Whole-genome landscape of pancreatic neuroendocrine tumours.

The diagnosis of pancreatic neuroendocrine tumours (PanNETs) is increasing owing to more sensitive detection methods, and this increase is creating challenges for clinical management. We performed whole-genome sequencing of 102 primary PanNETs and defined the genomic events that characterize their pathogenesis. Here we describe the mutational signatures they harbour, including a deficiency in G:C > T:A base excision repair due to inactivation of MUTYH, which encodes a DNA glycosylase. Clinically sporadic PanNETs contain a larger-than-expected proportion of germline mutations, including previously unreported mutations in the DNA repair genes MUTYH, CHEK2 and BRCA2. Together with mutations in MEN1 and VHL, these mutations occur in 17% of patients. Somatic mutations, including point mutations and gene fusions, were commonly found in genes involved in four main pathways: chromatin remodelling, DNA damage repair, activation of mTOR signalling (including previously undescribed EWSR1 gene fusions), and telomere maintenance. In addition, our gene expression analyses identified a subgroup of tumours associated with hypoxia and HIF signalling.

Corrigendum: Whole-genome landscape of pancreatic neuroendocrine tumours.

This corrects the article DOI: 10.1038/nature21063.

Genomic analyses identify molecular subtypes of pancreatic cancer.

Integrated genomic analysis of 456 pancreatic ductal adenocarcinomas identified 32 recurrently mutated genes that aggregate into 10 pathways: KRAS, TGF-ß, WNT, NOTCH, ROBO/SLIT signalling, G1/S transition, SWI-SNF, chromatin modification, DNA repair and RNA processing. Expression analysis defined 4 subtypes: (1) squamous; (2) pancreatic progenitor; (3) immunogenic; and (4) aberrantly differentiated endocrine exocrine (ADEX) that correlate with histopathological characteristics. Squamous tumours are enriched for TP53 and KDM6A mutations, upregulation of the TP63∆N transcriptional network, hypermethylation of pancreatic endodermal cell-fate determining genes and have a poor prognosis. Pancreatic progenitor tumours preferentially express genes involved in early pancreatic development (FOXA2/3, PDX1 and MNX1). ADEX tumours displayed upregulation of genes that regulate networks involved in KRAS activation, exocrine (NR5A2 and RBPJL), and endocrine differentiation (NEUROD1 and NKX2-2). Immunogenic tumours contained upregulated immune networks including pathways involved in acquired immune suppression. These data infer differences in the molecular evolution of pancreatic cancer subtypes and identify opportunities for therapeutic development.

Single-cell RNA-seq of human induced pluripotent stem cells reveals cellular heterogeneity and cell state transitions between subpopulations.

Heterogeneity of cell states represented in pluripotent cultures has not been described at the transcriptional level. Since gene expression is highly heterogeneous between cells, single-cell RNA sequencing can be used to identify how individual pluripotent cells function. Here, we present results from the analysis of single-cell RNA sequencing data from 18,787 individual WTC-CRISPRi human induced pluripotent stem cells. We developed an unsupervised clustering method and, through this, identified four subpopulations distinguishable on the basis of their pluripotent state, including a core pluripotent population (48.3%), proliferative (47.8%), early primed for differentiation (2.8%), and late primed for differentiation (1.1%). For each subpopulation, we were able to identify the genes and pathways that define differences in pluripotent cell states. Our method identified four transcriptionally distinct predictor gene sets composed of 165 unique genes that denote the specific pluripotency states; using these sets, we developed a multigenic machine learning prediction method to accurately classify single cells into each of the subpopulations. Compared against a set of established pluripotency markers, our method increases prediction accuracy by 10%, specificity by 20%, and explains a substantially larger proportion of deviance (up to threefold) from the prediction model. Finally, we developed an innovative method to predict cells transitioning between subpopulations and support our conclusions with results from two orthogonal pseudotime trajectory methods.

Whole genomes redefine the mutational landscape of pancreatic cancer.

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

A High-Fat Diet Increases Influenza A Virus-Associated Cardiovascular Damage.

