Genetic imputation of kidney transcriptome, proteome and multi-omics illuminates new blood pressure and hypertension targets.

Genetic mechanisms of blood pressure (BP) regulation remain poorly defined. Using kidney-specific epigenomic annotations and 3D genome information we generated and validated gene expression prediction models for the purpose of transcriptome-wide association studies in 700 human kidneys. We identified 889 kidney genes associated with BP of which 399 were prioritised as contributors to BP regulation. Imputation of kidney proteome and microRNAome uncovered 97 renal proteins and 11 miRNAs associated with BP. Integration with plasma proteomics and metabolomics illuminated circulating levels of myo-inositol, 4-guanidinobutanoate and angiotensinogen as downstream effectors of several kidney BP genes (SLC5A11, AGMAT, AGT, respectively). We showed that genetically determined reduction in renal expression may mimic the effects of rare loss-of-function variants on kidney mRNA/protein and lead to an increase in BP (e.g., ENPEP). We demonstrated a strong correlation (r = 0.81) in expression of protein-coding genes between cells harvested from urine and the kidney highlighting a diagnostic potential of urinary cell transcriptomics. We uncovered adenylyl cyclase activators as a repurposing opportunity for hypertension and illustrated examples of BP-elevating effects of anticancer drugs (e.g. tubulin polymerisation inhibitors). Collectively, our studies provide new biological insights into genetic regulation of BP with potential to drive clinical translation in hypertension.

Nab-Paclitaxel and Gemcitabine as First-Line Treatment of Metastatic Ampullary Adenocarcinoma with a Novel R-Spondin2 RNA Fusion and NTRK3 Mutation.

Ampullary adenocarcinoma is a rare malignancy that lacks standard systemic treatment. We describe a case of recurrent metastatic ampullary adenocarcinoma of the pancreaticobiliary subtype treated with nanoparticle albumin-bound (nab)-paclitaxel and gemcitabine as first-line treatment. This report also highlights the molecular profile of the ampullary adenocarcinoma and circulating tumor DNA (ctDNA). This is a case of pancreaticobiliary ampullary adenocarcinoma in a 67-year-old woman who initially presented with painless jaundice. Endoscopic and imaging evaluation revealed biliary ductal dilation secondary to an ampullary mass. Pathology confirmed the diagnosis of ampullary adenocarcinoma of the pancreaticobiliary subtype. She underwent surgical resection of the tumor, followed by adjuvant chemotherapy with gemcitabine and capecitabine. The tumor subsequently recurred in the liver. She received palliative chemotherapy with nab-paclitaxel and gemcitabine, resulting in an objective tumor response for 14 months. Molecular profiling of the tumor and ctDNA revealed a novel MATN2-RSPO RNA fusion and a novel NTRK3 mutation, respectively. Our report suggests that long-term durable response can be achieved in metastatic pancreaticobiliary ampullary adenocarcinoma using nab-paclitaxel and gemcitabine. Molecular profiling of the tumor identified a novel R-Spondin2 RNA fusion and NTRK3 mutation that can be potentially targeted for treatment.

Genome-wide analysis of aberrant position and sequence of plasma DNA fragment ends in patients with cancer.

Genome-wide fragmentation patterns in cell-free DNA (cfDNA) in plasma are strongly influenced by cellular origin due to variation in chromatin accessibility across cell types. Such differences between healthy and cancer cells provide the opportunity for development of novel cancer diagnostics. Here, we investigated whether analysis of cfDNA fragment end positions and their surrounding DNA sequences reveals the presence of tumor-derived DNA in blood. We performed genome-wide analysis of cfDNA from 521 samples and analyzed sequencing data from an additional 2147 samples, including healthy individuals and patients with 11 different cancer types. We developed a metric based on genome-wide differences in fragment positioning, weighted by fragment length and GC content [information-weighted fraction of aberrant fragments (iwFAF)]. We observed that iwFAF strongly correlated with tumor fraction, was higher for DNA fragments carrying somatic mutations, and was higher within genomic regions affected by copy number amplifications. We also calculated sample-level means of nucleotide frequencies observed at genomic positions spanning fragment ends. Using a combination of iwFAF and nine nucleotide frequencies from three positions surrounding fragment ends, we developed a machine learning model to differentiate healthy individuals from patients with cancer. We observed an area under the receiver operative characteristic curve (AUC) of 0.91 for detection of cancer at any stage and an AUC of 0.87 for detection of stage I cancer. Our findings remained robust with as few as 1 million fragments analyzed per sample, demonstrating that analysis of fragment ends can become a cost-effective and accessible approach for cancer detection and monitoring.

Refined characterization of circulating tumor DNA through biological feature integration.

