Single Cell and Plasma RNA Sequencing for RNA Liquid Biopsy for Hepatocellular Carcinoma.

BACKGROUND: Human plasma contains RNA transcripts released by multiple cell types within the body. Single-cell transcriptomic analysis allows the cellular origin of circulating RNA molecules to be elucidated at high resolution and has been successfully utilized in the pregnancy context. We explored the application of a similar approach to develop plasma RNA markers for cancer detection. METHODS: Single-cell RNA sequencing was performed to decipher transcriptomic profiles of single cells from hepatocellular carcinoma (HCC) samples. Cell-type-specific transcripts were identified and used for deducing the cell-type-specific gene signature (CELSIG) scores of plasma RNA from patients with and without HCC. RESULTS: Six major cell clusters were identified, including hepatocyte-like, cholangiocyte-like, myofibroblast, endothelial, lymphoid, and myeloid cell clusters based on 4 HCC tumor tissues as well as their paired adjacent nontumoral tissues. The CELSIG score of hepatocyte-like cells was significantly increased in preoperative plasma RNA samples of patients with HCC (n = 14) compared with non-HCC participants (n = 49). The CELSIG score of hepatocyte-like cells declined in plasma RNA samples of patients with HCC within 3 days after tumor resection. Compared with the discriminating power between patients with and without HCC using the abundance of ALB transcript in plasma [area under curve (AUC) 0.72)], an improved performance (AUC: 0.84) was observed using the CELSIG score. The hepatocyte-specific transcript markers in plasma RNA were further validated by ddPCR assays. The CELSIG scores of hepatocyte-like cell and cholangiocyte trended with patients' survival. CONCLUSIONS: The combination of single-cell transcriptomic analysis and plasma RNA sequencing represents an approach for the development of new noninvasive cancer markers.

Characteristics of Fetal Extrachromosomal Circular DNA in Maternal Plasma: Methylation Status and Clearance.

BACKGROUND: Although the characterization of cell-free extrachromosomal circular DNA (eccDNA) has gained much research interest, the methylation status of these molecules is yet to be elucidated. We set out to compare the methylation densities of plasma eccDNA of maternal and fetal origins, and between small and large molecules. The clearance of fetal eccDNA from maternal circulation was also investigated. METHODS: We developed a sequencing protocol for eccDNA methylation analysis using tagmentation and enzymatic conversion approaches. A restriction enzyme-based approach was applied to verify the tagmentation results. The efficiency of cell-free fetal eccDNA clearance was investigated by fetal eccDNA fraction evaluations at various postpartum time points. RESULTS: The methylation densities of fetal eccDNA (median: 56.3%; range: 40.5-67.6%) were lower than the maternal eccDNA (median: 66.7%; range: 56.5-75.7%) (P = 0.02, paired t-test). In addition, eccDNA molecules from the smaller peak cluster (180-230 bp) were of lower methylation levels than those from the larger peak cluster (300-450 bp). Both of these findings were confirmed using the restriction enzyme approach. We also observed comparable methylation densities between linear and eccDNA of both maternal and fetal origins. The average half-lives of fetal linear and eccDNA in the maternal blood were 30.2 and 29.7 min, respectively. CONCLUSIONS: We found that fetal eccDNA in plasma was relatively hypomethylated compared to the maternal eccDNA. The methylation densities of eccDNA were positively correlated with their sizes. In addition, fetal eccDNA was found to be rapidly cleared from the maternal blood after delivery, similar to fetal linear DNA.

Jagged Ends of Urinary Cell-Free DNA: Characterization and Feasibility Assessment in Bladder Cancer Detection.