BACKGROUND: Influenza A virus (IAV) causes a wide range of extrarespiratory complications. However, the role of host factors in these complications of influenza virus infection remains to be defined. METHODS: Here, we sought to use transcriptional profiling, virology, histology, and echocardiograms to investigate the role of a high-fat diet in IAV-associated cardiac damage. RESULTS: Transcriptional profiling showed that, compared to their low-fat counterparts (LF mice), mice fed a high-fat diet (HF mice) had impairments in inflammatory signaling in the lung and heart after IAV infection. This was associated with increased viral titers in the heart, increased left ventricular mass, and thickening of the left ventricular wall in IAV-infected HF mice compared to both IAV-infected LF mice and uninfected HF mice. Retrospective analysis of clinical data revealed that cardiac complications were more common in patients with excess weight, an association which was significant in 2 out of 4 studies. CONCLUSIONS: Together, these data provide the first evidence that a high-fat diet may be a risk factor for the development of IAV-associated cardiovascular damage and emphasizes the need for further clinical research in this area.

Hypermutation In Pancreatic Cancer.

Pancreatic cancer is molecularly diverse, with few effective therapies. Increased mutation burden and defective DNA repair are associated with response to immune checkpoint inhibitors in several other cancer types. We interrogated 385 pancreatic cancer genomes to define hypermutation and its causes. Mutational signatures inferring defects in DNA repair were enriched in those with the highest mutation burdens. Mismatch repair deficiency was identified in 1% of tumors harboring different mechanisms of somatic inactivation of MLH1 and MSH2. Defining mutation load in individual pancreatic cancers and the optimal assay for patient selection may inform clinical trial design for immunotherapy in pancreatic cancer.

Integrated genomic and transcriptomic analysis of human brain metastases identifies alterations of potential clinical significance.

Treatment options for patients with brain metastases (BMs) have limited efficacy and the mortality rate is virtually 100%. Targeted therapy is critically under-utilized, and our understanding of mechanisms underpinning metastatic outgrowth in the brain is limited. To address these deficiencies, we investigated the genomic and transcriptomic landscapes of 36 BMs from breast, lung, melanoma and oesophageal cancers, using DNA copy-number analysis and exome- and RNA-sequencing. The key findings were as follows. (a) Identification of novel candidates with possible roles in BM development, including the significantly mutated genes DSC2, ST7, PIK3R1 and SMC5, and the DNA repair, ERBB-HER signalling, axon guidance and protein kinase-A signalling pathways. (b) Mutational signature analysis was applied to successfully identify the primary cancer type for two BMs with unknown origins. (c) Actionable genomic alterations were identified in 31/36 BMs (86%); in one case we retrospectively identified ERBB2 amplification representing apparent HER2 status conversion, then confirmed progressive enrichment for HER2-positivity across four consecutive metastatic deposits by IHC and SISH, resulting in the deployment of HER2-targeted therapy for the patient. (d) In the ERBB/HER pathway, ERBB2 expression correlated with ERBB3 (r(2) = 0.496; p < 0.0001) and HER3 and HER4 were frequently activated in an independent cohort of 167 archival BM from seven primary cancer types: 57.6% and 52.6% of cases were phospho-HER3(Y1222) or phospho-HER4(Y1162) membrane-positive, respectively. The HER3 ligands NRG1/2 were barely detectable by RNAseq, with NRG1 (8p12) genomic loss in 63.6% breast cancer-BMs, suggesting a microenvironmental source of ligand. In summary, this is the first study to characterize the genomic landscapes of BM. The data revealed novel candidates, potential clinical applications for genomic profiling of resectable BMs, and highlighted the possibility of therapeutically targeting HER3, which is broadly over-expressed and activated in BMs, independent of primary site and systemic therapy.

Genome-wide DNA methylation patterns in pancreatic ductal adenocarcinoma reveal epigenetic deregulation of SLIT-ROBO, ITGA2 and MET signaling.

