B cells in tertiary lymphoid structures are associated with favorable prognosis in gastric cancer

j o u r n a l o f s u r g i c a l r e s e a r c h  j u l y 2 0 1 7 ( 2 1 5 ) 7 4 e8 2

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B cells in tertiary lymphoid structures are associated with favorable prognosis in gastric cancer Chie Sakimura, MD,a Hiroaki Tanaka, PhD,a,* Takahiro Okuno, MD,b Soichiro Hiramatsu, MD,a Kazuya Muguruma, PhD,a Kosei Hirakawa, PhD,a Hideki Wanibuchi, PhD,b and Masaichi Ohira, PhDa a b

Department of Surgical Oncology, Osaka City University Graduate School of Medicine, Osaka, Osaka, Japan Department of Molecular Pathology, Osaka City University Graduate School of Medicine, Osaka, Osaka, Japan

article info

abstract

Article history:

Background: The role of tumor-infiltrating B cells in the tumor microenvironment is still

Received 1 September 2016

unclear. Recent studies have reported that B cells and tertiary lymphoid structures (TLSs)

Received in revised form

that contain B cell follicles correlate with the favorable prognosis of cancer patients. The

10 March 2017

aim of this study was to investigate the association between tumor-infiltrating B cells and

Accepted 24 March 2017

clinicopathological features in gastric cancer.

Available online 1 April 2017

Methods: Tumor blocks were obtained from 226 patients with stage Ib to stage IV gastric cancer. The density of CD20þ B cells within the tumor and in the invasive margin area was

Keywords:

assessed using immunohistochemistry. We also evaluated CD3þ T cells, CD21þ follicular

Gastric cancer

dendritic cells, Bcl6þ germinal center B cells, and PNAdþ high endothelial venules to show

Tertiary lymphoid structure

the presence of TLSs.

B cell

Results: Tumor-infiltrating B cells were mostly organized as clusters that were surrounded by CD3þ T cells. The B cell area contained follicular dendritic cells and some clusters contained Bcl6þ B cells. High endothelial venules were present around follicles. We identified these follicles as TLSs. A high number of CD20þ B cells were associated with significantly better overall survival, and multivariate analysis also showed that CD20 high was one of the independent predictors of prognosis. In addition, there was a significant correlation between CD20þ B cell and CD8þ T cell infiltration. Conclusions: B cells mostly infiltrated tumors as TLSs and were associated with better prognosis in patients with gastric cancer. ª 2017 Elsevier Inc. All rights reserved.

Introduction The host immune system is one of the key players in host antitumor functions. High numbers of tumor-infiltrating T lymphocytes have been shown to be a predictor of favorable clinical outcome in several solid cancers.1 However, the role of

B cells in the tumor microenvironment is still unclear and a matter of debate. B cells are the main effector cells of humoral immunity and ultimately differentiate into plasma cells secreting antibodies. In addition, B cells serve as antigenpresenting cells of cellular immunity. Recent studies have reported a correlation between the densities of B cells and the

* Corresponding author. Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka, Osaka 545-8585, Japan. Tel.: þ81 6 6645 3838; fax: þ81 6 6646 6450. E-mail address: [email protected] (H. Tanaka). 0022-4804/$ e see front matter ª 2017 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2017.03.033

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sakimura et al  b cells in gastric cancer

favorable clinical outcome of patients with cancers such as breast cancer, lung cancer, melanoma, and ovarian cancer.2-5 Moreover, some studies have considered that the organization of tumor-infiltrating B cells, in addition to the cell density, is a criterion for a favorable outcome. Tertiary lymphoid structures (TLSs) are defined as transient ectopic lymphoid organizations that can develop in nonlymphoid tissues at the site of chronic inflammation and that are anatomically and functionally similar to secondary lymphoid organs.6,7 Similar to lymphoid follicles, TLSs are comprised of B cell aggregates in a meshwork of follicular dendritic cells (FDCs), and the B cells are surrounded by T cells, as well as by specialized blood vessels that are called high endothelial venules (HEVs). The presence of these structures in tumors has also been reported to be associated with prolonged cancer patient survival.8-10 Our hypothesis was that these B cells might undergo terminal differentiation into effector cells such as antibodyproducing plasma cells at the tumor site and contribute to antitumor immunity. Although the favorable role of T cell populations in gastric cancer has been established, a role for B cells has never been reported.11 The aim of this study was to investigate the density and the organization of tumorinfiltrating B cells and to identify the association between B cells and various clinicopathological factors and the prognostic significance of gastric cancer.

