scholarly journals PIK3CA Mutations in Diffuse Gliomas: An Update on Molecular Stratification, Prognosis, Recurrence, and Aggressiveness

2022 ◽  
Vol 16 ◽  
pp. 117955492110688
Author(s):  
Cheila Brito ◽  
Ana Tomás ◽  
Ana Azevedo ◽  
Susana Esteves ◽  
Manuela Mafra ◽  
...  
Keyword(s):  
Immune Cell ◽  
Synergistic Effects ◽  
Pik3ca Mutation ◽  
Response To Therapy ◽  
The Cancer Genome Atlas ◽  
Immune Microenvironment ◽  
Egfr Amplification ◽  
Pik3ca Mutations ◽  
Glioma Recurrence ◽  
Pten Deletion

Introduction: PIK3CA is one of the most mutated oncogenes in solid tumors. In breast cancer (ER-positive, HER2-negative), these events represent a predictive biomarker of response to alpelisib. In glioblastomas (GBM), PIK3CA mutations were described as early constitutive events. Here, we investigated PIK3CA mutational profile across glioma molecular subgroups and its relevance during glioma recurrence. Furthermore, PIK3CA mutations’ effect in PI3K pathway, prognosis, and response to therapy was also explored. Material and Methods: Exons 10 and 21 of PIK3CA mutations were evaluated in 394 gliomas and 19 glioma recurrences from Instituto Português de Oncologia Lisboa Francisco Gentil (IPOLFG) and compared with The Cancer Genome Atlas (TCGA) data. TIMER2.0 and NetMHCpan4.1 were used to assess the immune-microenvironment contribution. Results: PIK3CA mutations were identified among all glioma subgroups, although with no impact on their stratification or prognosis. In both cohorts (IPOLFG and TCGA), PIK3CA mutation frequencies in IDH-mutant and IDH-wild-type GBM were similar (IPOLFG: 9% and 3%; TCGA: 8% and 2%). These mutations were not mutually exclusive with PTEN deletion and EGFR amplification. Despite their reduced frequency, we discovered PIK3CA mutations were maintained during glioma recurrence regardless of administered therapies. The immune microenvironment might not contribute to this phenotype as PIK3CA mutations did not influence immune cell infiltration. Conclusions: Despite the absence of a predominant effect in glioma stratification, PIK3CA mutations were maintained during glioma recurrence, possibly contributing to glioma cell survival, representing promising therapeutic targets in recurrent glioma. Nevertheless, understanding the potential synergistic effects between PIK3CA mutations, PTEN deletion, and EGFR amplification is pivotal to targeted therapies’ efficiency.

scholarly journals P03.13 Evaluation of PIK3CA mutational status in glioma molecular subgroups

2019 ◽  
Vol 21 (Supplement_3) ◽  
pp. iii27-iii28
Author(s):  
C Brito ◽  
A Azevedo ◽  
S Esteves ◽  
C Martins ◽  
M Mafra ◽  
...  
Keyword(s):  
Who Classification ◽  
Response To Therapy ◽  
World Health ◽  
Diffuse Glioma ◽  
Molecular Subgroups ◽  
Egfr Amplification ◽  
Pik3ca Mutations ◽  
Mutational Status ◽  
Idh Mutations ◽  
Pten Deletion

