The precise molecular pathogenesis of splenic marginal zone lymphoma (SMZL) is still unknown. Clinical and epidemiological data suggest that chronic hepatitis C virus (HCV) infection may have an etiological role in a subset of cases.
We performed a large-scale microRNA (miRNA) expression profiling analysis of 381 miRNAs by quantitative reverse transcription PCR (Q-RT-PCR) of 26 microdissected splenic tissue samples (7 HCV+ SMZL; 8 HCV− SMZL and 11 non-neoplastic splenic controls). Single assay Q-RT-PCR and miRNA in situ hybridization (miRNA-ISH) were used to confirm the results in an independent cohort. Unsupervised hierarchical clustering of miRNA expression profiles demonstrated a distinct signature of SMZL compared with the normal splenic marginal zone. Supervised analysis revealed differentially expressed miRNAs, including miRNAs with previously recognized tumor suppressive or oncogenic potential. Five miRNAs were found significantly overexpressed in SMZL, including miR-21, miR-155 and miR-146a, whereas seven miRNAs showed significantly reduced expression, including miR-139, miR-345, miR-125a and miR-126. Furthermore, we identified miR-26b, a miRNA known to have tumor suppressive properties, as significantly downregulated in SMZL arising in HCV-positive patients (P=0.0016). In conclusion, there is a characteristic dysregulation of miRNA expression in SMZL with a possible implication in its molecular tumorigenesis.
Splenic marginal zone lymphoma (SMZL) is a rare low-grade B-cell lymphoma listed as distinct entity in the World Health Organization classification of lymphoid neoplams, accounting for <2% of non-Hodgkin lymphomas.1, 2 It commonly follows an indolent course exceeding a median 10-year survival. However, in a minority of cases it can pursue a more aggressive course with the possibility of transformation into a diffuse large B-cell lymphoma.3
microRNAs (miRNAs) are members of a class of small, noncoding RNAs that modulate gene expression at the post-transcriptional level in a sequence-specific manner. They have a role in controlling a variety of biological functions, including developmental patterning, cell differentiation, cell proliferation, genome rearrangements and transcriptional regulation.4 Dysregulation of miRNA expression is thought to have a pivotal role in carcinogenesis (reviewed in Calin and Croce5). In addition, viral infection, for example, with the hepatitis C virus (HCV), has been shown to distinctively influence miRNA expression in vivo and in vitro.6, 7
Over 175 million people worldwide are chronically infected with HCV, a hepatotropic and lymphotropic virus.8 HCV infection frequently leads to chronic hepatitis and is a major cause for liver cirrhosis and its sequela such as hepatocellular carcinoma.9 There is also evidence for an association with lymphoproliferative diseases, such as mixed cryoglobulinemia and B-cell non-Hodgkin lymphoma.10, 11, 12 In epidemiological studies SMZL was among the lymphoma entities found associated with HCV infection in subgroup analyses, though numbers of included SMZL patients were low.13, 14 A sustained virological response to antiviral treatment with interferon alfa and ribavirin has been shown to induce regression of HCV-associated lymphomas, and a viral relapse after initial virological response is associated with lymphoma recurrence.15 These findings suggest a causal relationship between HCV and the development of malignant lymphomas. The mechanisms by which HCV may contribute to this transformation process are poorly understood.
Previous research on miRNAs in SMZL analyzed miRNAs extracted from whole-tissue sections. One study measured the expression of miR-29a and miR-29b, two miRNAs located on chromosome 7q, a frequently deleted region in SMZL.16 In another recent study, a global miRNA screening was performed from whole tissue of six cases of SMZL in comparison with non-neoplastic splenic controls.17 Moreover, in a study using next-generation sequencing for miRNA analysis a singular case of SMZL was included.18 None of the above mentioned studies detected miRNAs differentially expressed between SMZL and non-neoplastic tissue at a statistical significant level. To our knowledge, neither miRNA expression profiling on microdissected SMZL tissue nor a correlation between miRNA expression of this lymphoma entity and HCV status has been reported to date.
