Cell-free RNA Research Articles

Evaluating the Effects of Storage Conditions on Multiple Cell-Free RNAs in Plasma by High-Throughput Sequencing.

Background: Plasma cell-free RNAs (cfRNAs) can serve as noninvasive biomarkers for the diagnosis and monitoring of diseases. However, the delay in blood processing may lead to unreliable results. Therefore, an unbiased evaluation based on the whole transcriptome under different storage conditions is needed. Methods: Here, blood samples were collected in ethylenediaminetetraacetic acid tubes and processed immediately (0 hour), or stored at room temperature (RT) or 4°C for different time intervals (2, 6, and 24 hours) before plasma separation. High-throughput sequencing was applied to assess the effects of storage conditions on the transcript profiles and fragment characteristics of plasma cell-free mRNA, long noncoding RNA (lncRNA), and small RNAs. Results: More genes changed their expression levels with time when blood was stored at RT compared with those at 4°C. Cell-free mRNA and lncRNA were relatively stable in blood preserved at 4°C for 6 hours, while cell-free microRNA (miRNA) and piwi-interacting RNA (piRNA) remained stable at 4°C for 24 hours. After 24 hours, more contamination of the leukocyte-derived RNAs occurred at RT, possibly due to apoptosis. Meanwhile, significant changes were also observed regarding the characteristics of the RNA fragments, including fragment size, the proportion of intron, and the pyrimidine frequency of the fragmented 3' end. Fifteen tissue-enriched genes were detected in the plasma but not expressed in leukocytes. The expression level and fragment length of these genes gradually decreased during storage, suggesting the degradation of the cfRNA and the dilution of leukocyte-derived RNA with other tissue-derived cfRNA. Conclusions: Our results suggest that the contamination of leukocyte-derived RNA and the degradation of original cfRNA contribute to the changes in the cfRNA expression profiles and the fragment characteristics during short-term storage. The storage of blood at 4°C for 6 hours allows plasma cfRNA to remain relatively stable, which will be useful for further studies or clinical applications where adequate quantification or the fragment signature of cfRNA is required.

Diagnostic and Therapeutic Potential of Circulating-Free DNA and Cell-Free RNA in Cancer Management.

Over time, molecular biology and genomics techniques have been developed to speed up the early diagnosis and clinical management of cancer. These therapies are often most effective when administered to the subset of malignancies harboring the target identified by molecular testing. Important advances in applying molecular testing involve circulating-free DNA (cfDNA)- and cell-free RNA (cfRNA)-based liquid biopsies for the diagnosis, prognosis, prediction, and treatment of cancer. Both cfDNA and cfRNA are sensitive and specific biomarkers for cancer detection, which have been clinically proven through multiple randomized and prospective trials. These help in cancer management based on the noninvasive evaluation of size, quantity, and point mutations, as well as copy number alterations at the tumor site. Moreover, personalized detection of ctDNA helps in adjuvant therapeutics and predicts the chances of recurrence of cancer and resistance to cancer therapy. Despite the controversial diagnostic values of cfDNA and cfRNA, many clinical trials have been completed, and the Food and Drug Administration has approved many multigene assays to detect genetic alterations in the cfDNA of cancer patients. In this review, we underpin the recent advances in the physiological roles of cfDNA and cfRNA, as well as their roles in cancer detection by highlighting recent clinical trials and their roles as prognostic and predictive markers in cancer management.

Low Impact of Clonal Hematopoiesis on the Determination of RAS Mutations by Cell-Free DNA Testing in Routine Clinical Diagnostics.

Targeted sequencing of circulating cell-free DNA (cfDNA) is used in routine clinical diagnostics for the identification of predictive biomarkers in cancer patients in an advanced stage. The presence of KRAS mutations associated with clonal hematopoiesis of indeterminate potential (CHIP) might represent a confounding factor. We used an amplicon-based targeted sequencing panel, covering selected regions of 52 genes, for circulating cell-free total nucleic acid (cfTNA) analysis of 495 plasma samples from cancer patients. The cfDNA test failed in 4 cases, while circulating cell-free RNA (cfRNA) sequencing was invalid in 48 cases. In the 491 samples successfully tested on cfDNA, at least one genomic alteration was found in 222 cases (45.21%). We identified 316 single nucleotide variants (SNVs) in 21 genes. The most frequently mutated gene was TP53 (74 variants), followed by KRAS (71), EGFR (56), PIK3CA (33) and BRAF (19). Copy number variations (CNVs) were detected in 36 cases, while sequencing of cfRNA revealed 6 alterations. Analysis with droplet digital PCR (ddPCR) of peripheral blood leukocyte (PBL)-derived genomic DNA did not identify any KRAS mutations in 39 cases that showed KRAS mutations at cfDNA analysis. These findings suggest that the incidence of CHIP-associated KRAS mutations is relatively rare in routine clinical diagnostics.

