The expression of versican and its role in pancreatic neuroendocrine tumors

Pancreatology xxx (xxxx) xxx

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The expression of versican and its role in pancreatic neuroendocrine tumors Heli Gao a, 1, Yuejuan Cheng a, 1, Yuanjia Chen b, Fei Luo a, Yajuan Shao a, Zhao Sun a, *, Chunmei Bai a, ** a b

Department of Medical Oncology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, 100730, China Department of Gastroenterology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences, Beijing, 100730, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 May 2019 Received in revised form 13 November 2019 Accepted 19 November 2019 Available online xxx

Background: Pancreatic neuroendocrine tumors (pNET) are rare and heterogeneous. New biomarkers are needed for better predicting the prognosis and for providing individualized treatment. Versican (VCAN) plays an important role in tumorigenesis. Therefore, we plan to investigate the role of VCAN in pNET prognosis. Method: The clinical and pathological data of pNET patients who underwent surgery between 2005 and 2010 were collected and evaluated. Radiologic tumor assessments with contrast computed tomography or magnetic resonance imaging were performed at baseline and follow up. The radiologic response was classified according to the RECIST 1.1 criteria. VCAN expression was assessed by immunohistochemical staining (IHC). Result: Among 155 pNET patients, 112 (72.3%) pNET patients were VCAN positive, and 43 (27.7%) were negative. Positive expression of VCAN in pNET was significantly associated with a longer disease-free survival (DFS) compared with VCAN negative pNET (p ¼ 0.038, HR 0.462, 95% CI 0.218e0.978). Subgroup analysis showed that VCAN positive expression was associated with a longer DFS in the G1 subgroup (p ¼ 0.031, HR 0.124, 95% CI 0.013e1.193), the tumor size>2 cm subgroup (p ¼ 0.047, HR 0.458, 95% CI 0.207e1.012) and the NF-pNET subgroup (p ¼ 0.003, HR 0.274, 95% CI 0.112e0.673). Multivariable analysis showed that VCAN negative expression, G2 and tumor size>2 cm were independent factors of poor prognosis of pNET (p ¼ 0.041, p < 0.001, p ¼ 0.008, respectively). Conclusion: Our data indicate that VCAN positive expression may serve as an independent factor of predicting DFS in pNET; its expression in pNET tissues was correlated with a longer DFS. © 2019 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Keywords: Pancreatic neuroendocrine tumor Versican Prognosis

Introduction Neuroendocrine tumors (NET) are a type of rare tumor that can occur in different organs of the body. According to the National Cancer Institute Surveillance, the incidence of NET has increased dramatically. Gastroenteropancreatic NET (GEP-NET) is the second most common cancer of the digestive system [1], and pancreas NET (pNET) is the most common form of GEP-NET and accounts for 49.8% of total NETs in China. Based on its clinical features and the presence or lack of hormone secretion, pNET can be classified into

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Z. Sun), [email protected] (C. Bai). 1 These authors have contributed equally to this work.

functioning-pNET (F-pNET) and nonfunctioning pNET (NF-pNET). pNET can also be classified into well-differentiated (G1-2) and poorly differentiated (G3). However, pNET is heterogeneous, and even for tumors with the same pathological stage and grade, patient survival time with metastatic pNET still varies widely from 10 months to more than 10 years. Thus, new biomarkers are needed for better prognostication and for providing individualized treatment. Studies have discovered some prognostic biomarkers of pNET, such as MEN1, ATRX/DAXX and the mTOR pathway. MEN1 mutations are related to more favorable outcomes of pNET [2,3], but one study showed that there was no relationship between MEN1 mutations and prognosis in pNET patients [4]. The relationship between ATRX/DAXX expression and the prognosis of pNET is highly controversial. Some studies showed that ATRX/DAXX loss was associated with metastasis and shortened survival time in patients

https://doi.org/10.1016/j.pan.2019.11.009 1424-3903/© 2019 IAP and EPC. Published by Elsevier B.V. All rights reserved.

Please cite this article as: Gao H et al., The expression of versican and its role in pancreatic neuroendocrine tumors, Pancreatology, https:// doi.org/10.1016/j.pan.2019.11.009

