BIOM-39. METHYLATION AND MUTATION PROFILES IN MENINGIOMA CELL-DERIVED EXTRACELLULAR VESICLE DNA REFLECT EPIGENETIC AND GENOMIC ALTERATIONS IN ORIGINAL TUMORS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi19-vi19
Author(s):  
Franz Ricklefs ◽  
Cecile Maire ◽  
Kathrin Wollmann ◽  
Lasse Dührsen ◽  
Krystian Fita ◽  
...  
Keyword(s):  
Tumor Resection ◽  
Extracellular Vesicle ◽  
Invasive Disease ◽  
Copy Number Variations ◽  
High Molecular Weight Dna ◽  
Molecular Alterations ◽  
Simpson Grade ◽  
Healthy Donors ◽  
Potential Biomarker ◽  
Almost All

Abstract The majority of meningiomas are benign but approximately 20% of display an aggressive behavior, resulting in significant patient morbidity and mortality. Standard monitoring after meningioma resection relies on serial MRI examinations, which are time-consuming, expensive and provide no information on molecular alterations that may indicate progression towards a more aggressive tumor. Extracellular vesicles (EVs) are released by tumor cells and contain high molecular weight DNA, rendering circulating EVs a potential biomarker source for non-invasive disease monitoring and for obtaining information on genetic and epigenetic alterations. We quantified EVs in plasma of 46 meningioma patients (n = 29 M1, 12 M2, 5 M3) by nanoparticle tracking analysis and detected significantly higher levels compared to age-matched healthy donors (n = 18). EV concentrations correlated with malignancy grade (p = 0.0049) and with the extent of peritumoral edema (p = 0.0031). Comparisons between paired pre- and postoperative samples revealed that EV levels counts dropped significantly the day after tumor resection and were reduced to normal levels after about one week. Completely resected patients (Simpson grade I) displayed a greater reduction of postoperative EV concentrations than incompletely resected patients. DNA methylation profiling was performed on EVs secreted by cultured meningioma cells, as well as matched cells and original tumors using 850k arrays (n = 7 M1, 5 M2, 3 M3). All EV samples were correctly identified as meningiomas by the Heidelberg classifier, and methylation subclasses were also correctly assigned in almost all cases. t-SNE analysis showed that EVs mapped in close proximity to their corresponding parental cells and tumor tissue. Tumor specific mutations and copy number variations were detected in EV-DNA with high accuracy. Differential quantitative proteomic analysis of EVs, cells and tumors identified shared proteins that could potentially be useful for enriching tumor-derived circulating EVs from biofluids.

scholarly journals GENE-22. GENOME-WIDE METHYLATION PROFILING OF GLIOBLASTOMA EXTRACELLULAR VESICLE DNA ALLOWS TUMOR CLASSIFICATION

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi102-vi102
Author(s):  
Franz Ricklefs ◽  
Cecile Maire ◽  
Katharina Kolbe ◽  
Mareike Holz ◽  
Manfred Westphal ◽  
...  
Keyword(s):  
Copy Number ◽  
Methylation Status ◽  
Extracellular Vesicle ◽  
Copy Number Variations ◽  
Tumor Classification ◽  
Cns Tumors ◽  
Original Tumor ◽  
Genome Wide ◽  
Potential Biomarker ◽  
Methylation Profiling

Abstract BACKGROUND Genome-wide methylation profiling has recently been developed into a tool that allows subtype tumor classification in central nervous system (CNS) tumors. Extracellular vesicles (EVs) are released by CNS tumor cells and contain high molecular weight tumor DNA, rendering EVs a potential biomarker source to identify tumor subgroups, stratify patients and monitor therapy by liquid biopsy. We investigated whether the DNA in glioma-derived EVs reflects genome-wide tumor methylation profiles and allows tumor subtype classification. METHODS DNA was isolated from EVs secreted by cultured glioma stem-like cells (GSC) as well as from the cells of origin and from the original tumor samples (n=3 patients). EVs were classified by nanoparticle analysis (NTA), immunoblotting, imaging flow cytometry (IFCM), multiplex EV assays and electron microscopy. Genome-wide DNA methylation profiling was performed using an 850k Illumina EPIC array and results were classified according to the DKFZ brain tumor classifier. RESULTS The size range of GSC-derived EVs was 120–150 nm, as measured by NTA. The majority of secreted EVs exhibited high expression of common EV markers (i.e. CD9, CD63 and CD81), as characterized by IFCM and multiplex EV assays. Genome-wide methylation profiling of GSC-derived EVs correctly identified the methylation class of the original tumor, including information on the IDH mutation status and subclass classification (RTK1, RTK2). In addition, copy number alterations and the MGMT metyhlation status matched the pattern of the parental GSCs and original tumor samples. CONCLUSION EV DNA faithfully reflects the tumor methylation class as well as the MGMT methylation status and copy number variations present in the parental cells and the original tumor. Methylation profiling of circulating tumor EV DNA could become a useful tool to detect and classify CNS tumors.