Cell-Free RNA Transcriptome and Prediction of Adverse Pregnancy Outcomes.

Pregnancy is a complex condition with maternal, placental, and fetal factors all contributing to outcomes. Comprehensive profiling of biological pathways underlying both healthy and adverse pregnancy outcomes is in its infancy, and tools to predict obstetric complications remain limited. Minimally invasive methods to monitor pregnancy health through sampling of the maternal blood, also known as liquid biopsies, could significantly advance pregnancy care. Whole-blood transcriptome profiling involves the sequencing of circulating gene transcripts—referred to as cell-free RNA (cfRNA)—and allows minimally invasive assessment of human tissues. Utilization of cfRNA has lagged behind other minimally invasive assessments (such as cell-free DNA [cfDNA]) due to concerns that circulating RNA would be highly unstable and fragmented. However, with optimized collection and extraction, most of the cfRNA in blood is protected from degradation (1). cfRNA consists primarily of ribosomal RNA (rRNA; >95% of transcripts) and mitochondrial RNA (mtRNA), with about 2% messenger RNA (mRNA) reflecting the composition in most cell types (1). A few high abundance transcripts dominate the mRNA signature, including hemoglobin subunits, major histocompatibility complex components, and red blood cell markers. Thus, to optimize detection of transcripts with lower levels of expression, these high abundance transcripts are best removed during RNA sequencing (RNA-seq) library preparation.

Liquid Biopsies or Therapeutic Drug Monitoring for CYP Activity Profile Determination.

During pharmacotherapy, knowledge about the actual drug and metabolite concentrations in plasma is often critical. Individual dose adjustments can be performed based on pre-emptive genotyping of certain absorption, distribution, metabolism, and excretion (ADME) genes but also using therapeutic drug monitoring (TDM). Analyses of liquid biopsies for tumor-derived components are well-established and have been found to be a good complement to biopsy examinations. Recently, liquid biopsy-based quantification of cell-free RNA (cfRNA) in plasma exosomes was proposed as a proxy measurement for the expression of different hepatic ADME genes and for the rate of drug metabolism, constituting an alternative to TDM. In this study, we validated these findings by examining the correlation between mRNA expression of eight different CYP genes in liver and the corresponding rate of enzyme-specific drug metabolism in 96 donor-matched liver samples. Analyses of CYP-dependent drug metabolism in liver microsomes in comparison to the level of mRNA expression for the different CYP genes revealed a mean Pearson correlation coefficient of 0.28. The highest correlations (0.33-0.34) were obtained for CYP2D6 and CYP3A4 and the weakest correlations were observed for CYP1A2 and CYP2B6 (0.18-0.21). In all cases, the correlations obtained were too weak to demonstrate a predictive relationship, likely due to different regulatory and post-translational events controlling the rate of enzyme activity. Our results reinforce the notion that, whilst liquid biopsy-based approaches might have utility for prediction of hepatic CYP protein expression, they are not currently an important substitute for TDM.

Applications of Liquid Biopsies in Non-Small-Cell Lung Cancer.

The concept of liquid biopsy as an analysis tool for non-solid tissue carried out for the purpose of providing information about solid tumors was introduced approximately 20 years ago. Additional to the detection of circulating tumor cells (CTCs), the liquid biopsy approach quickly included the analysis of circulating tumor DNA (ctDNA) and other tumor-derived markers such as circulating cell-free RNA or extracellular vesicles. Liquid biopsy is a non-invasive technique for detecting multiple cancer-associated biomarkers that is easy to obtain and can reflect the characteristics of the entire tumor mass. Currently, ctDNA is the key component of the liquid biopsy approach from the point of view of the prognosis assessment, prediction, and monitoring of the treatment of non-small-cell lung cancer (NSCLC) patients. ctDNA in NSCLC patients carries variants or rearrangements that drive carcinogenesis, such as those in EGFR, KRAS, ALK, or ROS1. Due to advances in pharmacology, these variants are the subject of targeted therapy. Therefore, the detection of these variants has gained attention in clinical medicine. Recently, methods based on qPCR (ddPCR, BEAMing) and next-generation sequencing (NGS) are the most effective approaches for ctDNA analysis. This review addresses various aspects of the use of liquid biopsy with an emphasis on ctDNA as a biomarker in NSCLC patients.

