ctDNA guiding adjuvant immunotherapy in urothelial carcinoma.

Minimally invasive approaches to detect residual disease after surgery are needed to identify patients with cancer who are at risk for metastatic relapse. Circulating tumour DNA (ctDNA) holds promise as a biomarker for molecular residual disease and relapse1. We evaluated outcomes in 581 patients who had undergone surgery and were evaluable for ctDNA from a randomized phase III trial of adjuvant atezolizumab versus observation in operable urothelial cancer. This trial did not reach its efficacy end point in the intention-to-treat population. Here we show that ctDNA testing at the start of therapy (cycle 1 day 1) identified 214 (37%) patients who were positive for ctDNA and who had poor prognosis (observation arm hazard ratio = 6.3 (95% confidence interval: 4.45-8.92); P < 0.0001). Notably, patients who were positive for ctDNA had improved disease-free survival and overall survival in the atezolizumab arm versus the observation arm (disease-free survival hazard ratio = 0.58 (95% confidence interval: 0.43-0.79); P = 0.0024, overall survival hazard ratio = 0.59 (95% confidence interval: 0.41-0.86)). No difference in disease-free survival or overall survival between treatment arms was noted for patients who were negative for ctDNA. The rate of ctDNA clearance at week 6 was higher in the atezolizumab arm (18%) than in the observation arm (4%) (P = 0.0204). Transcriptomic analysis of tumours from patients who were positive for ctDNA revealed higher expression levels of cell-cycle and keratin genes. For patients who were positive for ctDNA and who were treated with atezolizumab, non-relapse was associated with immune response signatures and basal-squamous gene features, whereas relapse was associated with angiogenesis and fibroblast TGFß signatures. These data suggest that adjuvant atezolizumab may be associated with improved outcomes compared with observation in patients who are positive for ctDNA and who are at a high risk of relapse. These findings, if validated in other settings, would shift approaches to postoperative cancer care.

Correlation between variant allele frequency and mean tumor molecules with tumor burden in patients with solid tumors.

Several studies have demonstrated the prognostic value of circulating tumor DNA (ctDNA); however, the correlation of mean tumor molecules (MTM)/ml of plasma and mean variant allele frequency (mVAF; %) with clinical parameters is yet to be understood. In this study, we analyzed ctDNA data in a pan-cancer cohort of 23 543 patients who had ctDNA testing performed using a personalized, tumor-informed assay (Signatera™, mPCR-NGS assay). For ctDNA-positive patients, the correlation between MTM/ml and mVAF was examined. Two subanalyses were performed: (a) to establish the association of ctDNA with tumor volume and (b) to assess the correlation between ctDNA dynamics and patient outcomes. On a global cohort, a positive correlation between MTM/ml and mVAF was observed. Among 18 426 patients with longitudinal ctDNA measurements, 13.3% had discordant trajectories between MTM/ml and mVAF at subsequent time points. In metastatic patients receiving immunotherapy (N = 51), changes in ctDNA levels expressed both in MTM/ml and mVAF showed a statistically significant association with progression-free survival; however, the correlation with MTM/ml was numerically stronger.

Combining Donor-derived Cell-free DNA Fraction and Quantity to Detect Kidney Transplant Rejection Using Molecular Diagnoses and Histology as Confirmation.

BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) fraction and quantity have both been shown to be associated with allograft rejection. The present study compared the relative predictive power of each of these variables to the combination of the two, and developed an algorithm incorporating both variables to detect active rejection in renal allograft biopsies. METHODS: The first 426 sequential indication biopsy samples collected from the Trifecta study ( ClinicalTrials.gov # NCT04239703) with microarray-derived gene expression and dd-cfDNA results were included. After exclusions to simulate intended clinical use, 367 samples were analyzed. Biopsies were assessed using the molecular microscope diagnostic system and histology (Banff 2019). Logistic regression analysis examined whether combining dd-cfDNA fraction and quantity adds predictive value to either alone. The first 149 sequential samples were used to develop a two-threshold algorithm and the next 218 to validate the algorithm. RESULTS: In regression, the combination of dd-cfDNA fraction and quantity was found to be significantly more predictive than either variable alone ( P = 0.009 and P < 0.0001). In the test set, the area under the receiver operating characteristic curve of the two-variable system was 0.88, and performance of the two-threshold algorithm showed a sensitivity of 83.1% and specificity of 81.0% for molecular diagnoses and a sensitivity of 73.5% and specificity of 80.8% for histology diagnoses. CONCLUSIONS: This prospective, biopsy-matched, multisite dd-cfDNA study in kidney transplant patients found that the combination of dd-cfDNA fraction and quantity was more powerful than either dd-cfDNA fraction or quantity alone and validated a novel two-threshold algorithm incorporating both variables.

