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Transfer RNAs (tRNAs) are small adaptor RNAs essential for mRNA translation. Alterations in the cellular tRNA population can directly affect mRNA decoding rates and translational efficiency during cancer development and progression. To evaluate changes in the composition of the tRNA pool, multiple sequencing approaches have been developed to overcome reverse transcription blocks caused by the stable structures of these molecules and their numerous base modifications. However, it remains unclear whether current sequencing protocols faithfully capture tRNAs existing in cells or tissues. This is specifically challenging for clinical tissue samples that often present variable RNA qualities. For this reason, we developed ALL-tRNAseq, which combines the highly processive MarathonRT and RNA demethylation for the robust assessment of tRNA expression, together with a randomized adapter ligation strategy prior to reverse transcription to assess tRNA fragmentation levels in both cell lines and tissues. Incorporation of tRNA fragments not only informed on sample integrity but also significantly improved tRNA profiling of tissue samples. Our data showed that our profiling strategy effectively improves classification of oncogenic signatures in glioblastoma and diffuse large B-cell lymphoma tissues, particularly for samples presenting higher levels of RNA fragmentation, further highlighting the utility of ALL-tRNAseq for translational research.

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Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets.Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)–educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography.Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients.Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721–33. ©2017 AACR.

Dysregulated extracellular vesicle (EV) release has been implicated in various pathologies, including cancer, neurodegenerative disease and osteoarthritis. Despite clear therapeutic potential, drug screening for EV release modulators has yielded limited success due to the lack of a sensitive and scalable EV read-out system. Here, we employed CRISPR-Cas9 to engineer HEK293 cells expressing HA-NanoLuciferase-(NL)-tagged endogenous CD63. We found that under basal culture conditions, CD63-containing EVs are released via a mechanism that is independent of the exocytic SNARE protein SNAP23, presumably by direct budding from the plasma membrane. Endo-lysosome inhibition by chemical or genetic perturbation of vATPase strongly increased SNAP23 and nSmase2-dependent exosome secretion from intracellular compartments. Proteomic analysis revealed these exosomes are enriched for early- and late endosomal markers, but also for autophagosomal proteins. This suggests that a proportion of these exosomes originate from amphisomes, although chemical inhibition of canonical autophagy did not affect exosome secretion upon lysosome inhibition. Using a broad- spectrum kinase inhibitor screen, we identified and subsequently validated the lipid kinase PI4KIIIβ as a critical mediator of exosome secretion and amphisome-mediated secretory autophagy, upon lysosome inhibition. We conclude that tagging of endogenous CD63 with NanoLuciferase represents a sensitive, scalable reporter strategy that enables identification of (druggable) modulators of EV biogenesis and release under physiological and pathological conditions.

Somatic copy number alterations can be detected in cell-free DNA (cfDNA) by shallow whole genome sequencing (sWGS). PCR is typically included in library preparations, but a PCR-free method could serve as a high-throughput alternative. To evaluate a PCR-free method for research and diagnostics, archival peripheral blood or bone marrow plasma samples, collected in EDTA- or lithium-heparin–containing tubes, were collected from patients with non–small-cell lung cancer (n = 10 longitudinal samples; 4 patients), B-cell lymphoma (n = 31), and acute myeloid leukemia (n = 15), or from healthy donors (n = 14). sWGS was performed on PCR-free and PCR library preparations, and the mapping quality, percentage of unique reads, genome coverage, fragment lengths, and copy number profiles were compared. The percentage of unique reads was significantly higher for PCR-free method compared with PCR method, independent of the type of collection tube: EDTA PCR-free method, 96.4% (n = 35); EDTA PCR method, 85.1% (n = 32); heparin PCR-free method, 94.5% (n = 25); and heparin PCR method, 89.4% (n = 10). All other evaluated metrics were highly comparable for PCR-free and PCR library preparations. These results demonstrate the feasibility of somatic copy number alteration detection by PCR-free sWGS using cfDNA from plasma collected in EDTA- or lithium-heparin–containing tubes and pave the way for an automated cfDNA analysis workflow for samples from cancer patients.

Cell-free DNA (cfDNA) can be isolated from blood and/or urine of cancer patients and analyzed with sequencing. Unfortunately, most conventional short-read sequencing methods are technically challenging, labor intensive and time consuming, requiring several days but more typically weeks to obtain interpretable data which are limited by a bias for short cfDNA fragments.Here, we demonstrate that with Oxford Nanopore Technologies sequencing we can achieve economical and ultra-fast delivery of clinical data from liquid biopsies.Our ‘ITSFASTR’ approach is able to deliver copy number aberrations, and cfDNA fragmentation profiles in less than 24 hours from sample collection. The tumorderived cfDNA fraction calculated from lung cancer patient plasma and urine from bladder cancer patients was highly correlated (R=0.98) to the tumor fraction calculated from conventional short-read sequencing of the same samples. cfDNA size profile and fragmentation patterns in plasma and urine exhibited the typical cfDNA features yet with a significantly higher proportion of fragments that exceed 300bp, exhibiting similar tumor fraction than shorter size fragments. Notably, comprehensive fragment-end composition and nucleosome profiling near transcription start sites can be determined from the same data.We propose that ITSFASTR is the first point-of-care solution for obtaining genomic and fragmentomic results from liquid biopsies.