Cancer type classification using plasma cell-free RNAs derived from human and microbes.

The utility of cell-free nucleic acids in monitoring cancer has been recognized by both scientists and clinicians. In addition to human transcripts, a fraction of cell-free nucleic acids in human plasma were proven to be derived from microbes and reported to have relevance to cancer. To obtain a better understanding of plasma cell-free RNAs (cfRNAs) in cancer patients, we profiled cfRNAs in ~300 plasma samples of 5 cancer types (colorectal cancer, stomach cancer, liver cancer, lung cancer, and esophageal cancer) and healthy donors (HDs) with RNA-seq. Microbe-derived cfRNAs were consistently detected by different computational methods when potential contaminations were carefully filtered. Clinically relevant signals were identified from human and microbial reads, and enriched Kyoto Encyclopedia of Genes and Genomes pathways of downregulated human genes and higher prevalence torque teno viruses both suggest that a fraction of cancer patients were immunosuppressed. Our data support the diagnostic value of human and microbe-derived plasma cfRNAs for cancer detection, as an area under the ROC curve of approximately 0.9 for distinguishing cancer patients from HDs was achieved. Moreover, human and microbial cfRNAs both have cancer type specificity, and combining two types of features could distinguish tumors of five different primary locations with an average recall of 60.4%. Compared to using human features alone, adding microbial features improved the average recall by approximately 8%. In summary, this work provides evidence for the clinical relevance of human and microbe-derived plasma cfRNAs and their potential utilities in cancer detection as well as the determination of tumor sites.

Polyadenylation ligation-mediated sequencing (PALM-Seq) characterizes cell-free coding and non-coding RNAs in human biofluids.

BackgroundCell‐free messenger RNA (cf‐mRNA) and long non‐coding RNA (cf‐lncRNA) are becoming increasingly important in liquid biopsy by providing biomarkers for disease prediction, diagnosis and prognosis, but the simultaneous characterization of coding and non‐coding RNAs in human biofluids remains challenging.MethodsHere, we developed polyadenylation ligation‐mediated sequencing (PALM‐Seq), an RNA sequencing strategy employing treatment of RNA with T4 polynucleotide kinase to generate cell‐free RNA (cfRNA) fragments with 5′ phosphate and 3′ hydroxyl and RNase H to deplete abundant RNAs, achieving simultaneous quantification and characterization of cfRNAs.ResultsUsing PALM‐Seq, we successfully identified well‐known differentially abundant mRNA, lncRNA and microRNA in the blood plasma of pregnant women. We further characterized cfRNAs in blood plasma, saliva, urine, seminal plasma and amniotic fluid and found that the detected numbers of different RNA biotypes varied with body fluids. The profiles of cf‐mRNA reflected the function of originated tissues, and immune cells significantly contributed RNA to blood plasma and saliva. Short fragments (<50 nt) of mRNA and lncRNA were major in biofluids, whereas seminal plasma and amniotic fluid tended to retain long RNA. Body fluids showed distinct preferences of pyrimidine at the 3′ end and adenine at the 5′ end of cf‐mRNA and cf‐lncRNA, which were correlated with the proportions of short fragments.ConclusionTogether, PALM‐Seq enables a simultaneous characterization of cf‐mRNA and cf‐lncRNA, contributing to elucidating the biology and promoting the application of cfRNAs.

P-204 Transcriptome analysis of cell-free RNAs in embryonic culture medium reveals their potentials on non-invasive testing

