Integrating extracellular vesicle and circulating cell-free DNA analysis using a single plasma aliquot improves the detection of HER2 positivity in breast cancer patients.

Multi-analyte liquid biopsies represent an emerging opportunity for non-invasive cancer assessment. We developed ONCE (ONe Aliquot for Circulating Elements), an approach for the isolation of extracellular vesicles (EV) and cell-free DNA (cfDNA) from a single aliquot of blood. We assessed ONCE performance to classify HER2-positive early-stage breast cancer (BrCa) patients by combining EV-associated RNA (EV-RNA) and cfDNA signals on n=64 healthy donors (HD) and non-metastatic BrCa patients. Specifically, we isolated EV-enriched samples by a charge-based (CB) method and investigated EV-RNA and cfDNA by next-generation sequencing (NGS) and by digital droplet PCR (ddPCR). Sequencing of cfDNA and EV-RNA from HER2- and HER2+ patients demonstrated concordance with in situ molecular analyses of matched tissues. Combined analysis of the two circulating analytes by ddPCR showed increased sensitivity in ERBB2/HER2 detection compared to single nucleic acid components. Multi-analyte liquid biopsy prediction performance was comparable to tissue-based sequencing results from TCGA. Also, imaging flow cytometry analysis revealed HER2 protein on the surface of EV isolated from the HER2+ BrCa plasma, thus corroborating the potential relevance of studying EV as companion analyte to cfDNA. This data confirms the relevance of combining cfDNA and EV-RNA for HER2 cancer assessment and supports the ONCE as a valuable tool for multi-analytes liquid biopsies' clinical implementation.

The Role of Physical Exercise and Rehabilitative Implications in the Process of Nerve Repair in Peripheral Neuropathies: A Systematic Review.

BACKGROUND: The various mechanisms involved in peripheral nerve regeneration, induced by exercise and electrical nerve stimulation, are still unclear. OBJECTIVE: The aim of this review was to summarize the influence of physical exercise and/or electrical stimulation on peripheral nerve repair and regeneration and the variation of impact of intervention depending on timing, as well as kind and dosage of the intervention. A literature survey was conducted on PubMed, Scopus, and Web of Science, between February 2021 to July 2021, with an update in September 2022. METHODOLOGY: The literature search identified 101,386 articles with the keywords: "peripheral nerve" OR "neuropathy" AND "sprouting" OR "neuroapraxia" OR "axonotmesis" OR "neurotmesis" OR "muscle denervation" OR "denervated muscle" AND "rehabilitation" OR "physical activity" OR "physical exercise" OR "activity" OR "electrical stimulation". A total of 60 publications were included. Eligible studies were focused on evaluating the process of nerve repair (biopsy, electromyographic parameters or biomarker outcomes) after electrical stimulation or physical exercise interventions on humans or animals with peripheral sensory or motor nerve injury. SYNTHESIS: This study shows that the literature, especially regarding preclinical research, is mainly in agreement that an early physical program with active exercise and/or electrical stimulation promotes axonal regenerative responses and prevents maladaptive response. This was evaluated by means of changes in electrophysiological recordings of CMAPs for latency amplitude, and the sciatic functional index (SFI). Furthermore, this type of activity can cause an increase in weight and in muscle fiber diameter. Nevertheless, some detrimental effects of exercising and electrical stimulation too early after nerve repair were recorded. CONCLUSION: In most preclinical studies, peripheral neuropathy function was associated with improvements after physical exercise and electrical stimulation. For humans, too little research has been conducted on this topic to reach a complete conclusion. This research supports the need for future studies to test the validity of a possible rehabilitation treatment in humans in cases of peripheral neuropathy to help nerve sprouting.

Translational enhancement by base editing of the Kozak sequence rescues haploinsufficiency.

A variety of single-gene human diseases are caused by haploinsufficiency, a genetic condition by which mutational inactivation of one allele leads to reduced protein levels and functional impairment. Translational enhancement of the spare allele could exert a therapeutic effect. Here we developed BOOST, a novel gene-editing approach to rescue haploinsufficiency loci by the change of specific single nucleotides in the Kozak sequence, which controls translation by regulating start codon recognition. We evaluated for translational strength 230 Kozak sequences of annotated human haploinsufficient genes and 4621 derived variants, which can be installed by base editing, by a high-throughput reporter assay. Of these variants, 149 increased the translation of 47 Kozak sequences, demonstrating that a substantial proportion of haploinsufficient genes are controlled by suboptimal Kozak sequences. Validation of 18 variants for 8 genes produced an average enhancement in an expression window compatible with the rescue of the genetic imbalance. Base editing of the NCF1 gene, whose monoallelic loss causes chronic granulomatous disease, resulted in the desired increase of NCF1 (p47phox) protein levels in a relevant cell model. We propose BOOST as a fine-tuned approach to modulate translation, applicable to the correction of dozens of haploinsufficient monogenic disorders independently of the causing mutation.

Zebrafish Melanoma-Derived Interstitial EVs Are Carriers of ncRNAs That Induce Inflammation.