BACKGROUND: Double-stranded DNA in plasma is known to carry single-stranded ends, called jagged ends. Plasma DNA jagged ends are biomarkers for pathophysiologic states such as pregnancy and cancer. It remains unknown whether urinary cell-free DNA (cfDNA) molecules have jagged ends. METHODS: Jagged ends of cfDNA were detected by incorporating unmethylated cytosines during a DNA end-repair process, followed by bisulfite sequencing. Incorporation of unmethylated cytosines during the repair of the jagged ends lowered the apparent methylation levels measured by bisulfite sequencing and were used to calculate a jagged end index. This approach is called jagged end analysis by sequencing. RESULTS: The jagged end index of urinary cfDNA was higher than that of plasma DNA. The jagged end index profile of plasma DNA displayed several strongly oscillating major peaks at intervals of approximately 165 bp (i.e., nucleosome size) and weakly oscillating minor peaks with periodicities of approximately 10 bp. In contrast, the urinary DNA jagged end index profile showed weakly oscillating major peaks but strongly oscillating minor peaks. The jagged end index was generally higher in nucleosomal linker DNA regions. Patients with bladder cancer (n = 46) had lower jagged end indexed of urinary DNA than participants without bladder cancer (n = 39). The area under the curve for differentiating between patients with and without bladder cancer was 0.83. CONCLUSIONS: Jagged ends represent a property of urinary cfDNA. The generation of jagged ends might be related to nucleosomal structures, with enrichment in linker DNA regions. Jagged ends of urinary DNA could potentially serve as a new biomarker for bladder cancer detection.

Plasma DNA End-Motif Profiling as a Fragmentomic Marker in Cancer, Pregnancy, and Transplantation.

Plasma DNA fragmentomics is an emerging area of research covering plasma DNA sizes, end points, and nucleosome footprints. In the present study, we found a significant increase in the diversity of plasma DNA end motifs in patients with hepatocellular carcinoma (HCC). Compared with patients without HCC, patients with HCC showed a preferential pattern of 4-mer end motifs. In particular, the abundance of plasma DNA motif CCCA was much lower in patients with HCC than in subjects without HCC. The aberrant end motifs were also observed in patients with other cancer types, including colorectal cancer, lung cancer, nasopharyngeal carcinoma, and head and neck squamous cell carcinoma. We further observed that the profile of plasma DNA end motifs originating from the same organ, such as the liver, placenta, and hematopoietic cells, generally clustered together. The profile of end motifs may therefore serve as a class of biomarkers for liquid biopsy in oncology, noninvasive prenatal testing, and transplantation monitoring. SIGNIFICANCE: Plasma DNA molecules originating from the liver, HCC and other cancers, placenta, and hematopoietic cells each harbor a set of characteristic plasma DNA end motifs. Such markers carry tissue-of-origin information and represent a new class of biomarkers in the nascent field of fragmentomics.This article is highlighted in the In This Issue feature, p. 627.

Methylation analysis of plasma DNA informs etiologies of Epstein-Barr virus-associated diseases.

Epstein-Barr virus (EBV) is associated with a number of diseases, including malignancies. Currently, it is not known whether patients with different EBV-associated diseases have different methylation profiles of circulating EBV DNA. Through whole-genome methylation analysis of plasma samples from patients with nasopharyngeal carcinoma (NPC), EBV-associated lymphoma and infectious mononucleosis, we demonstrate that EBV DNA methylation profiles exhibit a disease-associated pattern. This observation implies a significant potential for the development of methylation analysis of plasma EBV DNA for NPC diagnostics. We further analyse the plasma EBV DNA methylome of NPC and non-NPC subjects from a prospective screening cohort. Plasma EBV DNA fragments demonstrate differential methylation patterns between NPC and non-NPC subjects. Combining such differential methylation patterns with the fractional concentration (count) and size of plasma EBV DNA, population screening of NPC is performed with an improved positive predictive value of 35.1%, compared to a count- and size-based only protocol.

Noninvasive Detection of Bladder Cancer by Shallow-Depth Genome-Wide Bisulfite Sequencing of Urinary Cell-Free DNA for Methylation and Copy Number Profiling.