Liquid biopsies contain multiple analytes that can be mined to improve the detection and management of cancer. Beyond cell-free DNA (cfDNA), mutations have been detected in DNA associated with extracellular vesicles (EV-DNA). The genome-wide composition and structure of EV-DNA are poorly characterized, and it remains undecided whether circulating EVs are enriched in tumor signal compared to unfractionated cfDNA.Here, using whole genome sequencing from selected lung cancer patients with a high cfDNA tumor content (>5%), we determined that the tumor fraction and heterogeneity are comparable between DNA associated with EVs and matched plasma cfDNA. DNA in EV fractions, obtained with standardized size-exclusion chromatography, are comprised of short ∼150-180 bp fragments and long >1000 bp fragments that are poor in tumor signal. Other fractions only exhibit short fragments with similar tumor DNA content. The composition in bases at the end of EV-DNA fragments, as well as their fragmentation patterns are similar to plasma cfDNA. Mitochondrial DNA is relatively enriched in EV fractions.Our results highlight that cfDNA in plasma is of dual nature, either bound to proteins (including the nucleosome) but also associated to EV. cfDNA associated to small EV (including exosomes) is however not preferentially enriched in tumor signal.

The discovery that cancer cells discharge vast quantities of extracellular vesicles (EVs), underscored the explosion of the EV field. A large body of evidence now supports their onco-functionality in an array of contexts; stromal crosstalk, immune evasion, metastatic site priming, and drug resistance - justifying therapeutic intervention. The current bottleneck is a lack of clear understanding of why and how EV biogenesis ramps up in cancer cells, and hence where exactly avenues for intervention may reside. We know that EVs also play an array of physiological roles, therefore effective anticancer inhibition requires a target distinct enough from physiology to achieve efficacy. Taking the perspective that EV upregulation may be a consequence of the tumor landscape, we examine classic mutational events and tumor characteristics for EV regulators. All the while, aiming to illuminate topics worth further research in therapeutic development.

BackgroundAssays that account for the biological properties and fragmentation of cell-free DNA (cfDNA) can improve the performance of liquid biopsy. However, preanalytic and physiological differences between individuals on fragmentomic analysis are poorly defined.MethodsWe analyzed the impact of collection tube, plasma processing time, and physiology on the size distribution of cfDNA, their genome-wide representation, and sequence diversity at the cfDNA fragment ends using shallow whole-genome sequencing.ResultsNeither different stabilizing collection tubes nor processing times affected the cfDNA fragment sizes, but could impact the genome-wide fragmentation patterns and fragment-end sequences of cfDNA. In addition, beyond differences depending on the gender, the physiological conditions tested between 63 individuals (age, body mass index, use of medication, and chronic conditions) minimally influenced the outcome of fragmentomic methods.ConclusionsFragmentomic approaches have potential for implementation in the clinic, pending clear traceability of analytical and physiological factors.

Objectives: Circulating tumor (ct)DNA analysis is rapidly gaining acceptance as a diagnostic tool to guide clinical management of advanced non-small cell lung cancer (NSCLC). Clinically-actionable EGFR mutations can be detected in ctDNA before or after first-line EGFR-Tyrosine Kinase Inhibitor (TKI) treatment, but data are limited for patients with a complex treatment history. This study aimed to explore the feasibility of ctDNA testing in a clinical setting of NSCLC patients receiving osimertinib as a second or third line EGFR-TKI. Materials and Methods: Twenty EGFR T790M-positive NSCLC patients, who had received osimertinib as a second or third line EGFR-TKI and had donated blood samples while attending routine follow-up consultations between April and November 2016, were retrospectively selected to test plasma cfDNA for tumor-guided EGFR mutations. We used EGFR mutations previously identified in tumor-tissue to retrospectively test plasma ctDNA from 20 patients who had received osimertinib as a second or third line EGFR-TKI. Both EGFR-TKI sensitising and T790 M resistance mutations were analysed by droplet digital PCR (ddPCR) in plasma taken alongside routine consultations and ctDNA detection was correlated with response under osimertinib. Follow-up solid-tissue biopsies were obtained after disease progression. Results: CtDNA was detected under osimertinib treatment in four out of the eight patients (50 %) who showed no response, two out of the seven (29 %) who showed an initial response and none of the five patients (0 %) who showed an ongoing response. The fraction of EGFR-mutant ctDNA in plasma tended to be higher in non-responders (0.1–68 %), compared to the initial responders (0.2–1.1 %). Blood samples were donated up to 34, 27 and 49 weeks after the start of osimertinib for the non-, initial and ongoing responders, respectively. Conclusions: These findings support a potential role for ctDNA analysis in response monitoring of NSCLC patients with a complex EGFR-TKI treatment history. The weak trend between ctDNA detection and disease progression warrants larger studies to further investigate potential clinical utility.