Materials and methods
Patients and controls
Formalin-fixed, paraffin-embedded splenic tissue from 15 patients with SMZL and 11 matched controls from patients without tumor (splenectomy was performed for blunt abdominal trauma) was selected for multiplex miRNA expression analysis from the Department of Pathology, Pavia, Italy; Department of Pathology, L’Aquila, Italy and the Department of Pathology, Frankfurt, Germany. Detailed clinical patient data are provided in Table 1. Additional 20 cases of SMZL and 9 non-neoplastic spleens (splenectomy performed for blunt abdominal trauma or accidental intraoperative organ laceration) were selected from the Department of Pathology, Pavia and the Department of Pathology, Frankfurt, Germany, for validation. Demographic data are provided in Supplementary Table 1. The diagnosis of SMZL was established by standard morphological, cytochemical and immunophenotypic methods according to the 2008 World Health Organization lymphoma classification and its diagnostic criteria.1, 2 All cases included in the study were classical SMZL, with a typical CD5−, CD10−, bcl-6−, CD23− phenotype and with a typical pattern of white pulp involvement. Local ethical guidelines were followed for the use of archival tissues for research, with the approval of the local Ethics Review Committees of the institutions involved, in accordance with the guidelines of the Declaration of Helsinki.
Tissue of the tumor-bearing marginal zone was laser-microdissected. For miRNA expression analysis 3-μm sections of the paraffin-embedded spleen from either lymphoma or control patients were mounted on membrane-covered slides (Zeiss, Jena, Germany) and dried at 37 °C for 3 h. Sections were then deparaffinized twice by 10 min xylene washes, rehydrated with a series of graded alcohol, incubated with hematoxylin containing 200 U/ml RNase inhibitor (Roche, Basel, Switzerland) for 2 min, washed in molecular biology grade water for 2 min, incubated in 2% eosin for 30 s washed again and finally dried at room temperature for 45 min. Microdissection was performed using the laser microdissection and pressure catapulting (LMPC) technique with an UV laser beam (PALM, Zeiss). The splenic marginal zone was selectively microdissected and the tissue was directly transferred into lysis buffer PKD (Qiagen, Hilden, Germany).
Global miRNA expression analysis
Total RNA (500–1000 ng per case) from microdissected tissue was extracted with the RNeasy formalin-fixed, paraffin-embedded Kit (Qiagen) following the manufacturer's protocol. miRNA expression was analyzed by multiplex quantitative stem-loop reverse transcription PCR (Q-RT-PCR) using TaqMan low-density array cards (TLDA-Cards) version 2.0 A (4400238 Applied Biosystems, Foster City, CA, USA). This technology allows simultaneous expression analysis of up to 384 miRNAs in a single Q-RT-PCR run. The probe composition of the arrays is available at www.appliedbiosystems.com. The TLDA-Card workflow was performed in accordance to the manufacturer's protocol. TLDAs were incubated in an Applied Biosystems 7900HT Fast Real-Time PCR System at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The cycle (Ct) threshold was set to 0.1.
Single assay miRNA expression analysis
TaqMan miRNA assays (Applied Biosystems) were used to quantify miRNAs according to the manufacturer's protocol. Expression was analyzed for six miRNAs (hsa-miR-139-5p, hsa-miR-126, hsa-miR-21, hsa-miR-146a, hsa-miR-34a and hsa-miR-26b) and one endogenous control (RNU48). Each sample was analyzed in triplicate, and ΔCt values were calculated using the endogenous control. Delta ΔCt (ΔCt SMZL–ΔCt splenic control) was calculated for comparison of groups.
miRNA in situ hybridization
miRNA in situ hybridization (miRNA-ISH) was carried out as reported elsewhere.19 Briefly, 11 digoxigenin-labled locked nucleic acid probes antisense to miR-139-5p or miR-146a (Exiqon, Vedbaek, Denmark) were used for overnight staining on tissue sections at 61 °C. Detection was accomplished with anti-digoxigenin alkaline phosphatase Fab fragment followed by nitro blue tetrazolium chloride/5-bromo-4-chlore-3-indolyl phosphate color development (Roche).
