scholarly journals Dipeptidyl peptidase like 6 promoter methylation is a potential prognostic biomarker for pancreatic ductal adenocarcinoma

2020 ◽  
Vol 40 (7) ◽  
Author(s):  
Xin Zhao ◽  
Di Cao ◽  
Zhangyong Ren ◽  
Zhe Liu ◽  
Shaocheng Lv ◽  
...  
Keyword(s):  
Regression Analysis ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Promoter Methylation ◽  
Methylation Level ◽  
Cox Regression ◽  
Prognostic Biomarker ◽  
Dipeptidyl Peptidase ◽  
Ductal Adenocarcinoma ◽  
Gene Promoters ◽  
Cox Regression Analysis

Abstract Background: Hypermethylation of gene promoters plays an important role in tumorigenesis. The present study aimed to identify and validate promoter methylation-driven genes (PMDGs) for pancreatic ductal adenocarcinoma (PDAC). Methods: Based on GSE49149 and the PDAC cohort of The Cancer Genome Atlas (TCGA), differential analyses of promoter methylation, correlation analysis, and Cox regression analysis were performed to identify PMDGs. The promoter methylation level was assessed by bisulfite sequencing polymerase chain reaction (BSP) in paired tumor and normal tissues of 72 PDAC patients. Kaplan−Meier survival analyses were performed to evaluate the clinical value of PMDGs. Results: In GSE49149, the β-value of the dipeptidyl peptidase like 6 (DPP6) promoter was significantly higher in tumor compared with normal samples (0.50 vs. 0.24, P<0.001). In the PDAC cohort of TCGA, the methylation level of the DPP6 promoter was negatively correlated with mRNA expression (r = −0.54, P<0.001). In a multivariate Cox regression analysis, hypermethylation of the DPP6 promoter was an independent risk factor for PDAC (hazard ratio (HR) = 543.91, P=0.002). The results of BSP revealed that the number of methylated CG sites in the DPP6 promoter was greater in tumor samples than in normal samples (7.43 vs. 2.78, P<0.001). The methylation level of the DPP6 promoter was moderately effective at distinguishing tumor from normal samples (area under ROC curve (AUC) = 0.74, P<0.001). Hypermethylation of the DPP6 promoter was associated with poor overall (HR = 3.61, P<0.001) and disease-free (HR = 2.01, P=0.016) survivals for PDAC patients. Conclusion: These results indicate that DPP6 promoter methylation is a potential prognostic biomarker for PDAC.

scholarly journals Prognostic Nomogram for Patients With Pancreatic Ductal Adenocarcinoma of Pancreatic Head After Pancreaticoduodenectomy

2021 ◽  
Vol 15 ◽  
pp. 117955492110241
Author(s):  
Hongkai Zhuang ◽  
Zixuan Zhou ◽  
Zuyi Ma ◽  
Shanzhou Huang ◽  
Yuanfeng Gong ◽  
...  
Keyword(s):  
Regression Analysis ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Perineural Invasion ◽  
Cox Regression ◽  
Area Under The Curve ◽  
Pancreatic Head ◽  
Ductal Adenocarcinoma ◽  
R0 Resection ◽  
Cox Regression Analysis ◽  
Ajcc Stage

Background: The prognosis of patients with pancreatic ductal adenocarcinoma (PDAC) of pancreatic head remains poor, even after potentially curative R0 resection. The aim of this study was to develop an accurate model to predict patients’ prognosis for PDAC of pancreatic head following pancreaticoduodenectomy. Methods: We retrospectively reviewed 112 patients with PDAC of pancreatic head after pancreaticoduodenectomy in Guangdong Provincial People’s Hospital between 2014 and 2018. Results: Five prognostic factors were identified using univariate Cox regression analysis, including age, histologic grade, American Joint Committee on Cancer (AJCC) Stage 8th, total bilirubin (TBIL), CA19-9. Using all subset analysis and multivariate Cox regression analysis, we developed a nomogram consisted of age, AJCC Stage 8th, perineural invasion, TBIL, and CA19-9, which had higher C-indexes for OS (0.73) and RFS (0.69) compared with AJCC Stage 8th alone (OS: 0.66; RFS: 0.67). The area under the curve (AUC) values of the receiver operating characteristic (ROC) curve for the nomogram for OS and RFS were significantly higher than other single parameter, which are AJCC Stage 8th, age, perineural invasion, TBIL, and CA19-9. Importantly, our nomogram displayed higher C-index for OS than previous reported models, indicating a better predictive value of our model. Conclusions: A simple and practical nomogram for patient prognosis in PDAC of pancreatic head following pancreaticoduodenectomy was established, which shows satisfactory predictive efficacy and deserves further evaluation in the future.

