Postdoc, Memorial Sloan Kettering Cancer Center
Finding RNA markers in the blood to detect cancer without a tumour biopsy or other invasive test
Analysis of cell-free DNA is exploding as a source of information in diagnostics in multiple fields, including cancer and neurological disorders, and is already in use in the clinic for prenatal testing to detect chromosomal imbalances such as trisomy 21. Until now, these types of studies have mostly required invasive procedures such as biopsies or amniocentesis in order to get testable material for diagnosis, however as technology has developed to become increasingly sensitive and the cost of next-generation sequencing has fallen, we are able to study smaller amounts of material and identify rarer mutations. Cell-free nucleic acids, such as DNA (cfDNA) and RNA (cfRNA), are regularly released into the blood stream during normal cell death processes, providing a source of information about hard to reach tissues and tumours where taking a biopsy can lead to trauma, such as in the brain, or can provide information about unknown metastatic sites. The cfDNA and cfRNA can easily be extracted from blood taken in the clinic during routine visits and removing the need for invasive procedures. cfDNA has been described in detail and methods for it’s extraction have been optimized by various groups, however there are relatively few publications on cfRNA. Here, we will compare the various methods currently available for the collection, processing and extraction of cfRNA and present the optimized methods determined for within our facility – a large cancer hospital in New York City where we anticipate a large number of samples. To validate the utility and usefulness of cfRNA analysis, a pilot project looking at a small group of patients with FGFR fusions present in their tumours will have cfRNA extracted and analyzed by RT-PCR. These types of rearrangements can be difficult to identify using cfDNA, and are informative for determining treatment options, potentially improving patient care and outcomes.
Abstract: The improvement in sensitive techniques has allowed the detection of tumor-specific aberrations in circulating tumor (ct) DNA. Amplification-refractory mutation system PCR has been used for the analysis of ctDNA to detect resistance-causing mutations in the epidermal growth factor receptor gene (eg, EGFR T790M) in lung cancer patients. So far, Streck tubes have been widely used as standard blood collection tubes. Here, we compared blood collection tubes manufactured by Streck with tubes from Roche and Qiagen regarding their utility in stabilizing ctDNA in plasma samples. Venous blood from healthy donors was collected in tubes from Streck, Roche, and Qiagen. Samples were spiked with artificially fragmented EGFR T790M-mutated DNA and stored for different periods of time or spiked with different DNA amounts before plasma preparation. Extracted ctDNA was analyzed by amplification-refractory mutation system PCR. Mutant DNA, spiked into blood samples from healthy donors at quantities of 1 or 3 ng, was still reliably detectable in all samples after 7 days. EGFR T790M could be detected when spiking was performed with an amount of artificial ctDNA of 0.5 ng when tubes from Roche and Qiagen were used. Blood collection tubes from Roche and Qiagen are highly suitable for ctDNA stabilization and subsequent liquid biopsy testing. Even low ctDNA concentrations allow the detection of somatic mutations.
Pub.: 22 Jul '17, Pinned: 22 Aug '17
Abstract: To investigate whether the plasma levels of cell-free RNA (cfRNA) and telomere-specific reverse transcriptase mRNA (hTERT) are associated with tumor response in rectal cancer patients who received preoperative chemoradiotherapy (pCRT).Patients who underwent pCRT for rectal cancer and for whom baseline and paired post-pCRT blood samples were available were studied. On the basis of tumor regression score, patients were classified as having response or having no response. Clinical variables and plasma levels of cfRNA and hTERT before and after the pCRT were evaluated. The association between each predictor and tumor response was assessed by univariate and multivariate analyses.Of 98 eligible patients, 45 were determined to respond to therapy, and 53 did not respond to therapy. In univariate analysis, gender (P = 0.040), baseline levels of cfRNA (P = 0.026), post-pCRT levels of both hTERT and cfRNA (P < 0.0001 and P = 0.001, respectively), and the difference between the post- and pre-pCRT levels of both hTERT and cfRNA (P = 0.009 and P = 0.001, respectively) were found to be significant predictors of tumor response. In multivariate analysis, using variables that were available before pCRT, cfRNA levels and gender independently predicted the tumor response, while in multivariate analysis, which used all of the variables available before the surgical procedure, the post-pCRT levels of cfRNA and the difference between the post- and pre-pCRT levels of cfRNA independently predicted tumor response.Plasma levels of cfRNA and hTERT are promising markers of tumor response to pCRT for rectal cancer.
