochemical study that uses a TMA block, including a subjective scoring system and the intrinsic heterogeneity of the immunoreactivity in the tumor. In summary, the present study revealed that Notch3 was expressed in 52 (29.9%) of the tumors and significantly correlated with the absence of lymphovascular invasion, lower T stage, and the histological subtype of tubular adenocarcinoma, and MUV5AC expression. Furthermore, Notch3 expression was a favorable prognostic factor in patients with SIACs. These findings suggest dual roles of Notch signaling in SICAs, including both the suppression
This research was supported by the Gangneung Asan Hospital Biomedical Research Center Promotion Fund & Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0004807). We thank to members of the Korean small intestinal cancer study groups: Dr. Hee-Kyung Chang, Kosin University College of Medicine, Pusan; Dr. Eun Sun Jung, The Catholic University of Korea College of Medicine, Seoul; Drs. Ghil Suk Yoon and Han-Ik Bae, Kyungpook National University, Dague; Dr. Joon Mee Kim, Inha University College of Medicine, Incheon; Dr. YoungHa Oh, Hanyang University College of Medicine, Seoul; Dr. Soo Jin Jung, Inje University College of Medicine, Busan; Dr. Mi Jin Gu, Fatima Hospital, Daegu; Dr. Jung Yeon Kim, Inje University Sanggye Paik Hospital, Seoul; Dr. Kyu Yun Jang, Chonbuk National University Medical School, Jeonju; Dr. Kye Won Kwon, Bundang Jesaeng General hospital, Seongnam; Dr. Gyeong Hoon Kang, Seoul National University College of Medicine, Seoul; Dr. Jae Bok Park, Catholic University of Daegu, Daegu; Dr. SoonWon Hong, Yonsei University College of Medicine, Seoul; Dr. Sun-Young Jun, Inchoen Saint Mary Hospital, The Catholic University of Korea College of Medicine, Incheon; and Dr. Ji Shin Lee, Chonnam National University Medical School, Gwangju, South Korea. References [1] H.K. Chang, E. Yu, J. Kim, et al., Adenocarcinoma of the small intestine: a multiinstitutional study of 197 surgically resected cases, Hum. Pathol. 41 (2010) 1087–1096. [2] J.C. Byrd, R.S. Bresalier, Mucins and mucin binding proteins in colorectal cancer, Cancer Metastasis Rev. 23 (2004) 77–99. [3] J.A. Gibson, H.P. Hahn, A. Shahsafaei, R.D. Odze, MUC expression in hyperplastic and serrated colonic polyps: lack of specificity of MUC6, Am. J. Surg. Pathol. 35 (2011) 742–749. [4] S.J. Gendler, A.P. Spicer, Epithelial mucin genes, Annu. Rev. Physiol. 57 (1995) 607–634. [5] S.K. Chang, A.F. Dohrman, C.B. Basbaum, et al., Localization of mucin (MUC2 and MUC3) messenger RNA and peptide expression in human normal intestine and colon cancer, Gastroenterology 107 (1994) 28–36. [6] A.E. Bartman, S.J. Sanderson, S.L. Ewing, et al., Aberrant expression of MUC5AC and MUC6 gastric mucin genes in colorectal polyps, Int. J. Cancer 80 (1999) 210–218. [7] G.R. Van den Brink, K.M. Tytgat, R.W. Van der Hulst, et al., H pylori colocalises with MUC5AC in the human stomach, Gut 46 (2000) 601–607. [8] N. Schroder, A. Gossler, Expression of Notch pathway components in fetal and adult mouse small intestine, Gene Expr. Patterns 2 (2002) 247–250. [9] M. Katoh, Notch signaling in gastrointestinal tract (review), Int. J. Oncol. 30 (2007) 247–251. [10] X.Y. Ding, J. Ding, K. Wu, et al., Cross-talk between endothelial cells and tumor via delta-like ligand 4/Notch/PTEN signaling inhibits lung cancer growth, Oncogene 31 (2012) 2899–2906. [11] K.L. VanDussen, A.J. Carulli, T.M. Keeley, et al., Notch signaling modulates proliferation and differentiation of intestinal crypt base columnar stem cells, Development 139 (2012) 488–497. [12] J.H. Kang, E.H. Lee, S.W. Park, I.Y. Chung, MUC5AC expression through bidirectional communication of Notch and epidermal growth factor receptor pathways, J. Immunol. 187 (2011) 222–229. [13] H.A. Kim, B.K. Koo, J.H. Cho, et al., Notch1 counteracts WNT/-catenin signaling through chromatin modification in colorectal cancer, J. Clin. Invest. 122 (2012) 3248–3259. [14] D. Bellavia, S. Checquolo, A.F. Campese, M.P. Felli, A. Gulino, I. Screpanti, Notch3: from subtle structural differences to functional diversity, Oncogene 27 (2008) 5092–5098. [15] M. Lardelli, J. Dahlstrand, U. Lendahl, The novel Notch homologue mouse Notch 3 lacks specific epidermal growth factor-repeats and is expressed in proliferating neuroepithelium, Mech. Dev. 46 (1994) 123–136. [16] F. Jundt, O. Acikgoz, S.H. Kwon, et al., Aberrant expression of Notch1 interferes with the B-lymphoid phenotype of neoplastic B cells in classical Hodgkin lymphoma, Leukemia 22 (2008) 1587–1594.
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