scholarly journals Development and Validation of a 7-Gene Prognostic Signature to Improve Survival Prediction in Pancreatic Ductal Adenocarcinoma

2021 ◽  
Vol 8 ◽  
Author(s):  
Zengyu Feng ◽  
Hao Qian ◽  
Kexian Li ◽  
Jianyao Lou ◽  
Yulian Wu ◽  
...  
Keyword(s):  
Regression Analysis ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Cox Regression ◽  
Predictive Accuracy ◽  
Disease Free Survival ◽  
Roc Curves ◽  
Gene Signature ◽  
Ductal Adenocarcinoma ◽  
Survival Prediction ◽  
Cox Regression Analysis

Background: Previous prognostic signatures of pancreatic ductal adenocarcinoma (PDAC) are mainly constructed to predict the overall survival (OS), and their predictive accuracy needs to be improved. Gene signatures that efficaciously predict both OS and disease-free survival (DFS) are of great clinical significance but are rarely reported.Methods: Univariate Cox regression analysis was adopted to screen common genes that were significantly associated with both OS and DFS in three independent cohorts. Multivariate Cox regression analysis was subsequently performed on the identified genes to determine an optimal gene signature in the MTAB-6134 training cohort. The Kaplan–Meier (K-M), calibration, and receiver operating characteristic (ROC) curves were employed to assess the predictive accuracy. Biological process and pathway enrichment analyses were conducted to elucidate the biological role of this signature.Results: Multivariate Cox regression analysis determined a 7-gene signature that contained ASPH, DDX10, NR0B2, BLOC1S3, FAM83A, SLAMF6, and PPM1H. The signature had the ability to stratify PDAC patients with different OS and DFS, both in the training and validation cohorts. ROC curves confirmed the moderate predictive accuracy of this signature. Mechanically, the signature was related to multiple cancer-related pathways.Conclusion: A novel OS and DFS prediction model was constructed in PDAC with multi-cohort and cross-platform compatibility. This signature might foster individualized therapy and appropriate management of PDAC patients.

scholarly journals An EMT-Related Gene Signature for Predicting Response to Adjuvant Chemotherapy in Pancreatic Ductal Adenocarcinoma

2021 ◽  
Vol 9 ◽  
Author(s):  
Zengyu Feng ◽  
Kexian Li ◽  
Jianyao Lou ◽  
Yulian Wu ◽  
Chenghong Peng
Keyword(s):  
Regression Analysis ◽  
Adjuvant Chemotherapy ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Cox Regression ◽  
Predictive Accuracy ◽  
Checkpoint Inhibitors ◽  
Gene Signature ◽  
Ductal Adenocarcinoma ◽  
Related Gene ◽  
Cox Regression Analysis

BackgroundFor pancreatic ductal adenocarcinoma (PDAC) patients, chemotherapy failure is the major reason for postoperative recurrence and poor outcomes. Establishment of novel biomarkers and models for predicting chemotherapeutic efficacy may provide survival benefits by tailoring treatments.MethodsUnivariate cox regression analysis was employed to identify EMT-related genes with prognostic potential for DFS. These genes were subsequently submitted to LASSO regression analysis and multivariate cox regression analysis to identify an optimal gene signature in TCGA training cohort. The predictive accuracy was assessed by Kaplan–Meier (K-M), receiver operating characteristic (ROC) and calibration curves and was validated in PACA-CA cohort and our local cohort. Pathway enrichment and function annotation analyses were conducted to illuminate the biological implication of this risk signature.ResultsLASSO and multivariate Cox regression analyses selected an 8-gene signature comprised DLX2, FGF9, IL6R, ITGB6, MYC, LGR5, S100A2, and TNFSF12. The signature had the capability to classify PDAC patients with different DFS, both in the training and validation cohorts. It provided improved DFS prediction compared with clinical indicators. This signature was associated with several cancer-related pathways. In addition, the signature could also predict the response to immune-checkpoint inhibitors (ICIs)-based immunotherapy.ConclusionWe established a novel EMT-related gene signature that was capable of predicting therapeutic response to adjuvant chemotherapy and immunotherapy. This signature might facilitate individualized treatment and appropriate management of PDAC patients.