Table 1 e Correlation of CD20D B cells with clinicopathological factors of patients with gastric cancer. Characteristics

No. of patients

CD20 low

CD20 high

226

121

105

<60

44

16

28

60

182

105

77

162

90

72

64

31

33

T1

19

12

7

T2

55

32

33

T3

54

24

30

T4

98

53

45

All cases

P value

Age (y)

0.011

Sex Male Female

0.334

pT category

0.392

pN category N0

76

43

33

169

78

72

Ib

45

28

17

II

68

34

34

III

78

38

40

IV

35

21

14

94

50

44

129

69

60

3

2

1

N1-N3

0.879

pStage

0.385

Histological type

Materials and methods Patients and samples Formalin-fixed, paraffin-embedded tumor blocks with representative tumor areas were collected from 226 consecutive patients with gastric cancer who had undergone initial surgical resection between 2007 and 2010 at Osaka City University Hospital, Japan. Patients who received preoperative chemotherapy and radiotherapy were excluded. TLSs have been described in chronic inflammatory diseases including in Helicobacter pylorieinduced gastritis.12,13 In some specimens, we found B cell aggregates in the mucosa in a nontumor area, and they were thought to be TLSs induced by H pylori. Since it was difficult to distinguish TLSs induced by H pylori from TLSs induced by cancer in the mucosa of intratumoral and peritumoral areas and since some of the patients with stage IA had undergone endoscopic submucosal dissection before surgery, which caused inflammation, we excluded stage IA in the present study. Dissected primary tumors and lymph nodes were stained with hematoxylin and eosin (HE), and pathological stage was determined according to the seventh edition of the Union for International Cancer Control TNM classification. Postoperative follow-up assessment was performed every 3 mo for the first 2 y, every 6 mo during the third to fourth years, and annually thereafter. The last day of followup was April 1, 2015, and the median follow-up period after surgery was 48.7 (0.6-92.2) mo. The clinicopathological characteristics of the patients are summarized in Table 1. This study was approved by the Osaka City University Ethics Committee. Informed consent was obtained from all patients.

tub1, tub2, pap por, sig, muc Others

0.896

Lymphatic invasion ly

42

17

25

lyþ

184

104

80

v

165

38

127

vþ

61

18

43

Yes

154

79

75

No

72

42

30

Positive

129

65

64

Negative

97

47

50

0.319

Venous invasion

0.181

Chemotherapy

0.322

Intratumor H pyroli

0.977

Immunohistochemistry Immunohistochemistry was carried out on 4-mm-thick sections from paraffin-embedded tumor blocks of patients with gastric cancer. A block with representative material, based on HE staining, was chosen from each patient without specific evaluation of the inflammatory infiltrate. After incubation at 60 C for 10 min, samples were deparaffinized in xylene and rehydrated with a graded series of ethanol. Endogenous peroxidase activity was blocked with absolute methanol

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containing 3% hydrogen peroxidase. Antigen was retrieved by microwave treatment. Nonspecific binding was blocked using a nonspecific staining blocking reagent (Dako, Denmark). The sections were then reacted with primary antibody at 4 C overnight. Sections were incubated with secondary antibody, and the signal was visualized using 3-30 -diaminobenzidine. For the detection of PNAdþ HEV, sections were incubated with HRP-labeled goat anti-rat light chain secondary antibody (#AP202P, Millipore, Temecula, CA) for 30 min prior to reaction with 3-30 -diaminobenzidine. Sections were counter stained with hematoxylin before mounting. All reactions were performed using appropriate positive and negative controls. Antibodies used for immunohistochemical analyses were as follows; mouse anti-CD3 (clone F7.2.38, 1:50, high pH retrieval), mouse anti CD4 (clone 4B12, 1:40, high pH), mouse anti-CD8 (clone F7.2.38, 1:50, high pH retrieval), mouse antiCD20 (clone IR604, prediluted, low pH), mouse anti-CD21 (clone 413771, prediluted, low pH), mouse anti-Bcl6 (clone M7211, 1:10, high pH), rat anti-PNAd (clone 120801, 1:25, high pH), mouse anti-CD24 (clone 8.B.76, 1:25, high pH), and rabbit anti-Helicobacter pylori (clone CH-20426, 1:50, high pH). AntiCD21 and anti-PNAd were obtained from Nichirei (Tokyo, Japan) and Biolegend (San Diego, CA). Anti-CD24 was from Abcam. Other antibodies were obtained from Dako, Denmark.