MicroRNAs (miRNAs) are a class of small non-coding RNA molecules that play a pivotal regulatory role in a broad variety of biological processes. Dysregulation of miRNAs is associated with several human diseases, particularly cancer. Extracellular vesicles (EVs) are crucial components in intercellular communication. As part of the cargo of EVs, miRNAs are involved in EV-mediated cell-to-cell interactions, including promotion or suppression of tumor development. The knowledge on the molecular mechanisms and clinical importance of EV-miRNAs in head and neck squamous cell carcinoma (HNSCC) has rapidly grown over the past years. In the present review, the current understanding regarding the effect of EV-miRNAs on HNSCC tumorigenesis is summarized, which includes effects on tumor proliferation, angiogenesis, invasion and metastasis, the tumor microenvironment, immune modulation, and treatment resistance. EV-miRNA-based biomarkers in liquid biopsies such as blood and saliva may open up new possibilities for employing EV-miRNAs for screening and early diagnostics as well as disease monitoring. Future perspectives include the promise of EV-miRNAs as a novel therapeutic target.

423 Background: Despite the advent of precision medicine, prediction of survival outcome of esophageal cancer patients remains a challenge. Here we aim to investigate the value of prediction models integrating multi-signal data including radiomics and circulating tumor DNA (ctDNA) data in addition to clinical data for the prediction of resectable esophageal adenocarcinoma (rEAC) related outcomes. Methods: In total n=111 rEAC patients treated with neoadjuvant chemoradiotherapy (nCRT; n=71) +/- anti-PD-L1 (n=40) were included. Baseline clinical variables (n=9) were based on the SOURCE survival prediction model (van den Boorn et al. JNCCN. 2021). The baseline ctDNA data from plasma was derived from fragmentomic and copy number aberrations (ichorCNA) from shallow whole genome sequencing (0.6). We evaluated the predictive performance of baseline ctDNA and radiomics features on overall survival (OS), progression free survival (PFS), and time to progression (TTP), through fitting Cox-regression models. Four ctDNA features were included in the models: P20-150, ichorCNA, fragment end score and mutation detection. For the radiomics features we performed an additional back- and forward variable selection based on Akaike’s Information Criterion. Using the likelihood ratio test we tested if the model fit changed after adding ctDNA and radiomics features to a model with clinical variables. Results: The addition of radiomics to clinical variables improved model fit for OS (p=0.017). Baseline prediction of OS resulted in a C-index of 0.65 using clinical variables only, 0.65 with ctDNA, 0.68 with radiomics and 0.68 with ctDNA and radiomics combined. For PFS model fit improved after adding radiomics (p=0.020) and ctDNA and radiomics combined (p=0.017). Baseline prediction of PFS resulted in a C-index of 0.64 using clinical variables, 0.65 with ctDNA, 0.67 with radiomics, and 0.68 with ctDNA and radiomics combined. For TTP model fit improved after adding radiomics (p=0.008) and radiomics and ctDNA combined (p=0.002). Baseline prediction of TTP resulted in a C-index of 0.64 with clinical variables, 0.65 with ctDNA, 0.71 with radiomics, and 0.72 with ctDNA and radiomics combined. Based on the cox-regression models using clinical variables and radiomics, risk stratification by splitting the cohort in a high and low risk group was possible for OS, PFS and TTP (p

Assays that account for the biological properties and fragmentation of cell-free DNA (cfDNA) can improve the performance of liquid biopsy. However, pre-analytic and physiological differences between individuals on fragmentomic analysis are poorly defined.We analyzed the impact of collection tube, plasma processing time and physiology on the size distribution of cfDNA, their genome-wide representation and sequence diversity at the cfDNA fragment-ends using shallow Whole Genome Sequencing.We observed that using different stabilizing collection tubes, or processing times does not affect the cfDNA fragment sizes, but can impact the genome-wide fragmentation patterns and fragment-end sequences of cfDNA. In addition, beyond differences depending on the gender, the physiological conditions tested between 63 individuals (age, body mass index, use of medication and chronic conditions) minimally influenced the outcome of fragmentomic methods.Our results highlight that fragmentomic approaches have potential for implementation in the clinic, pending clear traceability of analytical and physiological factors.

Exosomes are small, single-membrane, secreted organelles of ∼30 to ∼200 nm in diameter that have the same topology as the cell and are enriched in selected proteins, lipids, nucleic acids, and glycoconjugates. Exosomes contain an array of membrane-associated, high-order oligomeric protein complexes, display pronounced molecular heterogeneity, and are created by budding at both plasma and endosome membranes. Exosome biogenesis is a mechanism of protein quality control, and once released, exosomes have activities as diverse as remodeling the extracellular matrix and transmitting signals and molecules to other cells. This pathway of intercellular vesicle traffic plays important roles in many aspects of human health and disease, including development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases. In addition, viruses co-opt exosome biogenesis pathways both for assembling infectious particles and for establishing host permissiveness. On the basis of these and other properties, exosomes are being developed as therapeutic agents in multiple disease models.

Using mouse models of osteoarthritis (OA), a new study finds that osteoclasts secrete exosomes that deliver miRNAs to chondrocytes, leading to an increase in metalloproteinase activity in cartilage. A bone-specific inhibitor of exosome production can halt this process, hinting at a new therapeutic strategy for patients with OA.