scholarly journals A novel epigenetic signature for predicting the prognosis of pancreatic ductal adenocarcinoma

2020 ◽  
Author(s):  
Zhiming Zhao ◽  
Mengyang Li ◽  
Xianglong Tan ◽  
Rong Liu
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Regression Analysis ◽  
Survival Analysis ◽  
Prognostic Factors ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Cox Regression ◽  
Risk Groups ◽  
Ductal Adenocarcinoma ◽  
Cox Regression Analysis

Abstract Background Aberrant DNA methylation is often involved in carcinogenesis. This study is designed to establish an epigenetic signature to predict overall survival (OS) of pancreatic ductal adenocarcinoma (PDAC). Methods DNA methylation and RNA-seq data of PDAC patients were downloaded from the Cancer Genome Atlas database, Genotype-Tissue Expression (GTEx), and International Cancer Genome Consortium (ICGC) database. Methylation-related differentially expressed genes (DEGs) were identified using an R package MethylMix. Epigenetic signature and nomogram were established by the LASSO and multivariate Cox regression analysis, respectively. In addition, a joint survival analysis of the gene expression and methylation was performed to identify potential prognostic factors for patients with PDAC. Results There were a total of 56 methylation-related DEGs by MethylMix criteria. After LASSO Cox regression analysis, we developed an epigenetic signature composed of five genes according to their methylation level. The signature was able to divide patients into high-risk and low-risk groups, and the OS between the high-and low-risk groups was more significantly different in both training and validation cohort. The signature is independent of clinicopathological variables and indicated better predictive power. Moreover, we developed a novel prognostic nomogram that combines risk scores with three clinicopathological factors. The joint survival analysis of gene expression and methylation revealed that 24 genes could be independent prognostic factors for OS in PDAC. Conclusions The qualitative signature and nomogram that predict OS at the individualized level and guide therapy for patients with PDAC.

scholarly journals A Novel Ferroptosis-Related Gene Signature Predicts Recurrence in Patients With Pancreatic Ductal Adenocarcinoma

2021 ◽  
Vol 8 ◽  
Author(s):  
Zengyu Feng ◽  
Peng Chen ◽  
Kexian Li ◽  
Jianyao Lou ◽  
Yulian Wu ◽  
...  
Keyword(s):  
Regression Analysis ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Cox Regression ◽  
Validation Cohort ◽  
Gene Signature ◽  
The Cancer Genome Atlas ◽  
Ductal Adenocarcinoma ◽  
Pathway Enrichment Analysis ◽  
Cox Regression Analysis ◽  
The Stability

Background: Recurrence after surgery is largely responsible for the extremely poor outcomes for patients with pancreatic ductal adenocarcinoma (PDAC). Ferroptosis is implicated in chemotherapy sensitivity and tumor recurrence, we aimed to find out survival-associated ferroptosis-related genes and use them to build a practical risk model with the purpose to predict PDAC recurrence.Methods: Univariate Cox regression analysis was conducted to obtain prognostic ferroptosis-related genes in The Cancer Genome Atlas (TCGA, N = 140) cohort. Multivariate Cox regression analysis was employed to construct a reliable and credible gene signature. The prognostic performance was verified in a MTAB-6134 (N = 286) validation cohort and a PACA-CA (N = 181) validation cohort. The stability of the signature was tested in TCGA and MTAB-6134 cohorts by ROC analyses. Pathway enrichment analysis was adopted to preliminary illuminate the biological relevance of the gene signature.Results: Univariate and multivariate Cox regression analyses identified a 5-gene signature that contained CAV1, DDIT4, SLC40A1, SRXN1 and TFAP2C. The signature could efficaciously stratify PDAC patients with different recurrence-free survival (RFS), both in the training and validation cohorts. Results of subgroup receiver operating characteristic curve (ROC) analyses confirmed the stability and the independence of this signature. Our signature outperformed clinical indicators and previous reported models in predicting RFS. Moreover, the signature was found to be closely associated with several cancer-related and drug response pathways.Conclusion: This study developed a precise and concise prognostic model with the clinical implication in predicting PDAC recurrence. These findings may facilitate individual management of postoperative recurrence in patients with PDAC.

