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S100A4 expression is a prognostic indicator in small intestine adenocarcinoma
  1. Jin Roh1,
  2. Spencer Knight2,
  3. Joon-Yong Chung3,
  4. Soo-Heang Eo4,
  5. Michael Goggins2,
  6. Jihoon Kim1,
  7. HyungJun Cho4,
  8. Eunsil Yu1,
  9. Seung-Mo Hong1
  1. 1Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea
  2. 2Johns Hopkins Medical Institutions, Baltimore, Maryland, USA
  3. 3Applied Molecular Pathology Laboratory, National Cancer Institute, Bethesda, Maryland, USA
  4. 4Department of Statistics, Korea University, Seoul, Republic of Korea
  1. Correspondence to Professor Seung-Mo Hong, Department of Pathology, Asan Medical Center, University of Ulsan College of Medicine, 88, Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Republic of Korea; smhong28{at}gmail.com

Abstract

Aims Due to the rarity of small intestine adenocarcinoma (SIAC), estimating the prognosis for patients with surgically resected SIAC is difficult. Overexpression of S100A4 has been linked to worse patient survival in several malignant neoplasms, but its significance in SIAC has not been determined.

Methods S100A4 protein expression was assessed in 197 surgically resected SIAC cases and compared with clinicopathological factors, including patient survival.

Results A progressive increase in S100A4 labelling was observed in normal intestinal epithelium, adenoma and adenocarcinoma (p<0.001), and 50 SIAC cases (26.2%) showed strong S100A4 expression. Patients with SIAC with strong S100A4 expression had a higher pT classification (p=0.05), as well as increased lymph node metastasis (p=0.009) and perineural invasion (p=0.002). Patients with SIAC with strong S100A4 expression had significantly worse survival (median survival, 21 months) than those with weak/no S100A4 expression (42.5 months) by univariable (p=0.04) and multivariable (p=0.01) analyses.

Conclusions S100A4 overexpression is observed in a subset of SIACs, is associated with advanced disease and can be used as a prognostic indicator of poor prognosis in patients with SIAC.

  • SMALL INTESTINE
  • CANCER
  • CARCINOMA

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Introduction

The small intestine is the longest organ of the gastrointestinal (GI) tract. Its average length is approximately 7 m in adults, and it constitutes approximately 75% of the length and 90% of the mucosal surface area of the entire GI tract.1 Despite this, the small intestine is a rare site for primary GI tract cancer. Only 5% of malignant neoplasms of the GI tract occur in the small intestine. Its frequency is about 0.5% in Korea and the USA.2 ,3 Small intestine adenocarcinoma (SIAC) is the second most common subtype of small intestine cancer, following neuroendocrine tumours.4 Due to its rarity and the non-specific signs and symptoms, patients with SIAC usually are diagnosed at an advanced disease stage.5 The prognosis of patients with SIAC is generally poor because of late detection.6 Some recent studies show an increasing incidence of SIAC.7 ,8 Recent technical advances in imaging and endoscopy, such as the use of capsule endoscopy and double ballooned enteroscopy, have enabled the detection of early small intestinal lesions, including SIACs. However, our knowledge of SIAC is still limited compared with our understainding of stomach or colorectal cancers. For example, only a few prognostic markers, including E-cadherin and β-catenin, are known in SIAC.9 The identification of prongostic biomakers for SIAC will provide patients with SIAC with more accurate information about survival.

S100A4 is a member of the calcium-binding S100 protein family, which consists of 20 famliy members, and it is localised to the cytoplasm and the nucleus. S100A4 is involved in calcium-mediated signal transduction by interacting with intracellular target proteins, and plays important roles in cellular motility, invasion, angiogenesis and survival.10 ,11 Increased S100A4 expression is reported to correlate with worse survival in patients with several malignant neoplasms, including colorectal, gallbladder, bladder, oesophageal, breast and non-small cell lung cancers.12–18 However, the prognostic implications of S100A4 protein expression have not been determined in SIAC. The aim of this study was to determine the value of S100A4 expression as a predictor of outcome in patients with SIAC using immunohistochemical staining.