Abstract Study question Could cell-free RNA (cfRNA) in culture medium be non-invasive strategy to select in vitro fertilized (IVF) embryos? Summary answer CfRNAs in culture medium could be potential markers correlated with embryo qualities. What is known already Infertility is a critical component of reproductive health and couples with infertility may choose assisted reproductive technology (ART) to increase their chances of conception. However, the effectiveness remains low. One way to improve the outcome is to screen the embryo prior to implantation. Noninvasive testing opens new perspectives in analyzing embryo qualities and current pending includes microscopy images coupled with artificial intelligence and omics analysis of the spent blastocyst medium (SBM). Cell-free DNA in SBM could be used to identify the embryo ploidy and metabolomic/proteomic markers could be correlated with embryo potential, while the research of cfRNA is lacking. Study design, size, duration We developed a new library method of polyadenylation ligation-mediated sequencing (PALM-Seq) to capture the low amount of cfRNA and collected twelve culture mediums of in vitro embryos on Day 3 and five samples of embryos on Day 5. Participants/materials, setting, methods We employed PALM-Seq on culture medium of embryos generated by IVF or Intracytoplasmic sperm injection (ICSI). Owing to the new method, we could comprehensively profile the cfRNAs, including miRNA, tRNA, piRNA, lncRNA and mRNA. The morphology and fragmentation of the recruited embryos were also recorded. Main results and the role of chance Generally, thousands of genes could be detected in all culture mediums and the number greatly increased in SBM. We identified several reported miRNA markers in our study, such as miR-21-5p and miR-92a-3p, while highly expressed miRNAs varied across samples and stages. We indeed found piRNA in mediums and several specific piRNAs had large amount of expression. In contrast, detected number and expression of tRNA were stable across samples. Besides of reported mRNA markers, we could profile the expression of all detected mRNAs and no differences were found between embryos with and without fragmentation. We found that the majority of mRNA fragments were shorter than 30 nucleotides. In addition, some granular-specific expressed mRNAs could be detected in medium on Day 3 and they decreased of medium on Day 5. Limitations, reasons for caution We have only conducted preliminary exploration in the culture medium of pre-implantation embryos and studies of larger sample size are needed. Wider implications of the findings Our research proved the feasibility of performing high-throughput sequencing of cfRNAs in SBM. It opens new perspectives on defining non-invasive markers and selection of embryos with the most potential. Our results also contribute a new insight on improving the treatment of infertility. Trial registration number 2018YFC1004900

The role of liquid biopsy in the diagnosis of glioblastoma progression

Purpose: to summarize available data on the diagnostic value of various circulating biomarkers for the detection of glioblastoma recurrence. Material and Methods. A literature search was conducted using PubMED ExoCarta and SILVA databases. Results. Glioblastoma multiforme (GBM) is the most common glioma in adults with an unfavorable prognosis. Treatment of tumor recurrence can improve the survival of patients. Neuroimaging is the standard method of diagnosing brain tumor recurrence. However, a neuroimaging method to clearly distinguish between pseudo progression and tumor progression has not been found to date. Current molecular tumor profling relies heavily on tissue resection or biopsy. Tissue profling has several disadvantages in the central nervous system’s tumors, including the challenge associated with invasive biopsy, the heterogeneous nature of many malignancies where a small biopsy can under represent the mutational profle. Liquid biopsy is a promising method in diagnosing malignant tumors. Blood collection is a simple, minimally invasive procedure, but cerebrospinal fuid allows tumor markers to be detected more confdently. However, collection of cerebrospinal fuid is a complex and invasive procedure that can be accompanied by serious complications. Conclusion. Biological fuid markers such as circulating tumor cells, extracellular vesicles, cell-free DNA and cell-free RNA allow for the detection of GMB, determination of molecular genetic features of cancer during response to therapy, and early detection of GBM recurrence.

O-314 Epigenetic Profiling of Seminal Plasma in NOA Men to Predict Successful Testicular Sperm Retrieval