Clinical Validation of a Plasma Donor-derived Cell-free DNA Assay to Detect Allograft Rejection and Injury in Lung Transplant.

Background: Lung transplant patients are vulnerable to various forms of allograft injury, whether from acute rejection (AR) (encompassing acute cellular rejection [ACR] and antibody-mediated rejection [AMR]), chronic lung allograft dysfunction (CLAD), or infection (INFXN). Previous research indicates that donor-derived cell-free DNA (dd-cfDNA) is a promising noninvasive biomarker for the detection of AR and allograft injury. Our aim was to validate a clinical plasma dd-cfDNA assay for detection of AR and other allograft injury and to confirm and expand on dd-cfDNA and allograft injury associations observed in previous studies. Methods: We measured dd-cfDNA fraction using a novel single-nucleotide polymorphism-based assay in prospectively collected plasma samples paired with clinical-pathologic diagnoses. dd-cfDNA fraction was compared across clinical-pathologic cohorts: stable, ACR, AMR, isolated lymphocytic bronchiolitis, CLAD/neutrophilic-responsive allograft dysfunction (NRAD), and INFXN. Performance characteristics were calculated for AR and combined allograft injury (AR + CLAD/NRAD + INFXN) versus the stable cohort. Results: The study included 195 samples from 103 patients. Median dd-cfDNA fraction was significantly higher for ACR (1.43%, interquartile range [IQR]: 0.67%-2.32%, P = 5 × 10-6), AMR (2.50%, IQR: 2.06%-3.79%, P = 2 × 10-5), INFXN (0.74%, IQR: 0.46%-1.38%, P = 0.02), and CLAD/NRAD (1.60%, IQR: 0.57%-2.60%, P = 1.4 × 10-4) versus the stable cohort. Area under the receiver operator characteristic curve for AR versus stable was 0.91 (95% confidence interval [CI]: 0.83-0.98). Using a ≥1% dd-cfDNA fraction threshold, sensitivity for AR was 89.1% (95% CI: 76.2%-100.0%), specificity 82.9% (95% CI: 73.3%-92.4%), positive predictive value, 51.9% (95% CI: 37.5%-66.3%), and negative predictive value, 97.3% (95% CI: 94.3%-100%). For combined allograft injury area under the receiver operator characteristic curve was 0.76 (95% CI: 0.66-0.85), sensitivity 59.9% (95% CI: 46.0%-73.9%), specificity 83.9% (95% CI: 74.1%-93.7%), positive predictive value, 43.6% (95% CI: 27.6%-59.6%), and negative predictive value, 91.0% (95% CI: 87.9%-94.0%). Conclusions: These results indicate that our dd-cfDNA assay detects AR and other allograft injury. dd-cfDNA monitoring, accompanied by standard clinical assessments, represents a valuable precision tool to support lung transplant health and is appropriate for further assessment in a prospective randomized-controlled study.

Donor-derived Cell-free DNA Shows High Sensitivity for the Diagnosis of Pancreas Graft Rejection in Simultaneous Pancreas-kidney Transplantation.