Evaluation of immunohistochemistry Tumor slides stained with anti-CD20 antibody were scanned at low magnification to select three fields (9.1 mm2; original magnification 20  ; 1600  1200 resolution) with the greatest number of intratumoral and peritumoral CD20 positive cells. The microscopic images were imported from the digital photo filing system DP-73 (Olympus, Tokyo, Japan). We measured the area (mm2) of CD20 positive cells and calculated the percentage area (%) of each field that was covered by the CD20 positive area with Image J software (NIH, Bethesda, MD). CD20 density was determined as the mean value of the percentage area in the three fields. The area under the curve for independent variables used to predict postoperative recurrence was calculated using receiver operating characteristic (ROC) curve analysis. The 5% false positive rates of predictors in the ROC curve analysis were set as cutoff values. Tumor slides stained with anti-CD8 antibody were scanned at high magnification, and five fields (original magnification 200 ) were selected with staining of intratumoral and peritumoral CD8 positive cells. The mean numbers of positively immunostained cells per field were counted using Image J software.

Statistical analysis Statistical analysis other than ROC curve was performed with JMP 11 (SAS Institute Inc, Cary, NC). ROC curve analysis was performed with SPSS software ver.22 (SPSS Japan, Tokyo, Japan). Chi-squared tests were used to assess the associations between different categorical variables. Overall survival curves were calculated by the Kaplan-Meier method, and significant differences in survival were determined using Wilcoxon test. Prognostic factors were examined by univariate and multivariate analyses using a Cox proportional

regression model. P values less than 0.05 were considered statistically significant.

Results Presence of TLSs in gastric cancer We microscopically observed both peritumoral and intratumoral regions of clinical samples that were stained with HE (Fig. 1A). Regardless of whether CD20 expression was high or low, CD20þ B cells mostly appeared as aggregates that were located mainly at the invasive margin of the tumor and easily recognized with loupe observation (Fig. 1B and D, Fig. 2A). To identify immune cells that were present in these aggregates, we performed immunohistochemistry of the same section. The clusters of CD20þ B cells were surrounded by a CD3þ T cell rich area, and CD21þ FDCs formed a tight network in the B cell zone of the follicle (Figs. 1C-D and 2B-C). The majority of CD3þ T cells in the T cell zone were CD4þ T cells although scattered CD8þ T cells were also observed around B cell zone (Fig. 2C-E). Bcl6þ germinal center B cells were detected in some of the organized lymphocyte aggregates (Fig. 2F). Moreover, HEVs were also found in these aggregates (Fig. 2G). Previous investigators reported that TLSs are highly organized structures that resemble secondary lymphoid organs, in which CD20þ B cells are present within the follicles and which contain an area of CD3þ T cells. In these TLSs, the B cell area contains FDCs, and there are HEVs adjacent to the B cell areas. We therefore defined these aggregated immune complexes as TLSs. However, CD24þ cells, a marker of regulatory B cells, were not found in most clusters (Fig. 3A and B). It was difficult to assess the association of TLSs themselves with clinical outcome because of the variation in the size and extent of the TLSs. We therefore assessed association of the density of CD20þ B cells instead of association of TLSs with clinical outcome in the present study.

