The use of circulating cell-free DNA (cfDNA) as a biomarker in transplant recipients offers advantages over invasive tissue biopsy as a quantitative measure for detection of transplant rejection and immunosuppression optimization. However, the fraction of donor-derived cfDNA (dd-cfDNA) in transplant recipient plasma is low and challenging to quantify. Previously reported methods to measure dd-cfDNA require donor and recipient genotyping, which is impractical in clinical settings and adds cost. We developed a targeted next-generation sequencing assay that uses 266 single-nucleotide polymorphisms to accurately quantify dd-cfDNA in transplant recipients without separate genotyping. Analytical performance of the assay was characterized and validated using 1117 samples comprising the National Institute for Standards and Technology Genome in a Bottle human reference genome, independently validated reference materials, and clinical samples. The assay quantifies the fraction of dd-cfDNA in both unrelated and related donor-recipient pairs. The dd-cfDNA assay can reliably measure dd-cfDNA (limit of blank, 0.10%; limit of detection, 0.16%; limit of quantification, 0.20%) across the linear quantifiable range (0.2% to 16%) with across-run CVs of 6.8%. Precision was also evaluated for independently processed clinical sample replicates and is similar to across-run precision. Application of the assay to clinical samples from heart transplant recipients demonstrated increased levels of dd-cfDNA in patients with biopsy-confirmed rejection and decreased levels of dd-cfDNA after successful rejection treatment. This noninvasive clinical-grade sequencing assay can be completed within 3 days, providing the practical turnaround time preferred for transplanted organ surveillance.

Multiple doses of bPIV3 vaccine were well tolerated and immunogenic in young infants.

BackgroundA single non-invasive gene expression profiling (GEP) test (AlloMap®) is often used to discriminate if a heart transplant recipient is at a low risk of acute cellular rejection at time of testing. In a randomized trial, use of the test (a GEP score from 0–40) has been shown to be non-inferior to a routine endomyocardial biopsy for surveillance after heart transplantation in selected low-risk patients with respect to clinical outcomes. Recently, it was suggested that the within-patient variability of consecutive GEP scores may be used to independently predict future clinical events; however, future studies were recommended. Here we performed an analysis of an independent patient population to determine the prognostic utility of within-patient variability of GEP scores in predicting future clinical events.MethodsWe defined the GEP score variability as the standard deviation of four GEP scores collected ≥315 days post-transplantation. Of the 737 patients from the Cardiac Allograft Rejection Gene Expression Observational (CARGO) II trial, 36 were assigned to the composite event group (death, re-transplantation or graft failure ≥315 days post-transplantation and within 3 years of the final GEP test) and 55 were assigned to the control group (non-event patients). In this case-controlled study, the performance of GEP score variability to predict future events was evaluated by the area under the receiver operator characteristics curve (AUC ROC). The negative predictive values (NPV) and positive predictive values (PPV) including 95 % confidence intervals (CI) of GEP score variability were calculated.ResultsThe estimated prevalence of events was 17 %. Events occurred at a median of 391 (inter-quartile range 376) days after the final GEP test. The GEP variability AUC ROC for the prediction of a composite event was 0.72 (95 % CI 0.6-0.8). The NPV for GEP score variability of 0.6 was 97 % (95 % CI 91.4-100.0); the PPV for GEP score variability of 1.5 was 35.4 % (95 % CI 13.5-75.8).ConclusionIn heart transplant recipients, a GEP score variability may be used to predict the probability that a composite event will occur within 3 years after the last GEP score.Trial registrationClinicaltrials.gov identifier NCT00761787Electronic supplementary materialThe online version of this article (doi:10.1186/s12872-015-0106-1) contains supplementary material, which is available to authorized users.