BACKGROUND: Pancreas graft status in simultaneous pancreas-kidney transplant (SPKTx) is currently assessed by nonspecific biochemical markers, typically amylase or lipase. Identifying a noninvasive biomarker with good sensitivity in detecting early pancreas graft rejection could improve SPKTx management. METHODS: Here, we developed a pilot study to explore donor-derived cell-free DNA (dd-cfDNA) performance in predicting biopsy-proven acute rejection (P-BPAR) of the pancreas graft in a cohort of 36 SPKTx recipients with biopsy-matched plasma samples. dd-cfDNA was measured using the Prospera test (Natera, Inc.) and reported both as a fraction of the total cfDNA (fraction; %) and as concentration in the recipient's plasma (quantity; copies/mL). RESULTS: In the absence of P-BPAR, dd-cfDNA was significantly higher in samples collected within the first 45 d after SPKTx compared with those measured afterward (median, 1.00% versus 0.30%; median, 128.2 versus 35.3 cp/mL, respectively with both; P = 0.001). In samples obtained beyond day 45, P-BPAR samples presented a significantly higher dd-cfDNA fraction (0.83 versus 0.30%; P = 0.006) and quantity (81.3 versus 35.3 cp/mL; P = 0.001) than stable samples. Incorporating dd-cfDNA quantity along with dd-cfDNA fraction outperformed dd-cfDNA fraction alone to detect active rejection. Notably, when using a quantity cutoff of 70 cp/mL, dd-cfDNA detected P-BPAR with a sensitivity of 85.7% and a specificity of 93.7%, which was more accurate than current biomarkers (area under curve of 0.89 for dd-cfDNA (cp/ml) compared with 0.74 of lipase and 0.46 for amylase). CONCLUSIONS: dd-cfDNA measurement through a simple noninvasive blood test could be incorporated into clinical practice to help inform graft management in SPKTx patients.

Detection of Molecular Residual Disease Using Personalized Circulating Tumor DNA Assay in Patients With Colorectal Cancer Undergoing Resection of Metastases.

PURPOSE: More than 50% of patients with stage IV colorectal cancer (metastatic colorectal cancer [mCRC]) relapse postresection. The efficacy of postoperative systemic treatment is limited in this setting. Thus, these patients would greatly benefit from the use of a reliable prognostic biomarker, such as circulating tumor DNA (ctDNA) to identify minimal or molecular residual disease (MRD). PATIENTS AND METHODS: We analyzed a cohort of 112 patients with mCRC who had undergone metastatic resection with curative intent as part of the PREDATOR clinical trial. The study evaluated the prognostic value of ctDNA, correlating MRD status postsurgery with clinical outcomes by using a personalized and tumor-informed ctDNA assay (bespoke multiple PCR, next-generation sequencing assay). Postresection, systemic therapy was given to 39.2% of the patients at the discretion of the treating physician. RESULTS: Postsurgical, MRD positivity was observed in 54.4% (61 of 112) of patients, of which 96.7% (59 of 61) progressed at the time of data cutoff (hazard ratio [HR]: 5.8; 95% CI, 3.5 to 9.7; P < .001). MRD-positive status was also associated with an inferior overall survival: HR: 16.0; 95% CI, 3.9 to 68.0; P < .001. At the time of analyses, 96% (49 of 51) of patients were alive in the MRD-negative arm compared with 52.4% (32 of 61) in the MRD-positive arm. Patients who did not receive systemic therapy and were MRD-negative in the combined ctDNA analysis at two time points had an overall survival of 100%. In the multivariate analysis, ctDNA-based MRD status was the most significant prognostic factor associated with disease-free survival (HR: 5.78; 95% CI, 3.34 to 10.0; P < .001). CONCLUSION: This study confirms that in mCRC undergoing resection of metastases, postoperative MRD analysis is a strong prognostic biomarker. It holds promises for being implemented in clinical decision making, informing clinical trial design, and further translational research.

Detection of increased amounts of cell-free fetal DNA with short PCR amplicons.

AIM: A digital PCR approach has recently been suggested to detect greater amounts of cell-free fetal DNA in maternal plasma than conventional real-time quantitative PCR (qPCR). Because the digital qPCR approach uses shorter PCR amplicons than the real-time qPCR assay, we investigated whether a real-time qPCR assay appropriately modified for such short amplicons would improve the detection of cell-free fetal DNA. METHOD: We developed a novel universal-template (UT) real-time qPCR assay that was specific for the DYS14 sequence on Y chromosome and had a short amplicon size of 50 bp. We examined this "short" assay with 50 maternal plasma samples and compared the results with those for a conventional real-time qPCR assay of the same locus but with a longer amplicon (84 bp). RESULTS: Qualitatively, both assays detected male cell-free fetal DNA with the same specificity and detection capability. Quantitatively, however, the new UT real-time qPCR assay for shorter amplicons detected, on average, almost 1.6-fold more cell-free fetal DNA than the conventional real-time qPCR assay with longer amplicons. CONCLUSIONS: The use of short PCR amplicons improves the detection of cell-free fetal DNA. This feature may prove useful in attempts to detect cell-free fetal DNA under conditions in which the amount of template is low, such as in samples obtained early in pregnancy.