BACKGROUND: The current diagnosis and monitoring of bladder cancer are heavily reliant on cystoscopy, an invasive and costly procedure. Previous efforts in urine-based detection of bladder cancer focused on targeted approaches that are predicated on the tumor expressing specific aberrations. We aimed to noninvasively detect bladder cancer by the genome-wide assessment of methylomic and copy number aberrations (CNAs). We also investigated the size of tumor cell-free (cf)DNA fragments. METHODS: Shallow-depth paired-end genome-wide bisulfite sequencing of urinary cfDNA was done for 46 bladder cancer patients and 39 cancer-free controls with hematuria. We assessed (a) proportional contribution from different tissues by methylation deconvolution, (b) global hypomethylation, (c) CNA, and (d) cfDNA size profile. RESULTS: Methylomic and copy number approaches were synergistically combined to detect bladder cancer with a sensitivity of 93.5% (84.2% for low-grade nonmuscle-invasive disease) and a specificity of 95.8%. The prevalence of methylomic and CNAs reflected disease stage and tumor size. Sampling over multiple time points could assess residual disease and changes in tumor load. Muscle-invasive bladder cancer was associated with a higher proportion of long cfDNA, as well as longer cfDNA fragments originating from genomic regions enriched for tumor DNA. CONCLUSIONS: Bladder cancer can be detected noninvasively in urinary cfDNA by methylomic and copy number analysis without previous knowledge or assumptions of specific aberrations. Such analysis could be used as a liquid biopsy to aid diagnosis and for potential longitudinal monitoring of tumor load. Further understanding of the differential size and fragmentation of cfDNA could improve the detection of bladder cancer.

Preferred end coordinates and somatic variants as signatures of circulating tumor DNA associated with hepatocellular carcinoma.

Circulating tumor-derived cell-free DNA (ctDNA) analysis offers an attractive noninvasive means for detection and monitoring of cancers. Evidence for the presence of cancer is dependent on the ability to detect features in the peripheral circulation that are deemed as cancer-associated. We explored approaches to improve the chance of detecting the presence of cancer based on sequence information present on ctDNA molecules. We developed an approach to detect the total pool of somatic mutations. We then investigated if there existed a class of ctDNA signature in the form of preferred plasma DNA end coordinates. Cell-free DNA fragmentation is a nonrandom process. Using plasma samples obtained from liver transplant recipients, we showed that liver contributed cell-free DNA molecules ended more frequently at certain genomic coordinates than the nonliver-derived molecules. The abundance of plasma DNA molecules with these liver-associated ends correlated with the liver DNA fractions in the plasma samples. Studying the DNA end characteristics in plasma of patients with hepatocellular carcinoma and chronic hepatitis B, we showed that there were millions of tumor-associated plasma DNA end coordinates in the genome. Abundance of plasma DNA molecules with tumor-associated DNA ends correlated with the tumor DNA fractions even in plasma samples of hepatocellular carcinoma patients that were subjected to shallow-depth sequencing analysis. Plasma DNA end coordinates may therefore serve as hallmarks of ctDNA that could be sampled readily and, hence, may improve the cost-effectiveness of liquid biopsy assessment.

Maternal plasma fetal DNA fractions in pregnancies with low and high risks for fetal chromosomal aneuploidies.

Recently published international guidelines recommend the clinical use of noninvasive prenatal test (NIPT) for aneuploidy screening only among pregnant women whose fetuses are deemed at high risk. The applicability of NIPT to aneuploidy screening among average risk pregnancies requires additional supportive evidence. A key determinant of the reliability of aneuploidy NIPT is the fetal DNA fraction in maternal plasma. In this report, we investigated if differences in fetal DNA fractions existed between different pregnancy risk groups. One hundred and ninety-five singleton pregnancies with male fetuses divided into 3 groups according to first trimester screening parameters were examined for fetal DNA percentage by counting Y chromosome DNA sequences using massively parallel sequencing. Fetal DNA fractions were compared between risk groups and assessed for correlations with first trimester screening parameters. There was no statistically significant difference in fetal DNA fractions across the high, intermediate and low risk groups. Fetal DNA fraction showed a strong negative correlation with maternal weight. Fetal DNA fraction also showed weak but significant correlations with gestational age, crown-rump length, multiple of medians of free ß-subunit of human chorionic gonadotropin and pregnancy-associated plasma protein A. Similar fetal DNA fractions in maternal plasma between high, intermediate and low risk pregnant women is a precondition for uniform performance of the aneuploidy NIPTs for the general population. This study thus shows that the aneuploidy screening by NIPT is likely to offer similar analytical reliability without respect to the a priori fetal aneuploidy risk.

Placenta-derived fetal specific mRNA is more readily detectable in maternal plasma than in whole blood.

