Allogeneic hematopoietic cell transplantation (HCT) provides effective treatment for hematologic malignancies and immune disorders. Monitoring of posttransplant complications is critical, yet current diagnostic options are limited. Here, we show that cell-free DNA (cfDNA) in blood is a versatile analyte for monitoring of the most important complications that occur after HCT: graft-versus-host disease (GVHD), a frequent immune complication of HCT, infection, relapse of underlying disease, and graft failure. We demonstrate that these therapeutic complications are informed from a single assay, low-coverage bisulfite sequencing of cfDNA, followed by disease-specific bioinformatic analyses. To inform GVHD, we profile cfDNA methylation marks to trace the cfDNA tissues-of-origin and to quantify tissue-specific injury. To inform infection, we implement metagenomic cfDNA profiling. To inform cancer relapse, we implement analyses of tumor-specific genomic aberrations. Finally, to detect graft failure, we quantify the proportion of donor- and recipient-specific cfDNA. We applied this assay to 170 plasma samples collected from 27 HCT recipients at predetermined timepoints before and after allogeneic HCT. We found that the abundance of solid-organ–derived cfDNA in the blood at 1 mo after HCT is predictive of acute GVHD (area under the curve, 0.88). Metagenomic profiling of cfDNA revealed the frequent occurrence of viral reactivation in this patient population. The fraction of donor-specific cfDNA was indicative of relapse and remission, and the fraction of tumor-specific cfDNA was informative of cancer relapse. This proof-of-principle study shows that cfDNA has the potential to improve the care of allogeneic HCT recipients by enabling earlier detection and better prediction of the complex array of complications that occur after HCT.

Differential host responses in coronavirus disease 2019 (COVID-19) and multisystem inflammatory syndrome in children (MIS-C) remain poorly characterized. Here we use next-generation sequencing to longitudinally analyze blood samples from pediatric patients with acute COVID-19 (n=70) or MIS-C (n=141) across three hospitals. Profiling of plasma cell-free nucleic acids uncovers distinct signatures of cell injury and death between these two disease states, with increased heterogeneity and multi-organ involvement in MIS-C encompassing diverse cell types such as endothelial and neuronal Schwann cells. Whole blood RNA profiling reveals upregulation of similar pro-inflammatory signaling pathways in COVID-19 and MIS-C, but also MIS-C specific downregulation of T cell-associated pathways. Profiling of plasma cell-free RNA and whole blood RNA in paired samples yields different yet complementary signatures for each disease state. Our work provides a systems-level, multi-analyte view of immune responses and tissue damage in COVID-19 and MIS-C and informs the future development of new disease biomarkers.

Tuberculosis (TB) remains a leading cause of death from an infectious disease worldwide. This is partly due to a lack of tools to effectively screen and triage individuals with potential TB. Whole blood RNA signatures have been extensively studied as potential biomarkers for TB, but they have failed to meet the World Health Organization's (WHOs) target product profiles (TPPs) for a non-sputum triage or diagnostic test. In this study, we investigated the utility of plasma cell-free RNA (cfRNA) as a host response biomarker for TB. We used RNA profiling by sequencing to analyze plasma samples from 182 individuals with a cough lasting at least two weeks, who were seen at outpatient clinics in Uganda, Vietnam, and the Philippines. Of these individuals, 100 were diagnosed with microbiologically-confirmed TB. Our analysis of the plasma cfRNA transcriptome revealed 541 differentially abundant genes, the top 150 of which were used to train 15 machine learning models. The highest performing model led to a 9-gene signature that had a diagnostic accuracy of 89.1% (95% CI: 83.6-93.4%) and an area under the curve of 0.934 (95% CI: 0.8674-1) for microbiologically-confirmed TB. This 9-gene signature exceeds the optimal WHO TPPs for a TB triage test (sensitivity: 96.2% [95% CI: 80.9-100%], specificity: 89.7% [95% CI: 72.4-100%]) and was robust to differences in sample collection, geographic location, and HIV status. Overall, our results demonstrate the utility of plasma cfRNA for the detection of TB and suggest the potential for a point-of-care, gene expression-based assay to aid in early detection of TB.

14 15 16 17 ABSTRACT: Allogeneic hematopoietic cell transplantation (HCT) provides effective treatment for 18 hematologic malignancies and immune disorders. Monitoring for immune complications and infection is a 19 critical component of post-HCT therapy, however, current diagnostic options are limited. Here, we propose 20a blood test that employs genome-wide profiling of methylation marks comprised within circulating cell-21 free DNA to trace the tissues-of-origin of cell-free DNA (cfDNA), to quantify tissue-specific injury, and to 22 35 36More than 30,000 patients undergo allogeneic hematopoietic cell transplants (HCT) worldwide 37 each year for treatment of a variety of malignant and nonmalignant hematologic diseases 1-3 . However, 38 immune related complications occur frequently after HCT. Up to 50% of patients experience graft-versus-39host disease (GVHD) in the first year after transplantation. GVHD occurs when donor immune cells attack 40 the patient's own tissues 2,4-6 . Early and accurate diagnosis of GVHD is critical to inform treatment decisions 41and to prevent serious long-term complications, including organ failure and death. Unfortunately, there are 42 few, noninvasive diagnostic options that reliably identify patients very early after onset of GVHD 43 symptoms. In current clinical practice, diagnosis of GVHD relies almost entirely on clinical criteria and 44 often requires confirmation with invasive procedures, such as a biopsy of the gastrointestinal tract, skin, or 45 liver 7 . 46 47Small fragments of cell-free DNA (cfDNA) circulate in blood. In the absence of disease, cfDNA 48originates primarily from apoptosis of cells of the hematopoietic lineage 8 . During disease, a significant 49proportion of cfDNA can be derived from affected tissues 9-15 . In solid-organ transplantation (SOT), we and 50 others have shown that transplant donor derived cfDNA in the blood is a quantitative noninvasive marker 51 of solid organ transplant injury 12,13,16,17 . Here, we sought to investigate the utility of circulating cfDNA as a 52minimally invasive analyte to detect and quantify tissue injury due to GVHD after HCT. To quantify the 53 contributions of any tissue to the mixture of cfDNA in plasma, and thereby the degree of injury to any 54 vascularized tissue in the setting of HCT, we implemented shallow, whole genome bisulfite sequencing 55(WGBS) to profile 5-methylcytosine (5mC) marks of cfDNA. These marks are cell, tissue and organ type 56 specific 18 , and can inform the tissues-of-origin of cell-free DNA 8,11,19 . 57 58We collected serial blood samples from a prospective cohort of allogeneic HCT recipients at 59 predetermined time points before and after HCT. We analyzed a total of 106 plasma samples from 18 HCT 60 recipients with and without GVHD in the first 3 months post HCT. We observed rich dynamics in the tissue-61 origin of cfDNA in response to pre-transplant conditioning chemotherapy and following HCT. The tissue-62 origin of cfDNA after transplantation was patient-specific and a function of the ma...