Electrochemical detection of low-copy number salivary RNA based on specific signal amplification with a hairpin probe.

We developed a technique for electrochemical detection of salivary mRNA employing a hairpin probe (HP). Steric hindrance (SH) suppresses unspecific signal and generates a signal-on amplification process for target detection. The stem-loop configuration brings the reporter end of the probe into close proximity with the surface and makes it unavailable for binding with the mediator. Target binding opens the hairpin structure of the probe, and the mediator can then bind to the accessible reporter. Horseradish peroxidase is utilized to generate electrochemical signal. This signal-on process is characterized by a low basal signal, a strong positive readout and a large dynamic range. The SH is controlled via hairpin design and electrical field. By applying electric field control to HPs, the limit of detection of RNA is about 0.4 fM, which is 10 000-fold more sensitive than conventional linear probes. Endogenous Interleukin-8 mRNA is detected with the HP, and good correlation with the quantitative PCR technique is obtained. The resultant process allows a simple setup and by reducing the number of steps it is suited for the point-of-care detection of specific nucleic acid sequences from complex body fluids such as saliva.

The effect of surgical trauma on circulating free DNA levels in cancer patients-implications for studies of circulating tumor DNA.

Detection of circulating tumor DNA (ctDNA) post-treatment is an emerging marker of residual disease. ctDNA constitutes only a minor fraction of the cell-free DNA (cfDNA) circulating in cancer patients, complicating ctDNA detection. This is exacerbated by trauma-induced cfDNA. To guide optimal blood sample timing, we investigated the duration and magnitude of surgical trauma-induced cfDNA in patients with colorectal or bladder cancer. DNA levels were quantified in paired plasma samples collected before and up to 6 weeks after surgery from 436 patients with colorectal cancer and 47 patients with muscle-invasive bladder cancer. To assess whether trauma-induced cfDNA fragments are longer than ordinary cfDNA fragments, the concentration of short (< 1 kb) and long (> 1 kb) fragments was determined for 91 patients. Previously reported ctDNA data from 91 patients with colorectal cancer and 47 patients with bladder cancer were used to assess how trauma-induced DNA affects ctDNA detection. The total cfDNA level increased postoperatively-both in patients with colorectal cancer (mean threefold) and bladder cancer (mean eightfold). The DNA levels were significantly increased up to 4 weeks after surgery in both patient cohorts (P = 0.0005 and P ≤ 0.0001). The concentration of short, but not long, cfDNA fragments increased postoperatively. Of 25 patients with radiological relapse, eight were ctDNA-positive and 17 were ctDNA-negative in the period with trauma-induced DNA. Analysis of longitudinal samples revealed that five of the negative patients became positive shortly after the release of trauma-induced cfDNA had ceased. In conclusion, surgery was associated with elevated cfDNA levels, persisting up to 4 weeks, which may have masked ctDNA in relapse patients. Trauma-induced cfDNA was of similar size to ordinary cfDNA. To mitigate the impact of trauma-induced cfDNA on ctDNA detection, it is recommended that a second blood sample collected after week 4 is analyzed for patients initially ctDNA negative.

Personalized Detection of Circulating Tumor DNA Antedates Breast Cancer Metastatic Recurrence.