Pub.: 08 Mar '12, Pinned: 22 Aug '17
Abstract: In this study, we examined several molecular markers in prostate and breast cancer patients and in normal individuals. The markers tested were: variations in the quantity of plasma DNA, glutathione-S-transferase P1 gene (GSTP1), Ras association domain family 1A (RASSF1A), and ataxia telangiectasia mutated (ATM) methylation status in plasma, carcinoembryonic antigen (CEA) and prostate-specific membrane antigen (PSMA) mRNA in peripheral blood mononuclear cells (PBMC) and plasma samples from prostate cancer patients. DNA quantification in plasma was performed using real-time PCR (RT-PCR). We assessed the methylation status of GSTP1 in plasma DNA using methylation-specific PCR (MSP) assay, while the methylation status of RASSF1A and ATM genes was examined by the MethyLight technology. RT-PCR analysis was used for the detection of mRNA, PSMA, and CEA. In 58.3% of newly diagnosed prostate cancer patients and 26.7% of prostate cancer patients under therapy, plasma DNA levels were increased. Additionally, 48.5% of breast cancer patients showed plasma DNA levels above the cutoff limit. GSTP1 Promotor hypermethylation was detectable in 75% of plasma samples obtained from patients with newly diagnosed prostate cancer and in 36.8% of patients under therapy, whereas 26% and 14% of the breast cancer patients tested were positive for RASSF1A and ATM methylation, respectively. The combination of DNA load and promotor methylation status identified 88% of prostate cancer patients and 54% of breast cancer patients. This study shows that free-circulating DNA can be detected in cancer patients compared with disease-free individuals, and suggests a new, noninvasive approach for early detection of cancer.
Pub.: 17 Nov '06, Pinned: 11 Jul '17
Abstract: To clarify the implications of cell-free nucleic acids (cfNA) in the plasma in neoplastic disease, it is necessary to determine the kinetics of their release into the circulation.To quantify non-tumor and tumor DNA and RNA in the plasma of tumor-bearing rats and to correlate such levels with tumor progression, we injected DHD/K12-PROb colon cancer cells subcutaneously into syngenic BD-IX rats. Rats were sacrificed and their plasma was analyzed from the first to the eleventh week after inoculation.The release of large amounts of non-tumor DNA into plasma was related to tumor development from its early stages. Tumor-specific DNA was detected in 33% of tumor-bearing rats, starting from the first week after inoculation and at an increasing frequency thereafter. Animals that were positive for tumor DNA in the plasma had larger tumors than those that were negative (p = 0.0006). However, the appearance of both mutated and non-mutated DNA fluctuated with time and levels of both were scattered among individuals in each group. The release of non-tumor mRNA was unaffected by tumor progression and we did not detect mutated RNA sequences in any animals.The release of normal and tumor cfDNA into plasma appeared to be related to individual-specific factors. The contribution of tumor DNA to the elevated levels of plasma DNA was intermittent. The release of RNA into plasma during cancer progression appeared to be an even more selective and elusive phenomenon than that of DNA.
Pub.: 05 Feb '13, Pinned: 11 Jul '17
Abstract: Despite recent advances in molecular profiling of colorectal cancer (CRC), as of yet this has not translated into an unbiased molecular liquid biopsy profile which can accurately screen for early CRC. In this study we depict the profile of early stage CRC as well as for advanced adenomas (AA) by combination of current molecular knowledge with microarray technology, using efficient circulating free plasma RNA purification from blood and RNA amplification technologies. We joined literature search with Affymetrix gene chip experimental procedure to draw the circulating free plasma RNA profile of colorectal cancer disease reflected in blood. The RNA panel was tested by two datasets comparing patients with CRC with healthy subjects and patients with AA to healthy subjects. For the CRC patient cohort (28 CRC cases vs. 41 healthy controls), the ROC analysis of the selected biomarker panel generated a sensitivity of 75% and a specificity of 93% for the detection of CRC using 8-gene classification model. For the AA patient cohort (28 subjects vs. 46 healthy controls), a sensitivity of 60% and a specificity of 87% were calculated using a 2-gene classification model. We have identified a panel of 8 plasma RNA markers as a preliminary panel for CRC detection and subset markers suitable for AA detection. Subjected to extensive clinical validation we suggest that this panel represents a feasible approach and a potential strategy for noninvasive early diagnosis, as a first-line screening test for asymptomatic, average-risk population before colonoscopy.
Pub.: 22 Jul '16, Pinned: 07 Jul '17
Abstract: Non-small cell lung cancer (NSCLC) is one of the most malignant cancers in the world. Early diagnosis of NSCLC has become especially important for patient treatment and prognosis. Increasing evidence suggest that long non-coding RNA GAS5 plays vital roles in cancer proliferation and differentiation in NSCLC. However, its clinical value in the diagnosis of NSCLC is unclear. The objective of this study was to evaluate the importance of circulating GAS5 as a biomarker for NSCLC diagnosis. In our study, quantitative real-time PCR (QRT-PCR) was applied to detect the GAS5 expression level in 80 pairs of cancer tissues and 57 pairs of plasma samples of NSCLC patients. Further analysis was performed to study the differential expression of circulating GAS5 in 111 NSCLC patients and 78 healthy controls in our study. The results showed that GAS5 decreased in NSCLC tissues compared to noncancerous tissues (P<0.001). Furthermore, the GAS5 expression level was statistically declined in early stage of NSCLC before surgery compared with healthy controls (P<0.05) and sharply increased in postoperative groups (P=0.026). ROC curve analysis for early stage of NSCLC with the combination of GAS5, CEA and CA199 showed that the area under the ROC curve (AUC) was 0.734 (95% CI, 0.628‑0.839; P<0.0005). In conclusion, circulating GAS5 could be functioned as a potential combined biomarker for screening NSCLC and patient monitoring after surgical treatment.
