Single-cell genomics details the maturation block in BCP-ALL and identifies therapeutic vulnerabilities in DUX4-r cases.

ABSTRACT: B-cell progenitor acute lymphoblastic leukemia (BCP-ALL) is the most common childhood malignancy and is driven by multiple genetic alterations that cause maturation arrest and accumulation of abnormal progenitor B cells. Current treatment protocols with chemotherapy have led to favorable outcomes but are associated with significant toxicity and risk of side effects, highlighting the necessity for highly effective, less toxic, targeted drugs, even in subtypes with a favorable outcome. Here, we used multimodal single-cell sequencing to delineate the transcriptional, epigenetic, and immunophenotypic characteristics of 23 childhood BCP-ALLs belonging to the BCR::ABL1+, ETV6::RUNX1+, high hyperdiploid, and recently discovered DUX4-rearranged (DUX4-r) subtypes. Projection of the ALL cells along the normal hematopoietic differentiation axis revealed a diversity in the maturation pattern between the different BCP-ALL subtypes. Although the BCR::ABL1+, ETV6::RUNX1+, and high hyperdiploidy cells mainly showed similarities to normal pro-B cells, DUX4-r ALL cells also displayed transcriptional signatures resembling mature B cells. Focusing on the DUX4-r subtype, we found that the blast population displayed not only multilineage priming toward nonhematopoietic cells, myeloid, and T-cell lineages, but also an activation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling that sensitized the cells to PI3K inhibition in vivo. Given the multilineage priming of DUX4-r blasts with aberrant expression of myeloid marker CD371 (CLL-1), we generated chimeric antigen receptor T cells, which effectively eliminated DUX4-r ALL cells in vivo. These results provide a detailed characterization of BCP-ALL at the single-cell level and reveal therapeutic vulnerabilities in the DUX4-r subtype, with implications for the understanding of ALL biology and new therapeutic strategies.

TLE3 Is a Novel Fusion Partner of JAK2 in Myeloid/Lymphoid Neoplasm With Eosinophilia Responding to JAK2 Inhibition.

Chromosomal rearrangements involving Janus kinase 2 (JAK2) are rare but recurrent findings in lymphoid or myeloid neoplasia. Detection of JAK2 fusion genes is important as patients with aberrantly activated JAK2 may benefit from treatment with tyrosine kinase inhibitors such as ruxolitinib. Here, we report a novel fusion gene between the transcriptional co-repressor-encoding gene transducin-like enhancer of split 3 (TLE3) and JAK2 in a patient initially diagnosed with chronic eosinophilic leukemia with additional mutations in PTPN11 and NRAS. The patient was successfully treated with the JAK2 inhibitor ruxolitinib for 8 months before additional somatic mutations were acquired and the disease progressed into an acute lymphoblastic T-cell leukemia/lymphoma. The present case shows similarities to previously reported cases with PCM1::JAK2 and BCR::JAK2 with regard to disease phenotype and response to ruxolitinib, and importantly, provides an example that also patients harboring other JAK2 fusion genes may benefit from treatment with JAK2 inhibitors.

Precision Diagnostics in Myeloid Malignancies: Development and Validation of a National Capture-Based Gene Panel.

Gene panel sequencing has become a common diagnostic tool for detecting somatically acquired mutations in myeloid neoplasms. However, many panels have restricted content, provide insufficient sensitivity levels, or lack clinically validated workflows. We here describe the development and validation of the Genomic Medicine Sweden myeloid gene panel (GMS-MGP), a capture-based 191 gene panel including mandatory genes in contemporary guidelines as well as emerging candidates. The GMS-MGP displayed uniform coverage across all targets, including recognized difficult GC-rich areas. The validation of 117 previously described somatic variants showed a 100% concordance with a limit-of-detection of a 0.5% variant allele frequency (VAF), achieved by utilizing error correction and filtering against a panel-of-normals. A national interlaboratory comparison investigating 56 somatic variants demonstrated highly concordant results in both detection rate and reported VAFs. In addition, prospective analysis of 323 patients analyzed with the GMS-MGP as part of standard-of-care identified clinically significant genes as well as recurrent mutations in less well-studied genes. In conclusion, the GMS-MGP workflow supports sensitive detection of all clinically relevant genes, facilitates novel findings, and is, based on the capture-based design, easy to update once new guidelines become available. The GMS-MGP provides an important step toward nationally harmonized precision diagnostics of myeloid malignancies.

Transcriptomics paving the way for improved diagnostics and precision medicine of acute leukemia.

