Author Affiliations: Department of Pancreatic Surgery (Yu XZ, Guo ZY and Jin C) and Department of Pathology (Ouyang Q), Huashan Hospital; Institute of Pancreatic Disease (Di Y, Yang F and Fu DL); Fudan University, Shanghai 200040, China

Corresponding Author: Chen Jin, MD, PhD, Department of Pancreatic Surgery, Huashan Hospital, Fudan University, 12# Middle Urumqi Road, Shanghai 200040, China (Email: galleyking@hotmail.com)

 

The abstract of this article was presented as a poster at the Annual Meeting of the American Society of Clinical Oncology (ASCO) 2015 and at the Joint Meeting of Pancreatic Cancer Committee of Chinese Anti-Cancer Association & International Association of Pancreatology 2015 (PCCA&IAP 2015).

 

© 2017, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(16)60168-6

Published online January 16, 2017.

 

 

Contributors: YXZ and DY proposed the study. YXZ, GZY and DY carried out the research. YXZ drafted the manuscript. DY, YF and OQ collected and analyzed the data. FDL and JC kindly offered valuable suggestions about the experiment design. YXZ and GZY contributed equally to this article. JC is the guarantor.

Funding: This study was supported by grants from the National Natural Science Foundation of China (81201896 and 81071884) and the Research Fund for the Doctoral Program of Higher Education of China (20130071110052).

Ethical approval: All specimens were collected in accordance with informed consents of patients, and all procedures complied with the protocol approved by the review board committee of Fudan University.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: Previous researches in pancreatic cancer demonstrated a negative correlation between secreted protein acidic and rich in cysteine (SPARC) expression in primary tumor and survival, but not for SPARC expression in lymph node. In the present study, we aimed to evaluate the SPARC expression in various types of tissues and its impact on patients’ prognosis.

 

METHODS: The expression of SPARC was examined by immunohistochemistry in resected pancreatic cancer specimens. Kaplan-Meier analyses and Cox proportional hazards regression were applied to assess the mortality risk.

 

RESULTS: A total of 222 tissue samples from 73 patients were collected to evaluate the SPARC expression, which included 73 paired primary tumor and adjacent normal tissues, 38 paired metastatic and normal lymph nodes. The proportion of positive SPARC expression in metastatic lymph node was high (32/38), whereas in normal lymph node it was negative (0/38). Positive SPARC expression in primary tumor cells was associated with a significantly decreased overall survival (P=0.007) and disease-free survival (P=0.003), whereas in other types of tissues it did not show a predictive role for prognosis. Univariate and multivariate analyses both confirmed this significance.

 

CONCLUSION: SPARC can serve a dual function role as both predictor for prognosis and potentially biomarker for lymph node metastasis in resected pancreatic cancer patients.

 

(Hepatobiliary Pancreat Dis Int 2017;16:104-109)

 

KEY WORDS: pancreatic cancer; SPARC expression; immunohistochemistry; prognostic marker; lymph node metastasis

Pancreatic cancer is the fourth most common cause of cancer death in the Western world.^[1] Despite the impressive progress in basic and clinical research, treating pancreatic cancer is still a therapeutic challenge with dismal 5-year survival rates less than 5%.^{[2, 3]} In fact, the difficult surgical approach, resistance to conventional therapies, and the subsequent low survival rate can all contribute to the high-frequency occurrence of lymph node metastasis.^[4-6] Thus, finding an effective biomarker for the lymph node metastasis is critically important.^[7]

 

Secreted protein acidic and rich in cysteine (SPARC), also known as the osteonectin and basement membrane-40 (BM-40) protein, is a member of the matricellular family of proteins.^{[8, 9]} SPARC was first identified in bone and later in platelets as well as tumor tissues,^[2] which plays a pivotal role in cell proliferation,^[10] angiogenesis^[11] and cell migration.^[12] SPARC has been reported to be either up-regulated or down-regulated depending on cancer types,^[13] suggesting its complex role in the tumorigenesis. In addition, a hypothesis is proposed that SPARC may act as an albumin “sticker”, which facilitates the delivery of nab-paclitaxel towards pancreatic cancer cells.^[14-16] Recently, SPARC overexpression has been described in colorectal cancer,^[17] gastric cancer^[18] and pancreatic cancer,^[19] and has been associated with poor prognosis.^[20-22] Normally, increased SPARC expression has often been found in the stromal area rather than tumor cells, especially in pancreatic cancer.^[23] However, no data are available in regards to the SPARC expression in metastatic lymph node and its correlation with survival benefit.

 

To the best of our knowledge, this study firstly reported data on SPARC expression in lymph node (normal and metastatic) as well as primary tumor of pancreatic cancer patients, and its correlation with the survival benefit. We aimed to explore the dual function role of SPARC as both prognostic predictor and biomarker for the lymph node metastasis in pancreatic cancer patients.