Abstract BACKGROUND Gliomas are the most common and lethal malignant tumors of central nervous system. In 2016, World Health Organization (WHO) classification included IDH mutations and 1p/19q codeletion as diagnostic criteria to define glioma entities. However, new biomarkers for diagnosis, prognosis and response to therapy are needed. In this context, PIK3CA mutations have been described as constitutive mutations, which highlights their relevance in gliomas. Here we clarified the clinical relevance of PIK3CA mutations according to the 2016 WHO classification, the potential impact on diagnosis, prognosis, response to therapy, as well as their correlation with EGFR amplification and PTEN deletion. MATERIAL AND METHODS A cohort of 444 adult diffuse glioma samples from Instituto Português de Oncologia Lisboa Francisco Gentil (IPOLFG) was classified according to the 2016 WHO Classification. The mutational status of exon 9 and 20 of PIK3CA was evaluated in molecular subgroups of gliomas by Sanger sequencing. PTEN deletion and EGFR amplification were identified by Fluorescent in situ hybridization (FISH). RESULTS PIK3CA mutations showed a higher frequency in the subgroup of gliomas with IDH mutations and 1p/19q codeletion - oligodendrogliomas (10%). In Glioblastoma (GBM) IDH-mutant and IDH-wildtype these oncogenic mutations were observed in 9% and 3% of cases, respectively. Similar results were obtained using The Cancer Genome Atlas (TCGA) data, which was 8% and 2%, respectively. H1047R and E542K were the most frequent mutations identified in the glioma molecular subgroups. Importantly, we found 3 unreported pathogenic variants in exon 20 of PIK3CA (c.3112T>C, c.2988T>C, c.3040C>T) and one polymorphic variant (c.3210A>G). In addition, PIK3CA mutations, PTEN deletion and EGFR amplification were not mutually exclusive alterations in glioma molecular subgroups. For the first time in gliomas, it was identified the rs45455192 polymorphism at a frequency of 16% in astrocytomas IDH-mutant, 24% in oligodendrogliomas and 18% in both molecular subgroups of GBM, although this polymorphism did not have prognostic value. The analysis of PIK3CA mutations in glioma recurrences showed that these mutations are maintained during glioma progression. CONCLUSION In two independent cohorts (IPOLFG and TCGA), it was obtained similar frequencies of PIK3CA mutations in GBM molecular subgroups. In addition, these mutations are more relevant in less aggressive gliomas (IDH-mutated and 1p/19q codeleted). These alterations seem to be important in tumor maintenance and progression, which makes this gene a potential therapeutic target. In the future, we will investigate the effect of the in vitro pharmacological inhibition of PIK3CA in GBM mutant cell lines.

scholarly journals RNA N6-Methyladenosine Regulators Contribute to Tumor Immune Microenvironment and Have Clinical Prognostic Impact in Breast Cancer

2022 ◽  
Vol 12 ◽  
Author(s):  
Lan-Xin Mu ◽  
You-Cheng Shao ◽  
Lei Wei ◽  
Fang-Fang Chen ◽  
Jing-Wei Zhang
Keyword(s):  
Breast Cancer ◽  
Immune Cell ◽  
Risk Model ◽  
Cox Regression ◽  
Gene Set Enrichment Analysis ◽  
The Cancer Genome Atlas ◽  
Prognostic Impact ◽  
Immune Microenvironment ◽  
Model Based ◽  
Tumor Immune Microenvironment

Purpose: This study aims to reveal the relationship between RNA N6-methyladenosine (m6A) regulators and tumor immune microenvironment (TME) in breast cancer, and to establish a risk model for predicting the occurrence and development of tumors.Patients and methods: In the present study, we respectively downloaded the transcriptome dataset of breast cancer from Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA) database to analyze the mutation characteristics of m6A regulators and their expression profile in different clinicopathological groups. Then we used the weighted correlation network analysis (WGCNA), the least absolute shrinkage and selection operator (LASSO), and cox regression to construct a risk prediction model based on m6A-associated hub genes. In addition, Immune infiltration analysis and gene set enrichment analysis (GSEA) was used to evaluate the immune cell context and the enriched gene sets among the subgroups.Results: Compared with adjacent normal tissue, differentially expressed 24 m6A regulators were identified in breast cancer. According to the expression features of m6A regulators above, we established two subgroups of breast cancer, which were also surprisingly distinguished by the feature of the immune microenvironment. The Model based on modification patterns of m6A regulators could predict the patient’s T stage and evaluate their prognosis. Besides, the low m6aRiskscore group presents an immune-activated phenotype as well as a lower tumor mutation load, and its 5-years survival rate was 90.5%, while that of the high m6ariskscore group was only 74.1%. Finally, the cohort confirmed that age (p < 0.001) and m6aRiskscore (p < 0.001) are both risk factors for breast cancer in the multivariate regression.Conclusion: The m6A regulators play an important role in the regulation of breast tumor immune microenvironment and is helpful to provide guidance for clinical immunotherapy.

Molecular characteristics of complete PTEN loss in metastatic colorectal cancer.