scholarly journals Establishment of a Prognostic Model for Hepatocellular Carcinoma Based on Endoplasmic Reticulum Stress-Related Gene Analysis

2021 ◽  
Vol 11 ◽  
Author(s):  
Peng Liu ◽  
Jinhong Wei ◽  
Feiyu Mao ◽  
Zechang Xin ◽  
Heng Duan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Endoplasmic Reticulum ◽  
Regression Analysis ◽  
Endoplasmic Reticulum Stress ◽  
Cox Regression ◽  
Prognostic Biomarker ◽  
Cancer Genome ◽  
Survival Prediction ◽  
Training Cohort ◽  
Cox Regression Analysis

Hepatocellular carcinoma (HCC) is one of the most common types of cancer worldwide and its incidence continues to increase year by year. Endoplasmic reticulum stress (ERS) caused by protein misfolding within the secretory pathway in cells and has an extensive and deep impact on cancer cell progression and survival. Growing evidence suggests that the genes related to ERS are closely associated with the occurrence and progression of HCC. This study aimed to identify an ERS-related signature for the prospective evaluation of prognosis in HCC patients. RNA sequencing data and clinical data of patients from HCC patients were obtained from The Cancer Genome Atlas (TCGA) and The International Cancer Genome Consortium (ICGC). Using data from TCGA as a training cohort (n=424) and data from ICGC as an independent external testing cohort (n=243), ERS-related genes were extracted to identify three common pathways IRE1, PEKR, and ATF6 using the GSEA database. Through univariate and multivariate Cox regression analysis, 5 gene signals in the training cohort were found to be related to ERS and closely correlated with the prognosis in patients of HCC. A novel 5-gene signature (including HDGF, EIF2S1, SRPRB, PPP2R5B and DDX11) was created and had power as a prognostic biomarker. The prognosis of patients with high-risk HCC was worse than that of patients with low-risk HCC. Multivariate Cox regression analysis confirmed that the signature was an independent prognostic biomarker for HCC. The results were further validated in an independent external testing cohort (ICGC). Also, GSEA indicated a series of significantly enriched oncological signatures and different metabolic processes that may enable a better understanding of the potential molecular mechanism mediating the progression of HCC. The 5-gene biomarker has a high potential for clinical applications in the risk stratification and overall survival prediction of HCC patients. In addition, the abnormal expression of these genes may be affected by copy number variation, methylation variation, and post-transcriptional regulation. Together, this study indicated that the genes may have potential as prognostic biomarkers in HCC and may provide new evidence supporting targeted therapies in HCC.

scholarly journals Transcriptomic Profiling Identifies DCBLD2 as a Diagnostic and Prognostic Biomarker in Pancreatic Ductal Adenocarcinoma

2021 ◽  
Vol 8 ◽  
Author(s):  
Zengyu Feng ◽  
Kexian Li ◽  
Yulian Wu ◽  
Chenghong Peng
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Extracellular Vesicles ◽  
Immune Cell ◽  
Cox Regression ◽  
Univariate Analysis ◽  
Functional Enrichment ◽  
Ductal Adenocarcinoma ◽  
Survival Curves ◽  
Cox Regression Analysis ◽  
Calibration Curves