Pub.: 25 Mar '17, Pinned: 07 Jul '17
Abstract: The recently discovered long noncoding RNAs have the potential to regulate many biological processes, which are aberrantly expressed in many tumor types. Our previous study showed that the long noncoding RNA-growth arrest-specific transcript 5 (GAS5) was decreased in lung cancer tissue, which contributed to the proliferation and apoptosis of nonsmall cell lung cancer (NSCLC). GAS5 was also associated with the prognosis of lung cancer patients. These results suggest that GAS5 may represent a novel prognostic indicator and a target for gene therapy in NSCLC. However, the expression and diagnosis significance of GAS5 in the plasma of NSCLC patients was unknown. The plasma samples were more readily available than the tissue samples in clinical, so we designed the study to investigate the diagnosis value of GAS5 in blood samples. In our study, 90 patients with NSCLC and 33 healthy controls were included. Blood samples were collected before surgery and therapy. We extracted the free RNA in the plasma and analyzed the expression of GAS5 with quantitative reverse transcription PCR. Suitable statistics methods were used to compare the plasma GAS5 levels of preoperative and postoperative plasma samples between the NSCLC patients and healthy controls. Receiver-operating characteristic curve analysis was used to evaluate the diagnostic sensitivity and specificity of plasma GAS5 in NSCLC. The results showed that GAS5 was detectable and stable in the plasma of NSCLC patients. Furthermore, the plasma levels of GAS5 were significantly down-regulated in NSCLC patients compared with healthy controls (P = 0.000). Moreover, GAS5 levels increased markedly on the seventh day after surgery compared with preoperative GAS5 levels in NSCLC patients (P = 0.003). GAS5 expression levels could be used to distinguish NSCLC patients from control patients with an area under the curve of 0.832 (P < 0.0001; sensitivity, 82.2%; specificity, 72.7%). The combination of the GAS5 and carcinoembryonic antigen could produce an area of 0.909 under the receiver-operating characteristic curve in distinguishing NSCLC patients from control subjects (95% confidence interval 0.857-0.962, P = 0.000). We have demonstrated that GAS5 expression was decreased in NSCLC Plasma. Plasma samples were more accessible than tissue samples in clinical; therefore, GAS5 could be an ideal biomarker for the diagnosis of NSCLC.
Pub.: 16 Sep '16, Pinned: 20 Jun '17
Abstract: microRNAs are promising candidate breast cancer biomarkers due to their cancer-specific expression profiles. However, efforts to develop circulating breast cancer biomarkers are challenged by the heterogeneity of microRNAs in the blood. To overcome this challenge, we aimed to develop a molecular profile of microRNAs specifically secreted from breast cancer cells. Our first step towards this direction relates to capturing and analyzing the contents of exosomes, which are small secretory vesicles that selectively encapsulate microRNAs indicative of their cell of origin. To our knowledge, circulating exosome microRNAs have not been well-evaluated as biomarkers for breast cancer diagnosis or monitoring.Exosomes were collected from the conditioned media of human breast cancer cell lines, mouse plasma of patient-derived orthotopic xenograft models (PDX), and human plasma samples. Exosomes were verified by electron microscopy, nanoparticle tracking analysis, and western blot. Cellular and exosome microRNAs from breast cancer cell lines were profiled by next-generation small RNA sequencing. Plasma exosome microRNA expression was analyzed by qRT-PCR analysis.Small RNA sequencing and qRT-PCR analysis showed that several microRNAs are selectively encapsulated or highly enriched in breast cancer exosomes. Importantly, the selectively enriched exosome microRNA, human miR-1246, was detected at significantly higher levels in exosomes isolated from PDX mouse plasma, indicating that tumor exosome microRNAs are released into the circulation and can serve as plasma biomarkers for breast cancer. This observation was extended to human plasma samples where miR-1246 and miR-21 were detected at significantly higher levels in the plasma exosomes of 16 patients with breast cancer as compared to the plasma exosomes of healthy control subjects. Receiver operating characteristic curve analysis indicated that the combination of plasma exosome miR-1246 and miR-21 is a better indicator of breast cancer than their individual levels.Our results demonstrate that certain microRNA species, such as miR-21 and miR-1246, are selectively enriched in human breast cancer exosomes and significantly elevated in the plasma of patients with breast cancer. These findings indicate a potential new strategy to selectively analyze plasma breast cancer microRNAs indicative of the presence of breast cancer.
Pub.: 10 Sep '16, Pinned: 20 Jun '17
Abstract: MicroRNAs (miRNAs) constitute a class of small cellular RNAs (typically 21-23nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the cellular transcriptome is regulated by miRNAs, although they are relatively few in number (less than 2000 human miRNAs). The high relative stability of miRNA in common clinical tissues and biofluids (e.g. plasma, serum, urine, saliva, etc.) and the ability of miRNA expression profiles to accurately classify discrete tissue types and disease states have positioned miRNA quantification as a promising new tool for a wide range of diagnostic applications. Furthermore miRNAs have been shown to be rapidly released from tissues into the circulation with the development of pathology. To facilitate discovery and clinical development of miRNA-based biomarkers, we developed a genome-wide Locked Nucleic Acid (LNA™)-based miRNA qPCR platform with unparalleled sensitivity and robustness. The platform allows high-throughput profiling of miRNAs from important clinical sources without the need for pre-amplification. Using this system, we have profiled thousands of biofluid samples including blood derived plasma and serum. An extensive quality control (QC) system has been implemented in order to secure technical excellence and reveal any unwanted bias coming from pre-analytical or analytical variables. We present our approaches to sample and RNA QC as well as data QC and normalization. Specifically we have developed normal reference ranges for circulating miRNAs in serum and plasma as well as a hemolysis indicator based on microRNA expression.