Transcriptional profiling of acute leukemia, specifically by RNA-sequencing or whole transcriptome sequencing (WTS), has provided fundamental insights into its underlying disease biology and allows unbiased detection of oncogenic gene fusions, as well as of gene expression signatures that can be used for improved disease classification. While used as a research tool for many years, RNA-sequencing is becoming increasingly used in clinical diagnostics. Here, we highlight key transcriptomic studies of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) that have improved our biological understanding of these heterogeneous malignant disorders and have paved the way for translation into clinical diagnostics. Recent single-cell transcriptomic studies of ALL and AML, which provide new insights into the cellular ecosystem of acute leukemia and point to future clinical utility, are also reviewed. Finally, we discuss current challenges that need to be overcome for a more wide-spread adoption of RNA-sequencing in clinical diagnostics and how this technology significantly can aid the identification of genetic alterations in current guidelines and of newly emerging disease entities, some of which are critical to identify because of the availability of targeted therapies, thereby paving the way for improved precision medicine of acute leukemia.

Clinical and genomic characterization of patients diagnosed with the provisional entity acute myeloid leukemia with BCR-ABL1, a Swedish population-based study.

Acute myeloid leukemia (AML) with t(9;22)(q34;q11), also known as AML with BCR-ABL1, is a rare, provisional entity in the WHO 2016 classification and is considered a high-risk disease according to the European LeukemiaNet 2017 risk stratification. We here present a retrospective, population-based study of this disease entity from the Swedish Acute Leukemia Registry. By strict clinical inclusion criteria we aimed to identify genetic markers further distinguishing AML with t(9;22) as a separate entity. Twenty-five patients were identified and next-generation sequencing using a 54-gene panel was performed in 21 cases. Interestingly, no mutations were found in NPM1, FLT3, or DNMT3A, three frequently mutated genes in AML. Instead, RUNX1 was the most commonly mutated gene, with aberrations present in 38% of the cases compared to around 10% in de novo AML. Additional mutations were identified in genes involved in RNA splicing (SRSF2, SF3B1) and chromatin regulation (ASXL1, STAG2, BCOR, BCORL1). Less frequently, mutations were found in IDH2, NRAS, TET2, and TP53. The mutational landscape exhibited a similar pattern as recently described in patients with chronic myeloid leukemia (CML) in myeloid blast crisis (BC). Despite the concomitant presence of BCR-ABL1 and RUNX1 mutations in our cohort, both features of high-risk AML, the RUNX1-mutated cases showed a superior overall survival compared to RUNX1 wildtype cases. Our results suggest that the molecular characteristics of AML with t(9;22)/BCR-ABL1 and CML in myeloid BC are similar and do not support a distinction of the two disease entities based on their underlying molecular alterations.

A dynamic mutational landscape associated with an inter-regionally diverse immune response in malignant rhabdoid tumour.

Malignant rhabdoid tumour (MRT) is a childhood neoplasm of high malignancy characterised by biallelic mutation and/or loss of the epigenetic master regulator SMARCB1, accompanied by no or few other oncogenic drivers. In spite of their generally low mutational burden, an intratumoural T-cell response has been reported in a subset of MRTs, indicating that immune checkpoint inhibition may be considered a viable therapy option for some patients. We assess here the evolution over time and space of predicted neoantigens and indicators of immune checkpoint status in two MRT patients who progressed under treatment. Both patients showed an accumulation of novel clonal and subclonal mutations, including predicted neoantigens, in metastases compared to their inferred ancestral clones in the primary tumours. The first patient had peritoneal metastases from an MRT of the liver. Clonal deconvolution revealed polyclonal seeding from the primary tumour to a single metastatic site, followed by a local subclonal burst of mutations. The second patient had a renal MRT with multiple pulmonary metastases, each of which could be traced back to a single genetically unique founder cell, with formation of novel subclones in two metastases. Both patients showed a regionally heterogeneous landscape of predicted neoantigens and of tumour-infiltrating lymphocytes expressing CD8 and PD1. In both patients, some tumour regions fulfilled established criteria for PD-L1 positivity (> 1% of tumour cells), while others did not. This suggests that even in a tumour type like MRT, with a single driver mutation, there can be heterogeneity in neoantigen repertoire, immune response, and biomarkers for checkpoint blockade among sampled locations. This must be taken into account when assessing progressed MRT patients for checkpoint inhibition therapy. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Arrayed molecular barcoding identifies TNFSF13 as a positive regulator of acute myeloid leukemia-initiating cells.