KWF Kankerbestrijding, Grant/AwardNumber: KWF-5510; Cancer Center Amsterdam Foundation, Grant/AwardNumber: CCA-2013; The Hodgkin Lymphoma MRD Foundation; Technology Foundation STW, Grant/AwardNumber: CANCER-ID Project, Minimally-invasive tools to assess tumour presence and burden may improve clinical management. FDG-PET (metabolic) imaging is the current gold standard for interim response assessment in patients with classical Hodgkin Lymphoma (cHL), but this technique cannot be repeated frequently. Here we show that microRNAs (miRNA) associated with tumour-secreted extracellular vesicles (EVs) in the circulation of cHL patients may improve response assessment. Small RNA sequencing and qRT-PCR reveal that the relative abundance of cHL-expressed miRNAs, miR-127-3p, miR-155- 5p, miR-21-5p, miR-24-3p and let-7a-5p is up to hundred-fold increased in plasma EVs of cHL patients pre-treatmentwhen compared to completemetabolic responders (CMR). Notably, in partial responders (PR) or treatment-refractory cases (n = 10) the EV-miRNA levels remain elevated. In comparison, tumour specific copy number variations (CNV) were detected in cell-free DNA of 8 out of 10 newly diagnosed cHL patients but not in patients with PR. Combining EV-miR-127-3p and/or EV-let- 7a-5p levels, with serum TARC (a validated protein cHL biomarker), increases the accuracy for predicting PET-status (n = 129) to an area under the curve of 0.93 (CI: 0.87-0.99), 93.5% sensitivity, 83.8/85.0% specificity and a negative predictive value of 96%. Thus the level of tumour-associated miRNAs in plasma EVs is predictive of metabolic tumour activity in cHL patients. Our findings suggest that plasma EV-miRNA are useful for detection of small residual lesions and may be applied as serial response prediction tool., KWF Kankerbestrijding KWF-5510, Cancer Center Amsterdam Foundation CCA-2013, Hodgkin Lymphoma MRD Foundation, Technologiestichting STW

10037 Background: Mesenchymal stem cells (MSCs) play a key role in the progression of osteosarcoma (OS). In response to tumor-associated signals, including tumor-secreted extracellular vesicles (EVs), MSCs increase the expression of inflammatory cytokines promoting tumor cell aggressiveness. Previous data has demonstrated that IL-6 and IL-8 signaling mediate metastasis in OS. The aim of this study was to evaluate tumor EV-induced alterations of MSCs and identify combination therapies that can counteract MSC-induced resistance to antimetastatic drugs. Methods: Tumor EV-induced alterations of the MSC transcriptome were analyzed by RNA-seq. Gene set enrichment analysis (GSEA) was applied to discriminate TGFβ-dependent and independent pathways. EV RNA-induced expression changes were identified by transfecting purified EV-RNA in MSCs and by using a selective dsRNA antagonist. We selected candidate targets to block MSC-induced drug resistance and evaluated their effect in an orthotopic xenograft model of OS. Ladarixin (an allosteric inhibitor of the CXCL8/IL-8 receptors CXCR1 and CXCR2; 30 mg/kg 6x per week i.p.) and tocilizumab (anti-IL6 receptor antibody; 100 µg/mouse every other day i.p.) were administered starting from day one until the experimental endpoint. Metastasis were quantified by histological examination. This study was funded by the Dutch Cancer Society (KWF), POR FESR Campania 2014‐2020 and Dompé Farmaceutici SpA. Results: EVs from aggressive cancer cell lines induced an inflammatory MSC (iMSC) phenotype, characterized by increased expression of chemokines, including IL-8 as the most upregulated. Apart from IL-6, these alterations were mostly independent from TGFβ signaling and related to pattern recognition receptor (PRR) activation. We demonstrated that tumor EV-associated non-coding RNAs trigger TLR3 signaling in MSCs activating an innate immune response leading to high induction of IL-8 and other chemokines. Ladarixin and tocilizumab combination significantly reduced metastasis formation in a spontaneous metastasis model and overcame iMSC-induced resistance observed with single antimetastatic treatments. No effect was observed on primary tumor growth. Conclusions: EV-associated TGFβ together with EV-RNA induce iMSCs development in OS. Ladarixin in combination with tocilizumab reduced metastasis formation in a xenograft mouse model of OS, and, importantly, may prevent the occurrence of iMSC-induced tumor resistance to antimetastatic drugs.

Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy (CRS-HIPEC) may be curative for colorectal cancer patients with peritoneal metastases (PMs) but it has a high rate of morbidity. Accurate preoperative patient selection is therefore imperative, but is constrained by the limitations of current imaging techniques. In this pilot study, we explored the feasibility of circulating tumor (ct) DNA analysis to select patients for CRS-HIPEC. Thirty patients eligible for CRS-HIPEC provided blood samples preoperatively and during follow-up if the procedure was completed. Targeted Next-Generation Sequencing (NGS) of DNA from PMs was used to identify bespoke mutations that were subsequently tested in corresponding plasma cell-free (cf) DNA samples using droplet digital (dd) PCR. CtDNA was detected preoperatively in cfDNA samples from 33% of patients and was associated with a reduced disease-free survival (DFS) after CRS-HIPEC (median 6.0 months vs median not reached, p = 0.016). This association could indicate the presence of undiagnosed systemic metastases or an increased metastatic potential of the tumors. We demonstrate the feasibility of ctDNA to serve as a preoperative marker of recurrence in patients with PMs of colorectal cancer using a highly sensitive technique. A more appropriate treatment for patients with preoperative ctDNA detection may be systemic chemotherapy in addition to, or instead of, CRS-HIPEC.