Pub.: 06 Oct '12, Pinned: 20 Jun '17
Abstract: Interest in circulating RNAs for monitoring and diagnosing human health has grown significantly. There are few datasets describing baseline expression levels for total cell-free circulating RNA from healthy control subjects. In this study, total extracellular RNA (exRNA) was isolated and sequenced from 183 plasma samples, 204 urine samples and 46 saliva samples from 55 male college athletes ages 18-25 years. Many participants provided more than one sample, allowing us to investigate variability in an individual's exRNA expression levels over time. Here we provide a systematic analysis of small exRNAs present in each biofluid, as well as an analysis of exogenous RNAs. The small RNA profile of each biofluid is distinct. We find that a large number of RNA fragments in plasma (63%) and urine (54%) have sequences that are assigned to YRNA and tRNA fragments respectively. Surprisingly, while many miRNAs can be detected, there are few miRNAs that are consistently detected in all samples from a single biofluid, and profiles of miRNA are different for each biofluid. Not unexpectedly, saliva samples have high levels of exogenous sequence that can be traced to bacteria. These data significantly contribute to the current number of sequenced exRNA samples from normal healthy individuals.
Pub.: 18 Mar '17, Pinned: 20 Jun '17
Abstract: MicroRNAs (miRNAs) are small non-coding RNA molecules. Reduced or increased levels of specific miRNAs are observed in colon and other cancers, supporting their role in carcinogenesis. Detection of colorectal polyps is the cornerstone of the Bowel Cancer Screening Programme in the UK. However, uptake of screening nationally remains under 60%. We aimed to see whether circulating plasma miRNAs can be used to screen for patients with colorectal polyps, adenomas, or both.Blood samples were taken from patients from the Bowel Cancer Screening Programme (asymptomatic but faecal occult blood testing [FOBt] positive). Plasma RNA was extracted, target miRNAs (19a, 98, 146b, 186, 191, 222*, 331-5p, 452, 625, 664, 1247) were identified on pooled case miRNA assay cards, and miRNA fraction was quantified by quantitative RT-PCR assay. Results were compared with endoscopy reports and with histology of any polyps identified and removed. Analysis was done with Excel (2011) and SPSS (version 20) software.210 patients were included (117 with polyps, 12 with cancer, 81 healthy controls [FOBt positive]). The miRNA panel showed significant differences in expression (on t testing) for patients compared with controls for those with polyps, cancer, or both (miR-19a, p=0·0184; miR-98, p=0·0206; miR-146b, p=0·0029; miR-186, p=0·0006; miR-62,5 p=0·0008), polyps (miR-19a, p=0·0233; miR-98, p=0·0224; miR-146b, p=0·003; miR-186, p=0·0004; miR-625, p=0·001), adenomas (miR-19a, p=0·0339; miR-98, p=0·0266; miR-146b, p=0·0045; miR-186, p=0·0008; miR-625, p=0·0049), multiple adenomas (both sides of colon; miR-146b, p=0·0194; miR-186, p=0·0226; miR-625, p=0·0013), and right-sided adenomas (miR-98, p=0·031; miR-146b, p=0·0076; miR-186, p=0·0041; miR-331-5p, p=0·0142; miR-625, p=0·0049). Receiver operating characteristic analysis showed sensitivity of 60% or more, and specificity of 86% or more for men with polyps, men with adenomas, all patients with haemorrhoids or diverticulosis and polyps, and all patients with haemorrhoids or diverticulosis and adenomas.The target miRNAs that we identified showed significant differences in expression levels for patients with polyps and patients with adenomas from controls. Use of this panel has potential as a screening test.Bowel Disease Research Foundation.
Pub.: 28 Aug '15, Pinned: 14 Jun '17
Abstract: Long non-coding RNAs (lncRNAs) have shown great potential as powerful and non-invasive tumor markers. However, little is known about their value as biomarkers in pancreatic cancer (PC). We applied an Arraystar Human LncRNA Microarray which targeting 7419 lncRNAs to determine the lncRNA expression profile in PC and to screen the potential biomarkers. The most increased lncRNAs in PC tissues were HOTTIP-005, XLOC_006390, and RP11-567G11.1. Increased HOTTIP-005 and RP11-567G11.1 expression were poor prognostic factors for patients with PC (n = 144, p < 0.0001). The expression patterns of HOTTIP splice variants in PC were also detected. HOTTIP-005 and HOTTIP-001 were the first and second most increased HOTTIP splice variants, respectively. Plasma HDRF and RDRF (HOTTIP-005 and RP11-567G11.1 derived RNA fragments in plasma/serum) were present in stable form. Their levels were significantly increased in the patients with PC as compared to the healthy controls (n = 127 and 122 respectively, p < 0.0001) and the high levels were derived from PC. HDRF and RDRF levels are promising indicators for distinguishing patients with PC from those without PC. This study identified HOTTIP-005 and RP11-567G11.1 and their plasma fragments with the potential to be used as prognostic and diagnostic biomarkers of PC. Further large-scale prospective studies are needed to confirm our findings.