Circulating tumor DNA (ctDNA) in blood plasma is present at very low concentrations compared to cell-free DNA (cfDNA) of non-tumor origin. To enhance ctDNA detection, recent studies have been focused on understanding the non-random fragmentation pattern of cfDNA. These studies have investigated fragment sizes, genomic position of fragment end points, and fragment end motifs. Although these features have been described and shown to be aberrant in cancer patients, there is a lack of understanding of how the individual and integrated analysis of these features enrich ctDNA fraction and enhance ctDNA detection. Using whole genome sequencing and copy number analysis of plasma samples from 5 high grade serious ovarian cancer patients, we observed that (1) ctDNA is enriched not only in fragments shorter than mono-nucleosomes (~ 167 bp), but also in those shorter than di-nucleosomes (~ 240-330 bp) (28-159% enrichment). (2) fragments that start and end at the border or within the nucleosome core are enriched in ctDNA (5-46% enrichment). (3) certain DNA motifs conserved in regions 10 bp up- and down- stream of fragment ends (i.e. cleavage sites) could be used to detect tumor-derived fragments (10-44% enrichment). We further show that the integrated analysis of these three features resulted in a higher enrichment of ctDNA when compared to using fragment size alone (additional 7-25% enrichment after fragment size selection). We believe these genome wide features, which are independent of genetic mutational changes, could allow new ways to analyze and interpret cfDNA data, as significant aberrations of these features from a healthy state could improve its utility as a diagnostic biomarker.

Plasma metagenomic sequencing to detect and quantify bacterial DNA in ICU patients suspected of sepsis: A proof-of-principle study.

BACKGROUND: Timely recognition of sepsis and identification of pathogens can improve outcomes in critical care patients but microbial cultures have low accuracy and long turnaround times. In this proof-of-principle study, we describe metagenomic sequencing and analysis of nonhuman DNA in plasma. We hypothesized that quantitative analysis of bacterial DNA (bDNA) levels in plasma can enable detection and monitoring of pathogens. METHODS: We enrolled 30 patients suspected of sepsis in the surgical trauma intensive care unit and collected plasma samples at the time of diagnostic workup for sepsis (baseline), and 7 days and 14 days later. We performed metagenomic sequencing of plasma DNA and used computational classification of sequencing reads to detect and quantify total and pathogen-specific bDNA fraction. To improve assay sensitivity, we developed an enrichment method for bDNA based on size selection for shorter fragment lengths. Differences in bDNA fractions between samples were evaluated using t test and linear mixed-effects model, following log transformation. RESULTS: We analyzed 72 plasma samples from 30 patients. Twenty-seven samples (37.5%) were collected at the time of infection. Median total bDNA fraction was 1.6 times higher in these samples compared with samples with no infection (0.011% and 0.0068%, respectively, p < 0.001). In 17 patients who had active infection at enrollment and at least one follow-up sample collected, total bDNA fractions were higher at baseline compared with the next sample (p < 0.001). Following enrichment, bDNA fractions increased in paired samples by a mean of 16.9-fold. Of 17 samples collected at the time when bacterial pathogens were identified, we detected pathogen-specific DNA in 13 plasma samples (76.5%). CONCLUSION: Bacterial DNA levels in plasma are elevated in critically ill patients with active infection. Pathogen-specific DNA is detectable in plasma, particularly after enrichment using selection for shorter fragments. Serial changes in bDNA levels may be informative of treatment response. LEVEL OF EVIDENCE: Epidemiologic/Prognostic, Level V.

Analysis of recurrently protected genomic regions in cell-free DNA found in urine.

Cell-free DNA (cfDNA) in urine is a promising analyte for noninvasive diagnostics. However, urine cfDNA is highly fragmented. Whether characteristics of these fragments reflect underlying genomic architecture is unknown. Here, we characterized fragmentation patterns in urine cfDNA using whole-genome sequencing. Size distribution of urine cfDNA fragments showed multiple strong peaks between 40 and 120 base pairs (bp) with a modal size of 81- and sharp 10-bp periodicity, suggesting transient protection from complete degradation. These properties were robust to preanalytical perturbations, such as at-home collection and delay in processing. Genome-wide sequencing coverage of urine cfDNA fragments revealed recurrently protected regions (RPRs) conserved across individuals, with partial overlap with nucleosome positioning maps inferred from plasma cfDNA. The ends of cfDNA fragments clustered upstream and downstream of RPRs, and nucleotide frequencies of fragment ends indicated enzymatic digestion of urine cfDNA. Compared to plasma, fragmentation patterns in urine cfDNA showed greater correlation with gene expression and chromatin accessibility in epithelial cells of the urinary tract. We determined that tumor-derived urine cfDNA exhibits a higher frequency of aberrant fragments that end within RPRs. By comparing the fraction of aberrant fragments and nucleotide frequencies of fragment ends, we identified urine samples from cancer patients with an area under the curve of 0.89. Our results revealed nonrandom genomic positioning of urine cfDNA fragments and suggested that analysis of fragmentation patterns across recurrently protected genomic loci may serve as a cancer diagnostic.

The Genomic and Immune Landscapes of Lethal Metastatic Breast Cancer.

The detailed molecular characterization of lethal cancers is a prerequisite to understanding resistance to therapy and escape from cancer immunoediting. We performed extensive multi-platform profiling of multi-regional metastases in autopsies from 10 patients with therapy-resistant breast cancer. The integrated genomic and immune landscapes show that metastases propagate and evolve as communities of clones, reveal their predicted neo-antigen landscapes, and show that they can accumulate HLA loss of heterozygosity (LOH). The data further identify variable tumor microenvironments and reveal, through analyses of T cell receptor repertoires, that adaptive immune responses appear to co-evolve with the metastatic genomes. These findings reveal in fine detail the landscapes of lethal metastatic breast cancer.