Materilas and methods

Inclusion and exclusion criteria of the cases

This study used the same cohort of SIAC cases as our previous study.6 After approval from institutional review boards, cases were collected from 195 patients with primary SIAC who underwent surgical resection between 1993 and 2008 at 22 South Korean institutions. Carcinomas that originated in the mucosa of the small intestine, from the duodenum to the ileum, were included regardless of tumour extension into the serosa. Carcinoma of the ampulla of Vater and carcinomas in adjacent organs, such as the stomach, pancreas, caecum, or appendix; and malignant epithelial tumours with epicentres in the serosa or subserosa without involvement of the mucosa were excluded from this study.

Tissue microarray construction

Tissue microarrays were constructed from archival formalin-fixed, paraffin-embedded tissue blocks of 197 surgically resected primary SIACs, using a manual tissue microarrayer (Beecher Instruments, Silver Spring, Maryland, USA) as previously described.9 ,19 A representative tumour area was carefully selected for each tumour from a H&E-stained section of the donor block. Four cores (2 mm in diameter) were punched from each tumour, including adenomas and 197 adenocarcinomas, and from non-neoplastic intestinal epithelial tissue and embedded into recipient blocks to construct the tissue microarray.

Immunohistochemistry and scoring

Sections were deparaffinised in xylene and dehydrated using an alcohol gradient. Antigen retrieval was performed in a steam pressure cooker with a preheated, high pH antigen retrieval buffer (DakoCytomation) at 95°C for 20 min. Sections were incubated with a protein blocker (DakoCytomation) for 10 min to minimise non-specific antigen binding and stained using the Labeled Streptavidin Biotin Detection method. A primary antirabbit S100A4 polyclonal antibody (clone A5114, DAKO; 1:200 dilution) was applied manually and the sections were incubated for 30 min at room temperature. Immunostained sections were counterstained in haematoxylin, hydrated in ethanol and washed with xylene, using an automated immunostainer (DakoCytomation). Although some tissues did not immunolabel successfully because the tissue cores were lost during the sectioning or labelling process, immunohistochemical staining was successfully performed on 191 of the 197 SIACs on the tissue microarray. Immunohistochemical labelling of S100A4 protein was scored using a previously described histological scoring system which takes into account the size of the stained area and the intensity of the labelling. To be included for analysis, a tumour had to have sufficient numbers of S100A4-labelled cells to permit quantification (>100 S100A4-positive tumour cells). The labelled area was scored 0 to 4 for <5%, 5–25%, 26–50%, 51–75% or >75% S100A4-positive cells. An intensity scale was categorised from 0 to 3 as follows; 0, corresponding to no labelling of tumour cells; 1, corresponding to weak labelling of tumour cells; 2, corresponding to equivocal labelling of epithelial cells, and 3, corresponding to intense labelling, which was previously described elsewhere.20 A total histological score was calculated by multiplying the area score and the intensity score. Labelling of lymphocytes was used as an internal positive control. Representative images of S100A4 expression in normal intestinal epithelium and in SIAC samples are depicted in figure 1. Immunohistochemical staining was evaluated by two independent pathologists (JR, S-MH). Any discrepancies in interpretation were resolved by simultaneous re-evaluation by two additional pathologists (JK, EY).

Figure 1

Representative images of S100A4 expression in normal intestinal epithelium (A), small intestinal adenoma (B) and adenocarcinoma (C). Normal intesinal epithelium does not stain for S100A4. Some tumour cells in adenoma cases are diffusely weakly positive for S100A4. Cancer cells are diffusely and strongly positive for S100A4.

Statistical analysis

Statistical analyses were performed with SPSS V.17 (IBM SPSS). The mean S100A4 protein expression scores were compared by Student's t-test and the Mann-Whitney U-test. Correlations between S100A4 protein expression and clinicopathological factors were examined using the χ2 and Fisher's exact tests. Overall survival time was analysed by the Kaplan-Meier method and significance was evaluated with the log-rank test. Cox proportional hazards regression models were used to investigate the significance of S100A4 expression as a prognostic factor. A p value of less than 0.05 was regarded as statistically significant.