Clinicopathological characteristics and prognostic value of TLSs Using ROC curve (area under the curve ¼ 0.63), we determined 1.927 as cutoff value of CD20 density for postoperative recurrence (Fig. 4). To examine the impact of tumor-associated TLSs on clinicopathological features, we divided the patients into two groups based on the density of CD20 positive cells: a CD20 high group (n ¼ 121) and a CD20 low group (n ¼ 105). Table 1 shows the correlation of the presence of CD20þ cells with clinicopathological data compared between the two groups. Although young patients had higher CD20 density than elderly, there were no significant association of high infiltration of CD20þ cells and factors regarding with tumor progression. We also examined H pylori around the tumor to assess the effect of existing inflammation (Fig. 3C). Although H pylori infection was common in type IV cancer, there was no definite relationship of CD20 infiltration (Table 1). Kaplan-Meier survival analysis showed that the survival of patients with high infiltration of CD20þ cells was significantly better than that of patients with low infiltration (P < 0.0005, Wilcoxon test; Fig. 5A). The 5-year survival rate was 66 and

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Fig. 1 e Representative overview of lymphocyte infiltration in gastric cancer. Histologic and immunohistochemical analyses showing lymphocyte aggregates in primary gastric cancer. (A) HE staining showing intratumoral tertiary lymphoid structures (TLSs). (B-D) Immunohistochemical analysis of CD20D B cells, CD3D T cells, and CD21D FDCs. (B) Cluster of CD20D B cells were easily found in the loupe observation. Black arrows showed invasive margin of the tumor. (C) CD20D B cells mostly infiltrated the tumor in the form of follicles. (D) The follicles of CD20D cells were surrounded by a CD3D T cell area. (E) The B cell area contained CD21D FDCs that resemble secondary lymphoid organs. Scale bars indicate 200 mm. (Color version of figure is available online.)

43 mo for high CD20 and low CD20 groups, respectively. For further understanding of the prognostic importance of B cell infiltration into the tumor, patient survival was assessed for each pathological stage. Patients with high CD20 had significantly better survival compared with those with low CD20 in stage II and III (P ¼ 0.0001 and P ¼ 0.0017, respectively). For patients with stage I and IV, prognosis of the patients with high CD20 tended to be better than that of the patients with low CD20 (P ¼ 0.0578; Fig. 5B-E). In univariate analyses, pathological T category (pT), pathological lymph node metastasis (pN), pathological stage, histological type, lymphatic invasion, chemotherapy, and CD20 infiltration were associated with prognosis (Table 2). Multivariate Cox regression analysis showed that CD20 high was one of the independent predictors of OS (Table 2). We then evaluated the number of CD8þ T cells in the tumor to examine the association of CD20þ B cells with

tumor-infiltrating CD8þ T cells. As shown in Figure 6, significant association was found for the presence of CD20þ B cells according to intratumoral and peritumoral CD8þ cells in the primary tumors of patients with stage II gastric cancer.

Discussion In the present study, we showed that the CD20þ B cells that were present in gastric cancer tissue were often present as an aggregated form. These aggregates were identified as TLSs because they were accompanied by a compartmentalized area of T cells and involved CD21þ follicular dendritic cells and HEVs. Our study is the report to histologically demonstrate the cellular composition of tumor associated TLSs in gastric cancer. We showed that an increase in TLSs was correlated with

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Fig. 2 e Tertiary lymphoid structures in gastric cancer. The pictures show representative immunohistochemistry on consecutive sections of the same gastric cancer tissue samples to identify TLSs. (A) Clusters of CD20D B cells contain a network of (B) CD21D FDCs. (C) T cell areas around the B cell follicle were detected by staining for CD3. Most T cells in the T cell zone were (D) CD4D T cells, but scattered (E) CD8D T cells were also observed. (F) Bcl6D germinal center B cells were also detected within the B cell zone. (G) PNAdD HEVs (arrows) were detected adjacent to the B cell follicle. The scale bar indicates 100 mm. (Color version of figure is available online.)

favorable prognosis in patients with gastric cancer. All immune cell types can infiltrate into tumors. The organization of the tumor immune infiltrate has been documented for a long time in cancer patients. In gastric cancer, high

numbers of tumor-infiltrating CD8þ cells have been associated with improved overall survival.14 We previously showed that intratumoral CD11bþ cells, which are capable of immune regulatory function, were associated with poor prognosis.15 The

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Fig. 3 e Expression of CD24 and H pylori by immunohistochemistry. (A) Staining with CD20D in TLS (x200). (B) CD24D cells were not detected in the same TLS. (C) H pylori infection on the surface of mucosa around the tumor (4003). (Color version of figure is available online.)