2013 ◽  
Vol 31 (4_suppl) ◽  
pp. 389-389
Author(s):  
Zhi-Qin Jiang ◽  
Laurel Deaton ◽  
Nastaran Neishaboori ◽  
Jean-Nicolas Vauthey ◽  
Michael J. Overman ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Metastatic Colorectal Cancer ◽  
Cpg Island ◽  
Pik3ca Mutation ◽  
The Cancer Genome Atlas ◽  
Akt Pathway ◽  
Molecular Characteristics ◽  
Braf Mutations ◽  
Pik3ca Mutations ◽  
Pten Loss

389 Background: Loss of expression of phosphatase and tensin homolog (PTEN) is associated with activation of the PI3K/AKT pathway, and has been identified as a potential modulator of response to targeted therapies in metastatic colorectal cancer (mCRC). The association of PTEN loss with other molecular characteristics and outcomes has not been described for mCRC. Methods: Tumor from 229 mCRC patients (pts) were included for analysis of PTEN staining by IHC from whole-mounts, across two cohorts of unresectable mCRC or resectable liver-limited disease. PTEN loss was defined as complete loss of staining with preservation of expression of stromal components. Mutation status was defined using mass-spectroscopy or next-generation sequencing platforms. CpG island methylation (CIMP) status was determined using a previously defined 6-marker panel, with methylation of ≥3 markers denoting CIMP-High. Methylation findings where confirmed in 193 pts from the Cancer Genome Atlas (TCGA) database, using previously defined classifications. Results: The overall frequency of complete PTEN loss was 12% in the primary tumor and 15% when assayed from metastatic sites (P=NS). There was no difference in clinical characteristics in patients with PTEN-loss tumors. Complete PTEN loss and PIK3CA mutations were not mutually exclusive, with PIK3CA mutation rate of 24% and 14% in PTEN loss and intact, respectively (p=0.3). There was no association of PTEN loss with KRAS mutations (p=0.3), although there were non-significant trends toward higher rates of CIMP-High, BRAF mutations, and R-sided tumors (OR 3.2, 2.7, and 1.6, respectively; p<0.2), consistent with an association with the serrated adenoma pathway and epigenetic inactivation. In the TCGA, PTEN protein loss (defined by the lowest 12% of expression) was associated with increased AKT signaling (77% increase in pAKT/AKT, p<0.01) and a similar trend with CIMP-H tumors (OR 3.1, p=0.13). Conclusions: In patient samples, PTEN loss is independent of KRAS and PIK3CA mutations, associated with robust AKT pathway activation, and may be more prominent in tumors with hypermethylation and BRAF mutations.

Heterogeneous prevalence of somatic mutations in KRAS, BRAF, and PIK3CA genes among patients with colorectal carcinoma: Clues from Sardinia.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e14094-e14094
Author(s):  
Grazia Palomba ◽  
Maria Colombino ◽  
Antonio Silverio Contu ◽  
Bruno Massidda ◽  
Giovanni Baldino ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Pik3ca Mutation ◽  
Population Based ◽  
Incidence Rates ◽  
Response To Therapy ◽  
Cancer Genes ◽  
Pik3ca Mutations ◽  
Sardinian Population ◽  
Formalin Fixed Paraffin Embedded ◽  
Southern Island

e14094 Background: Role of KRAS, BRAF and PIK3CA mutations in pathogenesis of colorectal cancer (CRC) has been mostly clarified. In this population-based study, we investigated the incidence rates and roles for such somatic mutations in genetically isolated population of Sardinia. Methods: From April 2009 to July 2011, formalin-fixed paraffin-embedded tissues (n=478) were prospectively collected from Sardinian CRC patients at clinics across the entire island. Genomic DNA was isolated from tissue sections and screened for mutations in KRAS, BRAF, and PIK3CA genes by automated DNA sequencing. Results: Overall, KRAS tumor mutation rate was 30% (145/478 positive cases). Distribution of mutation carriers was however different within the island: 87/204 (43%) in North Sardinia vs. 58/274 (21%) in Middle-South Sardinia (p=0.023). Among 384 CRC cases whose DNA was available, only one (0.3%) mutation in BRAF gene was observed; conversely, PIK3CA was found mutated in 158/384 (41%) patients. An inverse distribution of PIK3CA mutation rates was observed within Sardinian population: 60/181 (33%) cases from northern vs. 98/203 (48%) cases from central-southern island. This heterogeneity in frequencies of KRAS/PIK3CA somatic mutations is consistent with already-reported discrepancies in distribution of germline mutations for other malignancies within Sardinian population. Preliminary clinical evaluation of 118 KRAS wild-type patients undergoing anti-EGFR-based treatment indicated lack of role for PIK3CA in predicting response to therapy in our series. Conclusions: Our findings further support the hypothesis that patients’ origin and genetic background may contribute to even determine the incidence rate of somatic mutations in candidate cancer genes.