Background: Accumulating evidence shows that the elevated expression of DCBLD2 (discoidin, CUB and LCCL domain-containing protein 2) is associated with unfavorable prognosis of various cancers. However, the correlation of DCBLD2 expression value with the diagnosis and prognosis of pancreatic ductal adenocarcinoma (PDAC) has not yet been elucidated. Methods: Univariate Cox regression analysis was used to screen robust survival-related genes. Expression pattern of selected genes was investigated in PDAC tissues and normal tissues from multiple cohorts. Kaplan–Meier (K–M) survival curves, ROC curves and calibration curves were employed to assess prognostic performance. The relationship between DCBLD2 expression and immune cell infiltrates was conducted by CIBERSORT software. Biological processes and KEGG pathway enrichment analyses were adopted to clarify the potential function of DCBLD2 in PDAC. Results: Univariate analysis, K–M survival curves and calibration curves indicated that DCBLD2 was a robust prognostic factor for PDAC with cross-cohort compatibility. Upregulation of DCBLD2 was observed in dissected PDAC tissues as well as extracellular vesicles from both plasma and serum samples of PDAC patients. Both DCBLD2 expression in tissue and extracellular vesicles had significant diagnostic value. Besides, DCBLD2 expression was correlated with infiltrating level of CD8+ T cells and macrophage M2 cells. Functional enrichment revealed that DCBLD2 might be involved in cell motility, angiogenesis, and cancer-associated pathways. Conclusion: Our study systematically analyzed the potential diagnostic, prognostic and therapeutic value of DCBLD2 in PDAC. All the findings indicated that DCBLD2 might play a considerably oncogenic role in PDAC with diagnostic, prognostic and therapeutic potential. These preliminary results of bioinformatics analyses need to be further validated in more prospective studies.

scholarly journals A Novel DNA Replication-Related Signature Predicting Recurrence After R0 Resection of Pancreatic Ductal Adenocarcinoma: Prognostic Value and Clinical Implications

2021 ◽  
Vol 9 ◽  
Author(s):  
Zengyu Feng ◽  
Kexian Li ◽  
Jianyao Lou ◽  
Mindi Ma ◽  
Yulian Wu ◽  
...  
Keyword(s):  
Dna Replication ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Risk Model ◽  
Cox Regression ◽  
External Validation ◽  
Gene Signature ◽  
Ductal Adenocarcinoma ◽  
R0 Resection ◽  
Pathway Enrichment Analysis ◽  
Cox Regression Analysis

The aim of any surgical resection for pancreatic ductal adenocarcinoma (PDAC) is to achieve tumor-free margins (R0). R0 margins give rise to better outcomes than do positive margins (R1). Nevertheless, postoperative morbidity after R0 resection remains high and prognostic gene signature predicting recurrence risk of patients in this subgroup is blank. Our study aimed to develop a DNA replication-related gene signature to stratify the R0-treated PDAC patients with various recurrence risks. We conducted Cox regression analysis and the LASSO algorithm on 273 DNA replication-related genes and eventually constructed a 7-gene signature. The predictive capability and clinical feasibility of this risk model were assessed in both training and external validation sets. Pathway enrichment analysis showed that the signature was closely related to cell cycle, DNA replication, and DNA repair. These findings may shed light on the identification of novel biomarkers and therapeutic targets for PDAC.

scholarly journals Development and Validation of a Nomogram Based on Nutritional Indicators and Tumor Markers for Prognosis Prediction of Pancreatic Ductal Adenocarcinoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Haoran Li ◽  
Fang Zhou ◽  
Zhifei Cao ◽  
Yuchen Tang ◽  
Yujie Huang ◽  
...  
Keyword(s):  
Regression Analysis ◽  
Tumor Markers ◽  
Lymphocyte Count ◽  
Cox Regression ◽  
External Validation ◽  
Training Group ◽  
Ductal Adenocarcinoma ◽  
Validation Group ◽  
Internal Validation ◽  
Cox Regression Analysis