 

 

We retrospectively reviewed 73 patients who underwent radical pancreatic cancer surgery from January 2007 to December 2013 in our hospital. All patients were confirmed to have pancreatic ductal adenocarcinoma by pathologists. Among the 73 patients, 50 were pancreatic head carcinoma, and 23 were pancreatic body or tail carcinoma. A total of 222 tissue samples from these patients were eligible to evaluate the SPARC expression, which included 73 paired primary tumor and matched adjacent normal tissues, 38 paired metastatic and matched normal lymph nodes. All of the clinical and pathologic information was acquired from a regular updated clinical database. The methods for follow-up included, but were not limited to, centers for disease control, outpatient, telephone, and mail. The study was approved by the review board committee of Fudan University.

 

Formalin-fixed, paraffin-embedded blocks of tissues from each patient were analyzed with immunohistochemistry, which was performed according to the standard protocol of previous report.^[24] Briefly, resected tissues were sliced into 4-5 µm thickness, de-paraffinized, and bathed in citrate buffer at 95 �� for 40 minutes for antigen retrieval. The slices were then incubated at 4 �� for 13 hours with a monoclonal SPARC antibody (Abcam plc, Cambridge, UK), and stained with an avidin-biotin system (Shanghai High-tech Inc., Shanghai, China). All of the nuclei were counterstained with hematoxylin.

 

SPARC staining analyses were evaluated by two observers (Di Y and Yang F) blinded to the clinical data and treatment arm. Any discrepancy was resolved by a third observer (Ouyang Q). The slices were evaluated for the distribution of staining in the tissues (cytoplasm or stroma). Five microscopic fields from each sample were randomly chosen. The positive cell number and total cell number in every field were counted. The percentage of positive cell=positive cells/total cells. SPARC immunolabeling was categorized as negative when the intensity was absent to weak (+) and if the extent was less than 10%; immunolabeling was positive if the intensity was moderate (++) to strong (+++), and the extent was ≥10%. SPARC was categorized as positive or negative for the primary tumor site, adjacent normal tissue and lymph node: Tumor-/-Stroma, Tumor+/-Stroma, Tumor-/+Stroma, Tumor+/+Stroma.

 

Overall survival (OS) was defined as the time period from surgery to death by any cause. Disease-free survival (DFS) was defined as the time from surgery to the development of either local recurrence or distant metastasis. The Pearson’s Chi-square test or Fisher’s exact probability test was used to compare clinicopathological characteristics of patients with positive and negative SPARC expression. Kaplan-Meier analysis and Cox proportional hazards regression modeling were used to assess the mortality risk associated with the presence or absence of tumor SPARC and stromal SPARC status. All of the statistical analyses were performed with SPSS software (version 22.0; IBM Inc., NY, USA). A P value of <0.05 was considered statistically significant.

 

 

A total of 73 pancreatic cancer patients were analyzed in the pool, with a median follow-up time of 30 months (the cut-off date for follow-up was December, 30th 2014). We collected 73 primary tumor site tissues, 73 adjacent normal tissues, 38 metastatic lymph nodes and 38 matched normal lymph nodes.

 

The SPARC expression was observed both in the cytoplasm of tumor cells and cytoplasm of fibroblasts in the stromal area (Fig. 1). From the results of the immunohistochemistry we found that the SPARC expression was strongly overexpressed in cancer cells and stromal fibroblasts immediately adjacent to cancer cells, suggesting its role in the tumor-stroma interaction. However, SPARC expression was weak or absent in normal pancreatic ductal cells. Among the 73 tissues from the primary tumor site: twenty-one samples (28.8%) were classified as Tumor-/+Stroma, and the other 52 samples (71.2%) were classified as Tumor+/+Stroma; among the 73 tissues from the adjacent normal tissues: fifteen samples (20.5%) were classified as Stroma+, the other 58 samples (79.5%) were classified as Stroma-; whereas in 38 tissues from the metastatic lymph node: eight samples (21%) were classified as Tumor-/+Stroma, six samples (15.8%) were classified as Tumor-/-Stroma, the other 24 samples (63.2%) were classified as Tumor+/+Stroma; however, in all tissues from the normal lymph node, there were none SPARC expression. Refer to previous reports,^[25-27] we examined SPARC expression in the primary tumor site and its association with major clinicopathological parameters. As shown in Table 1, baseline data and risk factors such as age, gender, tumor size, nodal status, resection margin were comparable between the Tumor-/+Stroma and Tumor+/+Stroma groups.

