P02.07 Characterization of the tumor immune microenvironment of pediatric posterior fossa A ependymomas

BackgroundEpendymoma is the third most common brain tumor in children. At the moment, surgery and radiotherapy are the only effective treatments that can be offered, and despite this, a significant part of the patients relapse with no therapeutic salvage options. Therefore, new treatment modalities are needed. To develop immunotherapies for these children, knowledge of the tumor microenvironment is crucial. The current study aims to unravel the tumor immune microenvironment (TIME) of pediatric posterior fossa A (PFA) ependymomas.Materials and MethodsWe used bulk RNA sequencing data of 22 pediatric ependymomas. We defined two groups, hereafter called PFA immune+ (PFAI+) and PFAI-, based on the RNA expression levels of the NanoString panel of Human PanCancer Immune Profiling genes. We performed gene set enrichment analysis and deconvoluted the bulk RNA samples with ependymoma-specific single-cell RNA sequencing datasets. To validate our findings on a protein level, we applied immunohistochemistry with antibodies recognizing tumor-infiltrating lymphocytes, tumor-associated macrophages and microglia.ResultsUnsupervised hierarchical clustering of RNA expression of immune-related genes revealed two distinct PFA groups. Differential gene expression analysis showed that PFAI+ have a significantly higher expression of genes associated with immune functions, such as CD3E, CCR2, GZMA, CXCL9 and TRBC2. Accordingly, gene set enrichment analysis demonstrated that several immune pathways, including T-cell signalling, interferon-gamma response and TNFα signalling are enriched in PFAI+ ependymomas. RNA expression of immune checkpoints was also higher in PFAI+ tumors, indicating that these tumors might be more responsive to combinational therapies including immune checkpoint inhibitors. While immunohistochemistry showed low amounts of infiltrating CD3+, CD8+ and CD20+ cells, high numbers of CD163+ and HLA-DRA+ cells were detected. These cells were mainly located in regions of tumor necrosis. Increased amounts of CD4+ and CD8+ lymphocytes were present in PFAI+ tumors compared to PFAI- tumors. Deconvolution of the bulk RNA samples based on single-cell RNA sequencing data revealed an enrichment of myeloid cell populations, especially microglia and macrophages. Furthermore, PFAI+ tumors were found to contain significantly higher relative proportions of T-cells compared to PFAI- tumors (median of 3.76% for PFAI+ compared to 0.03% for PFAI-).ConclusionsWe suggest that pediatric posterior fossa A ependymomas can be divided into two groups based on the expression of immune-related genes, in which PFAI+ ependymomas are characterized by higher RNA expression levels of these genes and greater amounts of tumor-infiltrating immune cells. Several techniques showed an enrichment of T-lymphocytes in the PFAI+ ependymomas relative to the PFAI- ependymomas.Disclosure InformationJ. Lammers: None. F. Calkoen: None. M. Kranendonk: None. A. Federico: None. M. Kool: None. L. Kester: None. J. van der Lugt: None.

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Benchmarking supervised signature-scoring methods for single-cell RNA sequencing data in cancer

10.1101/2021.06.29.450404 ◽

2021 ◽

Author(s):

Siyuan Zheng ◽

Noureen Nighat ◽

Zhenqing Ye ◽

Yidong Chen ◽

Xiaojing Wang

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Enrichment Analysis ◽

Bulk Sample ◽

Gene Set Enrichment Analysis ◽

Sequencing Data ◽

Gene Set Enrichment ◽

Single Cell Rna Sequencing ◽

Supervised Methods ◽

Cell Data

Quantifying the activity of gene expression signatures is common in analyses of single-cell RNA sequencing data. Methods originally developed for bulk samples are often used for this purpose without accounting for contextual differences between bulk and single-cell data. More broadly, these methods have not been benchmarked. Here we benchmark four such supervised methods, including single sample gene set enrichment analysis (ssGSEA), AUCell, Single Cell Signature Explorer (SCSE), and a new method we developed, Jointly Assessing Signature Mean and Inferring Enrichment (JASMINE). Using cancer as an example, we show cancer cells consistently express more genes than normal cells. This imbalance leads to bias in performance by bulk-sample-based ssGSEA in gold standard tests and down sampling experiments. In contrast, single-cell-based methods are less susceptible. Our results suggest caution should be exercised when using bulk-sample-based methods in single-cell data analyses, and cellular contexts should be taken into consideration when designing benchmarking strategies.