PURPOSE: Up to 30% of patients with breast cancer relapse after primary treatment. There are no sensitive and reliable tests to monitor these patients and detect distant metastases before overt recurrence. Here, we demonstrate the use of personalized circulating tumor DNA (ctDNA) profiling for detection of recurrence in breast cancer. EXPERIMENTAL DESIGN: Forty-nine primary patients with breast cancer were recruited following surgery and adjuvant therapy. Plasma samples (n = 208) were collected every 6 months for up to 4 years. Personalized assays targeting 16 variants selected from primary tumor whole-exome data were tested in serial plasma for the presence of ctDNA by ultradeep sequencing (average >100,000X). RESULTS: Plasma ctDNA was detected ahead of clinical or radiologic relapse in 16 of the 18 relapsed patients (sensitivity of 89%); metastatic relapse was predicted with a lead time of up to 2 years (median, 8.9 months; range, 0.5-24.0 months). None of the 31 nonrelapsing patients were ctDNA-positive at any time point across 156 plasma samples (specificity of 100%). Of the two relapsed patients who were not detected in the study, the first had only a local recurrence, whereas the second patient had bone recurrence and had completed chemotherapy just 13 days prior to blood sampling. CONCLUSIONS: This study demonstrates that patient-specific ctDNA analysis can be a sensitive and specific approach for disease surveillance for patients with breast cancer. More importantly, earlier detection of up to 2 years provides a possible window for therapeutic intervention.

Analysis of Plasma Cell-Free DNA by Ultradeep Sequencing in Patients With Stages I to III Colorectal Cancer.

IMPORTANCE: Novel sensitive methods for detection and monitoring of residual disease can improve postoperative risk stratification with implications for patient selection for adjuvant chemotherapy (ACT), ACT duration, intensity of radiologic surveillance, and, ultimately, outcome for patients with colorectal cancer (CRC). OBJECTIVE: To investigate the association of circulating tumor DNA (ctDNA) with recurrence using longitudinal data from ultradeep sequencing of plasma cell-free DNA in patients with CRC before and after surgery, during and after ACT, and during surveillance. DESIGN, SETTING, AND PARTICIPANTS: In this prospective, multicenter cohort study, ctDNA was quantified in the preoperative and postoperative settings of stages I to III CRC by personalized multiplex, polymerase chain reaction-based, next-generation sequencing. The study enrolled 130 patients at the surgical departments of Aarhus University Hospital, Randers Hospital, and Herning Hospital in Denmark from May 1, 2014, to January 31, 2017. Plasma samples (n = 829) were collected before surgery, postoperatively at day 30, and every third month for up to 3 years. MAIN OUTCOMES AND MEASURES: Outcomes were ctDNA measurement, clinical recurrence, and recurrence-free survival. RESULTS: A total of 130 patients with stages I to III CRC (mean [SD] age, 67.9 [10.1] years; 74 [56.9%] male) were enrolled in the study; 5 patients discontinued participation, leaving 125 patients for analysis. Preoperatively, ctDNA was detectable in 108 of 122 patients (88.5%). After definitive treatment, longitudinal ctDNA analysis identified 14 of 16 relapses (87.5%). At postoperative day 30, ctDNA-positive patients were 7 times more likely to relapse than ctDNA-negative patients (hazard ratio [HR], 7.2; 95% CI, 2.7-19.0; P < .001). Similarly, shortly after ACT ctDNA-positive patients were 17 times (HR, 17.5; 95% CI, 5.4-56.5; P < .001) more likely to relapse. All 7 patients who were ctDNA positive after ACT experienced relapse. Monitoring during and after ACT indicated that 3 of the 10 ctDNA-positive patients (30.0%) were cleared by ACT. During surveillance after definitive therapy, ctDNA-positive patients were more than 40 times more likely to experience disease recurrence than ctDNA-negative patients (HR, 43.5; 95% CI, 9.8-193.5 P < .001). In all multivariate analyses, ctDNA status was independently associated with relapse after adjusting for known clinicopathologic risk factors. Serial ctDNA analyses revealed disease recurrence up to 16.5 months ahead of standard-of-care radiologic imaging (mean, 8.7 months; range, 0.8-16.5 months). Actionable mutations were identified in 81.8% of the ctDNA-positive relapse samples. CONCLUSIONS AND RELEVANCE: Circulating tumor DNA analysis can potentially change the postoperative management of CRC by enabling risk stratification, ACT monitoring, and early relapse detection.

Real-time quantitative PCR for the detection of fetal aneuploidies.