Statistical analysis was performed with the statistical computing environment R.20 Additional software packages (HTqPCR, geneplotter, gplots) were taken from the Bioconductor project.21 Probe level normalization was conducted using a quantile normalization method implemented in HTqPCR, a software package designed for analysis of multiplex Q-PCR data.22 The quantile normalization method, using the signal of all detected miRNA as basis proved superior over methods using only selected housekeeping RNAs for normalization (e.g., RNU44, RNU48 and U6). For pairwise comparisons, a global filter was used to reduce the dimension of the data. Furthermore, a variance filter (for interquartile range intensities >1) was applied. Endogenous controls were excluded in pairwise comparisons. Also excluded were miRNAs with Ct values >35 in >5 samples, as miRNAs with Ct values between 35 and 40 did not show significant variance throughout the population studied. After global filtering and testing for normal distribution of data,23 a two-sample t-test was applied to identify differentially expressed miRNAs between two groups. To account for multiple testing adjusted P-values were calculated for multiplex Q-RT-PCR experiments.24
Unsupervised hierarchical clustering was performed for miRNAs with an interquartile range >1 across all samples using the Euclidean distance and the average linkage method after exclusion of miRNAs with CT values >35 in >5 samples. The corresponding heat maps are shown with a false color display of a matrix of numerical values. P-values <0.05 were considered significant.
SMZL demonstrate a distinct miRNA expression profile as shown by unsupervised hierarchical clustering analysis
Using ABI TaqMan Array miRNA cards, miRNA expression profiles were generated from microdissected splenic marginal zones of 26 biopsy samples, obtained from 7 HCV-positive and 8 HCV-negative patients with SMZL and 11 splenectomized patients without lymphoma (Table 1). Of 381 miRNAs analyzed, an average of 84 were found expressed in the tumor samples or non-neoplastic controls. Results were further filtered by an interquartile range algorithm and miRNA probesets with a Ct value of 35 or greater in >5 samples were excluded from the analysis. More than 75% of the miRNAs screened were either not expressed throughout most of the samples (Ct values >35) or showed only a small variability (interquartile range <1). The miRNA expression profiles of the 26 samples were divided by unsupervised hierarchical clustering into two distinct branches, largely comprising the malignant lymphomas and the normal spleens, respectively (Figure 1). Global expression profiles of patients with and without chronic HCV infection did not separate and were intermingled in the branch containing the lymphoma tissue.
Twelve miRNAs are differentially expressed between SMZL and the normal splenic marginal zone
In a supervised analysis comparing the profiles of all SMZL (combined HCV-positive and HCV-negative samples) and normal splenic tissue, 12 miRNAs were found differentially expressed (fold change (FC)>± 2) in a statistically significant manner (adjusted P-value <0.05) (Figure 2). Table 2 shows the miRNAs up and downregulated in SMZL as well as genes that have been experimentally validated as targets of the respective miRNAs.
Differential expression of miRNAs in SMZL patients with chronic HCV infection
Global unsupervised analysis did not reveal a characteristic pattern of miRNAs in SMZL in HCV-positive patients compared with HCV-negative patients (Figure 1). However, a supervised comparison of these two groups identified one miRNA, miR-26b, to be significantly downregulated in the HCV+ lymphoma group (P=0.0016, adjusted P=0.07) with a FC of −2.35 (Table 2, Supplementary Figure 1).
Results from TLDA-fluid cards could be confirmed by single-assay Q-RT-PCR and miRNA-ISH
Representative miRNAs with previously described roles in hemato–oncologic malignancies and high fold change or low P-value in the multiplex analysis were selected for validation of the experimental method and the bioinformatic normalization process with an independent sample set. Five miRNAs with differential expression comparing SMZL tissue vs normal controls, and miRNA-26b, which demonstrated differential expression between HCV-positive and -negative SMZL patients, were reanalyzed by single-assay Q-RT-PCR on whole-tissue sections. A total of 29 cases (5 HCV+ SMZL, 15 HCV− SMZL, 5 HCV− controls and 4 HCV+ controls) were analyzed by triplicate experiments (Supplementary Table 1). Downregulation of two miRNAs in SMZL, miR-139-5p and miR-126, was confirmed (mean ΔΔCT −3.80 and −3.67; P=0.02 and 0.03, respectively; Figure 3a). High expression of miR-21 and miR-146a could be confirmed while upregulation of miR-34a was not statistically significant (mean ΔΔCT 2.49, 1.76 and 1.21; P=0.04, 0.05 and 0.29, respectively; Figure 3a). Downregulation of miR-26b in HCV-positive vs -negative SMZL was confirmed (mean ΔΔCT −1.39; P=0.01). Notably, miR-26b was also expressed at significantly reduced levels in HCV+ SMZL samples in comparison with HCV+ or HCV− spleens without lymphoma involvement (Figure 3b).
