Cell-free DNA analysis in healthy individuals by next-generation sequencing: a proof of concept and technical validation study.

Pre-symptomatic screening of genetic alterations might help identify subpopulations of individuals that could enter into early access prevention programs. Since liquid biopsy is minimally invasive it can be used for longitudinal studies in healthy volunteers to monitor events of progression from normal tissue to pre-cancerous and cancerous condition. Yet, cell-free DNA (cfDNA) analysis in healthy individuals comes with substantial challenges such as the lack of large cohort studies addressing the impact of mutations in healthy individuals or the low abundance of cfDNA in plasma. In this study, we aimed to investigate the technical feasibility of cfDNA analysis in a collection of 114 clinically healthy individuals. We first addressed the impact of pre-analytical factors such as cfDNA yield and quality on sequencing performance and compared healthy to cancer donor samples. We then confirmed the validity of our testing strategy by evaluating the mutational status concordance in matched tissue and plasma specimens collected from cancer patients. Finally, we screened our group of healthy donors for genetic alterations, comparing individuals who did not develop any tumor to patients who developed either a benign neoplasm or cancer during 1-10 years of follow-up time. To conclude, we have established a rapid and reliable liquid biopsy workflow that allowed us to study genomic alterations with a limit of detection as low as 0.08% of variant allelic frequency in healthy individuals. We detected pathogenic cancer mutations in four healthy donors that later developed a benign neoplasm or invasive breast cancer up to 10 years after blood collection. Even though larger prospective studies are needed to address the specificity and sensitivity of liquid biopsy as a clinical tool for early cancer detection, systematic screening of healthy individuals will help understanding early events of tumor formation

Integrated analysis of the prostate cancer small-nucleolar transcriptome reveals <i>SNORA55</i> as a driver of prostate cancer progression

Metastasis is the primary cause of death in prostate cancer (PCa) patients. Small nucleolar RNAs (snoRNAs) have long been considered "housekeeping" genes with no relevance for cancer biology. Emerging evidence has challenged this assumption, suggesting that snoRNA expression is frequently modulated during cancer progression. Despite this, no study has systematically addressed the prognostic and functional significance of snoRNAs in PCa. We performed RNA Sequencing on paired metastatic/non-metastatic PCa xenografts derived from clinical specimens. The clinical significance of differentially expressed snoRNAs was further investigated in two independent primary PCa cohorts (131 and 43 patients, respectively). The snoRNA demonstrating the strongest association with clinical outcome was quantified in PCa patient-derived serum samples and its functional relevance was investigated in PCa cells via gene expression profiling, pathway analysis and gene silencing. Our comparison revealed 21 differentially expressed snoRNAs in the metastatic vs. non-metastatic xenografts. Of those, 12 were represented in clinical databases and were further analyzed. SNORA55 emerged as a predictor of shorter relapse-free survival (results confirmed in two independent databases). SNORA55 was reproducibly detectable in serum samples from PCa patients. SNORA55 silencing in PCa cell lines significantly inhibited cell proliferation and migration. Pathway analysis revealed that SNORA55 expression is significantly associated with growth factor signaling and pro-inflammatory cytokine expression in PCa. Our results demonstrate that SNORA55 up-regulation predicts PCa progression and that silencing this non-coding gene affects PCa cell proliferation and metastatic potential, thus positioning it as both a novel biomarker and therapeutic target

Psip1/p52 regulates posterior Hoxa genes through activation of lncRNA Hottip

Long noncoding RNAs (lncRNAs) have been implicated in various biological functions including the regulation of gene expression, however, the functionality of lncRNAs is not clearly understood and conflicting conclusions have often been reached when comparing different methods to investigate them. Moreover, little is known about the upstream regulation of lncRNAs. Here we show that the short isoform (p52) of a transcriptional co-activator—PC4 and SF2 interacting protein (Psip1), which is known to be involved in linking transcription to RNA processing, specifically regulates the expression of the lncRNA Hottip–located at the 5’ end of the Hoxa locus. Using both knockdown and knockout approaches we show that Hottip expression is required for activation of the 5’ Hoxa genes (Hoxa13 and Hoxa10/11) and for retaining Mll1 at the 5’ end of Hoxa. Moreover, we demonstrate that artificially inducing Hottip expression is sufficient to activate the 5’ Hoxa genes and that Hottip RNA binds to the 5’ end of Hoxa. By engineering premature transcription termination, we show that it is the Hottip lncRNA molecule itself, not just Hottip transcription that is required to maintains active expression of posterior Hox genes. Our data show a direct role for a lncRNA molecule in regulating the expression of developmentally-regulated mRNA genes in cis