Pub.: 09 Oct '15, Pinned: 14 Jun '17
Abstract: Mammalian extracellular fluids, such as plasma of blood or plasma of milk, contain cell-free RNAs. Fragments of ubiquitously expressed mRNAs as well as tumor-specific and viral RNAs have been previously revealed in human plasmas by template-defined RT-PCR. In the present work we aimed to detect major forms of human blood plasma RNAs or to reveal new forms by using two approaches which were independent of context of RNA templates. The first approach was based on ligation of total plasma RNAs with adapter-oligoribonucleotides, reverse transcription of RNA-constructs, and amplification and cloning of cDNA. The second EST-like approach involved reverse transcription of RNAs with 3'-N6 degenerate primer, ligation of cDNA with dsDNA-adapter, amplification and cloning. Using these approaches we determined tens of tiny RNAs of human plasma and identified several long (>100 nt) RNAs. Most of tiny RNAs could be attributed to the fragments of rRNAs, whereas longer forms are representatives of noncoding RNAs.
Pub.: 08 Oct '08, Pinned: 14 Jun '17
Abstract: Circulating RNA in plasma and serum is a newly developed area for molecular diagnosis. To date, increasing numbers of studies show that plasma and serum RNA could serve as both tumor- and fetal-specific markers for cancer detection and prenatal diagnosis, respectively. Recently, by introducing the highly sensitive one-step real-time quantitative reverse-transcription (RT)-polymerase chain reaction (PCR), these potentially valuable RNA species, which often only exist at low concentrations in plasma and serum, can now be readily detected and quantified. Following the successful quantification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in plasma of normal individuals, several placenta-derived mRNA species, including the mRNA transcripts of human placental lactogen (hPL), the beta-subunit of human chorionic gonadotropin (betahCG), and corticotropin-releasing hormone (CRH) were also quantified in plasma of pregnant women. These circulating placental RNA species have provided the fetal-polymorphism-independent markers for prenatal diagnosis. The achievement in detecting the placental RNA in maternal plasma represents a significant step toward the development of RNA markers for noninvasive prenatal gene expression profiling. This detection technique can be extended to access a wide range of disease conditions, such as cancer and trauma. The one-step, real-time quantitative RT-PCR is a highly sensitive and specific, yet practically simple, RNA detection technique. This powerful technology may allow the practical employment of circulating RNA in the high-throughput clinical screening and monitoring applications.
Pub.: 19 Aug '06, Pinned: 14 Jun '17
Abstract: The analysis of cell-free DNA (cfDNA) as a sensitive biomarker for cancer diagnosis and monitoring has resulted in a need for efficient and standardized cfDNA isolation. In this study, we compared the isolation efficiency of the QIAamp circulating nucleic acid kit (QIA) with four other cfDNA isolation kits: the PME free-circulating DNA Extraction Kit (PME), the Maxwell RSC ccfDNA Plasma Kit (RSC), the EpiQuick Circulating Cell-Free DNA Isolation Kit (EQ), and two consecutive versions of the NEXTprep-Mag cfDNA Isolation Kit (NpMV1/2). cfDNA was isolated from 10 plasma samples, of which five contained KRAS mutated cell-free tumor DNA (ctDNA). Digital droplet PCR was used to quantify the total cfDNA concentration as well as the KRAS mutated ctDNA fraction. cfDNA integrity was assessed with real-time quantitative PCR. The QIA and the RSC kits displayed similar isolation efficiencies of both KRAS mutated ctDNA and nonmutated cfDNA, whereas the yield generated by the PME and NpMV2 kits was significantly lower. Real-time quantitative PCR indicated the presence of digital droplet PCR inhibiting agents in the eluates of the NpMV1 and EQ kits. To conclude, this study presents two highly efficient isolation kits for cfDNA isolation, of which the RSC kit has the advantage of a fully automated protocol over the labor-intensive QIA kit.
Pub.: 21 Nov '16, Pinned: 14 Jun '17
Abstract: The detection of cell-free RNA in plasma and serum of human subjects has found increasing applications in the field of medical diagnostics. However, many questions regarding the biology of circulating RNA remain to be addressed. One issue concerns the molecular nature of these circulating RNA species. We have recently developed a simple and quantitative method to investigate the integrity of plasma RNA. Our results have suggested that cell-free RNA in plasma is generally present as fragmented molecules instead of intact transcripts, with a predominance of 5' fragments. In this article, we summarize the basic principles in the experimental design for plasma RNA integrity analysis and highlight some of the important technical considerations for this type of investigation.