In prenatal analysis, one of the major concerns is the detection of fetal aneuploidies. Several molecular methods have been described recently for the rapid analysis of amniotic fluid and chorionic villi. Fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) of short tandem repeats are already implemented in prenatal laboratories and permit the evaluation of the major chromosomal aberrations within 24 h. However, both methods have their disadvantages. The advent of real-time PCR has revolutionized the measurement of nucleic acid copy numbers in recent years. Quantitative PCR, a term that was only 10 years ago considered to be an oxymoron, is now widely accepted. We demonstrated previously the feasibility of detection of trisomy 21 by real-time PCR, and here we describe the modified test that permits simultaneous analysis of trisomies 18 and 21. This approach has been demonstrated in a recent large-scale analysis, and it is presented in detail in this chapter.

Quantification of circulatory fetal DNA in the plasma of pregnant women.

The analysis of cell-free fetal DNA in the circulation of the pregnant woman plays the pivotal role in noninvasive prenatal research. Here, we describe an improved method for the quantification of male DNA, which is a valuable research tool for the quantification of fetal DNA. The quantification of fetal DNA serves two main purposes. First, the levels may indicate certain pregnancy-related disorders such as preeclampsia even before onset of the disease; thus, the quantification may serve as a marker for early detection. Second, extraction and enrichment strategies of the fetal DNA compartment are important factors in the development and implementation of clinical tests, such as detection of fetal sex, Rhesus D status, point mutations, and aneuploidies. In this context, the quantification of fetal DNA is an important tool for the evaluation of protocols.

Salivary mRNA targets for cancer diagnostics.

Head and neck squamous cell carcinoma (HNSCC) affects almost 1 million people worldwide per year. Despite therapeutic advances the overall survival rate remains low because diagnosis often occurs only at advanced stages with poor prognosis. Like in most cancers, the implementation of an early detection scheme would have a positive impact on this disease. Similarly, as oral cancer has a very high recurrence rate, the early identification of recurrence or second primary tumors is an important challenge. HNSCC detection is currently based on expert clinical examination of the upper aerodigestive tract and histologic analysis of suspicious areas, but it may be undetectable in hidden sites, and unfortunately visual screening for oral lesions is an often neglected part of dental healthcare. Our group is actively pursuing the assembly of a toolbox for the molecular analysis of oral fluid. Here we present our current status utilizing the salivary transcriptome for oral cancer diagnostics.

Genomic targets in saliva.

Saliva, the most accessible and noninvasive biofluid of our body, harbors a wide spectrum of biological analytes informative for clinical diagnostic applications. While proteomic constituents are a logical first choice as salivary diagnostic analytes, genomic targets have emerged as highly informative and discriminatory. This awareness, coupled with the ability to harness genomic information by high-throughput technology platforms such as genome-wide microarrays, ideally positions salivary genomic targets for exploring the value of saliva for detection of specific disease states and augmenting the diagnostic and discriminatory value of the saliva proteome for clinical applications. Buccal cells and saliva have been used as sources of genomic DNA for a variety of clinical and forensic applications. For discovery of disease targets in saliva, the recent realization that there is a transcriptome in saliva presented an additional target for oral diagnostics. All healthy subjects evaluated have approximately 3,000 different mRNA molecules in their saliva. Almost 200 of these salivary mRNAs are present in all subjects. Exploration of the clinical utility of the salivary transcriptome in oral cancer subjects shows that four salivary mRNAs (OAZ, SAT, IL8, and IL1b) collectively have a discriminatory power of 91% sensitivity and specificity for oral cancer detection. Data are also now in place to validate the presence of unique diagnostic panels of salivary mRNAs in subjects with Sjögren's disease.

Characterization of salivary RNA by cDNA library analysis.