Transmembrane receptors, of which G protein-coupled receptors (GPCRs) constitute the largest group, typically act as cellular antennae that reside at the plasma membrane (PM) to collect and interpret information from the extracellular environment. The discovery of cell-released extracellular vesicles (EVs) has added a new dimension to intercellular communication. These unique nanocarriers reflect cellular topology and can systemically transport functionally competent transmembrane receptors, ligands, and a cargo of signal proteins. Recent developments hint at roles for GPCRs in the EV life cycle and, conversely, at roles for EVs in GPCR signal transduction. We highlight key points of convergence, discuss their relevance to current GPCR and EV paradigms, and speculate on how this intersection could lend itself to future therapeutic avenues.

International audience; Exosomes are small extracellular vesicles with a diameter of 40-150 nm, and are implicated in cellular homeostasis and cell-cell communication. They can be secreted in bulk in response to cell-extrinsic and cell-intrinsic signals that cause multivesicular body (MVB) fusion with the plasma membrane (PM). However, research on the regulation of exosome release is hampered by the failure of current methods to capture the dynamics of exosome release. Here we describe how live imaging with tetraspanin-based pH-sensitive fluorescent reporters can quantify the MVB-PM fusion rate of single cells. Our approach enables identification of exogenous stimuli, signaling pathways, and fusion complexes, and can map subcellular sites of fusion events. In addition, dual-color imaging can be used to assess simultaneous release of different cargo by MVB exocytosis. This protocol describes the complete imaging experiment, consisting of transient expression of tetraspanin reporters (2 d), live-cell (dual-color) total internal reflection fluorescence microscopy (30-60 min per condition), and semiautomatic image analysis by using a newly developed ImageJ macro (±30 min per condition).

Introduction Patients with high grade B cell lymphoma that harbor a MYC rearrangement with concomitant BCL2 and/or BCL6 rearrangements (double hit and triple hit (HGBL-DH/TH)) face a poor prognosis upon standard treatment with R-CHOP [Rosenwald, JCO 2019]. DA-EPOCH-R might yield higher complete metabolic remission (CMR) rates and longer disease free survival (DFS) as compared to R-CHOP, but improvement of overall survival (OS) has not been demonstrated [Dunleavy, Lancet Haematol 2018]. Tumors with MYC overexpression may be susceptible for immune checkpoint inhibition (CI) [Casey, Science 2016], providing a rationale for CI after reaching CMR to improve tumor immune surveillance for minimal residual disease (MRD) positive disease. Here, we present data of the planned interim analysis of 33/97 patients included in the HOVON-152 trial (NCT03620578). Methods HOVON-152 is a prospective, multi-center, single arm phase II trial. Inclusion criteria are newly diagnosed HGBL-DH/TH; age ≥ 18 year; WHO performance status 0-3; Ann Arbor stage II-IV. During the screening period for rearrangements patients receive 1 cycle of R-CHOP followed by 5 cycles of DA-EPOCH-R induction treatment. All patients receive intrathecal prophylaxis. All diagnostic lymphoma samples are centrally reviewed. PET-CT scans are performed at diagnosis, midterm and end-of-induction. Patients in CMR after induction treatment (Deauville 1-3 or a negative lymphoma biopsy in case of Deauville 4) proceed with Nivolumab consolidation (480 mg iv every 4 weeks) for one year. The primary objective is to improve 12 months DFS with Nivolumab consolidation from 70% to 85% in patients in CMR after induction treatment. Secondary objectives include evaluation of CMR rates, OS and safety. Exploratory side studies investigate blood-based biomarkers for response prediction by immune profiling using multicolor flow cytometry after 1 cycle of R-CHOP and molecular circulating tumor DNA (ctDNA) analyses using ClonoSEQ (Adaptive Biotechnologies, Seattle) after 1 cycle of R-CHOP and at midterm. Results From August 2018 to June 2021, 69 of planned 97 patients have been enrolled. Baseline characteristics of the first 33 patients included in the interim analysis are shown in Table 1. Dose adjustments of EPOCH (according to protocol) resulted in 48% of patients receiving dose level ≤1 and 52% dose level ≥ 2 at the last cycle. After induction, 20/33 patients (61%; 95% CI 42%-77%) reached CMR. 11/33 patients (33%) did not reach CMR and 2/33 (6%) patients went off protocol (due to progression). During DA-EPOCH-R, one patient (3%) experienced grade 5 AE (sepsis), 9 patients (27%) experienced a grade 4 AE, and 9 (27%) patients grade 3. Neurotoxicity led to dose adjustments or discontinuation of vincristine in 52% of the patients. In an amendment the vincristine dose was capped at 2 mg/cycle. Twenty patients received 6 cycles of Nivolumab consolidation. One patient had a grade 4 AE (neutropenia); 2 patients had a grade 3 AE (one lung infection and one colitis (reason for going off protocol)). The Data Safety Monitoring Board recommended to continue the trial. Exploratory biomarker analyses show that patients achieving CMR after DA-EPOCH-R (data available for 19/20 patients) have higher percentages of T cells (p=0.04) after 1 cycle of R-CHOP than patients that do not achieve CMR (data available for 12/13 patients). ctDNA analysis was possible in 16/28 patients (in 12/28 patients no clone could be detected for monitoring). 10/16 patients achieved CMR, of which 9/10 were negative for minimal residual disease (MRD) at midterm. The patient that was MRD positive at midterm relapsed shortly after CMR. 4/5 patients that did not achieve CMR were MRD positive at midterm. Conclusions Interim analysis of the HOVON-152 trial demonstrates that 61% (95% CI 42%-77%) of the patients with HGBL-DH/TH achieve CMR after induction with DA-EPOCH-R. Toxicity of DA-EPOCH-R consists mainly of neurotoxicity leading to a protocol amendment (dose cap of vincristine). Nivolumab consolidation is safe with only one patient going off protocol due to colitis. Biomarkers for CMR after induction with DA-EPOCH-R point out to ctDNA-based MRD negativity at midterm and to higher T percentages after 1 cycle of R-CHOP, supporting the hypothesis of contributive immune surveillance for response in patients with HGBL-DH/TH. The trial is ongoing. Figure 1 Figure 1. Disclosures Nijland: Roche: Research Funding; Nordic Nanovector: Research Funding; Takeda: Research Funding. Van Der Poel: Roche, Janssen, Abbvie: Honoraria. Klerk: Van Lanschot Kempen: Other: I have an investment porfolio which is managed by Van Lanschot Kempen as a portfolio manager (vermogensbeheerder). Van Lanschot Kempen invests on my behalf, and takes investment decisions on a discretionary basis. I am not involved in the investment decis. Pegtel: Exbiome BV: Current holder of individual stocks in a privately-held company; Takeda: Other: Travel compensation. Mutis: Novartis: Research Funding; ONK Therapeutics: Research Funding; Janssen: Honoraria; Takeda: Research Funding; Genmab: Research Funding. Zijlstra: Takeda: Research Funding. Kersten: BMS/Celgene: Consultancy; Kite/Gilead: Consultancy, Honoraria, Research Funding; Milteny Biotech: Consultancy; Novartis: Consultancy, Honoraria; Roche: Honoraria; Takeda: Consultancy. Chamuleau: Gilead: Research Funding; Genmab: Research Funding; Celgene: Research Funding. OffLabel Disclosure: Nivolumab as immune checkpoint inhibitor (inhibiting PD-1) in consolidation phase for the treatment of DH/TH-HGBL patients.

Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.

B cell lymphomas are heterogeneous malignancies of hematological origin with vastly different biology and clinical outcomes. Histopathology of tissue biopsies and image-based assessment guide clinical decisions. Given that tissue biopsies cannot be frequently repeated and will not inform on systemic responses to the treatment, more accessible biomarkers, such as circulating miRNAs, are considered. Aberrant miRNA expression in lymphoma tissues and ongoing immune reactions may lead to miRNA alterations in circulation. miRNAs bound to extracellular vesicles (EVs) are of interest because of their role in intercellular communication and organ crosstalk. Herein, we highlight the role of miRNAs and EVs in B cell lymphomagenesis and explain how circulating miRNAs may be turned into robust liquid biopsy tests for aggressive B cell lymphoma.

Extracellular vesicles (EVs) are released by most cell types but providing evidence for their physiological relevance remains challenging due to a lack of appropriate model organisms. Here, we developed an in vivo model to study EV function by expressing CD63-pHluorin in zebrafish embryos. A combination of imaging methods and proteomic analysis allowed us to study biogenesis, composition, transfer, uptake, and fate of individual endogenous EVs. We identified a subpopulation of EVs with exosome features, released in a syntenin-dependent manner from the yolk syncytial layer into the blood circulation. These exosomes are captured, endocytosed, and degraded by patrolling macrophages and endothelial cells in the caudal vein plexus (CVP) in a scavenger receptor- and dynamin-dependent manner. Interference with exosome biogenesis affected CVP growth, suggesting a role in trophic support. Altogether, our work represents a system for studying endogenous EV function in vivo with high spatiotemporal accuracy, demonstrating functional inter-organ communication by exosomes. ispartof: DEVELOPMENTAL CELL vol:48 issue:4 pages:573-+ ispartof: location:United States status: published

4033 Background: ctDNA is becoming an established marker to assess tumor burden, relapse after surgery, and to identify responders in immunotherapy studies. In the phase II PERFECT trial rEAC patients were treated with neoadjuvant chemoradiotherapy (nCRT) and a PD-L1 inhibitor (van den Ende et al. CCR. 2021). Here we evaluated the potential of cell-free DNA (cfDNA) to predict pathological complete response (pCR) and recurrence. Methods: The cohort consisted of 40 patients and 145 plasma samples. EDTA blood samples were drawn at baseline (B, N = 40), in week 5 of nCRT (W5, N = 40), before surgery (OR, N = 33) and 3 months after surgery (FU, N = 32). cfDNA was isolated by affinity columns (CNAkit, QIAgen) quantified by spectrofluorometer (BioAnalyzer, Agilent), sequencing libraries were prepared for lcWGS ( < 5-fold coverage, Tag-seq, Takara) and sequenced on a NovaSeq (S4, PE150). Sequencing data were processed with an in-house pipeline. Copy number aberrations (CNA) and the tumor fraction were estimated using the ichorCNA tool. Insert sizes were recovered and we determined a Tumor Enriched Fragment Fraction (TEFF), calculated by doing the ratio of fragments between 90-150 bp and 250-320 bp (enriched in tumor signal) and fragments between 150-250 bp and 320-360 bp (poor in tumor signal). ichorCNA and TEFF were used to quantify the ctDNA fraction in plasma samples. pCR was defined as ypT0N0. Residual tumor, progression or death before surgery were considered non-pCR. Relapse-free survival (RFS) was defined as the time after surgery until recurrence. Results: The pCR rate was 25% (10/40). The median fold change TEFF between B and W5 was -0.15 (range -0.67 to 0.44) in the pCR group and 0.16 (range -1.40 to 0.76) in the non-pCR group (Mann–Whitney U; p = 0.047). Of the 17 patients in whom ctDNA was detected (TEFF≥0.3 and/or ichorCNA≥0.03) in the FU sample, 13 (76%) showed a recurrence. Of the 15 patients with no ctDNA detected 5 (33%) showed a recurrence. Patients with ctDNA detected at FU had worse RFS, HR = 2.72 (95%CI 0.96-7.71; p = 0.050). Recurrences were detected earlier by FU ctDNA than by imaging due to physical complaints with a median of 312 days (163-798 days). Conclusions: lcWGS appears to be a useful tool to predict pCR and recurrence in resectable esophageal cancer. These lcWGS results will be further combined with fragmentomics analysis and targeted mutational data (Ion Torrent next-generation sequencing) in order to assess response to immunotherapy. Clinical trial information: NCT03087864.