The importance of epigenetic modifications such as DNA methylation in tumorigenesis is increasingly being appreciated. To define the genome-wide pattern of DNA methylation in pancreatic ductal adenocarcinomas (PDAC), we captured the methylation profiles of 167 untreated resected PDACs and compared them to a panel of 29 adjacent nontransformed pancreata using high-density arrays. A total of 11,634 CpG sites associated with 3,522 genes were significantly differentially methylated (DM) in PDAC and were capable of segregating PDAC from non-malignant pancreas, regardless of tumor cellularity. As expected, PDAC hypermethylation was most prevalent in the 5' region of genes (including the proximal promoter, 5'UTR and CpG islands). Approximately 33% DM genes showed significant inverse correlation with mRNA expression levels. Pathway analysis revealed an enrichment of aberrantly methylated genes involved in key molecular mechanisms important to PDAC: TGF-ß, WNT, integrin signaling, cell adhesion, stellate cell activation and axon guidance. Given the recent discovery that SLIT-ROBO mutations play a clinically important role in PDAC, the role of epigenetic perturbation of axon guidance was pursued in more detail. Bisulfite amplicon deep sequencing and qRT-PCR expression analyses confirmed recurrent perturbation of axon guidance pathway genes SLIT2, SLIT3, ROBO1, ROBO3, ITGA2 and MET and suggests epigenetic suppression of SLIT-ROBO signaling and up-regulation of MET and ITGA2 expression. Hypomethylation of MET and ITGA2 correlated with high gene expression, which was associated with poor survival. These data suggest that aberrant methylation plays an important role in pancreatic carcinogenesis affecting core signaling pathways with potential implications for the disease pathophysiology and therapy.

Acquisition of murine splenic myeloid cells for protein and gene expression profiling by advanced flow cytometry and CITE-seq.

Here, we outline detailed protocols to isolate and profile murine splenic dendritic cells (DCs) through advanced flow cytometry of the myeloid compartment and single-cell transcriptomic profiling with integrated cell surface protein expression through CITE-seq. This protocol provides a general transferrable road map for different tissues and species. For complete details on the use and execution of this protocol, please refer to Lukowski et al. (2021).

Absence of Batf3 reveals a new dimension of cell state heterogeneity within conventional dendritic cells.

Conventional dendritic cells (cDCs) are traditionally subdivided into cDC1 and cDC2 lineages. Batf3 is a cDC1-required transcription factor, and we observed that Batf3-/- mice harbor a population of cDC1-like cells co-expressing cDC2-associated surface molecules. Using single-cell RNA sequencing with integrated cell surface protein expression (CITE-seq), we found that Batf3-/- mitotic immature cDC1-like cells showed reduced expression of cDC1 features and increased levels of cDC2 features. In wild type, we also observed a proportion of mature cDC1 cells expressing surface features characteristic to cDC2 and found that overall cDC cell state heterogeneity was mainly driven by developmental stage, proliferation, and maturity. We detected population diversity within Sirpa+ cDC2 cells, including a Cd33+ cell state expressing high levels of Sox4 and lineage-mixed features characteristic to cDC1, cDC2, pDCs, and monocytes. In conclusion, these data suggest that multiple cDC cell states can co-express lineage-overlapping features, revealing a level of previously unappreciated cDC plasticity.

Single-Cell Transcriptomic Analysis of Cardiac Differentiation from Human PSCs Reveals HOPX-Dependent Cardiomyocyte Maturation.

Cardiac differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic gene regulatory networks during stepwise fate transitions but often generates immature cell types that do not fully recapitulate properties of their adult counterparts, suggesting incomplete activation of key transcriptional networks. We performed extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during cardiac differentiation of hPSCs and identified strategies to improve in vitro cardiomyocyte differentiation. Utilizing genetic gain- and loss-of-function approaches, we found that hypertrophic signaling is not effectively activated during monolayer-based cardiac differentiation, thereby preventing expression of HOPX and its activation of downstream genes that govern late stages of cardiomyocyte maturation. This study therefore provides a key transcriptional roadmap of in vitro cardiac differentiation at single-cell resolution, revealing fundamental mechanisms underlying heart development and differentiation of hPSC-derived cardiomyocytes.

A workflow to increase verification rate of chromosomal structural rearrangements using high-throughput next-generation sequencing.

Somatic rearrangements, which are commonly found in human cancer genomes, contribute to the progression and maintenance of cancers. Conventionally, the verification of somatic rearrangements comprises many manual steps and Sanger sequencing. This is labor intensive when verifying a large number of rearrangements in a large cohort. To increase the verification throughput, we devised a high-throughput workflow that utilizes benchtop next-generation sequencing and in-house bioinformatics tools to link the laboratory processes. In the proposed workflow, primers are automatically designed. PCR and an optional gel electrophoresis step to confirm the somatic nature of the rearrangements are performed. PCR products of somatic events are pooled for Ion Torrent PGM and/or Illumina MiSeq sequencing, the resulting sequence reads are assembled into consensus contigs by a consensus assembler, and an automated BLAT is used to resolve the breakpoints to base level. We compared sequences and breakpoints of verified somatic rearrangements between the conventional and high-throughput workflow. The results showed that next-generation sequencing methods are comparable to conventional Sanger sequencing. The identified breakpoints obtained from next-generation sequencing methods were highly accurate and reproducible. Furthermore, the proposed workflow allows hundreds of events to be processed in a shorter time frame compared with the conventional workflow.

