Whole-exome sequencing reveals novel cancer genes and actionable targets in biliary tract cancers in primary sclerosing cholangitis.

BACKGROUND: People with primary sclerosing cholangitis (PSC) have a 20% lifetime risk of biliary tract cancer (BTC). Using whole-exome sequencing, we characterized genomic alterations in tissue samples from BTC with underlying PSC. METHODS: We extracted DNA from formalin-fixed, paraffin-embedded tumor and paired nontumor tissue from 52 resection or biopsy specimens from patients with PSC and BTC and performed whole-exome sequencing. Following copy number analysis, variant calling, and filtering, putative PSC-BTC-associated genes were assessed by pathway analyses and annotated to targeted cancer therapies. RESULTS: We identified 53 candidate cancer genes with a total of 123 nonsynonymous alterations passing filtering thresholds in 2 or more samples. Of the identified genes, 19% had not previously been implicated in BTC, including CNGA3, KRT28, and EFCAB5. Another subset comprised genes previously implicated in hepato-pancreato-biliary cancer, such as ARID2, ELF3, and PTPRD. Finally, we identified a subset of genes implicated in a wide range of cancers such as the tumor suppressor genes TP53, CDKN2A, SMAD4, and RNF43 and the oncogenes KRAS, ERBB2, and BRAF. Focal copy number variations were found in 51.9% of the samples. Alterations in potential actionable genes, including ERBB2, MDM2, and FGFR3 were identified and alterations in the RTK/RAS (p = 0.036), TP53 (p = 0.04), and PI3K (p = 0.043) pathways were significantly associated with reduced overall survival. CONCLUSIONS: In this exome-wide characterization of PSC-associated BTC, we delineated both PSC-specific and universal cancer genes. Our findings provide opportunities for a better understanding of the development of BTC in PSC and could be used as a platform to develop personalized treatment approaches.

Combining Solid and Liquid Biopsy for Therapy Monitoring in Esophageal Cancer.

Esophageal cancer (EC) has one of the highest mortality rates among cancers, making it imperative that therapies are optimized and dynamically adapted to individuals. In this regard, liquid biopsy is an increasingly important method for residual disease monitoring. However, conflicting detection rates (14% versus 60%) and varying cell-free circulating tumor DNA (ctDNA) levels (0.07% versus 0.5%) have been observed in previous studies. Here, we aim to resolve this discrepancy. For 19 EC patients, a complete set of cell-free DNA (cfDNA), formalin-fixed paraffin-embedded tumor tissue (TT) DNA and leukocyte DNA was sequenced (139 libraries). cfDNA was examined in biological duplicates and/or longitudinally, and TT DNA was examined in technical duplicates. In baseline cfDNA, mutations were detected in 12 out of 19 patients (63%); the median ctDNA level was 0.4%. Longitudinal ctDNA changes were consistent with clinical presentation. Considerable mutational diversity was observed in TT, with fewer mutations in cfDNA. The most recurrently mutated genes in TT were TP53, SMAD4, TSHZ3, and SETBP1, with SETBP1 being reported for the first time. ctDNA in blood can be used for therapy monitoring of EC patients. However, a combination of solid and liquid samples should be used to help guide individualized EC therapy.

High-throughput method for the hybridisation-based targeted enrichment of long genomic fragments for PacBio third-generation sequencing.

Hybridisation-based targeted enrichment is a widely used and well-established technique in high-throughput second-generation short-read sequencing. Despite the high potential to genetically resolve highly repetitive and variable genomic sequences by, for example PacBio third-generation sequencing, targeted enrichment for long fragments has not yet established the same high-throughput due to currently existing complex workflows and technological dependencies. We here describe a scalable targeted enrichment protocol for fragment sizes of >7 kb. For demonstration purposes we developed a custom blood group panel of challenging loci. Test results achieved > 65% on-target rate, good coverage (142.7×) and sufficient coverage evenness for both non-paralogous and paralogous targets, and sufficient non-duplicate read counts (83.5%) per sample for a highly multiplexed enrichment pool of 16 samples. We genotyped the blood groups of nine patients employing highly accurate phased assemblies at an allelic resolution that match reference blood group allele calls determined by SNP array and NGS genotyping. Seven Genome-in-a-Bottle reference samples achieved high recall (96%) and precision (99%) rates. Mendelian error rates were 0.04% and 0.13% for the included Ashkenazim and Han Chinese trios, respectively. In summary, we provide a protocol and first example for accurate targeted long-read sequencing that can be used in a high-throughput fashion.

Detection of Cancer Mutations by Urine Liquid Biopsy as a Potential Tool in the Clinical Management of Bladder Cancer Patients.