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Uncovering the immune-modulating role of anti-RANKL therapy for cervical cancer: Preliminary results.

Journal of Clinical Oncology ◽

10.1200/jco.2020.38.15_suppl.e18028 ◽

2020 ◽

Vol 38 (15_suppl) ◽

pp. e18028-e18028

Author(s):

Peter Van Dam ◽

Yannick Verhoeven ◽

Jorrit De Waele ◽

Julie Jacobs ◽

Pieter-Jan Van Dam ◽

...

Keyword(s):

Cervical Cancer ◽

Rna Sequencing ◽

Immune Checkpoints ◽

Clinicopathological Parameters ◽

Sequencing Data ◽

Immune Microenvironment ◽

Preliminary Results ◽

Risk Of Death ◽

Increased Risk ◽

Tumor Immune Microenvironment

e18028 Background: Conventional treatments for cervical cancer (CC) have reached a plateau and only limited progress for targeted therapy has been made over the last decades, resulting in a meager five-year survival rate of only 17% for the advanced stages. To improve long-term benefits for the patient, a promising hot field of research in oncology that opens new perspectives is immunotherapy. Even though CC has shown to be immunogenic, only a minority of patients respond to this type of treatment. In recent years, the RANKL/RANK signaling pathway has been implicated as a key immune modulating factor in the tumor microenvironment, allowing the cancer cells to evade the immune response by disrupting the immune-intrinsic crosstalk. Both RANKL and RANK are highly co-expressed in CC, which correlates with inferior clinicopathological parameters and an increased risk of death. Targeting this pathway may therefore be of great value in the treatment of CC and the quest to release the brakes on the immune system, thereby reinvigorating the tumors’ susceptibility to immunotherapy. Hence, we aim to elucidate the effects of anti-RANKL therapy on the tumor-immune microenvironment in CC. Methods: Two cervical biopsies were taken before and after anti-RANKL therapy in CC patients. One fresh biopsy was immediately processed to a single cell suspension for flow cytometry (FCM) using enzymatic digestion, while the other was formalin-fixed and paraffin-embedded for immunohistochemistry (IHC) and RNA sequencing. For FCM and IHC, the samples were stained with different markers for RANK/L signaling, the immune infiltrate and immune checkpoints. FCM was performed on a BD FACSAria IIä cytometer and analyzed with FlowJo. IHC staining was performed on a Ventana Benchmark Ultra and Ventana Discovery Ultra and scored by a pathologist or by HistoScientist using Visiopharm, while RNA sequencing was performed with the Truseq RNA exome panel on the NextSeq 500 system. Results: Our preliminary results show a relative increase of the CD8+ population, while a trend is observed in increased lymphocyte activation after anti-RANKL therapy. Updated results will be presented in more detail at the conference, including RNA sequencing data. Conclusions: Preliminary findings indicate that anti-RANKL therapy modifies the tumor-immune microenvironment in CC. Higher patient accrual will allow to dissect targets for combination therapy with anti-RANKL to further optimize this treatment strategy.

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EPEN-10. UNRAVELLING THE TUMOR IMMUNE MICROENVIRONMENT OF POSTERIOR FOSSA A EPENDYMOMAS ON RNA AND PROTEIN EXPRESSION LEVELS

Neuro-Oncology ◽

10.1093/neuonc/noab090.060 ◽

2021 ◽

Vol 23 (Supplement_1) ◽

pp. i15-i15

Author(s):

Fenna F. Feenstra ◽

Friso Calkoen ◽

Johan M Kros ◽

Lennart Kester ◽

Mariëtte Kranendonk ◽

...