Abstract Study question Can epigenetic profiling of seminal plasma be used to predict successful testicular sperm retrieval for men with non-obstructive azoospermia (NOA)? Summary answer Epigenetic screening of cell-free seminal RNA identified gene imbalances in NOA men, with specific correlation to those who failed to yield spermatozoa at testicular biopsy. What is known already Although the chance of a successful microdissection testicular sperm extraction (micro-TESE) in men with NOA can be up to 60%, the procedure may still fail to yield spermatozoa. Several factors have been proposed to predict a successful retrieval, including FSH, inhibin B, genetics, and histopathology. Although histopathology would be the most reliable of these to predict successful micro-TESE, it is equally invasive to perform. Indeed, cell-free RNA extracted from testicular biopsy specimens has been shown to be differentially expressed in infertile men according to the origin of their azoospermia, whether obstructive or nonobstructive, and in relation to a normozoospermic control. Study design, size, duration Over a 2-year period, we identified men in whom no spermatozoa were identified despite extensive semen analyses conducted by multiple embryologists. These patients, who were negative for Y microdeletion, subsequently underwent micro-TESE. For consenting men, we performed epigenetic analyses on their seminal plasma by RNAseq. Significant differentially expressed gene (DEG) profiles were then assessed and compared according to whether surgical sperm retrieval successfully yielded spermatozoa (+TESE) or not (-TESE). Participants/materials, setting, methods RNA was isolated from the ejaculates for RNAseq using a commercially available spin column kit. RNA isolates were sequenced by Illumina HiSeq at 2x150bp. An absolute log2fold change of &amp;gt; 1 and a P-value of &amp;lt; 0.05 was considered significant. DEG profiles were compared within, as well as between, the +TESE and -TESE cohorts in comparison to a donor control. Main results and the role of chance All 12 men (37.3±6yrs) had normal peripheral karyotypes. Six (38.0±7yrs) underwent successful testicular sperm retrievals, defining the +TESE cohort. These men exclusively shared 10 significantly imbalanced genes involved in processes such as spermatogenesis (n = 4), sperm function (n = 2), and testis development (n = 1). For the 6 men (36.6±5 yrs) who underwent testicular sperm retrievals that failed to yield spermatozoa (-TESE), we identified 16 significantly imbalanced genes, exclusively shared by these patients. These genes are mainly involved in spermatogenesis (n = 9), sperm maturation (n = 1), and cell cycle regulation (n = 4). We then compared the DEG profiles between the +TESE and -TESE cohorts and identified 8 imbalanced genes that were shared among all 12 NOA men. Of interest, TPTE2 was partially (67%) expressed in patients from the +TESE group, while IGSF11-AS1 was underexpressed in all men from the -TESE group. Both of these genes are implicated in spermatogenic defects and are normally highly expressed in the testis. Interestingly, we identified a gene (NA) that was solely and specifically underexpressed in all men from the -TESE group, yet simultaneously overexpressed in all men from the +TESE group. NA, which is well known for its role in sialic acid metabolism, is also present on the sperm acrosome. Limitations, reasons for caution Using non-invasive RNAseq on the seminal plasma of NOA men, we were able to identify DEGs according to whether spermatozoa were successfully retrieved or had failed retrieval with micro-TESE. Although intriguing, these are preliminary results that should be further validated in a larger study cohort. Wider implications of the findings RNAseq identified genes shared within the same prognostic cohort. Moreover, differential expression of some specific genes predicted micro-TESE outcome. This epigenetic assessment, carried out on the ejaculate, can therefore be used as a non-invasive biomarker tool to predict loss of spermatogenesis in NOA men, sparing them from unnecessary surgery. Trial registration number N/A

Abstract 5174: Utilization of Cell free messenger RNA Sequencing for non-invasive molecular profiling of multiple myeloma and Acute Myeloid Leukemia

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.

Abstract 5996: Single tube PCR-based NGS assay for detection of multiple gene fusions from cell free total nucleic acid

Abstract Introduction: Recently, liquid biopsy has begun to be adopted for treatment selection and drug-resistance assessment for cancers. However, currently liquid biopsy is mostly focused on analyzing cell-free DNA (cfDNA). Though analyzing RNA provides more accurate analysis for functional gene fusions, little is known about the stability and availability of cfRNA (cell free RNA). Here we report our one-tube robust cfTNA (cell free total nucleic acids) assay to analyze ctDNA and ctRNA simultaneously. Methods: We assessed Pillar’s cell free TNA panel performance on 2 confirmed fusion positive and 5 fusion negative samples that were serially diluted to obtain a total of 88 samples including replicates. An input of 10ng cfTNA was used for fusion positive samples with fusion frequency ranging from 0.2% to 20%. A range of 5 to 30ng input was used for fusion negative samples. The amount of input cfRNA was estimated as equal to the cfDNA input. Total nucleic acids were extracted and cfRNA was reverse transcribed. Amplicons targeted the specific breakpoints of 36 known fusion transcripts. Fusion positive samples were diluted for library prep to achieve fusion frequency of 0.5%, 1%, 4%, 5% and 20%. Novaseq was used for sequencing with an average depth of 200,000X and downsampled by 25%. Pillar Variant Analysis Toolkit (PiVAT®) was used to map and call fusions. Results: Among dilutions of &amp;gt;=4%, PiVAT achieved &amp;gt;=99% sensitivity and &amp;gt;=99.9% specificity in calling RNA fusions from 10ng input. Even at extremely low dilutions of &amp;lt;=1%, PiVAT achieved &amp;gt;= 50% sensitivity. Conclusion: Pillar’s cell free TNA panel offers a simple, rapid, and reliable workflow that enables detection of a broad range of clinically relevant gene fusions. Detection of fusion events in cell-free samples from noninvasive liquid biopsies represents an important step forward in cancer diagnosis and monitoring. Citation Format: Tejashree Modak, Jilong Li, Sean Polvino, Yue Ke, Zhaohui Wang, Alex V. Kotlar. Single tube PCR-based NGS assay for detection of multiple gene fusions from cell free total nucleic acid [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 5996.

