Complex Patterns of Genomic Heterogeneity Identified in 42 Tumor Samples and ctDNA of a Pulmonary Atypical Carcinoid Patient.

Tumor evolution underlies many challenges facing precision oncology, and improving our understanding has the potential to improve clinical care. This study represents a rare opportunity to study tumor heterogeneity and evolution in a patient with an understudied cancer type. A patient with pulmonary atypical carcinoid, a neuroendocrine tumor, metastatic to 90 sites, requested and consented to donate tissues for research. 42 tumor samples collected at rapid autopsy from 14 anatomically distinct sites were analyzed through DNA whole-exome sequencing and RNA sequencing, and five analyzed through linked-read sequencing. Targeted DNA sequencing was completed on two clinical tissue biopsies and one blood plasma sample. Chromosomal alterations and gene variants accumulated over time, and specific chromosomal alterations preceded the single predicted gene driver variant (ARID1A). At the time of autopsy, all sites shared the gain of one copy of Chr 5, loss of one copy of Chr 6 and 21, chromothripsis of one copy of Chr 11, and 39 small variants. Two tumor clones (carrying additional variants) were detected at metastatic sites, and occasionally in different regions of the same organ (e.g., within the pancreas). Circulating tumor DNA (ctDNA) sequencing detected shared tumor variants in the blood plasma and captured marked genomic heterogeneity, including all metastatic clones but few private tumor variants. This study describes genomic tumor evolution and dissemination of a pulmonary atypical carcinoid donated by a single generous patient. It highlights the critical role of chromosomal alterations in tumor initiation and explores the potential of ctDNA analysis to represent genomically heterogeneous disease. Significance: DNA sequencing data from tumor samples and blood plasma from a single patient highlighted the critical early role of chromosomal alterations in atypical carcinoid tumor development. Common tumor variants were readily detected in the blood plasma, unlike emerging tumor variants, which has implications for using ctDNA to capture cancer evolution.

The amyotrophic lateral sclerosis-linked protein TDP-43 regulates interleukin-6 cytokine production by human brain pericytes.

Amyotrophic lateral sclerosis (ALS) is a fatal movement disorder involving degeneration of motor neurons through dysfunction of the RNA-binding protein TDP-43. Pericytes, the perivascular cells of the blood-brain, blood-spinal cord, and blood-CSF barriers also degenerate in ALS. Indeed, pericytes are among the earliest cell types to show gene expression changes in pre-symptomatic animal models of ALS. This suggests that pericyte degeneration precedes neurodegeneration and may involve pericyte cell-autonomous TDP-43 dysfunction. Here we determined the effect of TDP-43 dysfunction in human brain pericytes on interleukin 6 (IL-6), a critical secreted inflammatory mediator reported to be regulated by TDP 43. Primary human brain pericytes were cultured from biopsy tissue from epilepsy surgeries and TDP-43 was silenced using siRNA. TDP-43 silencing of pericytes stimulated with pro-inflammatory cytokines, interleukin-1ß or tumour necrosis factor alpha, robustly suppressed the induction of IL-6 transcript and protein. IL-6 regulation by TDP-43 did not involve the assembly of TDP-43 nuclear splicing bodies, and did not occur via altered splicing of IL6. Instead, transcriptome-wide analysis by RNA-Sequencing identified a poison exon in the IL6 destabilising factor HNRNPD (AUF1) as a splicing target of TDP-43. Our data support a model whereby TDP-43 silencing favours destabilisation of IL6 mRNA, via enhanced AU-rich element-mediated decay by HNRNP/AUF1. This suggests that cell-autonomous deficits in TDP-43 function in human brain pericytes would suppress their production of IL-6. Given the importance of the blood-brain and blood-spinal cord barriers in maintaining motor neuron health, TDP-43 in human brain pericytes may represent a cellular target for ALS therapeutics.

Side-Population Trophoblasts Exhibit the Differentiation Potential of a Trophoblast Stem Cell Population, Persist to Term, and are Reduced in Fetal Growth Restriction.