Furthermore, miRNA-ISH for representative miRNAs shown to be differentially expressed in SMZL (miR-139-5p, miR-146a) was performed as a complementary validation method in a total of 20 cases (15 SMZL, 5 reactive spleens; Supplementary Table 1). MiRNA-ISH proved to be challenging in the splenic formalin-fixed, paraffin-embedded tissue. In evaluable cases (4 of 15 SMZL, 3 of 5 non-neoplastic spleens) the low expression of miR-139-5p and high expression of miR-146a in tumor cells in contrast to their surrounding tissue and to follicles of the normal spleen could be clearly confirmed (Figure 4).
mRNA targets of differentially expressed miRNAs
Table 2 shows the miRNAs differentially expressed in SMZL as well as corresponding experimentally validated target genes from the literature with an important role in cancerogenesis.
Furthermore, we used gene chip data from a large study on mRNA expression in SMZL, including 27 SMZL samples and 5 non-neoplastic splenic controls (Gene Expression Omnibus Database Series GSE9327).25 The original normalized data set was kindly provided by Ruiz-Ballesteros et al. and was reanalyzed to provide correction for multiple testing. Of 4834 analyzed genes, 27 were found to be downregulated and 16 to be upregulated in SMZL with statistical significance (FC>±2; adjusted P-value <0.05) compared with the non-neoplastic controls. We used in silico target prediction software for miRNAs to analyze if the signature of miRNAs we found dysregulated in the current investigation may potentially target exactly those genes found differentially expressed in SMZL by Ruiz-Ballesteros et al.25 There is a multitude of available programs predicting miRNA targets, using different algorithms to score the probability of miRNA–mRNA interaction. Therefore, matching predictions produced by multiple programs were selected. miRWalk algorithm was used to define high probability targets by scanning several target prediction platforms (including miRanda, TARGETScan, RNAHybrid, PITA and PICTAR)26, 27, 28, 29, 30, 31 selecting only predictions with a P-value <0.05 or seed length >7. MiRNA targets meeting these criteria in three or more of the platforms were selected. Of 43 genes differentially expressed in SMZL, 23 are high fidelity predicted target genes of miRNAs we found up- or downregulated in SMZL. In the majority, but not all of these genes mRNA downregulation corresponds with miRNA upregulation and vice versa. The differentially expressed miRNAs and their corresponding mRNA targets are shown in Table 2.
We performed a global miRNA expression analysis profiling 26 microdissected samples—15 cases of SMZL and 11 non-neoplastic splenic controls—and consecutively confirmed our findings on an independent sample set. MiRNAs have a pivotal role in the molecular pathogenesis of various cancer types and indeed, we could demonstrate characteristic deregulation of miRNA expression in SMZL.
Unsupervised hierarchical clustering analysis demonstrates that SMZL shows a distinct and characteristic miRNA expression profile compared with non-neoplastic splenic tissue. We used cells from the normal splenic marginal zone as non-tumorous counterpart for SMZL. Although this choice seems obvious, the cell of origin of SMZL is still not entirely clear.32 Therefore, the results obtained may in some respect be only an approximation to the true SMZL phenotype.
Future analysis of miRNA profiles of other lymphoma entities, especially those with high morphological similarity to SMZL (for example, lymphoplasmocytic lymphoma, splenic B-cell lymphoma unclassifiable) may enable the identification of entity-specific miRNAs or miRNA profiles. This might improve differential diagnosis, as highly specific markers for SMZL are lacking.