Abstract LB109: Circulating miRNA as biomarkers for pancreatic cancer early detection

Abstract Pancreatic cancer is one of the most fatal human cancers, with an overall 5-year survival rate of 10.8%. Early detection is critical for improving pancreatic cancer prognosis, but biomarkers for early detection are lacking. We conducted a two-stage study to identify circulating miRNAs as biomarkers for pancreatic cancer early detection using pre-diagnostic plasma samples, collected within 5 years prior to cancer diagnosis, from case-control studies nested in five prospective studies. The discovery stage included 185 case-control pairs from the Prostate, Lung, Colorectal and Ovarian Cancer (PLCO) Screening Trial. Replication stage samples comprised 277 case-control pairs from diverse cohorts: Shanghai Women’s and Men’s Health Studies, Southern Community Cohort Study (SCCS), and Multiethnic Cohort. Controls were individually matched on age at enrollment, sex, recruitment site (SCCS), race/ethnicity, and date of blood draw in each cohort. Cell-free small RNAs were extracted from plasma samples and miRNAs were measured by the NanoString nCounter Analysis System using the Human v3 miRNA Expression panel (a total of 798 miRNAs). Normalized miRNAs were categorized by decile. For miRNAs that have ≥10% samples with an undetectable level (0), the non-zero level was categorized by approximately 10% increment. Associations of circulating miRNAs with pancreatic cancer risk, measured in odds ratios (ORs) and 95% confidence intervals (CIs) per decile change, were calculated using conditional logistic regression analyses in discovery and replication studies, separately within each cohort, and results meta-analyzed. We identified three miRNAs, hsa-miR-199a-3p+/hsa-miR-199b-3p, hsa-miR-191-5p, hsa-miR-767-5p, being consistently associated with pancreatic risk in both discovery and replication sets, with combined ORs (95% CIs) of 0.89 (0.84-0.95), 0.90 (0.85-0.95), and 1.08 (1.02-1.13), and P of 9.09E-05, 6.95E-05 and 4.03E-03, respectively. Adjustment for age, BMI, smoking, diabetes and family history of pancreatic cancer did not change the associations. Stratified analyses by age at diagnosis found five additional replicated miRNAs: hsa-miR-640, hsa-miR-1299, hsa-miR-22-3p, hsa-miR-874-5p, and hsa-miR-449b-5p among those 65 years or older, with combined ORs (95% CIs) of 1.33 (1.16-1.52), 1.28 (1.12-1.46), 0.76 (0.65-0.89), 1.25 (1.09-1.43), and 1.22 (1.07-1.39), and P-value ranging from 4.75E-05 to 0.003. These results suggest that circulating miRNA biomarkers may be useful in identifying individuals with high risk of developing pancreatic cancer for close surveillance and/or a screening test. Citation Format: Cong Wang, Hui Cai, Qiuyin Cai, Jie Wu, Rachael Stolzenberg-Solomon, Clair Zhu, Yu-Tang Gao, Jordan Berlin, Fei Ye, Wei Zheng, Veronica W. Setiawan, Xiao-Ou Shu. Circulating miRNA as biomarkers for pancreatic cancer early detection [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 LB109.

Circulating biomarkers in the diagnosis and management of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most prevalent and lethal causes of cancer-related death worldwide. The treatment of HCC remains challenging and is largely predicated on early diagnosis. Surveillance of high-risk groups using abdominal ultrasonography, with or without serum analysis of α-fetoprotein (AFP), can permit detection of early, potentially curable tumours, but is limited by its insensitivity. Reviewed here are two current approaches that aim to address this limitation. The first is to use old re-emerged empirically derived biomarkers such as AFP, now applied within statistical models. The second is to use circulating nucleic acid biomarkers, which include cell-free DNA (for example, circulating tumour DNA, cell-free mitochondrial DNA and cell-free viral DNA) and cell-free RNA, applying modern molecular biology-based technologies and machine learning techniques closely allied to the underlying biology of cancer. Taken together, these approaches are likely to be complementary. Both hold considerable promise for achieving earlier diagnosis as well as offering additional functionalities including improved monitoring of therapy and prediction of response thereto.

Detection of Embryonic Trisomy 21 in the First Trimester Using Maternal Plasma Cell-Free RNA.