Dysregulation of cytokines in the bone marrow (BM) microenvironment promotes acute myeloid leukemia (AML) cell growth. Due to the complexity and low throughput of in vivo stem-cell based assays, studying the role of cytokines in the BM niche in a screening setting is challenging. Here, we developed an ex vivo cytokine screen using 11 arrayed molecular barcodes, allowing for a competitive in vivo readout of leukemia-initiating capacity. With this approach, we assessed the effect of 114 murine cytokines on MLL-AF9 AML mouse cells and identified the tumor necrosis factor ligand superfamily member 13 (TNFSF13) as a positive regulator of leukemia-initiating cells. By using Tnfsf13-/- recipient mice, we confirmed that TNFSF13 supports leukemia initiation also under physiological conditions. TNFSF13 was secreted by normal myeloid cells but not by leukemia mouse cells, suggesting that mature myeloid BM cells support leukemia cells by secreting TNFSF13. TNFSF13 supported leukemia cell proliferation in an NF-κB-dependent manner by binding TNFRSF17 and suppressed apoptosis. Moreover, TNFSF13 supported the growth and survival of several human myeloid leukemia cell lines, demonstrating that our findings translate to human disease. Taken together, using arrayed molecular barcoding, we identified a previously unrecognized role of TNFSF13 as a positive regulator of AML-initiating cells. The arrayed barcoded screening methodology is not limited to cytokines and leukemia, but can be extended to other types of ex vivo screens, where a multiplexed in vivo read-out of stem cell functionality is needed.

Risk of childhood asthma is associated with CpG-site polymorphisms, regional DNA methylation and mRNA levels at the GSDMB/ORMDL3 locus.

Single-nucleotide polymorphisms (SNPs) in GSDMB (Gasdermin B) and ORMDL3 (ORMDL sphingolipid biosynthesis regulator 3) are strongly associated with childhood asthma, but the molecular alterations contributing to disease remain unknown. We investigated the effects of asthma-associated SNPs on DNA methylation and mRNA levels of GSDMB and ORMDL3. Genetic association between GSDMB/ORMDL3 and physician-diagnosed childhood asthma was confirmed in the Swedish birth-cohort BAMSE. CpG-site SNPs (rs7216389 and rs4065275) showed differences in DNA methylation depending on carrier status of the risk alleles, and were significantly associated with methylation levels in two CpG sites in the 5' UTR (untranslated region) of ORMDL3. In the Swedish Search study, we found significant differences in DNA methylation between asthmatics and controls in five CpG sites; after adjusting for lymphocyte and neutrophil cell counts, three remained significant: one in IKZF3 [IKAROS family zinc finger 3 (Aiolos); cg16293631] and two in the CpG island (CGI) of ORMDL3 (cg02305874 and cg16638648). Also, cg16293631 and cg02305874 correlated with mRNA levels of ORMDL3. The association between methylation and asthma was independent of the genotype in rs7216389, rs4065275 and rs12603332. Both SNPs and CpG sites showed significant associations with ORMDL3 mRNA levels. SNPs influenced expression independently of methylation, and the residual association between methylation and expression was not mediated by these SNPs. We found a differentially methylated region in the CGI shore of ORMDL3 with six CpG sites less methylated in CD8(+) T-cells. In summary, this study supports that there are differences in DNA methylation at this locus between asthmatics and controls; and both SNPs and CpG sites are independently associated with ORMDL3 expression.

Transcriptome analysis of controlled and therapy-resistant childhood asthma reveals distinct gene expression profiles.

BACKGROUND: Children with problematic severe asthma have poor disease control despite high doses of inhaled corticosteroids and additional therapy, leading to personal suffering, early deterioration of lung function, and significant consumption of health care resources. If no exacerbating factors, such as smoking or allergies, are found after extensive investigation, these children are given a diagnosis of therapy-resistant (or therapy-refractory) asthma (SA). OBJECTIVE: We sought to deepen our understanding of childhood SA by analyzing gene expression and modeling the underlying regulatory transcription factor networks in peripheral blood leukocytes. METHODS: Gene expression was analyzed by using Cap Analysis of Gene Expression in children with SA (n = 13), children with controlled persistent asthma (n = 15), and age-matched healthy control subjects (n = 9). Cap Analysis of Gene Expression sequencing detects the transcription start sites of known and novel mRNAs and noncoding RNAs. RESULTS: Sample groups could be separated by hierarchical clustering on 1305 differentially expressed transcription start sites, including 816 known genes and several novel transcripts. Ten of 13 tested novel transcripts were validated by means of RT-PCR and Sanger sequencing. Expression of RAR-related orphan receptor A (RORA), which has been linked to asthma in genome-wide association studies, was significantly upregulated in patients with SA. Gene network modeling revealed decreased glucocorticoid receptor signaling and increased activity of the mitogen-activated protein kinase and Jun kinase cascades in patients with SA. CONCLUSION: Circulating leukocytes from children with controlled asthma and those with SA have distinct gene expression profiles, demonstrating the possible development of specific molecular biomarkers and supporting the need for novel therapeutic approaches.