Results

Patient characteristics

SIAC samples were analysed from 119 men (62.3%) and 72 women (37.7%). The patients’ ages ranged from 23 years to 86 years (mean, 58.9±14.1 years). There were 104 duodenal (54.5%), 57 jejunal (29.8%) and 30 ileal (15.7%) tumours. Information regarding the tumour growth pattern was available in 183 cases; of these, 34 were polypoid (18.6%), 12 were nodular (6.6%)and 137 were infiltrative (74.9%) tumours. By histological type, there were 175 tubular adenocarcinomas (91.6%), eight mucinous carcinomas (4.2%), four signet ring cell carcinomas (2.1%) and four undifferentiated carcinomas (2.1%). Using the pT staging system, tumours were classified as pTis in three cases (1.6%), pT1 in seven (3.7%), pT2 in nine (4.7%), pT3 in 62 (32.5%) and pT4 in 110 (57.6%) cases. Information regarding regional lymph node metastasis was available in 173 out of 191 cases; of these, 89 cases (51.4%) showed lymph node metastasis. Vascular and lymphatic invasion was identified in 50 (26.2%) and 93 (48.7%) cases, resepectively. The median follow-up period was 28.2 months (range, 0.3–158.2).

S100A4 protein expression in normal intestinal epithelium, adenoma and adenocarcinoma

The mean histological scores for S100A4 staining in normal intestinal epithelium, adenoma and SIAC were 1.26±1.44, 2.00±2.77 and 3.38±2.87, respectively. There were significant differences in the histological scores among normal intestinal epithelium, adenoma and SIAC (p<0.001). When compared in a pairwise manner, there was a significant difference in S100A4 expression between normal intestinal epithelium and SIAC (p<0.001). However, there was no difference between normal epithelium and adenoma or between adenoma and SIAC. Strong S100A4 expression was observed in 50 cases (26.2%).

Correlation between S100A4 protein expression and clinicopathological factors

Relationships between S100A4 protein expression and clinicopathological factors were compared after dicotomising S100A4 expression into strong and weak/no expression. A greater percentage of patients with lymph node metastasis had strong S100A4 expression (31/89 cases, 34.8%) than patients who were negative for lymph node metastasis (14/84 cases, 16.6%; p=0.01). SIACs with perineural invasion (23/63 cases, 37.1%) were more likely to have strong S100A4 expression than those without perinueral invasion (27/129 cases, 20.9%; p=0.02). Patients with strong S100A4 expression showed a modest tendency towards deeper invasion (pT1+pT2, 0/16 cases, 0%; pT3, 17/62 cases, 27.4%; pT4, 32/110 cases, 29.1%; p=0.05; table 1). However, there was no significant correlation between S100A4 expression and other clinicopathological factors, including gender, tumour growth pattern, histological subtype, differentiation, pancreas invasion, other loop invasion, retroperitoneal seeding, or vascular and lymphatic invasion.

Table 1

S100A4 expression and association with clinicopathological factors in patients with small intestine adenocarcinoma

Patients survival and S100A4 expression

Patient survival was analysed with respect to S100A4 expression. The median survival time for patients with strong S100A4 expression was significantly worse (median, 21.0 months) than for those with weak/no expression (median, 42.5 months; p=0.04; figure 2).

Figure 2

A Kaplan-Meier survival analysis of small intestine adenocarcinomas was performed according to S100A4 expression status. The median survival time for patients with strong S100A4 expression was significantly worse (median, 21 months) than for those with weak/no expression (median, 42.5 months; log rank test, p=0.04).

By univariable analysis, the following clinicopathological factors were associated with worse patient survival (table 2): histological subtype (p=0.02), tumour location (proximal vs distal; p=0.03), differentiation (p=0.02), pT classificaion (p=0.03), pN classification (p<0.001), other intestinal loop involvement (p=0.03), retroperitoneal seeding (p<0.001), perineural invasion (p=0.009), vascular invasion (p=0.02), lymphatic invasion (p<0.001), sporadic adenoma (p=0.008) and peritumoral dysplasia (p=0.001). Univariable analysis revealed no significant survival differences according to gender, growth pattern, pancreas involvement or marginal status.