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Posterior Fossa ◽

Antigen Processing ◽

Sequence Data ◽

Expression Profiles ◽

Immune Microenvironment ◽

Expression Levels ◽

Tumor Immune Microenvironment ◽

Immune Related Genes

Abstract Background Ependymomas account for 8–10% of pediatric brain tumors, and the standard therapy of surgery and radiation has not changed for the past two decades. Characterization of the tumor immune microenvironment (TIME) is of great importance in order to find better therapies. However, the TIME of ependymomas is still not defined. In this retrospective observational study we aimed to unravel the TIME of ependymomas at mRNA and protein expression levels. Methods Ependymoma samples from two locations were selected: Posterior Fossa (PF-A, n=8), and supratentorial (ST, n=5). Targeted gene expression profile using the PanCancer immune profile panel of NanoString technology was performed. Data were analyzed using the nSolver software. In addition, 8 samples were subjected to RNA bulk sequencing, and the sequenced data were connected to the expression data of the same samples. To validate some of the findings, immunohistochemistry was performed. Results Unsupervised hierarchical clustering showed that PF-A ependymomas can be divided into two groups based on the expression of their immune-related genes. PF-A that showed high immune-expression clustered closely to the ST ependymomas. Significant expressions of genes related to “antigen-processing” and “adhesion” pathways were found in the immune-active groups. On the contrary, the PF-A that had low expressions of immune-related genes showed a high expression of BMI1 that has a prognostic and therapeutic value. Connecting gene expression to bulk sequence data validated the findings. In addition, immunohistochemical analysis confirmed that protein expression for some of the findings. Conclusion The TIME varies in ependymomas based on the location of the tumor. Moreover, the immune-related expression profiles indicated that PF-A ependymomas can be divided into two groups: immune-active and immune-not active PF-A. The prognostic and therapeutic values of the immune activity of PF-A should be further studied.

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Infiltration of Immunoinflammatory Cells and Related Chemokine/Interleukin Expression in Different Gastric Immune Microenvironments

Journal of Immunology Research ◽

10.1155/2020/2450569 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Ang Wang ◽

Siru Nie ◽

Zhi Lv ◽

Jing Wen ◽

Yuan Yuan

Keyword(s):

Gastric Cancer ◽

Gastric Mucosa ◽

Immune Cell ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Upward Trend ◽

Immune Microenvironment ◽

Gene Set Enrichment ◽

Expression Characteristic ◽

Precancerous Stage

Gastric mucosal immune microenvironment plays an important role in the occurrence and development of diseases such as inflammation and cancer. In the present study, single-sample gene set enrichment analysis (ssGSEA) was used to evaluate the expression of cytokines and the degree of immune cell infiltration in four different gastric mucosa tissues from normal gastric mucosa, simple gastritis, and atrophic gastritis to gastric cancer. Here, we show the immune microenvironments of these four gastric mucosae were significantly different. From inflammation to gastric cancer, most immunoinflammatory cells showed a downward trend such as central memory CD4 T cell. Instead, several cells showed an upward trend such as macrophage. Additionally, we found some chemokines/interleukins were illustrated to be low expressed (or highly expressed) in precancerous stage and highly expressed (or low expressed) in postcancerous stage, which demonstrated an opposite expression characteristic in pre-/postcancerous stage.

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LncRNA FOXP4-AS1 Promotes Progression of Ewing Sarcoma and Is Associated With Immune Infiltrates

Frontiers in Oncology ◽

10.3389/fonc.2021.718876 ◽

2021 ◽

Vol 11 ◽

Author(s):

Jiachao Xiong ◽

Liang Wu ◽

Lu Huang ◽

Chunyang Wu ◽

Zhiming Liu ◽

...

Keyword(s):

