Extracellular Vesicles: A Brief Overview and Its Role in Precision Medicine.

Precision medicine has emerged as an approach to tailor therapies for an individual at the time of diagnosis and/or treatment. This emergence has been fueled by the ability to profile nucleic acids, along with proteins and lipids isolated from biofluids, a method called "liquid biopsy ," either by or in combination of one of the following components: circulating tumor cells (CTCs), cell-free DNA (cfDNA), and/or extracellular vesicles (EVs) . EVs are membrane-surrounded structures released by cells in an evolutionarily conserved manner. EVs have gained much attention from both the basic and clinical research areas, as EVs appear to play a role in many diseases; however, the well-known case is cancer. The hallmark of EVs in cancer is their role as mediators of communication between cells both at physiological and pathophysiological levels; hence, EVs are thought to contribute to the creation of a microenvironmental niche that promotes cancer cell survival, as well as reprogramming distant tissue for invasion. It is important to understand the mechanistic and functional aspects at the basic science level of EVs to get a better grasp on their role in healthy and disease states. EVs range from 30-1000 nm membrane-enclosed vesicles that are released by many mammalian cell types and present in a variety of biofluids. EVs have emerged as an area of clinical interest in the era of Precision Medicine, from their role in liquid biopsy (tissue biopsy free) approach for screening, assessing tumor heterogeneity, monitoring therapeutic responses, and minimal residual disease detection to EV-based therapeutics . EVs' diagnostic and therapeutic exploitation is under intense investigation in various indications. This chapter highlights EV biogenesis , composition of EVs, and their potential role in liquid biopsy diagnostics and therapeutics in the area of cancer.

Cancer Liquid Biopsy: Is It Ready for Clinic?

The management of cancer relies on a combination of imaging and tissue biopsy for diagnosis, monitoring, and molecular classification-based patient stratification to ensure appropriate treatment. Conventional tissue biopsy harvests tumor samples with invasive procedures, which are often difficult for patients with advanced disease. Given the well-recognized intratumor genetic heterogeneity [1], the biopsy of small tumor fragments does not necessarily represent all the genetic aberrations in the tumor, but sampling the entire tumor in each patient is not realistic. Moreover, tumors evolve all the time from local to advanced disease and by adapting to selective pressure from treatment.

Molecular analysis of gastric cancer identifies subtypes associated with distinct clinical outcomes.

Gastric cancer, a leading cause of cancer-related deaths, is a heterogeneous disease. We aim to establish clinically relevant molecular subtypes that would encompass this heterogeneity and provide useful clinical information. We use gene expression data to describe four molecular subtypes linked to distinct patterns of molecular alterations, disease progression and prognosis. The mesenchymal-like type includes diffuse-subtype tumors with the worst prognosis, the tendency to occur at an earlier age and the highest recurrence frequency (63%) of the four subtypes. Microsatellite-unstable tumors are hyper-mutated intestinal-subtype tumors occurring in the antrum; these have the best overall prognosis and the lowest frequency of recurrence (22%) of the four subtypes. The tumor protein 53 (TP53)-active and TP53-inactive types include patients with intermediate prognosis and recurrence rates (with respect to the other two subtypes), with the TP53-active group showing better prognosis. We describe key molecular alterations in each of the four subtypes using targeted sequencing and genome-wide copy number microarrays. We validate these subtypes in independent cohorts in order to provide a consistent and unified framework for further clinical and preclinical translational research.

The draft genome of the large yellow croaker reveals well-developed innate immunity.

The large yellow croaker, Larimichthys crocea, is one of the most economically important marine fish species endemic to China. Its wild stocks have severely suffered from overfishing, and the aquacultured species are vulnerable to various marine pathogens. Here we report the creation of a draft genome of a wild large yellow croaker using a whole-genome sequencing strategy. We estimate the genome size to be 728 Mb with 19,362 protein-coding genes. Phylogenetic analysis shows that the stickleback is most closely related to the large yellow croaker. Rapidly evolving genes under positive selection are significantly enriched in pathways related to innate immunity. We also confirm the existence of several genes and identify the expansion of gene families that are important for innate immunity. Our results may reflect a well-developed innate immune system in the large yellow croaker, which could aid in the development of wild resource preservation and mariculture strategies.

Genomic landscape and genetic heterogeneity in gastric adenocarcinoma revealed by whole-genome sequencing.

