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PO-523 Unveiling the microRNA signature of gastric cancer exosomes: longitudinal and cross-sectional perspectives

Introduction There is no consensus regarding follow-up of Gastric Cancer (GC) patients. The available imaging tools and serological markers are poorly sensitive to monitor treatment response, minimal residual disease and tumour regrowth in a time-effective manner. We hypothesised that exosomes isolated from GC patients’ plasma contain specific small RNAs, useful to monitor tumour dynamics, likely to change during the therapy, anticipating disease relapse. Material and methods We designed a prospective study of the small RNA profile of exosomes isolated from plasma of four GC patients collected at three timepoints: before, soon- and late-after surgery. Exosomes were isolated and characterised by ultracentrifugation and nanoparticle tracking analysis. Exosome and tumour RNA was extracted and profiled using small-RNA sequencing technology (Ion Torrent). Bioinformatics analysis was performed using the tools bowtie2 and cufflinks for genome alignment, annotation and quantification. Analysis was focused on microRNAs (miRs), the most abundant class of small RNAs in these samples. Informed consent was given by all patients. Results and discussions On average, we detected 233 miRs in tumours and 175 in exosomes that were classified as present/absent and interrogated across samples. Cross-sectional analysis: same timepoint across patients. Longitudinal analysis: tumour and exosomes from the same patient. Cross-sectional analysis identified 49 miRs shared by all tumours, 33 by all exosomes collected before surgery, 83 by all exosomes collected after surgery. To pinpoint miRs useful to monitor tumour dynamics in each patient, three criteria were defined: 1) miRs present both in tumours and exosomes before surgery, and; 2) miRs present in tumours and absent in exosomes soon-after surgery. We found 133 miRs in patients A, 50 in B, 34 in C and 11 in D. Combining the cross-sectional and longitudinal analysis, we found two miRs fulfilling the above criteria that were shared by three out of four patients. These two miRs were still detected in Patient D exosomes soon after surgery, likely reflecting non-curative surgery. Validation of these data is currently ongoing in a larger series of patients. Conclusion Overall, this study pinpointed two miRs that may prove useful to monitor tumour dynamics and response to treatment in GC. The longitudinal analysis holds the promise of revealing a set of miRs with clinical utility for anticipating disease relapse, on a personalised manner.

Introduction Meningiomas are the most common primary intracranial brain tumour arising from meningeal tissue. Despite the majority of them displaying benign features, they can cause mild to severe morbidity. The current main therapeutic approach is complete tumour resection commonly with adjunct radiation therapy. However, tumour location can hamper complete resection and chemotherapies are ineffective. In this study we aim to elucidate the pathogenic signature of these tumours and identify novel molecular targets by deciphering the global proteome and phosphoprotein profile of different grades of meningiomas. Material and methods Tumour lysates were collected from grade I, II and III frozen meningioma specimens and three normal healthy human meninges. Phosphoprotein purification was performed using Qiagen ® PhosphoProtein Purification Kit.
Proteins were separated by SDS-PAGE followed by in-gel tryptic digestion. Extracted peptides were purified and analysed by electrospray ionisation LC-MS/MS. Raw mass spectrometry files were analysed using MaxQuant TM . Expression data were validated by Western blot and immunohistochemistry. In silico functional annotation of expression data was completed using Perseus 1.5.0.31 software suite, Ingenuity Pathway Analysis (IPA ® ) and DAVID 6.8.