scholarly journals Identification and validation of a hypoxia-related prognostic and immune microenvironment signature in bladder cancer

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Xianchao Sun ◽  
Zhen Zhou ◽  
Ying Zhang ◽  
Jinyou Wang ◽  
Xiaofeng Zhao ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Cancer Patients ◽  
Immune Cell ◽  
Immune Escape ◽  
Risk Group ◽  
The Cancer Genome Atlas ◽  
Pcr Analysis ◽  
Rna Seq ◽  
Immune Microenvironment ◽  
Related Risk

Abstract Background Bladder cancer is the leading causes of cancer-associated mortality and seriously affects population health. Hypoxia plays a key role in tumor development and immune escape, which contributes to malignant behaviors. Methods In this study, we analyzed the RNA-seq and clinical information of bladder cancer patients from The Cancer Genome Atlas (TCGA) database. To investigate the hypoxia-related prognostic and immune microenvironment in bladder cancer, we constructed a hypoxia-related risk model for overall survival (OS). The RNA-seq and clinical data of bladder cancer patients from the Gene Expression Omnibus (GEO) database were used as validation sets. Results The hypoxia-related risk signature was significantly correlated with clinical outcomes and could independently predict OS outcomes. Furthermore, the hypoxia-related risk signature could effectively reflected the levels of immune cell type fractions and the expression of critical immune checkpoint genes were higher in the high-risk group compared to the low-risk group. We also validated the expression levels of the prognostic genes in bladder cancer and paracancerous tissue samples through qRT-PCR analysis. Conclusion We established a 7 hypoxia-related gene (HRG) signature that can be used as an independent clinical predictor and provided a potential mechanism in bladder cancer immunotherapy.

scholarly journals An m6A-Related Prognostic Biomarker Associated With the Hepatocellular Carcinoma Immune Microenvironment

2021 ◽  
Vol 12 ◽  
Author(s):  
Yingxi Du ◽  
Yarui Ma ◽  
Qing Zhu ◽  
Tongzheng Liu ◽  
Yuchen Jiao ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Immune Checkpoint ◽  
Immune Cell ◽  
Cancer Genome ◽  
The Cancer Genome Atlas ◽  
Risk Scores ◽  
Survival Prediction ◽  
Consensus Clustering ◽  
Immune Microenvironment ◽  
Cluster 2

Background: N6-methyladenosine (m6A) is related to the progression of multiple cancers. However, the underlying influences of m6A-associated genes on the tumor immune microenvironment in hepatocellular carcinoma (HCC) remain poorly understood. Therefore, we sought to construct a survival prediction model using m6A-associated genes to clarify the molecular and immune characteristics of HCC.Methods: HCC case data were downloaded from The Cancer Genome Atlas (TCGA). Then, by applying consensus clustering, we identified two distinct HCC clusters. Next, four m6A-related genes were identified to construct a prognostic model, which we validated with Gene Expression Omnibus (GEO) and International Cancer Genome Consortium (ICGC) datasets. Additionally, the molecular and immune characteristics in different subgroups were analyzed.Results: m6A RNA methylation regulators were differentially expressed between HCC and normal samples and linked with immune checkpoint expression. Using consensus clustering, we divided HCC samples into two subtypes with distinct clinical features. Cluster 2 was associated with unfavorable prognosis, higher immune checkpoint expression and immune cell infiltration levels. In addition, the immune and carcinogenic signaling pathways were enriched in cluster 2. Furthermore, we constructed a risk model using four m6A-associated genes. Patients with different risk scores had distinct survival times, expression levels of immunotherapy biomarkers, TP53 mutation rates, and sensitivities to chemotherapy and targeted therapy. Similarly, the model exhibited an identical impact on overall survival in the validation cohorts.Conclusion: The constructed m6A-based signature may be promising as a biomarker for prognostics and to distinguish immune characteristics in HCC.