Genomic catastrophes frequently arise in esophageal adenocarcinoma and drive tumorigenesis.

Oesophageal adenocarcinoma (EAC) incidence is rapidly increasing in Western countries. A better understanding of EAC underpins efforts to improve early detection and treatment outcomes. While large EAC exome sequencing efforts to date have found recurrent loss-of-function mutations, oncogenic driving events have been underrepresented. Here we use a combination of whole-genome sequencing (WGS) and single-nucleotide polymorphism-array profiling to show that genomic catastrophes are frequent in EAC, with almost a third (32%, n=40/123) undergoing chromothriptic events. WGS of 22 EAC cases show that catastrophes may lead to oncogene amplification through chromothripsis-derived double-minute chromosome formation (MYC and MDM2) or breakage-fusion-bridge (KRAS, MDM2 and RFC3). Telomere shortening is more prominent in EACs bearing localized complex rearrangements. Mutational signature analysis also confirms that extreme genomic instability in EAC can be driven by somatic BRCA2 mutations. These findings suggest that genomic catastrophes have a significant role in the malignant transformation of EAC.

Somatic point mutation calling in low cellularity tumors.

Somatic mutation calling from next-generation sequencing data remains a challenge due to the difficulties of distinguishing true somatic events from artifacts arising from PCR, sequencing errors or mis-mapping. Tumor cellularity or purity, sub-clonality and copy number changes also confound the identification of true somatic events against a background of germline variants. We have developed a heuristic strategy and software (http://www.qcmg.org/bioinformatics/qsnp/) for somatic mutation calling in samples with low tumor content and we show the superior sensitivity and precision of our approach using a previously sequenced cell line, a series of tumor/normal admixtures, and 3,253 putative somatic SNVs verified on an orthogonal platform.

Development and characterization of new inhibitors of the human and mouse hematopoietic prostaglandin D(2) synthases.

The hematopoietic prostaglandin D(2) synthase has a proinflammatory effect in a range of diseases, including allergic asthma, where its product prostaglandin D(2) (PGD(2)) has a role in regulating many of the hallmark disease characteristics. Here we describe the development and characterization of a novel series of hematopoietic prostaglandin D(2) synthase inhibitors with potency similar to that of known inhibitors. Compounds N-benzhydryl-5-(3-hydroxyphenyl)thiophene-2-carboxamide (compound 8) and N-(1-amino-1-oxo-3-phenylpropan-2-yl)-6-(thiophen-2-yl)nicotinamide (compound 34) demonstrated low micromolar potency in the inhibition of the purified enzyme, while only 34 reduced Toll-like receptor (TLR) inducible PGD(2) production in both mouse primary bone marrow-derived macrophages and the human megakaryocytic cell line MEG-01S. Importantly, 34 demonstrated a greater selectivity for inhibition of PGD(2) synthesis versus other eicosanoids that lie downstream of PGH(2) (PGE(2) and markers of prostacyclin (6-keto PGF(1alpha)) and thromboxane (TXB(2))) when compared to the known inhibitors HQL-79 (compound 1) and 2-phenyl-5-(1H-pyrazol-3-yl)thiazole (compound 2). Compound 34 therefore represents a selective hematopoietic prostaglandin D(2) synthase inhibitor.

Identification and characterisation of new inhibitors for the human hematopoietic prostaglandin D2 synthase.

Prostaglandin D(2) synthesised by the hematopoietic prostaglandin D(2) synthase has a pro-inflammatory effect in allergic asthma, regulating many hallmark characteristics of the disease. Here we describe identification of hematopoietic prostaglandin D(2) synthase inhibitors including cibacron blue, bromosulfophthalein and ethacrynic acid. Expansion around the drug-like ethacrynic acid identified a novel inhibitor, nocodazole, and a fragment representing its aromatic core. Nocodazole binding was further characterised by docking calculations in combination with conformational strain analysis. The benzyl thiophene core was predicted to be buried in the active site, binding in the putative prostaglandin binding site, and a likely hydrogen bond donor site identified. X-ray crystallographic studies supported the predicted binding mode.