ABSTRACT Despite the increasing prevalence of neurodegenerative diseases, the molecular characterization of the brain remains challenging due to limited access to the tissue. Cerebrospinal fluid (CSF) contains a significant proportion of molecular contents originating from the brain, and characterizing these molecules has served as a surrogate to evaluate molecular dysregulation in the brain. Here we performed cell-free messenger RNA (cf-mRNA) RNA-sequencing on 52 human CSF samples, and further compared their transcriptomic profiles to matched plasma samples. In addition, we evaluated the molecular dysregulation of cf-mRNA in CSF between individuals with Alzheimer’s disease (AD) and non-cognitively impaired (NCI) controls. The molecular content of CSF cf-mRNA was distinct from plasma cf-mRNA, with a substantially higher number of brain-associated genes identified in CSF. We identified a large set of dysregulated gene transcripts in the CSF cf-mRNA population of individuals with AD, and these gene transcripts were used to establish a diagnostic classifier to discriminate AD from NCI subjects. Notably, the gene transcripts were enriched in biological processes closely associated with AD, such as brain development and synaptic signaling. We also discovered a subset of gene transcripts within AD subjects that exhibit a strong correlation between CSF and plasma cf-mRNA. This study not only reveals the novel cf-mRNA content of CSF but also highlights the potential of CSF cf-mRNA profiling as a tool to garner pathophysiological insights into AD.

Despite gene-expression profiling being one of the most common methods to evaluate molecular dysregulation in tissues, the utilization of cell-free messenger RNA (cf-mRNA) as a blood-based non-invasive biomarker analyte has been limited compared to other RNA classes. Recent advancements in low-input RNA-sequencing and normalization techniques, however, have enabled characterization as well as accurate quantification of cf-mRNAs allowing direct pathological insights. The molecular profile of the cell-free transcriptome in multiple diseases has subsequently been characterized including, prenatal diseases, neurological disorders, liver diseases and cancers suggesting this biological compartment may serve as a disease agnostic platform. With mRNAs packaged in a myriad of extracellular vesicles and particles, these signals may be used to develop clinically actionable, non-invasive disease biomarkers. Here, we summarize the recent scientific developments of extracellular mRNA, biology of extracellular mRNA carriers, clinical utility of cf-mRNA as disease biomarkers, as well as proposed functions in cell and tissue pathophysiology.

Background: Primary sclerosing cholangitis (PSC) is a rare chronic cholestatic liver disease characterized by multifocal bile duct strictures. To date, underlying molecular mechanisms of PSC remain unclear, and therapeutic options are limited. Methods: We performed cell-free messenger RNA (cf-mRNA) sequencing to characterize the circulating transcriptome of PSC and noninvasively investigate potentially bioactive signals that are associated with PSC. Serum cf-mRNA profiles were compared among 50 individuals with PSC, 20 healthy controls, and 235 individuals with NAFLD. Tissue and cell type-of-origin genes that are dysregulated in subjects with PSC were evaluated. Subsequently, diagnostic classifiers were developed using PSC dysregulated cf-mRNA genes. Results: Differential expression analysis of the cf-mRNA transcriptomes of PSC and healthy controls resulted in identification of 1407 dysregulated genes. Furthermore, differentially expressed genes between PSC and healthy controls or NAFLD shared common genes known to be involved in liver pathophysiology. In particular, genes from liver- and specific cell type-origin, including hepatocyte, HSCs, and KCs, were highly abundant in cf-mRNA of subjects with PSC. Gene cluster analysis revealed that liver-specific genes dysregulated in PSC form a distinct cluster, which corresponded to a subset of the PSC subject population. Finally, we developed a cf-mRNA diagnostic classifier using liver-specific genes that discriminated PSC from healthy control subjects using gene transcripts of liver origin. Conclusions: Blood-based whole-transcriptome cf-mRNA profiling revealed high abundance of liver-specific genes in sera of subjects with PSC, which may be used to diagnose patients with PSC. We identified several unique cf-mRNA profiles of subjects with PSC. These findings may also have utility for noninvasive molecular stratification of subjects with PSC for pharmacotherapy safety and response studies.

BackgroundInflammatory and immune responses are essential and dynamic biological processes that protect the body against acute and chronic adverse stimuli. While conventional protein markers have been used to evaluate systemic inflammatory response, the immunological response to stimulation is complex and involves modulation of a large set of genes and interacting signalling pathways of innate and adaptive immune systems. There is a need for a non-invasive tool that can comprehensively evaluate and monitor molecular dysregulations associated with inflammatory and immune responses in circulation and in inaccessible solid organs.MethodsHere we utilized cell-free messenger RNA (cf-mRNA) RNA-Seq whole transcriptome profiling and computational biology to temporally assess lipopolysaccharide (LPS) induced and JAK inhibitor modulated inflammatory and immune responses in mouse plasma samples.FindingsCf-mRNA profiling displayed a pattern of systemic immune responses elicited by LPS and dysregulation of associated pathways. Moreover, attenuation of several inflammatory pathways, including STAT and interferon pathways, were observed following the treatment of JAK inhibitor. We further identified the dysregulation of liver-specific transcripts in cf-mRNA which reflected changes in the gene-expression pattern in this generally inaccessible biological compartment.InterpretationUsing a preclinical mouse model, we demonstrated the potential of plasma cf-mRNA profiling for systemic and organ-specific characterization of drug-induced molecular alterations that are associated with inflammatory and immune responses.FundingMolecular Stethoscope.