 

We performed survival analyses in dependence of SPARC expression within all tissue groups. Fig. 2 indicated that negative SPARC expression in tumor cell was associated with a significantly increased OS (HR=0.44; 95% CI: 0.24-0.82; P=0.007) and DFS (HR=0.38; 95% CI: 0.20-0.71; P=0.003). However, there seemed no connection between SPARC expression and survival benefits in the metastatic lymph node group (Fig. 3). It was also the same situation in adjacent normal tissue group.

 

Univariate analysis revealed that the following parameters were associated with increased mortality: positive SPARC expression in the primary tumor (HR=2.28; 95% CI: 1.22-4.25; P=0.007); metastatic lymph node (HR=2.09; 95% CI: 1.22-3.58; P=0.005); advanced tumor grade (HR=2.23; 95% CI: 1.23-4.04; P=0.008); positive resection margin (HR=1.80; 95% CI: 1.05-3.08; P=0.026); without adjuvant chemotherapy (HR=2.96; 95% CI: 1.56-5.61; P=0.001). In a further step of multivariate analysis, only SPARC expression in the primary tumor (HR=2.37; 95% CI: 1.20-4.67; P=0.013) and without adjuvant chemotherapy (HR=3.41; 95% CI: 1.62-7.18; P=0.001) retained their negative impact on survival (Table 2).

 

 

SPARC positive or overexpression has often been related to bad prognosis in various malignant tumors,^[28-31] several studies in vivo and in vitro also confirmed this.^{[32, 33]} However, the exact mechanism of action remains unclear.^{[34, 35]}

 

This study shows that positive SPARC expression in the primary tumor cell is associated with a significantly decreased OS and DFS, which is generally in accordance with Infante et al’s report.^[25] In their study, pancreatic cancer patients with positive SPARC expression in primary tumor had a significant worse prognosis than those with negative SPARC expression (P<0.001). Notably, this difference was restricted to SPARC expression in the stromal area, they did not find a correlation between tumor cell SPARC expression and survival (P=0.13). In another report by Sinn et al,^[36] both positive stromal and cytoplasmic SPARC expression was associated with worse DFS and OS. In our study, however, SPARC staining within stromal area was observed in all samples from the primary tumor site; patients with Tumor-/+Stroma had a significant longer median survival compared with those with Tumor+/+Stroma. For other type of tissues, including adjacent normal tissues, metastatic lymph nodes, and normal lymph nodes, we did not find a correlation between SPARC expression and survival.

 

Univariate analysis indicated that positive SPARC expression in the primary tumor, metastatic lymph node, advanced tumor grade, positive resection margin, without adjuvant chemotherapy were all risk factors of overall survival. Multivariate regression analysis showed that positive SPARC expression in the primary tumor was still an independent prognostic factor (HR=2.37; 95% CI: 1.20-4.67; P=0.013) after adjustment for metastatic lymph node, advanced tumor grade, without adjuvant chemotherapy and positive resection margin.

 

A highlight of this study is that SPARC can potentially serve as a biomarker for the lymph node metastasis. SPARC expression in the metastatic lymph node is higher than that in the matched normal lymph node. Comparing with CA19-9, a generally accepted biomarker for early diagnosis and efficacy evaluation, with a sensitivity of 78.6% and specificity of 87.3%,^[37] SPARC in our study yield a sensitivity of 84.2% (32/38) and specificity of 100% (38/38). Nevertheless, this result should be treated with caution due to our limited sample size and a retrospective feature. Further validation is needed in a larger cohort study.

 

For resectable pancreatic cancer patients, chemotherapy always plays a pivotal role. Whether chemotherapy will have an impact on the relationship between SPARC and survival remains controversial. Sinn and coworkers^[36] found that the negative impact of SPARC on survival was only restricted to patients who were treated with gemcitabine. Infante et al^[25] demonstrated that chemotherapy did not affect the correlation between SPARC and survival.

 

Our study has several limitations. First one is a retrospective feature with a relatively small size. However, all of the patients and samples were recruited on a consecutive time basis to maximally avoid selection bias. Second, only 38 metastatic lymph nodes were acquired for analysis which may cause type II error. Third, we only used immunohistochemical staining which is not sensitive for protein detection, other methods such as Western blotting and ELISA may improve the sensitivity. Another limitation is that, according to Von Hoff et al,^[14] in patients with pancreatic cancer who treated with nab-paclitaxel, SPARC improves chemotherapy response. None of our patient received nab-paclitaxel treatment because nab-paclitaxel is recommended for advanced pancreatic cancer rather than resectable cancer; nab-paclitaxel is expensive and there is no insurance coverage in China now.

 

In summary, positive SPARC expression predicts poor prognosis, additionally, SPARC has the potential to serve as a biomarker for lymph node metastasis in resected pancreatic cancer patients. Future randomized controlled trials are needed to validate our results.

 

 

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