BACKGROUND: Fetal growth restriction often results from poor placental function and is a major cause of stillbirth. Clinically, fetal growth restriction is difficult to diagnose and currently has no effective treatment. Trophoblasts are unique placental cells that form the feto-maternal interface and facilitate nutrient and gas exchange. Fetal growth restriction is linked to inadequate trophoblast function. However, our understanding of the mechanisms underlying this dysfunction are poor, in part because of our inability to isolate and study the trophoblast stem cells from which mature trophoblasts arise in pathologic pregnancies. METHODS: Cells isolated from first-trimester placentae using the Hoechst side-population technique were propagated or differentiated into mature trophoblasts. Side-population trophoblasts were isolated from normal third-trimester and growth restricted placentae using the same technique. First and third-trimester side-population trophoblasts were compared by microarray analysis. RESULTS: First-trimester side-population trophoblasts could be propagated in an undifferentiated state or differentiated, via intermediate cytotrophoblasts, into syncytiotrophoblast or extravillous trophoblasts. Using the same technique, side-population trophoblasts could be isolated from term placentae for the first time, demonstrating that while they were present at consistent levels throughout gestation (~3·5%), side-population trophoblasts were significantly depleted in growth restricted pregnancies (0·32%). CONCLUSIONS: Our novel method of isolating a population of human trophoblast stem cell-like cells directly from human placental tissue throughout gestation provides the first insights into trophoblast dysfunction in pregnancy pathologies. The depletion of side-population trophoblasts in growth restricted placentae may contribute to poor placental function.

Ex Vivo Human Adipose Tissue Derived Mesenchymal Stromal Cells (ASC) Are a Heterogeneous Population That Demonstrate Rapid Culture-Induced Changes.

Human adipose-derived mesenchymal stromal cells (ASC) are showing clinical promise for the treatment of a range of inflammatory and degenerative conditions. These lipoaspirate-derived cells are part of the abundant and accessible source of heterogeneous stromal vascular fraction (SVF). They are typically isolated and expanded from the SVF via adherent cell culture for at least 2 weeks and as such represent a relatively undefined population of cells. We isolated ex vivo ASC directly from lipoaspirate using a cocktail of antibodies combined with immunomagnetic bead sorting. This method allowed for the rapid enrichment of a defined and untouched ex vivo ASC population (referred to as MACS-derived ASC) that were then compared to culture-derived ASC. This comparison found that MACS-derived ASC contain a greater proportion of cells with activity in in vitro differentiation assays. There were also significant differences in the secretion levels of some key paracrine molecules. Moreover, when the MACS-derived ASC were subjected to adherent tissue culture, rapid changes in gene expression were observed. This indicates that culturing cells may alter the clinical utility of these cells. Although MACS-derived ASC are more defined compared to culture-derived ASC, further investigations using a comprehensive multicolor flow cytometry panel revealed that this cell population is more heterogeneous than previously appreciated. Additional studies are therefore required to more precisely delineate phenotypically distinct ASC subsets with the most therapeutic potential. This research highlights the disparity between ex vivo MACS-derived and culture-derived ASC and the need for further characterization.

Recurrent loss of heterozygosity correlates with clinical outcome in pancreatic neuroendocrine cancer.

Pancreatic neuroendocrine tumors (pNETs) are uncommon cancers arising from pancreatic islet cells. Here we report the analysis of gene mutation, copy number, and RNA expression of 57 sporadic well-differentiated pNETs. pNET genomes are dominated by aneuploidy, leading to concordant changes in RNA expression at the level of whole chromosomes and chromosome segments. We observed two distinct patterns of somatic pNET aneuploidy that are associated with tumor pathology and patient prognosis. Approximately 26% of the patients in this series had pNETs with genomes characterized by recurrent loss of heterozygosity (LoH) of 10 specific chromosomes, accompanied by bi-allelic MEN1 inactivation and generally poor clinical outcome. Another ~40% of patients had pNETs that lacked this recurrent LoH pattern but had chromosome 11 LoH, bi-allelic MEN1 inactivation, and universally good clinical outcome. The somatic aneuploidy allowed pathogenic germline variants (e.g., ATM) to be expressed unopposed, with RNA expression patterns showing inactivation of downstream tumor suppressor pathways. No prognostic associations were found with tumor morphology, single gene mutation, or expression of RNAs reflecting the activity of immune, differentiation, proliferative or tumor suppressor pathways. In pNETs, single gene mutations appear to be less important than aneuploidy, with MEN1 the only statistically significant recurrently mutated driver gene. In addition, only one pNET in the series had clearly actionable single nucleotide variants (SNVs) (in PTEN and FLCN) confirmed by corroborating RNA expression changes. The two clinically relevant patterns of LoH described here define a novel oncogenic mechanism and a plausible route to genomic precision oncology for this tumor type.

Massively Parallel Sequencing of Genes Implicated in Heritable Cardiac Disorders: A Strategy for a Small Diagnostic Laboratory.