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with pNET [5,6], but others showed that DAXX loss was positively correlated with a lower Ki67 index and a higher degree of differentiation [7]. In the mTOR pathway, pNET cases with PTEN/TSC2 deleted often have a higher Ki67 proliferation index and a shorter survival time [8]. However, these molecular markers cannot fully explain the heterogeneity of pNET. Therefore, we still need to find new molecular biological markers for prognostication and developing targeted therapy in pNET. Our previous study of pNET proteomics found that VCAN was specifically expressed in pNET tumor tissues (it was expressed in 5 out of 5 tumors evaluated), but the clinical role of VCAN in NET has not been reported. Therefore, we planned to study the prognostic and clinicopathological role of VCAN for pNET patients. Patients and methods

based on both the proportion of positively stained tumor cells and the intensity of staining. The intensity score was classified as follows: (no staining); ± (light yellow), þ (yellow), þþ (yellow brown) and þþþ (brown). Tumors with positive staining (þ, þþ, þþþ) in >20% of tumor cells were defined as positive. Statistical analysis Statistical analyses were carried out using the SPSS statistical package (19.0; SPSS Inc., Chicago, Illinois, USA). The relationship between VCAN expression and clinical characteristics of the patients were analyzed by Chi square. Kaplan-Meier was used to analyze the disease-free survival (DFS) of the patients. Subgroup analyses were conducted for tumors based on function, grade and tumor size. Cox regression was used to analyze the influence of multiple factors on DFS.

Study design and patient selection Results This is a retrospective cohort study. A total of 155 pNET patients were recruited in this study. Eighty-six patients were from Peking Union Medical College Hospital (PUMCH) databases from 2005 to 2010. Sixty-Nine patient records and tumor tissues were from USA Memorial Sloan Kettering Cancer Center (MSKCC). This research was approved by the Scientific Ethics Committee of PUMCH and MSKCC; written informed consent was obtained from all patients. All patients were R0 resected and pathologically diagnosed with pNET. The patients were list in Supplementary Table 1. Tumor characteristics The clinicopathological characteristics were all retrospectively reviewed from the patients’ medical records. Data included age at diagnosis, sex, date of diagnosis, clinical symptoms, tumor site, metastatic site and number, functionality, type of tissue specimen, TNM stage, Ki67 index/mitotic count. Tumor grade is in accordance with the 2010 World Health Organization (WHO) classification: Grade 1 (G1) tumor with Ki67 index 2%, MI(mitosis/ 10HPF) < 2,and Grade 2 (G2) is Ki67 index 3e20%,MI 2e20, If Ki67 and MI are not consistent, high lever results should be adopted [9], and TNM stage is based on the 2006 European Neuroendocrine Tumor Society ENETs pNET TNM staging [10]. Follow-up ended on 30th May 2017. Radiologic tumor assessments with contrastenhanced computed tomography or magnetic resonance imaging was performed at baseline, every 6 months after surgery for 2 years and then every 12 months for 2e10 years. Radiologic response was classified according to the RECIST 1.1 criteria [11]. Immunohistochemistry (IHC) and assessment IHC staining was performed on 4 mm sections from paraffinembedded specimens. Slides were baked at 65  C for 30 min, then deparaffinized with xylene and rehydrated with ethanol. For antigen retrieval, slides were microwave-treated and boiled in 0.01 M citrate buffer (pH 6.0) for 10 min at 37  C. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 min. The slides were incubated with anti-VCAN antibody (1/100; Abcam, CA) overnight at 4  C in a humidified chamber. The tissue sections were treated with biotinylated anti-rabbit secondary antibody (Zymed), followed by further incubation with streptavidin-horseradish peroxidase complex (Zymed). The antigen-antibody complexes were visualized using 3, 30 -diaminobenzidine (DAB) and counterstained with 10% Mayer’s hematoxylin, dehydrated, and mounted in Crystal Mount. The degree of immunostaining of the formalin-fixed, paraffin-embedded sections was reviewed and scored independently by two observers,

Clinicopathological features of the pNET patients A total of 155 pNET patients were included in this study. Their clinicopathological features are summarized in Table 1. There were 67 men and 86 women, with a median age of 52 years old. Among them, 81 patients were diagnosed with NF-pNET and 74 patients were diagnosed with F-pNET (67 insulinoma, 4 gastrinoma, 1 vipoma and 2 glucagonoma); 86 of the tumors were G1, 69 G2. According to ENETs staging, 47 patients were stage I, 84 patients stage II, and 24 patients stage III. The end of follow-up was December 2017. The median follow-up time was 67 months. During the follow-up period, 28 patients developed a recurrence or metastasis, 9 patients died due to disease progression, and 12 died due to reasons unrelated to this disease. Relationship between VCAN expression and pNET prognosis We analyzed the relationship between VCAN expression and DFS in patients with pNET. The IHC results showed 112 cases (72.3%) were positive for VCAN expression and 43 cases (27.7%) were negative (Fig. 1). There were 16 patients who developed a recurrence or died among the 112 VCAN positive expression

Table 1 Expression of VCAN and the clinicopathological factors in pNET tumor tissue.