Abstract Background and Aims: Hematologic malignancies account for approximately 10% of all cancers in the US. In order to improve the treatment and outcome of patients with hematologic malignancies, integration of accurate noninvasive molecular biomarkers to diagnose, monitor therapeutic response and identify disease subtype are critical. Recent advances in next generation sequencing (NGS) have accelerated the utilization of circulating nucleic acids for the development of non-invasive disease biomarkers. We have developed an RNA-Seq platform that allows robust quantification of the circulating cell-free messenger RNA (cf-mRNA) whole transcriptome. Here, we conducted cf-mRNA profiling on plasma samples collected from multiple myeloma and acute myeloid leukemia patients to investigate the feasibility of cell-free transcriptomic profiling as a monitoring tool for hematologic malignancies following bone marrow transplantation and therapeutic treatment. Methods: We performed cf-mRNA RNA-Sequencing on 96 longitudinally collected plasma samples from 3 multiple myeloma and 3 acute myeloid leukemia patients. Cf-mRNA profiles of patients were evaluated using GTEx (tissue type) and Blueprint (cell type), and dysregulation of gene-expression signatures were assessed following bone marrow transplantation and therapeutic treatment. In addition, we sequenced corresponding whole blood cells and buffy coat for comparison. Results: Plasma cf-mRNA profiling showed substantially higher proportion of transcripts from megakaryocyte and non-hematopoietic cells relative to whole blood cellular transcripts, suggesting that the molecular profile of cf-mRNA is more suitable for the assessment of gene-expression dysregulation of bone marrow than whole blood. We further report that non-invasive cf-mRNA profiling captures gene-expression alterations of cancer patients following chemotherapy and bone marrow transplantation. And we observed dysregulation of hematopoietic lineage-specific transcripts in circulation during bone marrow ablation and reconstitution. Lastly, we used cf-mRNA profiling to identify a malignant Ig clone for multiple myeloma patients and were able to monitor bone marrow reconstitution. Conclusions: We demonstrated that cf-mRNA profiling is an effective means to non-invasively assess molecular characteristics that are associated with multiple clinical parameters of hematological malignancies. Our study highlights the potential of cf-mRNA Seq as a platform to develop patient monitoring biomarkers for hematologic malignancies. Citation Format: Shusuke Toden, David A. Ross, Stephen R. Quake, John J. Sninsky. Utilization of Cell free messenger RNA Sequencing for non-invasive molecular profiling of multiple myeloma and Acute Myeloid Leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5174.

Hepatic fibrosis stage is the most important determinant of outcomes in patients with nonalcoholic fatty liver disease (NAFLD). There is an urgent need for noninvasive tests that can accurately stage fibrosis and determine efficacy of interventions. Here, we describe a novel cell-free (cf)-mRNA sequencing approach that can accurately and reproducibly profile low levels of circulating mRNAs and evaluate the feasibility of developing a cf-mRNA-based NAFLD fibrosis classifier. Using separate discovery and validation cohorts with biopsy-confirmed NAFLD (n = 176 and 59, respectively) and healthy subjects (n = 23), we performed serum cf-mRNA RNA-Seq profiling. Differential expression analysis identified 2,498 dysregulated genes between patients with NAFLD and healthy subjects and 134 fibrosis-associated genes in patients with NAFLD. Comparison between cf-mRNA and liver tissue transcripts revealed significant overlap of fibrosis-associated genes and pathways indicating that the circulating cf-mRNA transcriptome reflects molecular changes in the livers of patients with NAFLD. In particular, metabolic and immune pathways reflective of known underlying steatosis and inflammation were highly dysregulated in the cf-mRNA profile of patients with advanced fibrosis. Finally, we used an elastic net ordinal logistic model to develop a classifier that predicts clinically significant fibrosis (F2-F4). In an independent cohort, the cf-mRNA classifier was able to identify 50% of patients with at least 90% probability of clinically significant fibrosis. We demonstrate a novel and robust cf-mRNA-based RNA-Seq platform for noninvasive identification of diverse hepatic molecular disruptions and for fibrosis staging with promising potential for clinical trials and clinical practice. NEW & NOTEWORTHY This work is the first study, to our knowledge, to utilize circulating cell-free mRNA sequencing to develop an NAFLD diagnostic classifier.

Previous studies have demonstrated that donor-derived cell-free DNA (dd-cfDNA) found in circulating blood of transplant recipients may serve as a non-invasive biomarker of allograft rejection. The D-OAR study was undertaken to characterize the performance of dd-cfDNA in heart transplant recipients who are receiving rejection surveillance with gene expression profiling (GEP) as part of their standard care. To better interpret the clinical meaning of dd-cfDNA, it is essential to understand the biological variation of this biomarker in stable healthy recipients. This planned report will define the threshold of dd-cfDNA for rejection and will also establish the biological variation and clinical reference intervals of dd-cfDNA in heart transplant recipients using an analytically validated assay.

Gene expression profiling (GEP) is enhanced by an understanding of the contribution to the final score by the individual genes reflective of various physiological pathways. This analysis compares the relative contributions of individual genes and metagenes to GEP scores for commercially provided results from 2005-2015.

Cell-free DNA (cfDNA) from lung transplant recipients was analyzed using a clinical-grade targeted next-generation sequencing (NGS) method to determine the correlation between donor-derived cell-free DNA (dd-cfDNA) and transbronchial histopathology grades. These results were compared to published results using a shotgun sequencing method with prior donor and recipient genotyping to determine the correlation between methods.

The variability of gene expression scores (GEP, AlloMap®) over serial tests has been established as predictive of long-term outcomes. The contribution of individual genes to the variability of the scores has not previously been analyzed. This analysis examines the relative contribution of gene expression variability to GEP score variability (GEPSV).