The analytical validation is reported for a targeted methylation-based cell-free DNA multi-cancer early detection test designed to detect cancer and predict the cancer signal origin (tissue of origin). A machine-learning classifier was used to analyze the methylation patterns of >105 genomic targets covering >1 million methylation sites. Analytical sensitivity (limit of detection [95% probability]) was characterized with respect to tumor content by expected variant allele frequency and was determined to be 0.07%-0.17% across five tumor cases and 0.51% for the lymphoid neoplasm case. Test specificity was 99.3% (95% confidence interval, 98.6–99.7%). In the reproducibility and repeatability study, results were consistent in 31/34 (91.2%) pairs with cancer and 17/17 (100%) pairs without cancer; between runs, results were concordant for 129/133 (97.0%) cancer and 37/37 (100%) non-cancer sample pairs. Across 3- to 100-ng input levels of cell-free DNA, cancer was detected in 157/182 (86.3%) cancer samples but not in any of the 62 non-cancer samples. In input titration tests, cancer signal origin was correctly predicted in all tumor samples detected as cancer. No cross-contamination events were observed. No potential interferent (hemoglobin, bilirubin, triglycerides, genomic DNA) affected performance. The results of this analytical validation study support continued clinical development of a targeted methylation cell-free DNA multi-cancer early detection test.

Introduction: A blood-based test using cell-free DNA (cfDNA) may address an unmet need for earlier detection of multiple cancers. Here, we report the analytical validation of a targeted methylation-based cfDNA test to detect cancer and tissue of origin (TOO). Methods: Since detection was determined by a machine-learning classifier based on methylation patterns of >105 genomic targets, defining analytical sensitivity by a single common limit of detection (LOD) for each genomic target was not applicable. Thus, we characterized sensitivity with respect to tumor fraction using dilution series of contrived cancer samples containing mixtures of cfDNA from 6 individuals without cancer and 6 participants in the Circulating Cell-free Genome Atlas study (CCGA; NCT02889978) with cancer (breast, colorectal, head and neck, lung, lymphoid neoplasm). Each dilution series was performed at 3 levels near the expected LOD95% (lowest admixture fraction predicted to have 95% detection probability). Analytical specificity was determined by the false positive (FP) rate in 1,204 samples without cancer. Reproducibility and repeatability were characterized by within- and between-run variability in 81 cancer samples and 45 noncancer samples across multiple reagent lots, instruments, and operators. Test performance as a function of cfDNA input (3-100 ng) was assessed by an input titration study with cfDNA from 6 CCGA participants with cancer (colorectal, lung, esophageal, renal, multiple myeloma, lymphoid neoplasm) and 5 individuals not known to have cancer. The effect of 4 potential interferents (hemoglobin, bilirubin, triglycerides, white blood cell genomic DNA) on test performance was also evaluated. Results: In the assessment of sensitivity by tumor fraction, LOD95% ranged from 0.2% (upper bound) to 0.5% across the 5 individual solid tumor cases and was 1.9% for the blood cancer case. Among noncancer samples from CCGA participants, 9 FPs were detected (99.3% specificity; 95% CI, 98.6%-99.7%). Repeatability and reproducibility results were correct in 31/34 (91.2%) and 17/17 (100%) sample pairs with or without cancer in within-run tests and in 77/81 (95.1%) and 45/45 (100%) cancer and noncancer samples in between-run tests. For the input titration study, cancer was detected in 5/6 cancer specimens (156/183 samples) and not detected in any noncancer samples (n = 62) across all input cfDNA levels tested. TOO was correctly localized in all but 2 samples with a cancer signal. None of the tested interferents affected test performance. Conclusions: A targeted methylation-based cfDNA test repeatedly and reproducibly detected cancer with high analytical sensitivity and specificity and localized TOO with high accuracy, which are critical for multi-cancer early detection applications. This validation study supports clinical development of this multi-cancer early detection test. Citation Format: Gregory Alexander, Wendy Lin, Madhuvanthi Ramaiah, Byoungsok Jung, Lijuan Ji, Ekaterina Revenkova, Payal Shah, Christian Croisetiere, Jennifer Berman, Lane Eubank, Gunjan Naik, Jacqueline Brooks, Andrea Mich, Seyedmehdi Shojaee, Neda Ronaghi, Hemanshi Chawla, Xinyi Hou, Qinwen Liu, Christopher Yakym, Patriss Wais Moradi, Meredith Halks-Miller, Nathan Hunkapiller, Sonya Parpart-Li, Alexander Aravanis. Analytical validation of a multi-cancer early detection test with tissue localization using a cell-free DNA-based targeted methylation assay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 721.