Gender Female Male Age <65 65 Function Function Non-Function Size <2 cm 2 cm Grade 1 2 ENETs 1 2 3 Location Head/Neck Body/Tail

VCAN Negative

VCAN Positive

28 15

58 54

35 8

86 26

24 19

50 62

17 26

48 64

23 20

63 49

10 23 10

37 61 14

19 23

4368

P value P ¼ 0.135

P ¼ 0.535

P ¼ 0.213

P ¼ 0.707

P ¼ 0.757

P ¼ 0.243

P ¼ 0.465

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Fig. 1. Immunohistochemistry assay of VCAN positive expression in pNET tissues. IHC staining of positive VCAN expression levels in pNET tissues (A) negative 10  , (B) positive 10  and (C) positive 40  , (D) relationship between VCAN expression and the patient DFS.

patients compared with 12 patients among the 43 VCAN negative expression patients. VCAN-positive expression patients had a longer DFS compared with VCAN-negative expression patients (mDFS: not reached in both groups, p ¼ 0.038, HR 0.462, 95% CI 0.218e0.978, Fig. 1D). The positive expression of VCAN in pNET patients was not related to sex, age, functionality, grade or tumor size (Table 1).

pNET patients. The detailed results are shown in Table 2 and Fig. 2. Subgroup analysis

Other potential factors affecting recurrence were evaluated using univariate analysis, and the results are summarized in Table 1. Several variables were associated with a positive prognosis (Fig. 2). Major factors for recurrence risk included tumor grade (G2 vs. G1, p < 0.001, HR 9.035, 95% CI 3.123e26.138), tumor size (>2 cm vs. <2 cm, p < 0.001, HR 7.458, 95% CI 2.249e24.732), lymph node metastasis (N1 vs. N0, p < 0.001, HR 8.965, 95% CI 4.259e18.870) and stage (stage III vs. stage II vs. stage I, p ¼ 0.001, HR 7.506, 95% CI 3.832e14.702), and patients with insulinomas had a better prognosis than those with NF-pNET and non-insulinoma F-pNET (HR 3.238, 95% CI 1.743e6.016, p < 0.001).

We then analyzed VCAN expression in subgroups of pNET patients. The results showed that in F-pNET, VCAN expression was not correlated with DFS; however, in NF-pNET, VCAN-positive patients had a significantly longer DFS than VCAN-negative patients (mDFS: not reached vs. 71 months(18.95e123.05 m), p ¼ 0.003, HR 0.274, 95% CI 0.112e0.673, Fig. 3B). In the G1 subgroup, patients with VCAN-positive expression had a longer DFS than VCAN-negative expression patients (p ¼ 0.031, HR 0.124, 95% CI 0.013e1.193, Fig. 3C). In the G2 subgroup, patients with VCAN-positive expression had a longer DFS than VCAN-negative expression patients, but this result is not statistically significant (mDFS: not reached vs. 71 months(32.764e109.236 m), Fig. 3D, p ¼ 0.104). In the tumor size2 cm subgroup, VACN expression had no relationship with DFS (Fig. 3E) but in the tumor size >2 cm subgroup, patients with VCAN-positive expression had a significantly longer DFS than VCAN-negative expression patients (mDFS: not reached vs. 71 months, p ¼ 0.047, HR 0.458, 95% CI 0.207e1.012, Fig. 3F).

Multivariable analysis of the prognosis of the pNET patients

Discussion

Multivariable analysis was used to analyze the relationship between clinicopathological factors and prognosis in patients with pNET. The results showed that tumor size (p ¼ 0.008, HR ¼ 5.599, 95% CI 1.566e20.011), tumor grade (p < 0.001, HR ¼ 8.144, 95% CI 2.758e24.045) and VCAN expression (p ¼ 0.041, HR ¼ 0.443, 95% CI 0.203e0.968) were independent risk factors for DFS prognosis in

VCAN is a large molecular weight chondroitin sulfate proteoglycan that forms part of the extracellular matrix (extracellular matrix, ECM). Previous studies showed that VCAN expression was increased in many malignancies, including ovarian cancer, nonsmall cell lung cancer, and breast cancer [12,13]. VCAN expression is associated with a poor prognosis in leukemia, oral squamous cell

Other clinicopathological features related to pNET prognosis

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Fig. 2. Survival analysis of the relationship between clinicopathological factors and mDFS of pNET patients. (A) Tumor Grade, (B) Tumor size, (C) Tumor functionality and (D) ENETS Stage.

Table 2 Cox analysis.

VCAN Garde Age Gender Size Type

B

SE

Wald

df

P value

Exp(B)

95%CI of Exp(B)

0.814 2.097 0.165 0.167 1.723 0.148

0.399 0.552 0.544 0.410 0.650 0.462

4.164 14.417 0.092 0.1165 7.026 0.103

1 1 1 1 1 1

0.041 0.000 0.762 0.684 0.008 0.748

0.443 8.144 0.848 0.847 5.599 1.160

0.203e0.968 2.758e24.045 0.292e2.464 0.379e1.890 1.566e20.011 0.469e2.870

carcinoma and cervical cancer [14e16], but in colon cancer and gastric cancer, VCAN expression is a positive prognostic biomarker [17,18]. Although there are studies that have analyzed VCAN mechanisms in tumors, the role of VCAN in NET, especially in pNET, is still unclear. Our previous study of pNET proteomics found that the VCAN expression level in pNET tissue is higher than in normal pancreatic tissue. Thus, we measured VCAN expression in 155 pNET tumors.