scholarly journals Serum IL-35 is decreased in overweight patients with rheumatoid arthritis: its correlation with Th1/Th2/Th17-related cytokines

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Yuxuan Li ◽  
Yang Jie ◽  
Xiaofei Wang ◽  
Jing Lu
Keyword(s):  
Rheumatoid Arthritis ◽  
Medical Records ◽  
Clinical Information ◽  
Serum Cytokines ◽  
Healthy Donors ◽  
Potential Biomarker ◽  
Tnf Α ◽  
Ifn Γ ◽  
Quantitative Changes ◽  
Anti Inflammatory Cytokine

Abstract Background Obesity is correlated with worse drug responses and high disease activity in patients with rheumatoid arthritis (RA). Interleukin (IL)-35 is a novel anti-inflammatory cytokine that mainly produced by regulatory T (Treg). This study was performed to analyze whether IL-35 was correlated with obesity in RA and investigate the correlation between other Th1/Th2/Th17-related cytokines and obesity in RA. Results The serum IL-35 level was analyzed in RA (n = 81) and healthy donors (n = 53) by ELISA assay, and was compared between three groups (body mass index (BMI) < 18.5,≥18.5 to 25, > 25). Serum cytokines including IL-2, IL-4, IL-10, IL-17, INF-γ, TNF-α levels were measured using Flowcytometry assay. Clinical information was extracted from medical records. Serum IL-35 level in overweight patients were significantly decreased than those in lean patients. Furthermore, Th1/Th2/Th17-related cytokines from overweight patients with RA showed the characteristic immunological features. Serum IL-6, IL-17 and TNF-α levels were positively correlated with BMI. However, serum IL-2, IL-4, IL-10 and IFN-γ concentrations were not correlated with BMI. Conclusions Quantitative changes in serum IL-35 level were characteristic in overweight patients with RA. These findings indicate that IL-35 plays an important role in the development of RA and may prove to be a potential biomarker of active RA.

scholarly journals Molecular Alterations in Sporadic and SOD1-ALS Immortalized Lymphocytes: Towards a Personalized Therapy

2021 ◽  
Vol 22 (6) ◽  
pp. 3007
Author(s):  
Isabel Lastres-Becker ◽  
Gracia Porras ◽  
Marina Arribas-Blázquez ◽  
Inés Maestro ◽  
Daniel Borrego-Hernández ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Therapeutic Interventions ◽  
Autophagic Flux ◽  
Metabolic Disturbances ◽  
Molecular Alterations ◽  
Healthy Donors ◽  
Inflammatory Profile ◽  
Als Patients

Amyotrophic lateral sclerosis (ALS) is a fatal neurological condition where motor neurons (MNs) degenerate. Most of the ALS cases are sporadic (sALS), whereas 10% are hereditarily transmitted (fALS), among which mutations are found in the gene that codes for the enzyme superoxide dismutase 1 (SOD1). A central question in ALS field is whether causative mutations display selective alterations not found in sALS patients, or they converge on shared molecular pathways. To identify specific and common mechanisms for designing appropriate therapeutic interventions, we focused on the SOD1-mutated (SOD1-ALS) versus sALS patients. Since ALS pathology involves different cell types other than MNs, we generated lymphoblastoid cell lines (LCLs) from sALS and SOD1-ALS patients and healthy donors and investigated whether they show changes in oxidative stress, mitochondrial dysfunction, metabolic disturbances, the antioxidant NRF2 pathway, inflammatory profile, and autophagic flux. Both oxidative phosphorylation and glycolysis appear to be upregulated in lymphoblasts from sALS and SOD1-ALS. Our results indicate significant differences in NRF2/ARE pathway between sALS and SOD1-ALS lymphoblasts. Furthermore, levels of inflammatory cytokines and autophagic flux discriminate between sALS and SOD1-ALS lymphoblasts. Overall, different molecular mechanisms are involved in sALS and SOD1-ALS patients and thus, personalized medicine should be developed for each case.