Myositis is a heterogeneous group of autoimmune diseases, with different pathogenic mechanisms contributing to the different subsets of disease. The aim of this study was to test whether the autoantibody profile in patients with myositis is associated with a type I interferon (IFN) signature, as in patients with systemic lupus erythematous (SLE). Patients with myositis were prospectively enrolled in the study and compared to healthy controls and to patients with SLE. Autoantibody status was analysed using an immunoassay system and immunoprecipitation. Type I IFN activity in whole blood was determined using direct gene expression analysis. Serum IFN-inducing activity was tested using peripheral blood cells from healthy donors. Blocking experiments were performed by neutralizing anti-IFNAR or anti-IFN-α antibodies. Patients were categorized into IFN high and IFN low based on an IFN score. Patients with autoantibodies against RNA-binding proteins had a higher IFN score compared to patients without these antibodies, and the IFN score was related to autoantibody multispecificity. Patients with dermatomyositis (DM) and inclusion body myositis (IBM) had a higher IFN score compared to the other subgroups. Serum type I IFN bioactivity was blocked by neutralizing anti-IFNAR or anti-IFN-α antibodies. To conclude, a high IFN score was not only associated with DM, as previously reported, and IBM, but also with autoantibody monospecificity against several RNA-binding proteins and with autoantibody multispecificity. These studies identify IFN-α in sera as a trigger for activation of the type I IFN pathway in peripheral blood and support IFN-α as a possible target for therapy in these patients.

Extracellular vesicles (EVs) are heterogeneous multi-signal messengers that support cancer growth and dissemination by mediating the tumor-stroma crosstalk. Exosomes are a subtype of EVs that originate from the limiting membrane of late endosomes, and as such contain information linked to both the intrinsic cell “state” and the extracellular signals cells received from their environment. Resolving the signals affecting exosome biogenesis, cargo sorting and release will increase our understanding of tumorigenesis. In this review we highlight key cell biological processes that couple exosome biogenesis to cargo sorting in cancer cells. Moreover, we discuss how the bidirectional communication between tumor and non-malignant cells affect cancer growth and metastatic behavior.

Contains fulltext : 193223.pdf (Publisher’s version ) (Open Access) Cancer cells release extracellular vesicles (EVs) that contain functional biomolecules such as RNA and proteins. EVs are transferred to recipient cancer cells and can promote tumour progression and therapy resistance. Through RNAi screening, we identified a novel EV uptake mechanism involving a triple interaction between the chemokine receptor CCR8 on the cells, glycans exposed on EVs and the soluble ligand CCL18. This ligand acts as bridging molecule, connecting EVs to cancer cells. We show that glioblastoma EVs promote cell proliferation and resistance to the alkylating agent temozolomide (TMZ). Using in vitro and in vivo stem-like glioblastoma models, we demonstrate that EV-induced phenotypes are neutralised by a small molecule CCR8 inhibitor, R243. Interference with chemokine receptors may offer therapeutic opportunities against EV-mediated cross-talk in glioblastoma.

All mammalian cells release small endosome-derived exosomes that function in intercellular communication, but the secretion process is poorly understood. Verweij et al. developed a live-imaging approach and demonstrate that external cues can trigger exosome release from a subpopulation of multivesicular bodies by phosphorylating the target membrane SNARE SNAP23 at serine residue 110., Exosomes are small endosome-derived extracellular vesicles implicated in cell–cell communication and are secreted by living cells when multivesicular bodies (MVBs) fuse with the plasma membrane (PM). Current techniques to study exosome physiology are based on isolation procedures after secretion, precluding direct and dynamic insight into the mechanics of exosome biogenesis and the regulation of their release. In this study, we propose real-time visualization of MVB–PM fusion to overcome these limitations. We designed tetraspanin-based pH-sensitive optical reporters that detect MVB–PM fusion using live total internal reflection fluorescence and dynamic correlative light–electron microscopy. Quantitative analysis demonstrates that MVB–PM fusion frequency is reduced by depleting the target membrane SNAREs SNAP23 and syntaxin-4 but also can be induced in single cells by stimulation of the histamine H1 receptor (H1HR). Interestingly, activation of H1R1 in HeLa cells increases Ser110 phosphorylation of SNAP23, promoting MVB–PM fusion and the release of CD63-enriched exosomes. Using this single-cell resolution approach, we highlight the modulatory dynamics of MVB exocytosis that will help to increase our understanding of exosome physiology and identify druggable targets in exosome-associated pathologies.