Pub.: 17 Nov '06, Pinned: 07 Jun '17
Abstract: Messenger RNA (mRNA) expression levels in blood cells are important in disease diagnosis, prognosis and biomarker discovery research. Accurate measurements of intracellular mRNA levels in blood cells depend upon several pre-analytical factors, including delays in RNA extraction from blood after phlebotomy. Dramatic changes in mRNA expression levels caused by delays in blood sample processing may render such samples unsuitable for gene expression analysis.This study was conducted to evaluate a blood collection tube, cell-free RNA-BCT(®) (RNA-BCT), for its ability to stabilize mRNA expression level in blood cells post-phlebotomy using indicator mRNAs in reverse transcription quantitative real-time PCR (RT-qPCR) assays.Blood samples from presumed healthy donors were drawn into both RNA-BCT and K3EDTA tubes and maintained at room temperature (18-22°C). The samples were processed to obtain white blood cells (WBCs) at days 0, 1, 2 and 3. Total cellular RNA was extracted from WBCs and mRNA concentrations were quantified by RT-qPCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), c-fos, and p53 transcripts.While blood cells isolated from K3EDTA tubes showed significant changes in cellular mRNA concentrations for GAPDH, c-fos, and p53, these mRNAs concentrations were stable in blood drawn into RNA-BCT.The reagent in the RNA-BCT device stabilizes cellular mRNA concentrations for GAPDH, c-fos and p53 for at least three days at room temperature.
Pub.: 03 Sep '14, Pinned: 07 Jun '17
Abstract: Since there is no validated assay to monitor disease in children with neuroblastoma (NB), we tested whether NB specific cell-free RNA could be detected in their plasma samples. Moreover, with the aim of reducing patients' discomfort, we compared this assay to a recently standardized procedure that uses a larger amount of whole blood.Using conditions that excluded RNA recovery from contaminating tumor cells, the total amount of cell-free RNA present in healthy children and patients with NB was quantified. Expression of tyrosine hydroxylase (TH) was assayed by quantitative RT-PCR.In patients with NB the amount of cell-free RNA was higher than in healthy children. However, it was less and more degraded than in healthy adults. The median amount of cell-free RNA that was reverse transcribed, measured through the use of standard curves for reference genes, was 0.03 (range 0-30) pg of input RNA, that is, always less than 1/10,000 of that reverse transcribed from total RNA extracted from whole cells. Despite the presence of disease and the positive results obtained with RNA extracted from peripheral blood cells, few cell-free RNA samples tested positive by the TH assay. Similar results were obtained also with TH primers specifically designed to amplify 50 bp RNA fragments.These findings suggest that for monitoring disease status detection of cell-free tumor-specific RNAs in patients with NB is not a reliable alternative to whole cell RNA.
Pub.: 21 Apr '10, Pinned: 07 Jun '17
Abstract: Noninvasive genetic tests that use cell-free fetal DNA (cffDNA) are used increasingly in prenatal care. A low amount of cffDNA can have detrimental effects on the reliability of these tests. A marker to confirm the presence of fetal nucleic acids is therefore required that is universally applicable and easy to incorporate.We developed a novel multiplex, single-tube, noninvasive fetal sex determination assay by combining amplification of AMELY cffDNA with one-step reverse transcription (RT)-PCR of trophoblast-derived cell-free RNA (cfRNA), which functions as a sex-independent fetoplacental marker. We tested plasma samples from 75 pregnant women in duplicate in a blinded fashion. The fetus was considered to be male in the case of a positive result for AMELY and cfRNA amplification in both RT-PCRs. The fetus was considered to be female in the case of negative AMELY and positive cfRNA result in both RT-PCRs. In other cases, the test was repeated. We compared the results with invasive prenatal testing and pregnancy outcomes.The AMELY cffDNA amplification and cfRNA result was unambiguous and identical in duplicate in 71 of 75 plasma samples (95%). Four samples (5%) required an extra replicate because of an absent fetoplacental marker. Thereafter, fetal sex was correctly determined in all 75 plasma samples.Amplification of trophoblast-derived cfRNA is a reliable marker for the confirmation of the presence of fetoplacentally derived nucleic acids in noninvasive fetal sex determination.
Pub.: 16 Oct '15, Pinned: 07 Jun '17
Abstract: Cell-free RNA (cfRNA) naturally occurs in blood and has clinical significance. Accurate quantification of these extracellular RNAs in whole blood is hindered by the simultaneous unintended release of cellular RNA and degradation of cfRNA after blood draw. An appropriate blood collection device is needed to stabilize cfRNA during blood processing, transportation and storage, which will ensure cfRNA test reliability. In this study we compared a novel blood collection device against traditional K3EDTA tubes for its ability to stabilize cfRNA in blood when subjected to conditions that can occur during sample storage and shipping.Shipping blood samples drawn into K3EDTA tubes showed a significant increase in mRNA copy numbers for β-actin, c-fos, and 18S rRNA in plasma. In contrast, shipping blood drawn into Cell-Free RNA BCT™s (BCTs) showed only a slight change in mRNA copy numbers for circulating β-actin, c-fos, and 18S rRNA. Moreover, blood stored in K3EDTA tubes at 6°C, 22°C and 30°C for 3 days showed a significant increase in mRNA copy numbers for c-fos and β-actin, whereas samples stored in BCTs only showed a slight increase.Our results show that BCTs minimize increases in background RNA levels caused by temperature fluctuations or agitation that can occur during blood sample storage and shipping. This novel blood collection tube could provide a method for obtaining high quality stabilized cfRNA samples for rare RNA target detection and determining accurate cfRNA concentrations.
