Turning the tide in aggressive lymphoma: liquid biopsy for risk-adapted treatment strategies.

Diffuse large B cell lymphoma (DLBCL) exhibits significant biological and clinical heterogeneity that presents challenges for risk stratification and disease surveillance. Existing tools for risk stratification, including the international prognostic index (IPI), tissue molecular analyses, and imaging, have limited accuracy in predicting outcomes. The therapeutic landscape for aggressive lymphoma is rapidly evolving, and there is a pressing need to identify patients at risk of refractory or relapsed (R/R) disease in the context of personalized therapy. Liquid biopsy, a minimally invasive method for cancer signal detection, has been explored to address these challenges. We review advances in liquid biopsy strategies focusing on circulating nucleic acids in DLBCL patients and highlight their clinical potential. We also provide recommendations for biomarker-guided trials to support risk-adapted treatment modalities.

Full-length tRNAs lacking a functional CCA tail are selectively sorted into the lumen of extracellular vesicles.

Small extracellular vesicles (sEVs) are heterogenous lipid membrane particles typically less than 200 nm in size and secreted by most cell types either constitutively or upon activation signals. sEVs isolated from biofluids contain RNAs, including small non-coding RNAs (ncRNAs), that can be either encapsulated within the EV lumen or bound to the EV surface. EV-associated microRNAs (miRNAs) are, despite a relatively low abundance, extensively investigated for their selective incorporation and their role in cell-cell communication. In contrast, the sorting of highly-structured ncRNA species is understudied, mainly due to technical limitations of traditional small RNA sequencing protocols. Here, we adapted ALL-tRNAseq to profile the relative abundance of highly structured and potentially methylated small ncRNA species, including transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and Y RNAs in bulk EV preparations. We determined that full-length tRNAs, typically 75 to 90 nucleotides in length, were the dominant small ncRNA species (>60% of all reads in the 18-120 nucleotides size-range) in all cell culture-derived EVs, as well as in human plasma-derived EV samples, vastly outnumbering 21 nucleotides-long miRNAs. Nearly all EV-associated tRNAs were protected from external RNAse treatment, indicating a location within the EV lumen. Strikingly, the vast majority of luminal-sorted, full-length, nucleobase modification-containing EV-tRNA sequences, harbored a dysfunctional 3' CCA tail, 1 to 3 nucleotides truncated, rendering them incompetent for amino acid loading. In contrast, in non-EV associated extracellular particle fractions (NVEPs), tRNAs appeared almost exclusively fragmented or 'nicked' into tRNA-derived small RNAs (tsRNAs) with lengths between 18 to 35 nucleotides. We propose that in mammalian cells, tRNAs that lack a functional 3' CCA tail are selectively sorted into EVs and shuttled out of the producing cell, offering a new perspective into the physiological role of secreted EVs and luminal cargo-selection.

Towards IVDR-compliance by implementing quality control steps in a quantitative extracellular vesicle-miRNA liquid biopsy assay for response monitoring in patients with classic Hodgkin lymphoma.

Previously, we showed that quantification of lymphoma-associated miRNAs miR-155-5p, -127-3p and let-7a-5p levels in plasma extracellular vesicles (EVs) report treatment response in patients with classic Hodgkin lymphoma (cHL). Prior to clinical implementation, quality control (QC) steps and validation are required to meet international regulatory standards. Most published EV-based diagnostic assays have yet to meet these requirements. In order to advance the assay towards regulatory compliance (e.g., IVDR 2017/746), we incorporated three QC steps in our experimental EV-miRNA quantitative real-time reverse-transcription PCR (q-RT-PCR) assay in an ISO-13485 certified quality-management system (QMS). Liposomes encapsulated with a synthetic (nematode-derived) miRNA spike-in controlled for EV isolation by automated size-exclusion chromatography (SEC). Additional miRNA spike-ins controlled for RNA isolation and cDNA conversion efficiency. After deciding on quality criteria, in total 107 out of 120 samples from 46 patients passed QC. Generalized linear mixed-effect modelling with bootstrapping determined the diagnostic performance of the quality-controlled data at an area under the curve (AUC) of 0.84 (confidence interval [CI]: 0.76-0.92) compared to an AUC of 0.87 (CI: 0.80-0.94) of the experimental assay. After the inclusion of QC steps, the accuracy of the assay was determined to be 78.5% in predicting active disease status in cHL patients during treatment. We demonstrate that a quality-controlled plasma EV-miRNA assay is technically robust, taking EV-miRNA as liquid biopsy assay an important step closer to clinical evaluation.

Tumor-secreted extracellular vesicles counteract therapy response by triggering inflammatory mesenchymal stem cell development.

PURPOSE: Therapy resistance is a major clinical hurdle in bone cancer treatment and seems to be largely driven by poorly understood microenvironmental factors. Recent evidence suggests a critical role for a unique subpopulation of mesenchymal stem cells with inflammatory features (iMSCs), though their origin and function remained unexplored. We demonstrate that cancer-secreted extracellular vesicles (EVs) trigger the development of iMSCs, which hinder therapy response in vivo, and set out to identify strategies to counteract their function. EXPERIMENTAL DESIGN: The role of iMSCs in therapy resistance was evaluated in an orthotopic xenograft mouse model of osteosarcoma. EV-induced alterations of the MSC transcriptome were analyzed and compared with scRNA-seq data of osteosarcoma and multiple myeloma patient biopsies. Functional assays identified EV components driving iMSC development. We assessed the efficacy of clinical drugs in blocking iMSC-induced resistance in vivo. RESULTS: We found that iMSCs are induced by interaction with cancer EVs and completely abrogate the antimetastatic effect of TGFb signaling inhibition. Importantly, EV-induced iMSCs faithfully recapitulate the inflammatory single-cell RNA signature of stromal cells enriched in multiple myeloma and osteosarcoma patient biopsies. Mechanistically, cancer EVs act through two distinct mechanisms. EV-associated TGFb induces IL6 production, while the EV-RNA cargo enhances TLR3-mediated chemokine production. We reveal that simultaneous blockade of downstream EV-activated pathways with ladarixin and tocilizumab disrupts metastasis formation and overcomes iMSC-induced resistance. CONCLUSIONS: Our observations establish iMSCs as major contributors to drug resistance, reveal EVs as physiological triggers of iMSC development and highlight a promising combination strategy to improve therapy response in bone cancer patients.