Sudden cardiac death (SCD) in people before the age of 35 years is a devastating event for any family. The causes of SCD in the young can be broadly divided into two groups: heritable cardiac disorders that affect the heart structure (cardiomyopathies) and primary electrical disorders (cardiac ion channelopathies). Genetic testing is vital as those suffering from cardiac ion channelopathies have structurally normal hearts, and those with cardiomyopathies may only show subtle abnormalities in the heart and these signs may not be detected during an autopsy. Post-mortem genetic testing of SCD victims is important to identify the underlying genetic cause. This is important as family cascade screening may be undertaken to identify those who may be at risk and provide vital information about risk stratification and clinical management. The development of massively parallel sequencing (MPS) has made it possible for the simultaneous screening of multiple patients for hundreds of genes. In light of this, we opted to develop an MPS approach for SCD analysis that would allow us to screen for mutations in genes implicated in cardiomyopathies and cardiac ion channelopathies. The rationale behind this panel was to limit it to genes carrying the greatest mutation load. If no likely pathogenic gene variant were found then testing could cascade to whole exome/genome sequencing as a gene-discovery exercise. The overarching aim was to design and validate a custom-cardiac panel that satisfies the diagnostic requirements of LabPLUS (Auckland City Hospital, Auckland, NZ) and the guidelines provided by the Royal College of Pathologists of Australasia and the Association for Clinical Genetic Science.

A cross comparison of technologies for the detection of microRNAs in clinical FFPE samples of hepatoblastoma patients.

Although formalin fixed paraffin embedded (FFPE) tissue is a major biological source in cancer research, it is challenging to work with due to macromolecular fragmentation and nucleic acid crosslinking. Therefore, it is important to characterise the quality of data that can be obtained from FFPE samples. We have compared three independent platforms (next generation sequencing, microarray and NanoString) for profiling microRNAs (miRNAs) using clinical FFPE samples from hepatoblastoma (HB) patients. The number of detected miRNAs ranged from 228 to 345 (median = 294) using the next generation sequencing platform, whereas 79 to 125 (median = 112) miRNAs were identified using microarrays in three HB samples, including technical replicates. NanoString identified 299 to 372 miRNAs in two samples. Between the platforms, we observed high reproducibility and significant levels of shared detection. However, for commonly detected miRNAs, a strong correlation between platforms was not observed. Analysis of 10 additional HB samples with NanoString identified significantly overlapping miRNA expression profiles, and an alternative pattern was identified in a poorly differentiated HB with an aggressive phenotype. This investigation serves as a roadmap for future studies investigating miRNA expression in clinical FFPE samples, and as a guideline for the selection of an appropriate platform.

MicroRNA regulation in human CD8+ T cell subsets--cytokine exposure alone drives miR-146a expression.

BACKGROUND: microRNAs (miRNAs) are emerging as key regulators of the immune system, but their role in CD8+ T cell differentiation is not well explored. Some evidence suggests that signals from cell surface receptors influence the expression of miRNAs in CD8+ T cells, and may have consequent effects on cell phenotype and function. We set out to investigate whether common gamma chain cytokines modulated human CD8+ T cell expression of miR-146a, which previous studies have associated with different stages of CD8+ differentiation. We also investigated how changes in miR-146a related to other miRNAs that alter with CD8+ differentiation status. METHODS: We treated human CD8+ T cells with the cytokines IL-2, IL-7 or IL-15 either at rest or after stimulation with anti-CD3 and anti-CD28. For some experiments we also purified human CD8+ T cell subsets ex vivo. Flow cytometry was used in parallel to assess cell surface memory marker expression. Total RNA from these cells was subjected to microarray analysis and real-time PCR for miRNA expression. Nucleofection studies were performed to assess potential mRNA targets of miR-146a. RESULTS: We find that miR-146a is up-regulated in naïve CD8+ T cells exposed to IL-2 or IL-15, even in the absence of an activating T cell receptor stimulus, but not when IL-7 is also present. miR-146a expression correlates with a memory phenotype in both ex vivo and in vitro cultured cells although in our hands overexpression of miR-146a was not sufficient alone to drive a full memory phenotype. In ex vivo analysis, miR-146a was one of a small number of miRNAs that was differentially expressed between naïve and memory CD8+ T cells. CONCLUSIONS: miR-146a is emerging as a critical regulator of immune system. Our data shows that miR-146a expression is strongly influenced by the cytokine milieu even in the absence of a T cell receptor stimulus. Our results have implications for studies designed to assess the function of miR-146a, help to define a fingerprint of miRNA expression in CD8+ T cell subsets and may be useful when designing optimal protocols for T cell expansion as efficacy of T cell immunotherapy is correlated with an 'early' memory phenotype.

Species Delimitation--a Geneious plugin for the exploration of species boundaries.

Species Delimitation is a plugin to the Geneious software to support the exploration of species boundaries in a gene tree. The user assigns taxa to putative species and the plugin computes statistics relating to the probability of the observed monophyly or exclusivity having occurred by chance in a coalescent process. It also assesses the within and between species genetic distances to infer the probability with which members of a putative species might be identified successfully with tree-based methods.