BACKGROUND: Placental mRNA was detected in maternal whole blood, raising the possibility of using maternal blood for noninvasive prenatal diagnosis. We investigated fetal mRNA detection in maternal whole blood and determined if it offered advantages over maternal plasma analysis. METHODOLOGY: The concentrations of placental expressed genes, CSH1, KISS1, PLAC4 and PLAC1 in plasma and whole blood from healthy pregnant and non-pregnant individuals were compared by real-time quantitative reverse-transcriptase polymerase chain reaction analysis. Their fetal specificity was investigated by comparing the transcript concentrations in pre- and post-delivery samples and through SNP genotyping by matrix-assisted laser-desorption and ionization time-of-flight mass spectrometry. The gene expression profiles of pregnant and non-pregnant whole blood were investigated by microarray analysis. Upregulated genes in pregnant whole blood were selected for further quantitative analysis. PRINCIPAL FINDINGS: The concentrations of the four transcripts were significantly higher in third trimester maternal whole blood than corresponding plasma without significant correlations. KISS1, PLAC4 and PLAC1 were detected in non-pregnant whole blood but not plasma. The transcripts remained detectable in some postpartum whole blood samples. The PLAC4 mRNA in maternal plasma showed fetal genotype while that in corresponding whole blood indicated both fetal and maternal contributions. Microarray analysis revealed upregulation of genes involved in neutrophil functions in pregnant whole blood including DEFA4, CEACAM8, OLFM4, ORM1, MMP8 and MPO. Though possibly pregnancy-related, they were not pregnancy-specific as suggested by the lack of post-delivery reduction in concentrations. CONCLUSIONS: Maternal plasma is preferred over maternal whole blood for placenta-derived fetal RNA detection. Most studied 'placental' mRNA molecules in maternal whole blood were of maternal origin and might be derived from processes such as 'illegitimate transcription'.

Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection.

Current methods for prenatal diagnosis of chromosomal aneuploidies involve the invasive sampling of fetal materials using procedures such as amniocentesis or chorionic villus sampling and constitute a finite risk to the fetus. Here, we outline a strategy for fetal chromosome dosage assessment that can be performed noninvasively through analysis of placental expressed mRNA in maternal plasma. We achieved noninvasive prenatal diagnosis of fetal trisomy 21 by determining the ratio between alleles of a single-nucleotide polymorphism (SNP) in PLAC4 mRNA, which is transcribed from chromosome 21 and expressed by the placenta, in maternal plasma. PLAC4 mRNA in maternal plasma was fetal derived and cleared after delivery. The allelic ratios in maternal plasma correlated with those in the placenta. Fetal trisomy 21 was detected noninvasively in 90% of cases and excluded in 96.5% of controls.

Genome-wide gene expression profiling of cervical cancer in Hong Kong women by oligonucleotide microarray.

An analysis of gene expression profiles obtained from cervical cancers was performed to find those genes most aberrantly expressed. Total RNA was prepared from 29 samples of cervical squamous cell carcinoma and 18 control samples, and hybridized to Affymetrix oligonucleotide microarrays with probe sets complementary to over 20,000 transcripts. Unsupervised hierarchical clustering of the expression data readily distinguished normal cervix from cancer. Supervised analysis of gene expression data identified 98 and 139 genes that exhibited >2-fold upregulation and >2-fold downregulation, respectively, in cervical cancer compared to normal cervix. Several of the genes that were differentially regulated included SPP1 (Osteopontin), CDKN2A (p16), RPL39L, Clorf1, MAL, p11, ARS and NICE-1. These were validated by quantitative RT-PCR on an independent set of cancer and control specimens. Gene Ontology analysis showed that the list of differentially expressed genes included ones that were involved in multiple biological processes, including cell proliferation, cell cycle and protein catabolism. Immunohistochemical staining of cancer specimens further confirmed differential expression of SPP1 in cervical cancer cells vs. nontumor cells. In addition, 2 genes, CTGF and RGS1 were found to be upregulated in late stage cancer compared to early stage cancer, suggesting that they might be involved in cancer progression. The pathway analysis of expression data showed that the SPP1, VEGF, CDC2 and CKS2 genes were coordinately differentially regulated between cancer and normal. The present study is promising and provides potential new insights into the extent of expression differences underlying the development and progression of cervical squamous cell cancer. This study has also revealed several genes that may be highly attractive candidate molecular markers/targets for cervical cancer diagnosis, prognosis and therapy.