The results showed that patients with positive VCAN expression had a significantly longer DFS than patients with negative VCAN expression. In multivariable analysis, VCAN expression was also an independent risk factor for pNET DFS. As the OS events were limited, we did not analyze the relationship between VCAN expression and OS. Other independent prognostic factors of pNET found in our study include tumor stage and grade, which is a finding similar to that in other studies. pNET is a heterogeneous tumor, and patients with insulinoma had a longer DFS than those with NF-pNET and other F-pNETs, and this may be due to the early onset of symptoms of insulinoma, which can lead to earlier diagnosis and treatment. The number of other functional pNETs in our study was limited, and gastrinoma, which usually has a poor prognosis, was the main type among the non-insulinoma F-pNET, [19]. We then performed a subgroup analysis. In the poor prognosis subgroup, such as G2, tumor size> 2 cm and NF-pNET, patients with VCAN positive expression had a significantly longer DFS than patients with negative VCAN expression, indicating that VCAN can be used as a

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Fig. 3. The relationship of VCAN expression and DFS in the subgroups. (A) In F-pNET subgroup; (B) In NF-pNET subgroup; (C) In G1 subgroup; (D) In G2 subgroup; (E) In tumor size<2 cm subgroup. (F) In tumor size>2 cm subgroup.

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prognostic factor in certain pNET subgroups. The role of VCAN in pNET is not clear yet. However, the possible mechanisms of VCAN in tumors are regulating cell proliferation, adhesion, migration and invasion; regulating angiogenesis; and regulating the epithelial-mesenchymal transition [13]. Upregulated VCAN promoted invasion of ovarian cancer by activating the NF-kB signaling pathway and by upregulating expression of CD44, matrix metalloproteinase-9 (MMP-9), and the hyaluronan-mediated motility receptor. VCAN is also a key upregulated target gene in cancer-associated fibroblasts (CAF), where its expression is regulated through the transforming growth factor-b (TGF-b) receptor [20]. TGF-b can upregulate VCAN expression in other tumors, such as gliomas, osteosarcomas, and fibrosarcomas [21]. In addition, VCAN expression in tumors is correlated with robust CD8þ T cell infiltration [22]. Depletion of VCAN inhibits breast cancer cell migration induced by Snail or PAPSS2 [23]. In conclusion, this study confirmed positive VCAN expression was associated with a longer DFS of patients with pNET, but the role of VCAN in OS of patients with pNET and the mechanism of action of VCAN in NET needs further study. Funding This work was supported by grants from the National Natural Science Foundation of China (No. 81472785; No. 61435001), CAMS Innovation Fund for Medical Sciences (No. 2017-I2M-1-001, No. 2016-I2M-1-001). Declaration of competing interest The authors declare no conflict of interest. Acknowledgments We thank MSKCC for donating the pNET tissue and clinicopathological data to Professor Chen Yuanjia. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.pan.2019.11.009. References [1] Yao JC, Hassan M, Phan A, Dagohoy C, Leary C, Mares JE, Abdalla EK, Fleming JB, Vauthey JN, Rashid A, Evans DB. One hundred years after "carcinoid": epidemiology of and prognostic factors for neuroendocrine tumors in 35,825 cases in the United States. J Clin Oncol 2008;26:3063e72. [2] Corbo V, Dalai I, Scardoni M, Barbi S, Beghelli S, Bersani S, Albarello L, Doglioni C, Schott C, Capelli P, Chilosi M, Boninsegna L, Becker KF, Falconi M, Scarpa A. MEN1 in pancreatic endocrine tumors: analysis of gene and protein status in 169 sporadic neoplasms reveals alterations in the vast majority of cases. Endocr Relat Cancer 2010;17:771e83. [3] Jiao Y, Shi C, Edil BH, de Wilde RF, Klimstra DS, Maitra A, Schulick RD, Tang LH, Wolfgang CL, Choti MA, Velculescu VE, Diaz LJ, Vogelstein B, Kinzler KW, Hruban RH, Papadopoulos N. DAXX/ATRX, MEN1, and mTOR pathway genes are frequently altered in pancreatic neuroendocrine tumors. Science 2011;331:1199e203.

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Please cite this article as: Gao H et al., The expression of versican and its role in pancreatic neuroendocrine tumors, Pancreatology, https:// doi.org/10.1016/j.pan.2019.11.009