scholarly journals Comprehensive Analysis of m6A Regulators Characterized by the Immune Cell Infiltration in Head and Neck Squamous Cell Carcinoma to Aid Immunotherapy and Chemotherapy

2021 ◽  
Vol 11 ◽  
Author(s):  
Zhiqiang Yang ◽  
Xiaoping Ming ◽  
Shuo Huang ◽  
Minlan Yang ◽  
Xuhong Zhou ◽  
...  
Keyword(s):  
Overall Survival ◽  
Immune Cell ◽  
Comprehensive Evaluation ◽  
Principal Component ◽  
Response To Therapy ◽  
The Cancer Genome Atlas ◽  
Mutation Burden ◽  
Number Variation ◽  
Clinical Benefits ◽  
Cancer Genome Atlas

BackgroundN6-Methyladenosine (m6A), which is a prevalent regulator of mRNA expression, has gathered increasing study interests. Though the role of m6A as being important in many biological processes (such as growth and proliferation of cancers) has been well documented, its potential role in tumor immune microenvironment (TIME) has rarely been analyzed.MethodsWe downloaded RNA expression, single nucleotide polymorphism (SNP), and copy number variation (CNV) data from The Cancer Genome Atlas (TCGA). We then curated 21 m6A regulators and clustered patients into three m6A subtypes and m6A-related gene subtypes and compared them based on overall survival (OS). The combination of CIBERSORT as well as ssGSEA quantified the infiltration levels of immune cells and immune-related functions. The m6A scores were determined by using principal component analysis (PCA) algorithm. Furthermore, we evaluate the correlation of m6A regulators with immune and response to therapy.ResultsThree m6A clusters were identified based on the TCGA-HNSCC cohort, and there were significant associations among them in overall outcomes and caner-related pathways. We found that three m6A clusters were consistent with three phenotypes: immune-inflamed, immune-dessert, and immune-excluded. HNSCC patients were divided into high– and low–m6A score groups based on the cutoff of m6A score. Patients with lower m6A score had better overall survival outcome. Further analysis indicated that patients with higher m6A score presented higher tumor mutation burden (TMB). In addition, patients in low–m6A score subgroup had high chemotherapeutics sensitivity. GEO cohort confirmed patients with low m6A score demonstrated significant overall survival advantages and clinical benefits. Low m6A score carry an increased neoantigen load, eliciting a response to immunotherapy, and its value in predicting survival outcomes of immunotherapy was also confirmed in three anti-PD-1 cohorts.ConclusionsOur study demonstrated that m6A regulators are closely related to TIME and the m6A score was an effective prognostic biomarker and predictive indicator for immunotherapy and chemotherapeutics. Comprehensive evaluation of m6A regulators in tumors will extend our understanding of TIME and effectively guide increasing study investigations on immunotherapy and chemotherapy strategies for HNSCC.

scholarly journals BRAF Mutation as a Potential Therapeutic Target for Checkpoint Inhibitors: A Comprehensive Analysis of Immune Microenvironment in BRAF Mutated Colon Cancer

2021 ◽  
Vol 9 ◽  
Author(s):  
Shuyi Cen ◽  
Kun Liu ◽  
Yu Zheng ◽  
Jianzhen Shan ◽  
Chao Jing ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Braf Mutation ◽  
Plasma Cells ◽  
Immune Cell ◽  
Checkpoint Inhibitors ◽  
Treatment Strategies ◽  
Gene Expression Omnibus ◽  
The Cancer Genome Atlas ◽  
Immune Microenvironment ◽  
Tissue Specimens