Prenatal trisomy 21 (T21) screening commonly involves testing a maternal blood sample for fetal DNA aneuploidy. It is reliable but poses a cost barrier to universal screening. We hypothesized maternal plasma RNA screening might provide similar reliability but at a lower cost. Discovery experiments used plasma cell-free RNA from 20 women 11–13 weeks tested by RNA and miRNA microarrays followed by qRT-PCR. Thirty-six mRNAs and 18 small RNAs of the discovery cDNA were identified by qPCR as potential markers of embryonic T21. The second objective was validation of the RNA predictors in 998 independent pregnancies at 11–13 weeks including 50 T21. Initial analyses identified 9–15 differentially expressed RNA with modest predictive power (AUC < 0.70). The 54 RNAs were then subjected to machine learning. Eleven algorithms were trained on one partition and tested on an independent partition. The three best algorithms were identified by Kappa score and the effects of training/testing partition size and dataset class imbalance on prediction were evaluated. Six to ten RNAs predicted T21 with AUCs up to 1.00. The findings suggest that maternal plasma collected at 11–13 weeks, tested by qRT-PCR, and classified by machine learning, may accurately predict T21 for a lower cost than plasma DNA, thus opening the door to universal screening.

Combining cell-free RNA (cfRNA) with cell-free total nucleic acid (cfTNA) as a new paradigm for liquid biopsy.

3048 Background: Expressed RNA can capture mutations, gene fusions, and biomarker profiles. In principle, each abnormal cell has one copy of mutated gene, but numerous copies of mutated RNA. Cell-free RNA (cfRNA) is not used due to the assumption that it is degraded. Next Generation Sequencing (NGS) by design is particularly adaptable for fragmented DNA and RNA. We developed an approach to isolate cell-free total nucleic acid (cfTNA) and cell-free RNA (cfRNA) from peripheral blood. Using targeted sequencing, we explored the potential of this approach to detect mutations, fusion mRNA, and copy number variation (CNV) in solid tumors and hematologic neoplasms. Methods: Peripheral blood cfTNA and cfRNA were extracted from B-cell lymphoid neoplasms (#105), T-cell neoplasms (#16), Myeloid neoplasms (#73), solid tumors (#44), and Normal individuals (#51), and sequenced using a targeted panel of 1459 genes. Results: Numbers of mutations detected in solid tumors and hematologic neoplasms were significantly (P &gt; 0.0001) higher in cfRNA (No. = 1229) than in cfTNA (No. = 1004). Overall variant allele frequency (VAF) was significantly higher in cfRNA than in cfTNA (P &lt; 0.0001). However, numerous mutations detected by RNA were not detected by cfTNA and vice versa. In general, nonsense mutations were more likely to be detected by cfTNA than by cfRNA and at higher VAF. Low-level mutations (VAF &lt; 10%) were more likely to be detected by cfRNA than by cfTNA. For example, 136 mutations in TP53 gene were detected using cfRNA and only 70 mutations were detected in cfTNA. KRAS mutations were also higher in cfRNA (#33) as compared with cfTNA (#21). In contrast, when most of the mutations were nonsense, as in ASXL1 gene, more mutations were detected by cfTNA (24 vs 23). When mutations were detected in both cfRNA and cfTNA, mutation load (level of mutant copies) was overall slightly higher in cfTNA (P = 0.06), likely due to higher degradation of RNA, but varied significantly dependent on the type of mutated gene and type of mutation. cfRNA was reliable in detecting fusion transcripts in solid tumors and in hematologic neoplasms (SLC34A2-ROS1, DDX5-BCL6, ETV6-RUNX1, RUNX1T1-RUNX1, PML-RARA, RUNX1-ZFPM2, DEK-NUP214, EP300-ZNF384) irrespective of the breakpoint or partner gene. The cfTNA detected various CNVs expected by cytogenetic analysis when tumor fraction was adequate (VAF &gt; 10%). Conclusions: This data demonstrates that using cfRNA and cfTNA provides complementary comprehensive information for evaluating mutations, fusion genes, and CNV. This approach increased sensitivity and reliability of liquid biopsy. Furthermore, the cfRNA provides critical information on relative expression of various genes that can be used as biomarkers in characterizing the neoplastic process (see ASCO abstract, Liquid Biopsy Based on Cell-Free RNA and Biomarkers profiling of hematologic and solid tumors).

Cell-free RNA in liquid biopsy and biomarkers profiling of hematologic and solid tumors.