1.00 0.90 0.80

SENSITIVITY

0.70 0.60 0.50 0.40 0.30 0.20

AUC=0.626 (95% CI 0.554-0.700, p=0.001)

0.10 0.00 0.00

0.20

0.40

0.60

0.80

1.00

1-SPECIFICITY

Fig. 4 e ROC curve of density of CD20 cells. We used the continuous variable density of CD24D cells as the test variable and postoperative recurrence as the state variable. An investigation of the cutoff value score showed the most appropriate cutoff value for the density of CD24D cells to be 1.927 (AUC [ 0.626, P [ 0.001, 95% CI: 0.554-0.700, the sensitivity was 0.657, and the specificity was 0.562.). Therefore, we set 1.927 as the cutoff value for analysis. (Color version of figure is available online.)

clinical relevance of B cells in tumors has not been clarified. However, several clinical studies showed a positive correlation between B cell density in the tumor and clinical outcomes.2,5,16,17 One of the main underlying mechanisms of such a positive correlation is the beneficial effects of B cells in antitumor immunity. Tumor-infiltrating B cells produced class-switched affinity-matured antitumor antibodies in breast cancer.18 TLSderived B cells from patients with autoimmune diseases such as RA or Sjo¨gren syndrome displayed oligoclonal B cell proliferation and somatic hypermutation of immunoglobulin variable genes.19,20 Tumor-associated TLS-derived B cells were demonstrated to be antigen experienced by clonal proliferation, somatic hypermutation, and isotype switching in metastatic melanoma patients.21 Germain et al.22 also detected all stages of B-cell differentiation within the tumors of non-small-cell lung carcinoma patients, including germinal center B cells, differentiated memory B cells and plasmablasts within TLSs, and PCs within the tumor stroma. These findings may represent B cells that can undergo terminal differentiation and produce antigen-specific antitumor antibodies at the tumor site. In addition to producing antibodies, B cells can also function as antigen-presenting cells for T-cell activation. It is known that B cells present processed antigens to CD4þ helper T cells, which can then stimulate the B cells to produce antibody. B cell depletion with an antiCD20 antibody impaired CD4þ T-cell activation and clonal expansion in response to protein antigen and pathogen challenge in mice, indicating that both B cells and dendritic cells were required for optimal antigen-specific CD4þ T cell priming.23 In accordance with the mouse model, high densities of both TLS dendritic cells and TLS B cells correlated with longer survival compared to a high density of dendritic cells or B cells

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Fig. 5 e Kaplan-Meier curves of the overall survival of gastric cancer patients in the CD20 high and CD20 low groups. (A) The overall survival rate of patients with CD20 high was significantly higher than that of patients with CD20 low in all stages (P [ 0.0002, Wilcoxon test). Survival curves of the two groups in different pathological stages of gastric cancer are shown in (B-E): (B) stage Ib, (C) stage II, (D) stage III, and (E) stage IV. CD20 high patients with stage II and III gastric cancer had a better prognosis than CD20 low patients, and CD20 high patients with stage I and IV gastric cancer tended to have a better prognosis than CD20 low patients, although the differences between the two groups were not statistically significant.

alone in non-small-cell lung carcinoma.8 Effector-memory and interferon gamma or tumor necrosis factor alphaesecreting CD4þ and CD8þ T cell induction was significantly impaired in B celledepleted mice with B16 melanoma.24 Hennequin et al. showed that B cell aggregates, which could be considered as TLSs, were associated with favorable prognosis of patients with gastric cancer.25 We found that B cells of tumor-associated TLSs were an independent prognostic factor. Importantly, B cells were not correlated with other

prognostic factors such as pT, pN, or lymphatic invasion, suggesting that B cells, maybe in the form of TLSs, might be novel prognostic factors that reflect the immunological condition of patients. There are several reports on the immunosuppressive function of B cells against cancer. For example, Gunderson et al. showed that inhibition of Bruton tyrosine kinase promoted by interaction of B cells with macrophages induced Th1 polarization and T cell cytotoxicity for pancreatic ductal