Ewing Sarcoma ◽

Poor Prognosis ◽

Es Cells ◽

Enrichment Analysis ◽

Extracellular Vesicle ◽

Gene Set Enrichment Analysis ◽

Immune Microenvironment ◽

Primary Bone Tumor ◽

Cerna Network ◽

Tumor Immune Microenvironment

Ewing sarcoma (ES) is a highly malignant primary bone tumor with poor prognosis. Studies have shown that abnormal expression of lncRNA influences the prognosis of tumor patients. Herein, we established that FOXP4-AS1 was up-regulated in ES and this correlated with poor prognosis. Further analysis illustrated that FOXP4-AS1 down-regulation repression growth, migration, along with invasion of ES. On the contrary, up-regulation of FOXP4-AS1 promoted the growth, migration, as well as invasion of ES. To explore the mechanism of FOXP4-AS1, Spearman correlation analysis was carried out to determine genes that were remarkably linked to FOXP4-AS1 expression. The potential functions and pathways involving FOXP4-AS1 were identified by GO analysis, Hallmark gene set enrichment analysis, GSEA, and GSVA. The subcellular fractionation results illustrated that FOXP4-AS1 was primarily located in the cytoplasm of ES cells. Then a ceRNA network of FOXP4-AS1 was constructed. Analysis of the ceRNA network and GSEA yielded two candidate mRNAs for FOXP4-AS1. Results of the combined survival analysis led us to speculate that FOXP4-AS1 may affect the expression of TMPO by sponging miR-298, thereby regulating the malignant phenotype of ES. Finally, we found that FOXP4-AS1 may modulates the tumor immune microenvironment in an extracellular vesicle-mediated manner. In summary, FOXP4-AS1 correlates with poor prognosis of ES. It promotes the growth, migration, as well as invasion of ES cells and may modulate the tumor immune microenvironment.

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NRAS Expression is Associated With Prognosis and Tumor Immune Microenvironment in Lung Adenocarcinoma

10.21203/rs.3.rs-862652/v1 ◽

2021 ◽

Author(s):

Yueren Yan ◽

Zhendong Gao ◽

Han Han ◽

Yue Zhao ◽

Yang Zhang ◽

...

Keyword(s):

T Cells ◽

Lung Adenocarcinoma ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

The Cancer Genome Atlas ◽

Expression Level ◽

Immune Microenvironment ◽

Kaplan Meier ◽

Tumor Immune Microenvironment ◽

Cox Analysis

Abstract Purpose: NRAS plays a pivotal role in progression of various kinds of somatic malignancies; however, the correlation between NRAS and lung adenocarcinoma is less known. We aim to analyze the prognostic value of NRAS expression in lung adenocarcinoma, and explore the relationship between NRAS and tumor immune microenvironment. Methods: We obtained the transcriptome pofiles and clinical data of LUAD from The Cancer Genome Atlas database and three Genome Expression Omnibus datasets. Specimens from 325 patients with completely resected lung adenocarcinoma were collected for immunohistochemical assays of NRAS, PD-L1, PD-1 and TIM-3. Then we performed gene set enrichment analysis to investigate cancer-related and immune-related signaling pathways. TIMER algorithms were performed to evaluate tumor immune infiltrating cells and immune-related biomarkers.Results: Compared with adjacent non-tumor tissue, NRAS expression was significantly upregulated in LUAD tissue. NRAS expression was significantly correlated with more advanced stage and positive lymph nodes. Kaplan-Meier curves and Cox analysis suggested that high NRAS expression led to a poor prognosis, and could be an independent prognostic factor in LUAD patients. Besides, NRAS expression was positively correlated with CD8+ T cells, macrophages, and neutrophils, and negatively correlated with B cells and CD4+ T cells. The expression level of NRAS was positively correlated with PD-L1, PD-1, and TIM-3 both at RNA and protein level. Conclusions: To conclude, we found NRAS a novel prognostic biomarker in LUAD. Besides, the expression level of NRAS may influence the prognosis of LUAD via various kinds of cancer-related pathways and remodeling TIM.

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An Immune Panel Signature Predicts Prognosis of Lung Adenocarcinoma Patients and Correlates With Immune Microenvironment

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.797984 ◽

2021 ◽

Vol 9 ◽

Author(s):

Yuan Zhou ◽

Lu Tang ◽

Yuqiao Chen ◽

Youyu Zhang ◽

Wei Zhuang

Keyword(s):