Pub.: 27 Sep '13, Pinned: 07 Jun '17
Abstract: The presence of extracellular circulating or cell-free RNA in biological fluids is becoming a promising diagnostic tool for non invasive and cost effective cancer detection. Extracellular RNA or miRNA as biological marker could be used either for the early detection and diagnosis of the disease or as a marker of recurrence patterns and surveillance. In this review article, we refer to the origin of the circulating extracellular RNA, we summarise the data on the biological fluids (serum/plasma, saliva, urine, cerebrospinal fluid and bronchial lavage fluid) of patients suffering from various types of malignancies reported to contain a substantial amount of circulating extracellular (or cell-free) RNAs and we discuss the appropriate reagents and methodologies needed to be employed in order to obtain RNA material of high quality and integrity for the majority of the experimental methods used in RNA expression analysis. Furthermore, we discuss the advantages and disadvantages of the RT-PCR or microarray methodology which are the methods more often employed in procedures of extracellular RNA analysis.
Pub.: 26 Apr '11, Pinned: 07 Jun '17
Abstract: Breast cancer metastases differ biologically from primary disease; therefore, metastatic biopsies may assist in treatment decision making. Commercial genomic testing of both tumor and circulating tumor DNA have become available clinically, but utility of these tests in breast cancer management remains unclear.Patients undergoing a clinically indicated metastatic tumor biopsy were consented to the ongoing METAMORPH registry. Tumor and blood were collected at the time of disease progression before subsequent therapy, and patients were followed for response on subsequent treatment. Tumor testing (n = 53) and concurrent cell-free DNA (n = 32) in a subset of patients was performed using CLIA-approved assays.The proportion of patients with a genomic alteration was lower in tumor than in blood (69 vs. 91%; p = 0.06). After restricting analysis to alterations covered on both platforms, 83% of tumor alterations were detected in blood, while 90% of blood alterations were detected in tumor. Mutational load specific for the panel genes was calculated for both tumor and blood. Time to progression on subsequent treatment was significantly shorter for patients whose tumors had high panel-specific mutational load (HR 0.31, 95% CI 0.12-0.78) or a TP53 mutation (HR 0.35, 95% CI 0.20-0.79), after adjusting for stage at presentation, hormone receptor status, prior treatment type, and number of lines of metastatic treatment.Treating oncologists must distinguish platform differences from true biological heterogeneity when comparing tumor and cfDNA genomic testing results. Tumor and concurrent cfDNA contribute unique genomic information in metastatic breast cancer patients, providing potentially useful biomarkers for aggressive metastatic disease.
Pub.: 14 May '17, Pinned: 07 Jun '17
Abstract: Authors: Tie, J ; Gibbs ; P. Article URL: http://www.clinchem.org/cgi/content/short/62/6/796?rss=1 Citation: Vol 62 No. 6 (2016) pp 796 798 Publication Date: 2016-05-27T09:00:35-07:00 Journal: Clinical Chemistry
Pub.: 27 May '16, Pinned: 07 Jun '17
Abstract: Non-invasive prenatal testing (NIPT) through the analysis of cell free (cf)DNA is revolutionizing prenatal screening for fetal aneuploidy. Current methods used in clinical practice include shotgun massively parallel sequencing (s-MPS); targeted (t-MPS); and an approach that takes advantage of single nucleotide polymorphism (SNP) differences between mother and fetus. Efficacy of cfDNA testing for the common autosomal trisomies far exceeds that of conventional screening. Depending on the methodology used, reasons for discordancy between cfDNA results and fetal karyotype can include true fetal mosaicism, confined placental mosaicism, presence of a maternal karyotype abnormality, insufficient counting due to low fetal fraction, and a vanishing twin. Among the possible cfDNA strategies a Primary test has the highest performance but is expensive, while a Contingent cfDNA test can achieve high performance at a relatively low cost. Practicalities to be considered in the provision of testing include pretest counseling about the scope and accuracy of the testing, the interpretation of results when there is a low fetal fraction and follow-up studies for positive test results. The role of first trimester nuchal translucency measurement and conventional biochemical testing needs to be reassessed in the context of the use of cfDNA.
Pub.: 04 Mar '15, Pinned: 07 Jun '17
Abstract: We compared the plasma DNA concentrations in patients with different prostate cancer (PCa) stages and with benign prostate hyperplasia (BPH) and in healthy persons. Patients with localized cancer had DNA plasma within the reference interval. Increased plasma DNA was found in patients with lymph node and distant metastases and also in BPH. The association between plasma DNA and the survival time was similarly strong as with prostate-specific antigen. It can be concluded that plasma DNA has a limited validity as metastatic marker in PCa patients but follow-up studies combined with the investigation of tumor-related characteristics of plasma DNA would be reasonable.
Pub.: 24 Mar '04, Pinned: 07 Jun '17
Abstract: Increased cell-free DNA (cf-DNA) and the integrity of cf-DNA in plasma of patients with cancer has been described. We investigated the clinical utility of cf-DNA in the detection and monitoring of progression of leukemia.Plasma samples from 60 patients with acute leukemia were analyzed in comparison to plasma from 30 healthy controls. Plasma DNA was determined by quantitative PCR (qPCR) by amplifying the β-actin gene (ACTB). The DNA integrity index was calculated as the ratio of qPCR results (ACTB384/106). Paired diagnostic/complete remission (CR)/relapse samples from eight of 60 patients were analyzed, and the minimum residual disease (MRD) situations were monitored.DNA concentrations (median: 8.80 ng/mL, p=0.004) and DNA integrity (median: 0.51, p<0.001) in cancer patients were significantly higher. Receiver operating characteristic (ROC) curve analysis showed that the area under the ROC curve of DNA and DNA integrity were 0.79 and 0.88, respectively. DNA integrity at CR had a distinct reduction and then an increase at relapse. DNA integrity in CR cases was higher than that observed in healthy controls.Our preliminary data suggest that plasma DNA integrity is increased in acute leukemia and may be a potential biomarker for monitoring MRD. However, more work is needed.