Proteomic Profile of Urinary Extracellular Vesicles Identifies AGP1 as a Potential Biomarker of Primary Aldosteronism

Endocrinology ◽  
2021 ◽  
Vol 162 (4) ◽  
Author(s):  
Eric R Barros ◽  
Juan Pablo Rigalli ◽  
Alejandra Tapia-Castillo ◽  
Andrea Vecchiola ◽  
Morag J Young ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Primary Aldosteronism ◽  
Proteomic Analysis ◽  
Characteristic Curve ◽  
Area Under The Curve ◽  
Extracellular Vesicle ◽  
Parent Cell ◽  
Spot Urine ◽  
Potential Biomarker ◽  
Aldosterone To Renin Ratio

Abstract Context Primary aldosteronism (PA) represents 6% to 10% of all essential hypertension patients and is diagnosed using the aldosterone-to-renin ratio (ARR) and confirmatory studies. The complexity of PA diagnosis encourages the identification of novel PA biomarkers. Urinary extracellular vesicles (uEVs) are a potential source of biomarkers, considering that their cargo reflects the content of the parent cell. Objective We aimed to evaluate the proteome of uEVs from PA patients and identify potential biomarker candidates for PA. Methods Second morning spot urine was collected from healthy controls (n = 8) and PA patients (n = 7). The uEVs were isolated by ultracentrifugation and characterized. Proteomic analysis on uEVs was performed using LC-MS Orbitrap. Results Isolated uEVs carried extracellular vesicle markers, showed a round shape and sizes between 50 and 150 nm. The concentration of uEVs showed a direct correlation with urinary creatinine (r = 0.6357; P = 0.0128). The uEV size mean (167 ± 6 vs 183 ± 4nm) and mode (137 ± 7 vs 171 ± 11nm) was significantly smaller in PA patients than in control subjects, but similar in concentration. Proteomic analysis of uEVs from PA patients identified an upregulation of alpha-1-acid glycoprotein 1 (AGP1) in PA uEVs, which was confirmed using immunoblot. A receiver operating characteristic curve analysis showed an area under the curve of 0.92 (0.82 to 1; P = 0.0055). Conclusion Proteomic and further immunoblot analyses of uEVs highlights AGP1 as potential biomarker for PA.

Retrosigmoid intradural suprameatal approach to Meckel's cave and the middle fossa: surgical technique and outcome

2000 ◽  
Vol 92 (2) ◽  
pp. 235-241 ◽  
Author(s):  
Madjid Samii ◽  
Marcos Tatagiba ◽  
Gustavo A. Carvalho
Keyword(s):  
Tumor Resection ◽  
Neurological Deficits ◽  
Subtotal Resection ◽  
Middle Fossa ◽  
Meckel’S Cave ◽  
Content Type ◽  
Simpson Grade ◽  
Petroclival Meningiomas ◽  
Meckel's Cave ◽  
Fine Print

Object. The goal of this study was to determine whether some petroclival tumors can be safely and efficiently treated using a modified retrosigmoid petrosal approach that is called the retrosigmoid intradural suprameatal approach (RISA).Methods. The RISA was introduced in 1983, and since that time 12 patients harboring petroclival meningiomas have been treated using this technique. The RISA includes a retrosigmoid craniotomy and drilling of the suprameatus petrous bone, which is located above and anterior to the internal auditory meatus, thus providing access to Meckel's cave and the middle fossa.Radical tumor resection (Simpson Grade I or II) was achieved in nine (75%) of the 12 patients. Two patients underwent subtotal resection (Simpson Grade III), and one patient underwent complete resection of tumor at the posterior fossa with subtotal resection at the middle fossa. There were no deaths or severe complications in this series; all patients did well postoperatively, being independent at the time of their last follow-up examinations (mean 5.6 years). Neurological deficits included facial paresis in one patient and worsening of hearing in two patients.Conclusions. Theapproach described here is a useful modification of the retrosigmoid approach, which allows resection of large petroclival tumors without the need for supratentorial craniotomies. Although technically meticulous, this approach is not time-consuming; it is safe and can produce good results. This is the first report on the use of this approach for petroclival meningiomas.

scholarly journals Carcinosarcoma of the prostate: case report with molecular and histological characterization

2018 ◽  
Vol 33 (4) ◽  
pp. 540-544 ◽  
Author(s):  
Samanta Salvi ◽  
Valentina Casadio ◽  
Filippo Martignano ◽  
Giorgia Gurioli ◽  
Maria Maddalena Tumedei ◽  
...  
Keyword(s):  
Copy Number Variation ◽  
Copy Number ◽  
Copy Number Variations ◽  
Molecular Characteristics ◽  
Glutathione S Transferase ◽  
Molecular Alterations ◽  
Positive Expression ◽  
Molecular Pattern ◽  
Programmed Death ◽  
Number Variation