We identified several miRNAs that are differentially expressed in SMZL compared with the non-tumorous spleen. These results were confirmed by single-assay Q-RT-PCR for four of five miRNAs selected for the validation analysis on an independent sample set. The variance in validation experiments was considerably higher than in the TLDA experiment. This may be explained by the fact that, only for validation, RNA was extracted from whole-tissue sections instead of microdissected tissue. Recently, Bouteloup et al.17 performed a global miRNA expression analysis in SMZL using a previous version of the TLDA-card used in our study. Bouteloup et al. used RNA extracted from whole tissue of six SMZL and nine non-neoplastic spleens for their analysis. They found overexpression of miR-21 in cases with an aggressive morphological or clinical phenotype. No miRNAs with differential expression between SMZL and non-neoplastic splenic tissue were detected, but three miRNAs showed a tendency of overexpression and four miRNAs a low expression in SMZL (median relative expression >1.5 and <0.3, respectively). Remarkably, these miRNAs included overexpression of miR-155 and low expression of miR-139, which we found similarly dysregulated in SMZL. Using whole-tissue sections for miRNA expression analysis may result in relevant inaccuracy of results. Especially in non-neoplastic splenic controls the fraction of marginal zone cells may be as low as 15–20% of total cells. Therefore, we decided to additionally use miRNA-ISH to confirm our results for two representative miRNAs.
The observed associations do not allow the conclusion that the deregulated miRNAs might be driving factors in the pathogenesis of SMZL. However, the literature provides ample evidence for several of the differentially expressed miRNAs being involved in various mechanisms of tumor formation. Several of the miRNAs found downregulated in SMZL in this study (miR-126, miR-139, miR-345) are consistently suppressed in other hematological malignancies as well.33, 34 The same holds true for part of the miRNAs identified here as overexpressed in SMZL (miR-146a, miR-155 and miR-21).33, 35, 36 These miRNAs are likely to be ‘global players’ in the development of certain lymphoma entities and their mechanism of action and specific target genes have partly been already identified.37, 38 Other miRNAs found to be differentially expressed in SMZL in this study have rarely been linked to malignant lymphoma to date, but a major part (for example, miR-494, miR-138) are dysregulated in other malignant disease models, for example, solid tumors.39, 40 The genes of those miRNAs differentially expressed in SMZL are located on various chromosomal locations and do not derive from the same clusters. Therefore, the respective up- and downregulation is unlikely to be explained by cytogenetic aberrations, which are frequently observed in SMZL, for example, del7q or gains in 3q.41, 42
Using gene chip analysis, Ruiz-Ballesteros et al.25 have identified genes specifically up- and downregulated in SMZL. We used computational target predictions to analyze if these genes may be the targets of those miRNAs we found differentially expressed in SMZL. This approach is to some extent artificial as it compares two independent cohorts of SMZL patients but nevertheless, the fact that globally more than half of the dysregulated genes in SMZL turned out to be high fidelity predicted targets of the miRNAs we characterized supports a functional role of these miRNAs in the SMZL pathogenesis.
Not much is known of the cellular functions and pathways specifically dysregulated in SMZL. The study by Ruiz-Ballesteros et al., which we reanalyzed in this paper to compare with our miRNA expression profiles, is the largest study on gene expression in SMZL.25 Notably, this gene expression analysis detected upregulation of several genes involved in NF-kappaB signaling. Furthermore, genetic alterations with involvement in the NF-kappaB pathway were found in 36% of SMZL cases.43 Two of the miRNAs upregulated in SMZL (miR-21, miR-155) have been shown to exhibit their oncogenic potential by promoting NF-kappaB activity,44 which may be one mechanism how these miRNAs could be involved in the pathogenesis of SMZL.