Extracellular vesicles (EVs) are membranous particles released by all cell types. Their role as functional carrier of bioactive molecules is boosted by cells that actively secrete them in biological fluids or in the intercellular space (interstitial EVs, iEVs). Here we have optimised a method for the isolation and characterization of zebrafish iEVs from whole melanoma tissues. Zebrafish melanoma iEVs are around 140 nm in diameter, as determined by nanoparticle tracking and transmission electron microscopy (TEM) analysis. Western blot analysis shows enrichment for CD63 and Alix in the iEV fraction, but not in melanoma cell lysates. Super resolution and confocal microscopy reveal that purified zebrafish iEVs are green fluorescent protein positive (GFP+), indicating that they integrate the oncogene GFP-HRASV12G used to induce melanoma in this model within their vesicular membrane or luminal content. Analysis of RNA-Seq data found 118 non-coding (nc)RNAs differentially distributed between zebrafish melanoma and their iEVs, with only 17 of them being selectively enriched in iEVs. Among these, the RNA components of RNAses P and MRP, which process ribosomal RNA precursors, mitochondrial RNAs, and some mRNAs, were enriched in zebrafish and human melanoma EVs, but not in iEVs extracted from brain tumours. We found that melanoma iEVs induce an inflammatory response when injected in larvae, with increased expression of interferon responsive genes, and this effect is reproduced by MRP- or P-RNAs injected into circulation. This suggests that zebrafish melanoma iEVs are a source of MRP- and P-RNAs that can trigger inflammation in cells of the innate immune system.

Ultrasensitive detection of cancer biomarkers by nickel-based isolation of polydisperse extracellular vesicles from blood.

BACKGROUND: Extracellular vesicles (EVs) are secreted membranous particles intensively studied for their potential cargo of diagnostic markers. Efficient and cost-effective isolation methods need to be established for the reproducible and high-throughput study of EVs in the clinical practice. METHODS: We designed the nickel-based isolation (NBI) to rapidly isolate EVs and combined it with newly-designed amplified luminescent proximity homogeneous assay or digital PCR to detect biomarkers of clinical utility. FINDINGS: From plasma of 46 healthy donors, we systematically recovered small EV (~250 nm of mean diameter; ~3 × 1010/ml) and large EV (~560 nm of mean diameter; ~5 × 108/ml) lineages ranging from 50 to 700 nm, which displayed hematopoietic/endothelial cell markers that were also used in spike-in experiments using EVs from tumor cell lines. In retrospective studies, we detected picomolar concentrations of prostate-specific membrane antigen (PSMA) in fractions of EVs isolated from the plasma of prostate cancer patients, discriminating them from control subjects. Directly from oil-encapsulated EVs for digital PCR, we identified somatic BRAF and KRAS mutations circulating in the plasma of metastatic colorectal cancer (CRC) patients, matching 100% of concordance with tissue diagnostics. Importantly, with higher sensitivity and specificity compared with immuno-isolated EVs, we revealed additional somatic alterations in 7% of wild-type CRC cases that were subsequently validated by further inspections in the matched tissue biopsies. INTERPRETATION: We propose NBI-combined approaches as simple, fast, and robust strategies to probe the tumor heterogeneity and contribute to the development of EV-based liquid biopsy studies. FUND: Associazione Italiana per la Ricerca sul Cancro (AIRC), Fondazione Cassa di Risparmio Trento e Rovereto (CARITRO), and the Italian Ministero Istruzione, Università e Ricerca (Miur).

Long-term survival and immunological parameters in metastatic melanoma patients who responded to ipilimumab 10 mg/kg within an expanded access programme.

BACKGROUND: Ipilimumab can result in durable clinical responses among patients with advanced melanoma. However, no predictive marker of clinical activity has yet been identified. We provide preliminary data describing the correlation between immunological parameters and response/survival among patients with advanced melanoma who received ipilimumab 10 mg/kg in an expanded access programme. METHODS: Patients received ipilimumab 10 mg/kg every 3 weeks (Q3W) for four doses (induction) and Q12W from week 24 (W24) as maintenance therapy. Tumor assessments were conducted Q12W. Expression of inducible T cell costimulator (ICOS) on CD4(+) and CD8(+) T cells was assessed at baseline, W7, W12 and W24, and the ratio between absolute neutrophils (N) and lymphocytes (L) determined at baseline, W4, W7 and W10. RESULTS: Median overall survival among 27 patients was 9.6 months (95 % CI 3.2-16.1), with 3- and 4-year survival rates of 20.4 %. Five patients survived >4 years. Patients with an increase in the number of circulating ICOS(+) T cells at W7 were more likely to experience disease control and have improved survival. An N/L ratio below the median at W7 and W10 was also associated with better survival compared with an N/L ratio above the median. CONCLUSIONS: Ipilimumab can induce long-term survival benefits in heavily pretreated patients with metastatic melanoma. Changes in the number of circulating ICOS(+) T cells or N/L ratio during ipilimumab treatment may represent early markers of response. However, given the limited sample size, further investigation is required.

The acquired immune response to the mucosal adjuvant LTK63 imprints the mouse lung with a protective signature.

LTK63, a nontoxic mutant of Escherichia coli heat labile enterotoxin (LT), is a potent and safe mucosal adjuvant that has also been shown to confer generic protection to several respiratory pathogens. To understand the mechanisms of action underlying the LTK63 protective effect, we analyzed the molecular and cellular events triggered by its administration in vivo. We show here that LTK63 intrapulmonary administration induced in the mouse lung a specific gene expression signature characterized by the up-regulation of cell cycle genes, several host defense genes, chemokines, chemokine receptors, and immune cell-associated genes. Such a transcriptional profile reflected the activation of alveolar macrophages and the recruitment to the lung of T and B cells and innate immune cells such as granulocytes, NK, and dendritic cells. All of these events were T cell dependent and specific for LTK63 because they were absent in SCID and nude mice. Additionally, we showed that LTK63 induces a potent adaptive immune response against itself directed to the lung. We propose that acquired response to LTK63 is the driving force for the local recruitment of both adaptive and innate immune cells. Our data suggest that LTK63 acts as an airway infection mimic that establishes a generic protective environment limiting respiratory infection by innate immune mechanisms and by improving adaptive responses to invading pathogens.