Lung Adenocarcinoma ◽

Risk Score ◽

Human Life ◽

Enrichment Analysis ◽

Progression Free Survival ◽

Roc Curves ◽

Gene Set Enrichment Analysis ◽

Sequencing Data ◽

Immune Microenvironment ◽

Immune Genes

Background: Lung cancer, especially lung adenocarcinoma (LUAD) with high incidence, seriously endangers human life. The immune microenvironment is one of the malignant foundations of LUAD, but its impact at the molecular level is incompletely understood.Method: A total of 34 LUAD samples from Xiangya Hospital were collected for immune oncology (IO) profiling. Univariate Cox analysis was performed to profile prognostic immune genes based on our immune panel sequencing data. The least absolute shrinkage and selection operator (LASSO) algorithm was applied to construct a risk signature. The cut-off threshold of risk score was determined using X-tile software. Kaplan–Meier survival curves and receiver operating characteristic (ROC) curves were employed to examine the performance of this risk signature for predicting prognosis. The immune infiltration was estimated using a single-sample gene set enrichment analysis (ssGSEA) algorithm.Result: Thirty-seven immune genes were profiled to be significantly correlated with the progression-free survival (PFS) in our cohort. Among them, BST2, KRT7, LAMP3, MPO, S100A8, and TRIM29 were selected to construct a risk signature. Patients with a higher risk score had a significantly shorter PFS (p = 0.007). Time-dependent ROC curves indicated that our risk signature had a robust performance in accurately predicting survival. Specifically, the 6-, 12-, and 18-month area under curve (AUC) was 0.800, 0.932, and 0.912, respectively. Furthermore, the risk signature was positively related to N stage, tumor stage, and tumor malignancy. These results were validated using two external cohorts. Finally, the risk signature was significantly and uniquely correlated with abundance of neutrophil.Conclusion: Our study revealed an immune panel-based signature that could predict the prognosis of LUAD patients and was associated with the infiltration of neutrophils.

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RNA sequencing in human HepG2 hepatocytes reveals PPAR-α mediates transcriptome responsiveness of bilirubin

Physiological Genomics ◽

10.1152/physiolgenomics.00028.2019 ◽

2019 ◽

Vol 51 (6) ◽

pp. 234-240 ◽

Cited By ~ 20

Author(s):

Darren M. Gordon ◽

Thomas M. Blomquist ◽

Scott A. Miruzzi ◽

Robert McCullumsmith ◽

David E. Stec ◽

...

Keyword(s):

Rna Sequencing ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

Acid Oxidation ◽

Peroxisome Proliferator ◽

Gene Set Enrichment ◽

Peroxisome Proliferator Activated Receptor ◽

False Discovery ◽

Oxidation Reduction ◽

Antioxidant Function

Bilirubin is a potent antioxidant that reduces inflammation and the accumulation of fat. There have been reports of gene responses to bilirubin, which was mostly attributed to its antioxidant function. Using RNA sequencing, we found that biliverdin, which is rapidly reduced to bilirubin, induced transcriptome responses in human HepG2 hepatocytes in a peroxisome proliferator-activated receptor (PPAR)-α-dependent fashion (398 genes with >2-fold change; false discovery rate P < 0.05). For comparison, a much narrower set of genes demonstrated differential expression when PPAR-α was suppressed via lentiviral shRNA knockdown (23 genes). Gene set enrichment analysis revealed the bilirubin-PPAR-α transcriptome mediates pathways for oxidation-reduction processes, mitochondrial function, response to nutrients, fatty acid oxidation, and lipid homeostasis. Together, these findings suggest that transcriptome responses from the generation of bilirubin are mostly PPAR-α dependent, and its antioxidant function regulates a smaller set of genes.

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Developmental programming in human umbilical cord vein endothelial cells following fetal growth restriction

Clinical Epigenetics ◽

10.1186/s13148-020-00980-9 ◽

2020 ◽

Vol 12 (1) ◽

Author(s):

Fieke Terstappen ◽

Jorg J. A. Calis ◽

Nina D. Paauw ◽

Jaap A. Joles ◽

Bas B. van Rijn ◽

...

Keyword(s):