PurposeThis study aimed to develop and validate a nomogram with preoperative nutritional indicators and tumor markers for predicting prognosis of patients with pancreatic ductal adenocarcinoma (PDAC).MethodsWe performed a bicentric, retrospective study including 155 eligible patients with PDAC. Patients were divided into a training group (n = 95), an internal validation group (n = 34), an external validation group (n = 26), and an entire validation group (n = 60). Cox regression analysis was conducted in the training group to identify independent prognostic factors to construct a nomogram for overall survival (OS) prediction. The performance of the nomogram was assessed in validation groups and through comparison with controlling nutritional status (CONUT) and prognostic nutrition index (PNI).ResultsThe least absolute shrinkage and selection operator (LASSO) regression, univariate and multivariate Cox regression analysis revealed that serum albumin and lymphocyte count were independent protective factors while CA19-9 and diabetes were independent risk factors. The concordance index (C-index) of the nomogram in the training, internal validation, external validation and entire validation groups were 0.777, 0.769, 0.759 and 0.774 respectively. The areas under curve (AUC) of the nomogram in each group were 0.861, 0.845, 0.773, and 0.814. C-index and AUC of the nomogram were better than those of CONUT and PNI in the training and validation groups. The net reclassification index (NRI), integrated discrimination improvement (IDI) and decision curve analysis showed improvement of accuracy of the nomogram in predicting OS and better net benefit in guiding clinical decisions in comparison with CONUT and PNI.ConclusionsThe nomogram incorporating four preoperative nutritional and tumor markers including serum albumin concentration, lymphocyte count, CA19-9 and diabetes mellitus could predict the prognosis more accurately than CONUT and PNI and may serve as a clinical decision support tool to determine what treatment options to choose.

scholarly journals Blood-based extracellular matrix biomarkers as predictors of survival in patients with metastatic pancreatic ductal adenocarcinoma receiving pegvorhyaluronidase alfa

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Song Wang ◽  
Cecilie L. Bager ◽  
Morten A. Karsdal ◽  
Dimitrios Chondros ◽  
Darin Taverna ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Pancreatic Ductal Adenocarcinoma ◽  
Cox Regression ◽  
Progression Free Survival ◽  
Predictive Biomarker ◽  
Ductal Adenocarcinoma ◽  
Type Iii Collagen ◽  
Ecm Remodeling ◽  
Cox Regression Analysis ◽  
Stage 1

Abstract Background Extensive extracellular matrix (ECM) remodeling is a hallmark of metastatic pancreatic ductal adenocarcinoma (mPDA). We investigated fragments of collagen types III (C3M, PRO-C3), VI (PRO-C6), and VIII (C8-C), and versican (VCANM) in plasma as biomarkers for predicting progression-free survival (PFS) and overall survival (OS) in patients with mPDA treated with pegvorhyaluronidase alfa, a biologic that degrades the ECM component hyaluronan (HA), in a randomized phase 2 study (HALO109-202). Methods HALO109-202 comprised a discovery cohort (Stage 1, n = 94) and a validation cohort (Stage 2, n = 95). Plasma ECM biomarkers were analyzed by ELISAs. Univariate Cox regression analysis and Kaplan–Meier plots evaluated predictive associations between biomarkers, PFS and OS in patients treated with pegvorhyaluronidase alfa plus nab-paclitaxel/gemcitabine (PAG) versus nab-paclitaxel/gemcitabine (AG) alone. Results PFS was improved with PAG vs. AG in Stage 1 patients with high C3M/PRO-C3 ratio (median cut-off): median PFS (mPFS) 8.0 vs. 5.3 months, P = 0.031; HR = 0.40; 95% CI 0.17–0.92). High C3M/PRO-C3 ratio was validated in Stage 2 patients by predicting a PFS benefit of PAG vs. AG (mPFS: 8.8 vs. 3.4 months, P = 0.046; HR = 0.46; 95% CI 0.21–0.98). OS was also improved in patients with high C3M/PRO-C3 ratio treated with PAG vs. AG (mOS 13.8 vs 8.5 months, P = 0.009; HR = 0.35; 95% CI 0.16–0.77). Interestingly, high C3M/PRO-C3 ratio predicted for a PFS benefit to PAG vs. AG both in patients with HA-low tumors (HR = 0.36; 95% CI 0.17–0.79) and HA-high tumors (HR = 0.20; 95% CI 0.06–0.69). Conclusions The C3M/PRO-C3 ratio measuring type III collagen turnover in plasma has potential as a blood-based predictive biomarker in patients with mPDA and provides additional value to a HA biopsy when applied for patient selection. Trial registration: NCT01839487. Registered 25 April 2016

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