Gastric cancer (GC) is the second most common cause of cancer-related deaths. It is known to be a heterogeneous disease with several molecular and histological subtypes. Here we perform whole-genome sequencing of 49 GCs with diffuse (N=31) and intestinal (N=18) histological subtypes and identify three mutational signatures, impacting TpT, CpG and TpCp[A/T] nucleotides. The diffuse-type GCs show significantly lower clonality and smaller numbers of somatic and structural variants compared with intestinal subtype. We further divide the diffuse subtype into one with infrequent genetic changes/low clonality and another with relatively higher clonality and mutations impacting TpT dinucleotide. Notably, we discover frequent and exclusive mutations in Ephrins and SLIT/ROBO signalling pathway genes. Overall, this study delivers new insights into the mutational heterogeneity underlying distinct histologic subtypes of GC that could have important implications for future research in the diagnosis and treatment of GC.

Development of a miR-26 companion diagnostic test for adjuvant interferon-alpha therapy in hepatocellular carcinoma.

BACKGROUND & AIMS: Adjuvant therapies for hepatocellular carcinoma (HCC) such as interferon-alpha are effective only in a subset of patients. Previously we found that HCC patients with low level of miR-26 have survival benefits from interferon-alpha. The purpose of this study is to develop a standardized miR-26 diagnostic test (referred as MIR26-DX) to assist identification of candidate HCC patients for adjuvant interferon-alpha therapy. METHODS: We developed a multiplex reverse-transcription quantitative polymerase-chain-reaction assay to determine the levels of two HCC-related miR-26 transcripts along with six small RNA reference transcripts. We evaluated archived paraffin-embedded tissues from three cohorts of HCC patients (n=248) who underwent radical resection at three different clinical centers. Fifty-two percent of them underwent adjuvant interferon-alpha therapy. We used Cox-Mantel log-rank test to evaluate patient survival. RESULTS: We found that the multiplexing assay was stable and reproducible regardless of differences in sample preparations and operators. We developed a matrix template and a scoring algorithm based on a training cohort (n=129) to assign HCC patients, and then applied the template in two test cohorts (n=119). The proportions of HCC patients assigned as low miR-26 by this algorithm were 68, 4, and 63 percent in the training cohort and two test cohorts, respectively. Consistently, HCC with low miR-26 had a favorable response to interferon-alpha with improved median overall survival (≥3 year). CONCLUSIONS: MIR26-DX is a simple and reliable companion diagnostic test to select HCC patients for adjuvant interferon-alpha therapy.

Peripheral arterial occlusive disease: global gene expression analyses suggest a major role for immune and inflammatory responses.

BACKGROUND: Peripheral arterial disease (PAD), a major manifestation of atherosclerosis, is associated with significant cardiovascular morbidity, limb loss and death. However, mechanisms underlying the genesis and progression of the disease are far from clear. Genome-wide gene expression profiling of clinical samples may represent an effective approach to gain relevant information. RESULTS: After histological classification, a total of 30 femoral artery samples, including 11 intermediate lesions, 14 advanced lesions and 5 normal femoral arteries, were profiled using Affymetrix microarray platform. Following real-time RT-PCR validation, different algorithms of gene selection and clustering were applied to identify differentially expressed genes. Under a stringent cutoff, i.e., a false discovery rate (FDR) <0.5%, we found 366 genes were differentially regulated in intermediate lesions and 447 in advanced lesions. Of these, 116 genes were overlapped between intermediate and advanced lesions, including 68 up-regulated genes and 48 down-regulated ones. In these differentially regulated genes, immune/inflammatory genes were significantly up-regulated in different stages of PAD, (85/230 in intermediate lesions, 37/172 in advanced lesions). Through literature mining and pathway analysis using different databases such as Gene Ontology (GO), and the Kyoto Encyclopedia of Gene and Genomics (KEGG), genes involved in immune/inflammatory responses were significantly enriched in up-regulated genes at different stages of PAD(p < 0.05), revealing a significant correlation between immune/inflammatory responses and disease progression. Moreover, immune-related pathways such as Toll-like receptor signaling and natural killer cell mediated cytotoxicity were particularly enriched in intermediate and advanced lesions (P < 0.05), highlighting their pathogenic significance during disease progression. CONCLUSION: Lines of evidence revealed in this study not only support previous hypotheses, primarily based on studies of animal models and other types of arterial disease, that inflammatory responses may influence the development of PAD, but also permit the recognition of a wide spectrum of immune/inflammatory genes that can serve as signatures for disease progression in PAD. Further studies of these signature molecules may eventually allow us to develop more sophisticated protocols for pharmaceutical interventions.