We found one miRNA, miR-26b, to be differentially expressed between SMZL arising in HCV-positive vs HCV-negative patients. Single assay Q-RT-PCR confirmed these findings and further demonstrated that this phenomenon was exclusive to HCV-associated SMZL and not simply a consequence of chronic HCV infection. Other studies investigating miRNA expression in patients with chronic HCV infection concentrated mainly on the study of blood compartments and liver or liver-cancer tissue. In these studies other miRNAs than identified here have been found up- or downregulated. Beside technical aspects of these various studies, these differences are likely to a large extent due to the different influences that HCV will have on various cell types (for example, hepatocytes and B cells), which have a very distinct mRNA and miRNA expression pattern. Moreover, it is difficult to attribute any of the discovered dysregulations of miRNA expression exclusively to HCV infection. MiRNA expression analysis in the liver tissue of patients with and without HCV infection could demonstrate dysregulation of miR-21 in correlation with HCV viral load, but at the same time miR-21 expression strongly correlated with markers of liver damage and the extend of liver fibrosis.45 Similar findings have been reported on miRNA expression in the serum of HCV-infected patients. Different serum-miRNAs were found to correlate with liver inflammation and fibrosis indirectly caused by the virus.46, 47 Various studies reported on miRNA expression in hepatocellular carcinoma tissue of HCV-positive patients, but again these findings are likely not exclusively caused by HCV infection but rather by the tumor itself or again by HCV-induced liver damage.7, 48, 49 The diminished expression of miR-26b, as seen in the HCV-positive lymphoma group, has been linked to a malignant tumor phenotype in hepatocellular carcinoma50 and lung carcinoma51 as well as in cell culture models of colon cancer and diffuse large B-cell lymphoma.52, 53 Moreover, overexpression of miR-26b in colon cancer cell lines led to significant suppression of cell growth and the induction of apoptosis.53
Epidemiological data clearly link HCV infection with a higher risk of lymphoma development.54, 55 Moreover, eradication of the virus by antiviral therapy can lead to lymphoma remission, which points to a causal role of HCV in malignant transformation, as reviewed by Arcaini et al.15 SMZL is one of the subentities of B-cell non-Hodgkin lymphoma found associated with HCV infection in a statistically significant manner.13, 14 Nevertheless, the mechanisms by which HCV might contribute to lymphoma development are poorly understood. HCV has been shown to influence host miRNA expression in hepatoma cells in vitro6 as well as in hepatocellular carcinoma tissue in vivo.7 One might therefore speculate that HCV infection might be involved in downregulation of the tumor-suppressing miRNA 26b in cases of SMZL. Under these conditions miR-26b could function as a potential mediator of malignant transformation in HCV-associated B-cell non-Hodgkin lymphoma.
One predicted target of miR-26b, which is upregulated SMZL,25 is the NIMA-related kinase NEK6 (Table 2). NEK6 is thought to have a critical role in mitotic cell cycle progression and is upregulated in various human cancers.56, 57, 58 Overexpression of NEK6 can promote tumorigenesis by inhibiting cellular senescence.59 Thus, a possible loss of repression of NEK6 by downregulated miR-26b may have oncogenic potential also in HCV-associated SMZL.
In summary, we performed, for the first time, a global miRNA expression profiling in SMZL identifying a subset of miRNAs dysregulated in SMZL with possible implication in the pathogenesis of this lymphoma entity. Moreover, in HCV-positive SMZL we found reduced expression of miR-26b, a miRNA with known traits of a tumor suppressor, pointing to a possible mechanism by which the virus might unfold its oncogenic potential in malignant lymphoma. With respect to the rather low number of cases investigated, prospective studies on larger populations are warranted.
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We thank Janine Bronckhorst, Ekaterini Hadzoglou Sabine Albrecht and Ralf Lieberz for excellent technical assistance. This work was supported by the ‘Patenschaftsmodell’ grant from the medical faculty of the J.W. Goethe-University Hospital, Frankfurt, and by the Deutsche Forschungsgemeinschaft (GRK 1431/2, TR60, KFO129).
J Peveling-Oberhag: designed and performed research, revised and analyzed clinical data and wrote the manuscript; G Crisman performed research and collected and characterized histological samples; A Schmidt designed and performed research and assisted in the correction of the manuscript; C Döring performed bioinformatics and statistical analysis; M Lucioni provided advice and collected and characterized histological samples; L Arcaini provided advice and collected and revised clinical data; S Rattotti collected and revised clinical data; S Hartmann designed research, provided advice and collected and characterized histological samples; A Piiper provided advice and assisted in the correction of the paper; WP Hofmann designed research and assisted in the correction of the paper; M Paulli provided advice and collected and characterized histological samples; R Küppers designed research, provided advice and revised the manuscript; S Zeuzem designed research, provided advice and revised the paper and ML Hansmann designed research, collected and characterized histological samples and revised the paper.
The authors declare no conflict of interest.
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Peveling-Oberhag, J., Crisman, G., Schmidt, A. et al. Dysregulation of global microRNA expression in splenic marginal zone lymphoma and influence of chronic hepatitis C virus infection. Leukemia 26, 1654–1662 (2012). https://doi.org/10.1038/leu.2012.29
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