BRAF mutated colon cancer presents with poor survival, and the treatment strategies are controversial. The tumor microenvironment, which plays a key role in tumorigenesis as well as responses to treatments, of this subtype is largely unknown. In the present study, we analyzed the differences of immune microenvironments between BRAF mutated and BRAF wild-type colon cancer utilizing datasets from The Cancer Genome Atlas and Gene Expression Omnibus and confirmed the findings by tissue specimens of patients. We found that BRAF mutated colon cancer had more stromal cells, more immune cell infiltration, and lower tumor purity. Many immunotherapeutic targets, including PD-1, PD-L1, CTLA-4, LAG-3, and TIM-3, were highly expressed in BRAF mutated patients. BRAF mutation was also correlated with higher proportions of neutrophils and macrophages M1, and lower proportions of plasma cells, dendritic cells resting, and T cells CD4 naïve. In conclusion, our study demonstrates a different pattern of the immune microenvironment in BRAF mutated colon cancer and provides insights into the future use of checkpoint inhibitors in this subgroup of patients.

scholarly journals Differential gene expression of tumor-infiltrating CD33+ myeloid cells in advanced- versus early-stage colorectal cancer

2020 ◽  
Author(s):  
Salman M. Toor ◽  
Rowaida Z. Taha ◽  
Varun Sasidharan Nair ◽  
Reem Saleh ◽  
Khaled Murshed ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Disease Progression ◽  
Immune Cell ◽  
Inflammatory Responses ◽  
Advanced Disease ◽  
Myeloid Cells ◽  
Gene Signature ◽  
Response To Therapy ◽  
The Cancer Genome Atlas ◽  
Disease Stages

Abstract Colorectal cancer (CRC) has high mortality rates, especially in patients with advanced disease stages, who often do not respond to therapy. The cellular components of the tumor microenvironment are essentially responsible for dictating disease progression and response to therapy. Expansion of different myeloid cell subsets in CRC tumors has been reported previously. However, tumor-infiltrating myeloid cells have both pro- and anti-tumor roles in disease progression. In this study, we performed transcriptomic profiling of cells of myeloid lineage (CD33+) from bulk CRC tumors at varying disease stages. We identified differentially expressed genes and pathways between CRC patients with advanced stage and early stages. We found that pro-angiogenic and hypoxia-related genes were upregulated, while genes related to immune and inflammatory responses were downregulated in CD33+ myeloid cells from patients with advanced stages, implying that immune cell recruitment and activation could be compromised in advanced disease stages. Moreover, we identified a unique “poor prognosis CD33+ gene signature” by aligning top upregulated and downregulated genes in tumor-infiltrating myeloid cells from our analyses with data from The Cancer Genome Atlas. Our results showed that this gene signature is an independent prognostic indicator for disease-specific survival in CRC patients, potentially reflecting its clinical importance.

scholarly journals Germline variants that influence the tumor immune microenvironment also drive response to immunotherapy

2021 ◽  
Author(s):  
Meghana Pagadala ◽  
Victoria H. Wu ◽  
Eva Perez-Guijarro ◽  
Hyo Kim ◽  
Andrea Castro ◽  
...  
Keyword(s):  
Cancer Risk ◽  
Immune Cell ◽  
Target Identification ◽  
Treatment Strategies ◽  
Immune Checkpoint Blockade ◽  
The Cancer Genome Atlas ◽  
Integrative Approach ◽  
Immune Microenvironment ◽  
Host Genetics ◽  
Tumor Immune Microenvironment

With the continued promise of immunotherapy as an avenue for treating cancer, understanding how host genetics contributes to the tumor immune microenvironment (TIME) is essential to tailoring cancer risk screening and treatment strategies. Using genotypes from over 8,000 European individuals in The Cancer Genome Atlas (TCGA) and 137 heritable tumor immune phenotype components (IP components), we identified and investigated 482 TIME associations and 475 unique TIME-associated variants. Many TIME-associated variants influence gene activities in specific immune cell subsets, such as macrophages and dendritic cells, and interact to promote more extreme TIME phenotypes. TIME-associated variants were predictive of immunotherapy response in human cohorts treated with immune-checkpoint blockade (ICB) in 3 cancer types, causally implicating specific immune-related genes that modulate myeloid cells of the TIME. Moreover, we validated the function of these genes in driving tumor response to ICB in preclinical studies. Through an integrative approach, we link host genetics to TIME characteristics, informing novel biomarkers for cancer risk and target identification in immunotherapy.

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