Genetic profiling using plasma-derived cell-free DNA in therapy-naïve hepatocellular carcinoma patients: a pilot study

Hepatocellular carcinomas (HCCs) are not routinely biopsied, resulting in a lack of tumor materials for molecular profiling. Here we sought to determine whether plasma-derived cell-free DNA (cfDNA) captures the genetic alterations of HCC in patients who have not undergone systemic therapy.; Frozen biopsies from the primary tumor and plasma were synchronously collected from 30 prospectively recruited, systemic treatment-naïve HCC patients. Deep sequencing of the DNA from the biopsies, plasma-derived cfDNA and matched germline was carried out using a panel targeting 46 coding and non-coding genes frequently altered in HCCs.; In 26/30 patients, at least one somatic mutation was detected in biopsy and/or cfDNA. Somatic mutations in HCC-associated genes were present in the cfDNA of 63% (19/30) of the patients and could be detected 'de novo' without prior knowledge of the mutations present in the biopsy in 27% (8/30) of the patients. Mutational load and the variant allele fraction of the mutations detected in the cfDNA positively correlated with tumor size and Edmondson grade. Crucially, among the seven patients in whom the largest tumor was ≥5 cm or was associated with metastasis, at least one mutation was detected 'de novo' in the cfDNA of 86% (6/7) of the cases. In these patients, cfDNA and tumor DNA captured 87% (80/92) and 95% (87/92) of the mutations, suggesting that cfDNA and tumor DNA captured similar proportions of somatic mutations.; In patients with high disease burden, the use of cfDNA for genetic profiling when biopsy is unavailable may be feasible. Our results support further investigations into the clinical utility of cfDNA in a larger cohort of patients

IgA in the horse: cloning of equine polymeric Ig receptor and J chain and characterization of recombinant forms of equine IgA

As in other mammals, immunoglobulin A (IgA) in the horse has a key role in immune defense. To better dissect equine IgA function, we isolated complementary DNA (cDNA) clones for equine J chain and polymeric Ig receptor (pIgR). When coexpressed with equine IgA, equine J chain promoted efficient IgA polymerization. A truncated version of equine pIgR, equivalent to secretory component, bound with nanomolar affinity to recombinant equine and human dimeric IgA but not with monomeric IgA from either species. Searches of the equine genome localized equine J chain and pIgR to chromosomes 3 and 5, respectively, with J chain and pIgR coding sequence distributed across 4 and 11 exons, respectively. Comparisons of transcriptional regulatory sequences suggest that horse and human pIgR expression is controlled through common regulatory mechanisms that are less conserved in rodents. These studies pave the way for full dissection of equine IgA function and open up possibilities for immune-based treatment of equine diseases

The evolutionarily conserved long non‐coding RNA <i>LINC00261</i> drives neuroendocrine prostate cancer proliferation and metastasis <i>via</i> distinct nuclear and cytoplasmic mechanisms

Metastatic neuroendocrine prostate cancer (NEPC) is a highly aggressive disease, whose incidence is rising. Long noncoding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, most of which are still functionally uncharacterized. Thus, we set out to identify the highly conserved lncRNAs that play a central role in NEPC pathogenesis. To this end, we performed transcriptomic analyses of donor-matched patient-derived xenograft models (PDXs) with immunohistologic features of prostate adenocarcinoma (AR+/PSA+) or NEPC (AR-/SYN+/CHGA+ ) and through differential expression analyses identified lncRNAs that were upregulated upon neuroendocrine transdifferentiation. These genes were prioritized for functional assessment based on the level of conservation in vertebrates. Here, LINC00261 emerged as the top gene with over 3229-fold upregulation in NEPC. Consistently, LINC00261 expression was significantly upregulated in NEPC specimens in multiple patient cohorts. Knockdown of LINC00261 in PC-3 cells dramatically attenuated its proliferative and metastatic abilities, which are explained by parallel downregulation of CBX2 and FOXA2 through distinct molecular mechanisms. In the cell cytoplasm, LINC00261 binds to and sequesters miR-8485 from targeting the CBX2 mRNA, while inside the nucleus, LINC00261 functions as a transcriptional scaffold to induce SMAD-driven expression of the FOXA2 gene. For the first time, these results demonstrate hyperactivation of the LINC00261-CBX2-FOXA2 axes in NEPC to drive proliferation and metastasis, and that LINC00261 may be utilized as a therapeutic target and a biomarker for this incurable disease