Table 2

Univariable analysis of pathological features affecting patient survival with small intestine adenocarcinoma

Multivariable analysis was performed to assess which factors remained independent predictors of survival after adjusting for factors which were found to be significant by univariable analysis. The results are summarised in table 3. Strong S100A4 protein expression (p=0.01) and the absence of sporadic adenoma (p=0.04; HR, 0.21; CI 0.05 to 0.91) were independent prognostic factors for poor outcome in our model. The HR for SIACs with strong S100A4 expression was 1.80 (95% CI 1.13 to 2.88) compared to those with weak/no S100A4 expression.

Table 3

Multivariable analysis of small intestine adenocarcinoma

Discussion

S100 proteins are low molecular weight (10–12 kDa) calcium-binding proteins that generally exist as homodimers or heterodimers.6 To date, over 20 S100 protein family members have been identified in humans.21–23 S100A4, one member of the S100 family, has also been called p9Ka, FSP1, CAPL, calvasculin, pEL98, metastasin (mts1), 18A2 and 42A.24 ,25 S100A4 protein is involved in cellular motility, invasion, angiogenesis and metastais.10 As one of its alternate names, metastasin, suggests, S100A4 is involved in the process of metastasis by increasing cell motility and remodelling of the extracellular matrix.24 Although the exact biological mechanisms through which S100A4 promotes metastasis were not fully understood, Matsuura and colleagues recently demonstrated that calcium-dependent S100A4 binding to Smad3 increased the transcriptional activity of Smad3 and increased the expression of matrix metalloproteinase 9 in breast cancer.26 Therefore, based on their observation, it could be postulated that S100A4 increases cancer cell invasion by inducing the TGF-β pathway.26

S100A4 expression has been reported in association with tumour progression in several carcinomas, such as stomach, pancreas, colorectal and thyroid cancers.27–31 In our study, strong S100A4 expression correlated with lymph node metastasis, perineural invasion and the depth of tumour cell infiltration. Therefore, as with other cancers, S100A4 expression is associated with tumour progression in SIAC.

In the present study, S100A4 expression was associated with worse prognosis in patients with SIAC. Similarly, S100A4 expression is reported to correlate with decreased patient survival in several malignant neoplasms, including colorectal, gallbladder, bladder, oesophageal, breast and non-small cell lung cancer.12–18

The S100 family consists of 20 family members, which have different functions. We previously reported that the loss of S100A14 was associated with lymph node metastasis and advanced clinical stage in SIAC.19 However, loss of S100A14 expression did not correlate with patient survival.19 In the present study, in contrast with S100A14, S100A4 overexpression was associated with increased tumour progression and worse survival. Therefore, unlike S100A14, which seems to function as a tumour suppressor, S100A4 may function as an oncoprotein in carcinogenesis of the small intestine. Currently, only a few biomakers, including E-cadherin and β-catenin, are reported to be prognostic markers in SIAC.9 We add S100A4 protein to the list of prognostic indicators in SIAC.

In summary, we performed an immunohistochemical study of S100A4 with 191 surgically resected SIAC samples. Our key findings are that strong S100A4 expression (1) is observed in a subset of of SIACs, (2) is associated with tumour progression and (3) can be used as a prognostic indicator in patients with SIAC. To the best of our knowledge, this is the first report on the expression of S100A4 in SIAC.

Take home messages

  • Only a few prognostic markers, such as E-cadherin and β-catenin, are currently known in small intestine adenocarcinoma (SIAC).

  • Strong S100A4 expression is observed in a subset of SIACs, and it is associated with tumour progression

  • Strong S100A4 expression can be used as a prognostic indicator in patients with SIAC.

Acknowledgments

This research was funded by a grant (2013-554) from the Asan Institute for Life Sciences, Seoul, Korea.

References

Footnotes

  • Contributors Conception and design: JR and S-MH. Administrative support: SK and J-YC. Collection and assembly of data: JR, JK, MG, EY, S-MH. Data analysis and interpretation: JR, S-HE, HCand S-MH. Manuscript writing: JR, J-YC, EY and S-MH.

  • Funding This research was funded by a grant (2013-554) from the Asan Institute for Life Sciences, Seoul, Korea.

  • Competing interests None.

  • Ethics approval This study was approved by the institutional review board of Asan Medical Center (No. 2012-0360).

  • Provenance and peer review Not commissioned; externally peer reviewed.

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