Dna Methylation ◽

Endothelial Cells ◽

Rna Sequencing ◽

Fetal Growth ◽

Fetal Growth Restriction ◽

Enrichment Analysis ◽

Growth Restriction ◽

Gene Set Enrichment Analysis ◽

Gene Set Enrichment ◽

Gene Sets

Abstract Background Fetal growth restriction (FGR) is associated with an increased susceptibility for various noncommunicable diseases in adulthood, including cardiovascular and renal disease. During FGR, reduced uteroplacental blood flow, oxygen and nutrient supply to the fetus are hypothesized to detrimentally influence cardiovascular and renal programming. This study examined whether developmental programming profiles, especially related to the cardiovascular and renal system, differ in human umbilical vein endothelial cells (HUVECs) collected from pregnancies complicated by placental insufficiency-induced FGR compared to normal growth pregnancies. Our approach, involving transcriptomic profiling by RNA-sequencing and gene set enrichment analysis focused on cardiovascular and renal gene sets and targeted DNA methylation assays, contributes to the identification of targets underlying long-term cardiovascular and renal diseases. Results Gene set enrichment analysis showed several downregulated gene sets, most of them involved in immune or inflammatory pathways or cell cycle pathways. seven of the 22 significantly upregulated gene sets related to kidney development and four gene sets involved with cardiovascular health and function were downregulated in FGR (n = 11) versus control (n = 8). Transcriptomic profiling by RNA-sequencing revealed downregulated expression of LGALS1, FPR3 and NRM and upregulation of lincRNA RP5-855F14.1 in FGR compared to controls. DNA methylation was similar for LGALS1 between study groups, but relative hypomethylation of FPR3 and hypermethylation of NRM were present in FGR, especially in male offspring. Absolute differences in methylation were, however, small. Conclusion This study showed upregulation of gene sets related to renal development in HUVECs collected from pregnancies complicated by FGR compared to control donors. The differentially expressed gene sets related to cardiovascular function and health might be in line with the downregulated expression of NRM and upregulated expression of lincRNA RP5-855F14.1 in FGR samples; NRM is involved in cardiac remodeling, and lincRNAs are correlated with cardiovascular diseases. Future studies should elucidate whether the downregulated LGALS1 and FPR3 expressions in FGR are angiogenesis-modulating regulators leading to placental insufficiency-induced FGR or whether the expression of these genes can be used as a biomarker for increased cardiovascular risk. Altered DNA methylation might partly underlie FPR3 and NRM differential gene expression differences in a sex-dependent manner.

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Infiltrative tumour growth pattern correlates with poor outcome in oesophageal cancer

BMJ Open Gastroenterology ◽

10.1136/bmjgast-2020-000431 ◽

2020 ◽

Vol 7 (1) ◽

pp. e000431

Author(s):

Maelle Anciaux ◽

Pieter Demetter ◽

Roland De Wind ◽

Maria Gomez Galdon ◽

Sylvie Vande Velde ◽

...

Keyword(s):

Rna Sequencing ◽

Oesophageal Cancer ◽

Growth Pattern ◽

Prognostic Value ◽

Epithelial To Mesenchymal Transition ◽

Enrichment Analysis ◽

Gene Set Enrichment Analysis ◽

The Cancer Genome Atlas ◽

Sequencing Data ◽

Mesenchymal Transition

ObjectiveOesophageal cancer (OEC) is an aggressive disease with a poor survival rate. Prognostic markers are thus urgently needed. Due to the demonstrated prognostic value of histopathological growth pattern (HGP) in other cancers, we performed a retrospective assessment of HGP in patients suffering from invasive OEC.DesignA first cohort composed of 89 treatment-naïve operated patients with OEC from The Cancer Genome Atlas (TCGA) public database was constituted, from which H&E images and RNA-sequencing data were retrieved. Next, a second cohort composed of 99 patients with OEC treated and operated in a Belgian hospital was established. H&E-stained sections and extracted tumorous RNA were obtained from the samples. HGP were assessed on H&E slides as infiltrative (IGP) or expansive (EGP). TCGA RNA-sequencing data were analysed through the gene set enrichment analysis and Cytoscape softwares. Real-time quantitative PCR (qPCR) experiments were performed to assess gene expression in the Belgian cohort.ResultsIGP patients displayed a grim prognosis compared with EGP patients, while IGP was found as associated with numerous lymphovascular emboli and perinervous infiltrations. Analyses of the TCGA expression data showed that angiogenesis, epithelial-to-mesenchymal transition (EMT) and inflammation were significantly upregulated in IGP compared with EGP samples. qPCR experiments of three genes appearing as highly upregulated in each pathway showed no difference in expression according to the HGP.ConclusionThe current study demonstrates the poor prognostic value carried by IGP in OC and suggests angiogenesis, EMT and inflammation as key carcinogenetic pathways upregulated in this pattern.

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