Background: We report a case of prostatic carcinosarcoma, a rare variant of prostatic cancer, which is composed of a mixture of epithelial and mesenchymal components with a generally poor outcome. Aims and methods: We aim to identify molecular alterations, in particular copy number variations of AR and c -MYC genes, methylation and expression of glutathione S-transferase P1 (GSTP1), programmed death-ligand 1 (PD-L1), AR, and phosphorylated AR expression. Results: We found a distinct molecular pattern between adenocarcinoma and carcinosarcoma, which was characterized by high AR copy number variation gain; positive expression of PD-L1, AR, and phosphorylated AR; low espression of GSTP1 in epithelial component. The sarcomatoid component had a lower gain of the AR gene, and no expression of PD-L1, AR, phosphorylated AR, or GSTP1. Both components had a gain of c-MYC copy number variation. Conclusions: Our findings suggest that carcinosarcoma has specific molecular characteristics that could be indicative for early diagnosis and treatment selection.

scholarly journals A Novel Microfluidic Chip for Fast, Sensitive Quantification of Plasma Extracellular Vesicles as Biomarkers in Patients With Osteosarcoma

2021 ◽  
Vol 11 ◽  
Author(s):  
Yi-Qi Xu ◽  
Qi-Yuan Bao ◽  
Sai-Xi Yu ◽  
Qi Liu ◽  
Yan Xie ◽  
...  
Keyword(s):  
Microfluidic Chip ◽  
Limit Of Detection ◽  
Zno Nanorods ◽  
Extracellular Vesicle ◽  
Fluorescent Labeling ◽  
Total Plasma ◽  
Sarcoma Cell ◽  
Diagnosis And Prognosis ◽  
Healthy Donors ◽  
Capture Antibody

Plasma circulating extracellular vesicle (EV) has emerged as a promising biomarker for diagnosis and prognosis of various epithelial tumors. However, fast and efficient capture of EVs with microfluidic chip in sarcoma remains to be established. Herein, we reported a ZnO-nanorods integrated (ZNI) microfluidic chip, where EV capture antibody was uniformly grafted to the surface of the ZnO-nanorods of the chip to enhance the plasma turbulence formation and the capture efficiency at the micro-scale. Based on osteosarcoma (OS) cell line, we demonstrated that a combination of CD81 and CD63 antibody on ZNI chip yielded the greatest amount of total EVs, with an extra sensitive limit of detection (LOD) of ~104 particles mL-1. Furthermore, the addition of fluorescent labeling of Vimentin (VIM), a previously reported sarcoma cell surface biomarker, could enabled the dual visualization of total plasma EVs and VIM-positive EVs from OS patients’ plasma. Based on our ZNI chip, we found that the amount of plasma total EVs was significantly different between OS and healthy donors (1562 a.u. versus 639 a.u., p&lt; 0.05), but not between metastatic and nonmetastatic OS (p&gt; 0.05). Interestingly, patients with metastatic disease had a significantly greater amount of VIM-positive EVs (1411 a.u. versus 231 a.u.., p&lt; 0.05) and increased VIM-positive/total EVs ratio (0.943 versus 0.211, p&lt; 0.05) in comparison with the nonmetastatic counterpart. Therefore, our ZNI microfluidic chip has great potential for the fast quantification of plasma EVs, and the microfluidic-based quantification of total and VIM-positive EVs might serve as a promising biomarker for the diagnosis and surveillance in OS patients.

scholarly journals Resistance to miltefosine results from amplification of the RTA3 floppase or inactivation of flippases in Candida parapsilosis

2021 ◽  
Author(s):  
Sean Bergin ◽  
Fang Zhao ◽  
Adam P Ryan ◽  
Carolin A Müller ◽  
Conrad A Nieduszynski ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Copy Number ◽  
Candida Parapsilosis ◽  
Copy Number Variations ◽  
Loss Of Function ◽  
Adaptive Laboratory Evolution ◽  
Pathogenic Yeast ◽  
Lipid Asymmetry ◽  
Evolution Experiment ◽  
Almost All