The standard diagnostic and follow-up examination for bladder cancer is diagnostic cystoscopy, an invasive test that requires compliance for a long period. Urine cytology and recent biomarkers come short of replacing cystoscopy. Urine liquid biopsy promises to solve this problem and potentially allows early detection, evaluation of treatment efficacy, and surveillance. A previous study reached 52-68% sensitivity using small-panel sequencing but could increase sensitivity to 68-83% by adding aneuploidy and promoter mutation detection. Here, we explore whether a large 127-gene panel alone is sufficient to detect tumor mutations in urine from bladder cancer patients. We recruited twelve bladder cancer patients, obtained preoperative and postoperative urine samples, and successfully analyzed samples from eleven patients. In ten patients, we found at least one mutation in bladder-cancer-associated genes, i.e., a promising sensitivity of 91%. In total, we identified 114 variants, of which 90 were predicted as nonbenign, 30% were associated with cancer, and 13% were actionable according to the CIViC database. Sanger sequencing of the patients' formalin-fixed, paraffin-embedded (FFPE) tumor tissues confirmed the findings. We concluded that incorporating urine liquid biopsy is a promising strategy in the management of bladder cancer patients.

Cross-tissue transcriptome-wide association studies identify susceptibility genes shared between schizophrenia and inflammatory bowel disease.

Genetic correlations and an increased incidence of psychiatric disorders in inflammatory-bowel disease have been reported, but shared molecular mechanisms are unknown. We performed cross-tissue and multiple-gene conditioned transcriptome-wide association studies for 23 tissues of the gut-brain-axis using genome-wide association studies data sets (total 180,592 patients) for Crohn's disease, ulcerative colitis, primary sclerosing cholangitis, schizophrenia, bipolar disorder, major depressive disorder and attention-deficit/hyperactivity disorder. We identified NR5A2, SATB2, and PPP3CA (encoding a target for calcineurin inhibitors in refractory ulcerative colitis) as shared susceptibility genes with transcriptome-wide significance both for Crohn's disease, ulcerative colitis and schizophrenia, largely explaining fine-mapped association signals at nearby genome-wide association study susceptibility loci. Analysis of bulk and single-cell RNA-sequencing data showed that PPP3CA expression was strongest in neurons and in enteroendocrine and Paneth-like cells of the ileum, colon, and rectum, indicating a possible link to the gut-brain-axis. PPP3CA together with three further suggestive loci can be linked to calcineurin-related signaling pathways such as NFAT activation or Wnt.

Detailed Transcriptional Landscape of Peripheral Blood Points to Increased Neutrophil Activation in Treatment-Naïve Inflammatory Bowel Disease.

BACKGROUND AND AIMS: Inflammatory bowel disease [IBD] is a chronic relapsing disorder of the gastrointestinal tract, which generally manifests as Crohn's disease [CD] or ulcerative colitis [UC]. These subtypes are heterogeneous in terms of disease location and histological features, while sharing common clinical presentation, genetic associations and, thus, common immune regulatory pathways. METHODS: Using miRNA and mRNA coupled transcriptome profiling and systems biology approaches, we report a comprehensive analysis of blood transcriptomes from treatment-naïve [n = 110] and treatment-exposed [n = 177] IBD patients as well as symptomatic [n = 65] and healthy controls [n = 95]. RESULTS: Broadly, the peripheral blood transcriptomes of CD and UC patients were similar. However, there was an extensive gene deregulation in the blood of IBD patients, while only a slight deregulation in symptomatic controls, when compared with healthy controls. The deregulated mRNAs and miRNAs are mainly involved in the innate immunity and are especially enriched in neutrophil activation-related pathways. Oxidative phosphorylation and neutrophil activation-related modules were found to be differentially co-expressed among treatment-naïve IBD as compared to healthy controls. In the deregulated neutrophil activation-related co-expression module, IL1B was identified as the central gene. Levels of co-expression among IL1B and chemosensing receptor [CXCR1/2 and FPR1/2] genes were reduced in the blood of IBD patients when compared with healthy controls. CONCLUSIONS: Immune dysregulation seen in peripheral blood transcriptomes of treatment-naïve IBD patients is mainly driven by neutrophil activation.

Quantifying sequencing error and effective sequencing depth of liquid biopsy NGS with UMI error correction.

Liquid biopsies are a minimally invasive method to diagnose and longitudinally monitor tumor mutations in patients when tissue biopsies are difficult (e.g., in lung cancer). The percentage of cell-free tumor DNA in blood plasma ranges from more than 65% to 0.1% or lower. To reliably diagnose tumor mutations at 0.1%, there are two options: unrealistically large volumes of patient blood or library preparation and sequencing depth optimized to low-input DNA. Here, we assess two library preparation methods and analysis workflows to determine feasibility and reliability based on standards with known allelic frequency (0 and 0.13% in PIK3CA). However, the implementation for patients is still costly and requires elaborate setups.