Extracellular RNA (exRNA) has emerged as an important transducer of intercellular communication. Advancing exRNA research promises to revolutionize biology and transform clinical practice. Recent efforts have led to cutting-edge research and expanded knowledge of this new paradigm in cell-to-cell crosstalk; however, gaps in our understanding of EV heterogeneity and exRNA diversity pose significant challenges for continued development of exRNA diagnostics and therapeutics. To unravel this complexity, the NIH convened expert teams to discuss the current state of the science, define the significant bottlenecks, and brainstorm potential solutions across the entire exRNA research field. The NIH Strategic Workshop on Extracellular RNA Transport helped identify mechanistic and clinical research opportunities for exRNA biology and provided recommendations on high priority areas of research that will advance the exRNA field.

Purpose: Human osteosarcoma is a genetically heterogeneous bone malignancy with poor prognosis despite the employment of aggressive chemotherapy regimens. Because druggable driver mutations have not been established, dissecting the interactions between osteosarcoma cells and supporting stroma may provide insights into novel therapeutic targets. Experimental Design: By using a bioluminescent orthotopic xenograft mouse model of osteosarcoma, we evaluated the effect of tumor extracellular vesicle (EV)–educated mesenchymal stem cells (TEMSC) on osteosarcoma progression. Characterization and functional studies were designed to assess the mechanisms underlying MSC education. Independent series of tissue specimens were analyzed to corroborate the preclinical findings, and the composition of patient serum EVs was analyzed after isolation with size-exclusion chromatography. Results: We show that EVs secreted by highly malignant osteosarcoma cells selectively incorporate a membrane-associated form of TGFβ, which induces proinflammatory IL6 production by MSCs. TEMSCs promote tumor growth, accompanied with intratumor STAT3 activation and lung metastasis formation, which was not observed with control MSCs. Importantly, intravenous administration of the anti-IL6 receptor antibody tocilizumab abrogated the tumor-promoting effects of TEMSCs. RNA-seq analysis of human osteosarcoma tissues revealed a distinct TGFβ-induced prometastatic gene signature. Tissue microarray immunostaining indicated active STAT3 signaling in human osteosarcoma, consistent with the observations in TEMSC-treated mice. Finally, we isolated pure populations of EVs from serum and demonstrated that circulating levels of EV-associated TGFβ are increased in osteosarcoma patients. Conclusions: Collectively, our findings suggest that TEMSCs promote osteosarcoma progression and provide the basis for testing IL6- and TGFβ-blocking agents as new therapeutic options for osteosarcoma patients. Clin Cancer Res; 23(14); 3721–33. ©2017 AACR.

Different types of cells infected with Epstein-Barr virus (EBV) can release exosomes containing viral components that functionally affect neighboring cells. Previously, we found that EBV was localized mostly in infiltrating lymphocytes within the stromal layer of cervical lesions. In this study, we aimed to determine effects of exosome-transferred EBV-encoded RNAs (EBERs) on keratinocytes expressing human papillomavirus (HPV) 16 E6/E7 (DonorI-HPV16 HFKs). Lipid transfection of in vitro-transcribed EBER1 molecules (ivt EBER1) into DonorI-HPV16 HFKs caused strong induction of interferon (IFN)-related genes and interleukin 6 (IL-6). To gain insights into the physiological situation, monocyte-derived dendritic cells (moDCs), low passage DonorI-HPV16 HFKs and primary keratinocytes were used as recipient cells for internalization of exosomes from wild-type EBV (wt EBV) or B95-8 EBV-infected lymphoblastoid cell lines (LCLs). qRT-PCR was used to determine the expression of EBER1, HPV16 E6/E7, IFN-related genes and IL-6 in recipient cells. The secretion of inflammatory cytokines was investigated using cytometric bead array. Wt EBV-modified exosomes induced both IFN-related genes and IL-6 upon uptake into moDCs, while exosomes from B95-8 EBV LCLs induced only IL-6 in moDCs. Internalization of EBV-modified exosomes was demonstrated in DonorI-HPV16 HFKs, yielding only EBER1 but not EBER2. However, EBER1 transferred by exosomes did not induce IFN-related genes or IL-6 expression and inflammatory cytokine secretion in DonorI-HPV16 HFKs and primary keratinocytes. EBER1 copy numbers in exosomes from wt EBV-infected LCLs were 10-fold higher than in exosomes from B95-8 LCLs (equal cell equivalent), whereas ivt EBER1 was used at approximately 100-fold higher concentration than in exosomes. These results demonstrated that the induction of IFN-related genes and IL-6 by EBER1 depends on quantity of EBER1 and type of recipient cells. High levels of EBER1 in cervical cells or infiltrating dendritic cells may play a role in the inflammation-to-oncogenesis transition of HPV-associated cervical cancer through modulation of innate immune signals.