Flippases and floppases are two classes of proteins that have opposing functions in the maintenance of lipid asymmetry of the plasma membrane. Flippases translocate lipids from the exoplasmic leaflet to the cytosolic leaflet, and floppases act in the opposite direction. Phosphatidylcholine (PC) is a major component of the eukaryotic plasma membrane and is asymmetrically distributed, being more abundant in the exoplasmic leaflet. Here we show that gene amplification of a putative PC floppase or double disruption of two PC flippases in the pathogenic yeast Candida parapsilosis results in resistance to miltefosine, an alkylphosphocholine drug that affects PC metabolism that has recently been granted orphan drug designation approval by the US FDA for treatment of invasive candidiasis. We analysed the genomes of 170 C. parapsilosis isolates and found that 107 of them have copy number variations (CNVs) at the RTA3 gene. RTA3 encodes a putative PC floppase whose deletion is known to increase the inward translocation of PC in Candida albicans. RTA3 copy number ranges from 2 to >40 across the C. parapsilosis isolates. Interestingly, 16 distinct CNVs with unique endpoints were identified, and phylogenetic analysis shows that almost all of them have originated only once. We found that increased copy number of RTA3 correlates with miltefosine resistance. Additionally, we conducted an adaptive laboratory evolution experiment in which two C. parapsilosis isolates were cultured in increasing concentrations of miltefosine over 26 days. Two genes, CPAR2_303950 and CPAR2_102700, gained homozygous protein-disrupting mutations in the evolved strains and code for putative PC flippases homologous to S. cerevisiae DNF1. Our results indicate that alteration of lipid asymmetry across the plasma membrane is a key mechanism of miltefosine resistance. We also find that C. parapsilosis is likely to gain resistance to miltefosine rapidly, because many isolates carry loss-of-function alleles in one of the flippase genes.

scholarly journals Genetic characterization of colorectal cancer using a next generation sequencing approach to identify a specific pattern of somatic alterations in tumors arising from different anatomic sites

2016 ◽  
Author(s):  
Carmelo Laudanna ◽  
Gianluca Santamaria ◽  
Simona Migliozzi ◽  
Duarte Mendes Oliveira ◽  
Donatella Malanga ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Copy Number ◽  
Treatment Options ◽  
Copy Number Variations ◽  
Specific Pattern ◽  
Surgical Approaches ◽  
Anatomical Location ◽  
Molecular Alterations ◽  
Somatic Alterations ◽  
Novel Biomarkers

Colorectal cancer (CRC) is the third leading cause of cancer-related deaths worldwide, with nearly 1.4 million new cases diagnosed in 2012. CRC results from the accumulation of multiple genetic and epigenetic aberrations. Tumor localization in the large intestine tract determines different surgical approaches and treatment options. Considering the heterogeneous nature of these tumors we hypothesized that different patterns of molecular alterations could be associated with a specific anatomical location. To identify distinct genomic alterations (e.g, copy number variations and mutations) associated to different CRC anatomical sites we sequenced 32 CRCs samples from different location (right-sided, left-sided etc.) using the Ion AmpliSeq™ Comprehensive Cancer Panel that covered the whole coding sequence of 409 tumor suppressor genes and oncogenes frequently altered in cancer. Interestingly left-sided tumors were generally more altered respect to right-sided ones. Cluster analysis of all samples allowed the identification of 21-gene core that were significantly mutated in all sample groups. As expected, KRAS and APC mutations were frequently in the tumors resected from different anatomical localizations. Unsupervised analysis of copy number variations reveals a core of 160-gene significantly altered. In addition to the expected SRC, MYC and CEBPA, we found interestingly genes in validation status. Despite missing a significant number of cases, gene panel provides a solid alternative approach to WES in order to characterize a signature of alterations correlated with CRC tumor and the identification of novel biomarkers in colorectal carcinoma that could be used as potential clinical target.

scholarly journals Systematic identification of mutations and copy number alterations associated with cancer patient prognosis

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Joan C Smith ◽  
Jason M Sheltzer
Keyword(s):  
Copy Number ◽  
Meta Analysis ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Driver Genes ◽  
Cancer Driver ◽  
Molecular Alterations ◽  
Patient Prognosis ◽  
Almost All ◽  
Systematic Identification

Successful treatment decisions in cancer depend on the accurate assessment of patient risk. To improve our understanding of the molecular alterations that underlie deadly malignancies, we analyzed the genomic profiles of 17,879 tumors from patients with known outcomes. We find that mutations in almost all cancer driver genes contain remarkably little information on patient prognosis. However, CNAs in these same driver genes harbor significant prognostic power. Focal CNAs are associated with worse outcomes than broad alterations, and CNAs in many driver genes remain prognostic when controlling for stage, grade, TP53 status, and total aneuploidy. By performing a meta-analysis across independent patient cohorts, we identify robust prognostic biomarkers in specific cancer types, and we demonstrate that a subset of these alterations also confer specific therapeutic vulnerabilities. In total, our analysis establishes a comprehensive resource for cancer biomarker identification and underscores the importance of gene copy number profiling in assessing clinical risk.

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