Pub.: 14 Sep '10, Pinned: 07 Jun '17
Abstract: To advance the characterization of tumor-associated cell-free DNA in blood and bone marrow (BM), a rapid profiling method using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was established. MS-MLPA detects genetic and epigenetic aberrations of 37 tumor suppressor genes (TSG) in a single reaction and might, therefore, avoid the cumbersome single gene analyses.The validity of MS-MLPA for using cell-free plasma DNA was assessed by analyzing blood and BM samples of 91 patients with prostate cancer. As reference analyses, the methylation patterns of 4 genes (CD44, E-cadherin, CDKN2A and PTEN) chosen from the TSG set of the MS-MLPA kit were investigated in single reactions by sodium bisulfite DNA sequencing.Copy number changes and aberrant DNA methylation of 37 circulating TSG could be analyzed in BM and blood of 30 and 13 of the 91 patients, respectively, whereas the DNA content in the remaining samples was too low (<50 ng/μl of eluted DNA). The copy number of 28 of the 37 TSG was altered, and most changes were found for APC, CHFR, TP73 and GSTP1 genes in BM plasma. Statistical evaluations showed an association between copy number changes of TP73 and a positive resection margin of the prostate (p = 0.05). Both MS-MLPA and sodium bisulfite sequencing techniques showed that all genes were unmethylated.Our results demonstrate the potential and limitation of MS-MLPA for multiplex characterization of TSG in cell-free plasma DNA as a new non-invasive approach to obtain information on the molecular tumor biology of individual cancer patients.
Pub.: 05 Aug '10, Pinned: 07 Jun '17
Abstract: Histologic analysis of the allograft biopsy specimen is the standard method used to differentiate rejection from other injury in kidney transplants. Donor-derived cell-free DNA (dd-cfDNA) is a noninvasive test of allograft injury that may enable more frequent, quantitative, and safer assessment of allograft rejection and injury status. To investigate this possibility, we prospectively collected blood specimens at scheduled intervals and at the time of clinically indicated biopsies. In 102 kidney recipients, we measured plasma levels of dd-cfDNA and correlated the levels with allograft rejection status ascertained by histology in 107 biopsy specimens. The dd-cfDNA level discriminated between biopsy specimens showing any rejection (T cell-mediated rejection or antibody-mediated rejection [ABMR]) and controls (no rejection histologically), P<0.001 (receiver operating characteristic area under the curve [AUC], 0.74; 95% confidence interval [95% CI], 0.61 to 0.86). Positive and negative predictive values for active rejection at a cutoff of 1.0% dd-cfDNA were 61% and 84%, respectively. The AUC for discriminating ABMR from samples without ABMR was 0.87 (95% CI, 0.75 to 0.97). Positive and negative predictive values for ABMR at a cutoff of 1.0% dd-cfDNA were 44% and 96%, respectively. Median dd-cfDNA was 2.9% (ABMR), 1.2% (T cell-mediated types ≥IB), 0.2% (T cell-mediated type IA), and 0.3% in controls (P=0.05 for T cell-mediated rejection types ≥IB versus controls). Thus, dd-cfDNA may be used to assess allograft rejection and injury; dd-cfDNA levels <1% reflect the absence of active rejection (T cell-mediated type ≥IB or ABMR) and levels >1% indicate a probability of active rejection.
Pub.: 11 Mar '17, Pinned: 07 Jun '17
Abstract: Cell-free DNA (cfDNA) testing is increasingly being used to screen pregnant women for fetal aneuploidies. This technology may also identify fetal sex and can be used to screen for sex chromosome aneuploidies (SCAs). Physicians offering this screening will need to be prepared to offer comprehensive prenatal counseling about these disorders to an increasing number of patients. The purpose of this article is to consider the source of information to use for counseling, factors in parental decision-making, and the performance characteristics of cfDNA testing in screening for SCAs. Discordance between ultrasound examination and cfDNA results regarding fetal sex is also discussed.
Pub.: 20 Jun '15, Pinned: 07 Jun '17
Abstract: Circulating tumor DNA (ctDNA) has garnered much excitement over the past few years for its potential clinical utility as a surrogate for tumor biopsies in early cancer detection and prognosis. Numerous studies have demonstrated that ctDNA is shed into the circulation and is elevated in disease states such as cancer. Despite the low levels of ctDNA in the "sea" of normal DNA, advances in next generation sequencing (NGS) and digital polymerase chain reaction (PCR) technologies have led to dramatic improvements in variant detection sensitivity and specificity. These technologies allow the quantification of ctDNA, providing both prognostic and predictive information. Here, we review the history of cell-free DNA and different technologies for the detection of ctDNA in cancer and describe the different modalities for using ctDNA in clinical oncology.
Pub.: 04 May '17, Pinned: 07 Jun '17
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