Results and discussions
We have quantified 3888 proteins and 3074 phosphoproteins across all grades of meningioma and normal meninges. Comparative analysis identified 181 proteins and 338 phosphoproteins to be commonly significantly upregulated (log 2 fold-change !1.5; p<0.05) among all grades vs. normal meninges, and further identified differential expression profiles between meningioma grades. Expression data was validated on samples analysed by MS and on an additional cohort of meningiomas for upregulated proteins including SET, EGFR, SRSF1, CKAP4 and HK2; and phosphoproteins including AKT1, AKT2 and RB1. Gene Ontology revealed commonly upregulated proteins to be enriched in terms including RNA helicase activity, whilst phosphoproteins were enriched in the phosphoprotein associated terms of signal complex assembly, Rho guanyl-nucleotide exchange factor activity and EGFR signalling.
Conclusion In summary, we performed a comprehensive quantitative proteomic analysis from meningioma tissue of all WHO grades compared to healthy meninges and have identified several potential candidates that may hold therapeutic potential for targeted treatment of these tumours.
Introduction There is no consensus regarding follow-up of Gastric Cancer (GC) patients. The available imaging tools and serological markers are poorly sensitive to monitor treatment response, minimal residual disease and tumour regrowth in a time-effective manner. We hypothesised that exosomes isolated from GC patients' plasma contain specific small RNAs, useful to monitor tumour dynamics, likely to change during the therapy, anticipating disease relapse. Material and methods We designed a prospective study of the small RNA profile of exosomes isolated from plasma of four GC patients collected at three timepoints: before, soon-and late-after surgery. Exosomes were isolated and characterised by ultracentrifugation and nanoparticle tracking analysis. Exosome and tumour RNA was extracted and profiled using small-RNA sequencing technology (Ion Torrent). Bioinformatics analysis was performed using the tools bowtie2 and cufflinks for genome alignment, annotation and quantification. Analysis was focused on microRNAs (miRs), the most abundant class of small RNAs in these samples. Informed consent was given by all patients.
Results and discussions On average, we detected 233 miRs in tumours and 175 in exosomes that were classified as present/ absent and interrogated across samples. Cross-sectional analysis: same timepoint across patients. Longitudinal analysis: tumour and exosomes from the same patient. Cross-sectional analysis identified 49 miRs shared by all tumours, 33 by all exosomes collected before surgery, 83 by all exosomes collected after surgery. To pinpoint miRs useful to monitor tumour dynamics in each patient, three criteria were defined: 1) miRs present both in tumours and exosomes before surgery, and; 2) miRs present in tumours and absent in exosomes soon-after surgery. We found 133 miRs in patients A, 50 in B, 34 in C and 11 in D. Combining the cross-sectional and longitudinal analysis, we found two miRs fulfilling the above criteria that were shared by three out of four patients. These two miRs were still detected in Patient D exosomes soon after surgery, likely reflecting non-curative surgery. Validation of these data is currently ongoing in a larger series of patients.
Conclusion Overall, this study pinpointed two miRs that may prove useful to monitor tumour dynamics and response to treatment in GC. The longitudinal analysis holds the promise of revealing a set of miRs with clinical utility for anticipating disease relapse, on a personalised manner. Introduction Breast cancer is the leading cause of cancer death among women. The number of human diagnosed with breast cancer has been increased specially in middle and low countries. Early detection is a key to reduce cancer deaths. Biological fluids such as blood hold good opportunities to identify new non-invasive biomarkers. One the new and the promising classes of circulating cancer biomarkers is the microRNAs (miRNAs). These molecules are tissue-specific, are remarkably stable in several biological samples and have others intrinsic characteristics that make attractive as biomarkers. Thus to identify molecular signatures of circulating miRNAs that can be correlated with molecular subtypes of breast cancer in early stages can be an useful in clinical practice. We applied a new screening technology to determine the expression profile of circulating RNAs and to correlate molecular subtypes of early stage breast cancer and compare with healthy women. Material and methods Fifty-four patients with early-stage breast cancer paired with twenty-seven women without breast cancer risk (controls). Total RNA was obtained from serum of cases and controls and the expression profile of differentially expressed serum miRNAs was obtained using the nCounter panel from NanoString Technology. Statistical analyses were performed in program R. The interaction networks miRNAsgenes predicted were constructed in the Cytoscape program and the functional enrichment analysis was applied to identify