Oral fluid (saliva) meets the demands for a noninvasive and accessible diagnostic medium. Recent reports by our group and others described the presence and use of human RNA in saliva as a diagnostic or forensic tool, including the use for oral cancer detection. To gain insights into the integrity of salivary RNA, we examined in detail the integrity of salivary RNA by generating a cDNA library from pooled supernatant saliva of 10 healthy donors. From a library with a primary library titer of 1.3 x 10(6) cfu/mL of which 95% of the clones had inserts, we successfully sequenced 117 random colonies containing recombinant clones. BLAST search results indicated that all of these clones contained sequences of human origin. Most of the salivary RNAs appeared to be endonucleolytically cleaved at random positions as indicated by comparisons to respective full length parental RNAs from the Genbank. Twelve of the insert sequences matched to the normal salivary core transcriptome sequences, which are highly abundant mRNAs present in healthy individuals. This study provides an in-depth molecular analysis of the saliva transcriptome and should be a useful resource for future basic and translational studies of RNA in human saliva. In addition, this paper presents unequivocal evidence for the presence of RNA in saliva as determined by the use of diverse techniques such as reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), in vitro translation, and the construction of a salivary cDNA library.

Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma.

DNA of fetal origin is present in the plasma of pregnant women. The quantitative measurement of circulatory fetal DNA (cfDNA) by real-time quantitative PCR (qPCR) has been applied to investigate a possible correlation between increased levels and pregnancy-related disorders. However, as the levels of cfDNA are close to the detection limit (LOD) of the method used, the measurements may not be reliable. This is also problematic for the evaluation of preanalytical steps, such as DNA extraction and cfDNA enrichment by size separation. We optimized a protocol for the qPCR analysis of the multi-copy sequence DYS14 on the Y chromosome. This was compared with an established assay for the single-copy SRY gene. Probit regression analysis showed that the limit of detection (LOD) of the DYS14 assay, (0.4 genome equivalents (GE)) and limit of quantification (LOQ) were 10-fold lower in comparison to SRY (4 GE). The levels of cfDNA obtained from the first trimester of pregnancy could be quantified with high precision by the DYS14 assay (CV below 25%) as opposed to the SRY measurements (26-140%). Additionally, fetal sex was correctly determined in all instances. The low copy numbers of fetal DNA in plasma of women in the first trimester of pregnancy can be measured reliably, targeting the DYS14 that is present in multiple copies per Y chromosome.

Improvement of methods for the isolation of cell-free fetal DNA from maternal plasma: comparison of a manual and an automated method.

The low amount of cell-free fetal DNA present in the maternal circulation poses significant challenges to its use in future diagnostic applications, and ways of increasing the yield of this potential marker extracted from maternal plasma are constantly being explored. In this study, we compared two methods of DNA extraction, a manual and an automated method. Our analysis revealed that although the manual method yielded overall more total cell-free DNA, the automated system yielded higher quantities of cell-free DNA of fetal origin. Furthermore, the DNA isolated using the automated system appeared to be of greater purity than that isolated by the manual method, with fewer inhibitors to downstream real-time PCR reactions.

Implementing prenatal diagnosis based on cell-free fetal DNA: accurate identification of factors affecting fetal DNA yield.

OBJECTIVE: Cell-free fetal DNA is a source of fetal genetic material that can be used for non-invasive prenatal diagnosis. Usually constituting less than 10% of the total cell free DNA in maternal plasma, the majority is maternal in origin. Optimizing conditions for maximizing yield of cell-free fetal DNA will be crucial for effective implementation of testing. We explore factors influencing yield of fetal DNA from maternal blood samples, including assessment of collection tubes containing cell-stabilizing agents, storage temperature, interval to sample processing and DNA extraction method used. METHODS: Microfluidic digital PCR was performed to precisely quantify male (fetal) DNA, total DNA and long DNA fragments (indicative of maternal cellular DNA). Real-time qPCR was used to assay for the presence of male SRY signal in samples. RESULTS: Total cell-free DNA quantity increased significantly with time in samples stored in K(3)EDTA tubes, but only minimally in cell stabilizing tubes. This increase was solely due to the presence of additional long fragment DNA, with no change in quantity of fetal or short DNA, resulting in a significant decrease in proportion of cell-free fetal DNA over time. Storage at 4 °C did not prevent these changes. CONCLUSION: When samples can be processed within eight hours of blood draw, K(3)EDTA tubes can be used. Prolonged transfer times in K(3)EDTA tubes should be avoided as the proportion of fetal DNA present decreases significantly; in these situations the use of cell stabilising tubes is preferable. The DNA extraction kit used may influence success rate of diagnostic tests.