3047 Background: Expressed RNA can capture mutations, changes in expression levels due to methylation, and provide information on cell of origin, growth, and proliferation status. We developed an approach to isolate fragmented RNA from peripheral blood plasma and explored its potential to be used in liquid biopsy. Methods: Peripheral blood cfRNA was extracted from patients with neoplasms in B-cell (#105), T-cell (#16), Myeloid (#73), and from solid tumors (#44), Normal individuals (#51), and reactive post-transplant (#137). RNA was sequenced using a 1459-gene panel. Expression profile was generated using Cufflinks. Results: cfRNA levels of various solid tumor biomarkers (CA-125, CA-15-3, CEA 8, Keratin19, Keratin6A...) were significantly higher (P &lt; 0.0001) in samples from solid tumors as compared with normal control. Similarly, cfRNA lymphoid markers (CD19, CD22, CD79A, and CD79B...) and cfRNA myeloid markers (CD33, CD14, CD117, CD56...) were all higher in B-cell lymphoid neoplasms and myeloid neoplasms, respectively (P &lt; 0.0001), as compared with control. In evaluating the host immune system, cfRNA CD4:CD8B and CD3D:CD19 ratios in normal controls were as expected (median: 5.92 and 6.87, respectively) and were significantly lower in solid tumors (median 3.40 and 2.23, respectively, P &lt; 0.0002). Solid tumor cfRNA showed CTLA4:CD8B ratio significantly higher in tumors than in normal (median 0.74 vs 0.19, P = 0.0001), while there was no difference in cfRNA PD-L1:CD8B ratio (median 1.45 vs 1.77, P = 0.96). Similar distinct patterns are noted for various cytokine and chemokines. cfRNA was highly predictive of diagnosis (AUC &gt; 0.98) of solid tumors, B-cell lymphoid neoplasms, T-cell lymphoid neoplasms, and myeloid neoplasms as compared with normal control. When a specific neoplastic disease was considered against all cases including control and other neoplasms, the AUC varied between 0.77 and 0.949. Conclusions: This data shows that liquid biopsy using targeted sequencing of cfRNA in patients with various types of cancer provides comprehensive and reliable information on the neoplastic disease as well as the host. [Table: see text]

Multimodality in liquid biopsy: does a combination uncover insights undetectable in individual blood analytes?

Abstract The heterogeneity of each individual oncologic disease can be mirrored by molecular analysis of a simple blood draw in real time. Liquid biopsy testing has been shown useable for cancer detection, proof of minimal residual disease, therapy decision making and monitoring. However, an individual blood analyte does not present a comprehensive picture of the disease. It was recently shown that multi-modal/multi-parametric/multi-analyte liquid biopsy testing has the advantage of generating a high-resolution snapshot of the disease complexity. The different blood analytes such as circulating tumor cells, circulating immune cells, tumor-educated platelets, extracellular vesicles, cell-free DNA, cell-free RNA and circulating proteins complement each other and have additive value for clinical cancer management. We, here, like to review the studies leading to these promising conclusions and like to, at the end, mention that many challenges lie ahead before the translation into the clinic can be accomplished, including issues concerning clinical utility, method standardization, cost reimbursement and data management.

Evaluation of a Maternal Plasma RNA Panel Predicting Spontaneous Preterm Birth and Its Expansion to the Prediction of Preeclampsia.

Preterm birth is the principal contributor to neonatal death and morbidity worldwide. We previously described a plasma cell-free RNA panel that between 16 and 20 weeks of pregnancy had potential to predict spontaneous preterm birth (sPTB) ≤ 32 weeks caused by preterm labor (PTL) or preterm premature rupture of membranes (PPROM). The present study had three objectives: (1) estimate the RNA panel prognostic accuracy for PTL/PPROM ≤ 32 weeks in a larger series; (2) improve accuracy by adding clinical characteristics to the predictive model; and (3) examine the association of the RNA panel with preeclampsia. We studied 289 women from Memphis TN prospectively sampled 16.0–20.7 weeks and found: (1) PSME2 and Hsa-Let 7g were differentially expressed in cases of PTL/PPROM ≤ 32 weeks and together provided fair predictive accuracy with AUC of 0.76; (2) combining the two RNAs with clinical characteristics improved good predictive accuracy for PTL/PPROM ≤ 32 weeks (AUC 0.83); (3) NAMPT and APOA1 were differentially expressed in women with ‘early-onset preeclampsia’ (EOP) and together provided good predictive accuracy with AUC of 0.89; and (4) combining the two RNAs with clinical characteristics provided excellent predictive accuracy (AUC 0.96). Our findings suggest an underlying common pathophysiological relationship between PTL/PPROM ≤ 32 weeks and EOP and open inroads for the prognostication of high-risk pregnancies.