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Table 2 e Univariate and multivariate cox regression analysis of clinicopathological and molecular features for overall survival in patients with gastric cancer. Variables

Univariate analysis HR (95% CI)

Multivariate analysis

P value

Age (<60 y/60 y)

1.336 (0.824-2.290)

Sex (male/female)

0.797 (0.808-2.020)

0.3206

pT category (T1þT2/T3þT4)

3.184 (1.962-5.465)

<0.0001

pN (N0/N1  3)

2.207 (1.422-3.557)

0.0003

pStage (Ib þ II/III þ IV)

3.100 (2.071-4.730)

<0.0001

Histological type (tub1, tub2, pap/por, sig, muc)

1.673 (1.119-2.435)

Lymphatic invasion (ly/lyþ)

5.813 (2.626-16.477)

HR (95% CI)

P value

3.436 (2.033-6.040)

<0.0001

0.2496

0.0117

1.719 (1.145-2.629)

0.004

<0.0001

3.304 (1.386-9.770)

0.0051

Venous invasion (v/vþ)

1.203 (0.776-1.818)

Chemotherapy (yes/no)

0.593 (0.368-0.922)

0.0195

0.624 (0.891-2.829)

0.1139

CD20

2.014 (1.357-3.036)

0.0005

2.132 (1.423-3.241)

<0.0001

adenocarcinoma.26 Another report is that CD24hiCD38hi regulatory B cells upregulated regulatory T cells in gastric cancer.27 However, we found CD24 expression was not detected in TLSs and showed the correlation of B cells with tumorinfiltrating CD8þ T cells, another important element for the initiation and maintenance of antitumor immune response by TLSs is the presence of HEV. Martinet et al.28 observed a strong correlation between the density of tumor HEVs and tumorinfiltrating CD3þ T cells, CD8þ T cells, and CD20þ B cells. It has been proposed that newly formed HEV promote antitumor immunity by recruiting naive and central memory lymphocytes into the tumor, thus allowing the local generation of tissue-destroying lymphocytes.29 B cells may home into TLSs through HEV and differentiate into memory B cells and plasmablasts at the tumor site that can bind tumor antigen or promote antitumor T cell activation.

0.3987

There are several limitations to this study. First, we did not show direct evidence that B cells in intratumoral and peritumoral TLSs have a cytotoxic function against tumors. Second, the question of whether TLSs already exist in normal mucosa due to H pylori infection remains unclear. However, we found that no correlation of density of CD20þ cells with H pylori infection by immunohistochemistry and that TLSs only exist in the mucosa in normal tissue. Thus, in this study we discriminated cancer-associated TLSs from those in normal tissue based on their location. Although we were not able to provide direct evidence, we found that cancer associated TLSs are predominantly at the tumor invasive margin rather than in mucosa.

Conclusion Tumor-infiltrating B cells, especially when present in TLSs, could be associated with favorable prognosis of patients with gastric cancer. In addition, TLSs in the tumor might be an important factor in assessment of the ability of patients with gastric cancer to mount an immunological antitumor response.

Acknowledgment

Fig. 6 e Correlation between CD20D B cells and CD8D T cell infiltration. The degree of infiltration of CD20D and CD8D cells of patients with stage II gastric cancer was plotted. There was a significant correlation between CD20D B cells and CD8D T cells. The Pearson correlation test was used for statistical analysis.

H.T. was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (grant no.26461990) for this study. Authors’ contributions: C.S. and S.H. performed experiments in the article. K.M. contributed to collect samples, analysis, and data management. T.O. and H.W. contributed to pathological reevaluation. H.T. contributed to interpretation of data and review of the article. K.H. and M.O. provided supervision of experiments.

Disclosure The authors report no proprietary or commercial interest in any product mentioned or concept discussed in this article.

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