Micro-costing of genetic diagnostics in acute leukemia in Sweden: from standard-of-care to whole-genome sequencing.

AIMS AND BACKGROUND: Whole-genome sequencing (WGS) is increasingly applied in clinical practice and expected to replace standard-of-care (SoC) genetic diagnostics in hematological malignancies. This study aims to assess and compare the fully burdened cost ('micro-costing') per patient for Swedish laboratories using WGS and SoC, respectively, in pediatric and adult patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). METHODS: The resource use and cost details associated with SoC, e.g. chromosome banding analysis, fluorescent in situ hybridization, and targeted sequencing analysis, were collected via activity-based costing methods from four diagnostic laboratories. For WGS, corresponding data was collected from two of the centers. A simulation-based scenario model was developed for analyzing the WGS cost based on different annual sample throughput to evaluate economy of scale. RESULTS: The average SoC total cost per patient was €2,465 for pediatric AML and €2,201 for pediatric ALL, while in adults, the corresponding cost was €2,458 for AML and €1,207 for ALL. The average WGS cost (90x tumor/30x normal; sequenced on the Illumina NovaSeq 6000 platform) was estimated to €3,472 based on an annual throughput of 2,500 analyses, however, with an annual volume of 7,500 analyses the average cost would decrease by 23% to €2,671. CONCLUSION: In summary, WGS is currently more costly than SoC, however the cost can be reduced by utilizing laboratories with higher throughput and by the expected decline in cost of reagents. Our data provides guidance to decision-makers for the resource allocation needed when implementing WGS in diagnostics of hematological malignancies.

Transcriptome analysis reveals upregulation of bitter taste receptors in severe asthmatics.

The causes of severe childhood asthma are poorly understood. Our aim was to define global patterns of gene expression in children with severe therapy-resistant and controlled asthma. White blood cells were isolated and the global transcriptome profile was characterised using the Affymetrix Human Gene ST 1.0 chip in children with severe, therapy-resistant asthma (n=17), controlled asthma (n=19) and healthy controls (n=18). Receptor expression was studied in separated leukocyte fractions from asthmatic adults (n=12). Overall, 1378 genes were differentially expressed between children with severe/controlled asthma and controls. Three significantly enriched Kyoto Encyclopedia of Genes and Genomes pathways were represented: natural killer cell-mediated cytotoxicity (upregulated in controlled asthma); N-glycan biosynthesis (downregulated in severe asthma); and bitter taste transduction receptors (TAS2Rs) (upregulated in severe asthma). Quantitative PCR experiments confirmed upregulation of TAS2Rs in severe asthmatics. TAS2R expression was replicated in leukocytes from adult asthmatics, in which TAS2R agonists also inhibited LPS-induced cytokine release. Significant correlations between expression of TAS2Rs and clinical markers of asthma severity were found in both adults and children. In conclusion, specific gene expression patterns were observed in children with severe, therapy-resistant asthma. The increased expression of bronchodilatory TAS2Rs suggests a new target for the treatment of asthma.

Feasibility to use whole-genome sequencing as a sole diagnostic method to detect genomic aberrations in pediatric B-cell acute lymphoblastic leukemia.

Introduction: The suitability of whole-genome sequencing (WGS) as the sole method to detect clinically relevant genomic aberrations in B-cell acute lymphoblastic leukemia (ALL) was investigated with the aim of replacing current diagnostic methods. Methods: For this purpose, we assessed the analytical performance of 150 bp paired-end WGS (90x leukemia/30x germline). A set of 88 retrospective B-cell ALL samples were selected to represent established ALL subgroups as well as ALL lacking stratifying markers by standard-of-care (SoC), so-called B-other ALL. Results: Both the analysis of paired leukemia/germline (L/N)(n=64) as well as leukemia-only (L-only)(n=88) detected all types of aberrations mandatory in the current ALLTogether trial protocol, i.e., aneuploidies, structural variants, and focal copy-number aberrations. Moreover, comparison to SoC revealed 100% concordance and that all patients had been assigned to the correct genetic subgroup using both approaches. Notably, WGS could allocate 35 out of 39 B-other ALL samples to one of the emerging genetic subgroups considered in the most recent classifications of ALL. We further investigated the impact of high (90x; n=58) vs low (30x; n=30) coverage on the diagnostic yield and observed an equally perfect concordance with SoC; low coverage detected all relevant lesions. Discussion: The filtration of the WGS findings with a short list of genes recurrently rearranged in ALL was instrumental to extract the clinically relevant information efficiently. Nonetheless, the detection of DUX4 rearrangements required an additional customized analysis, due to multiple copies of this gene embedded in the highly repetitive D4Z4 region. We conclude that the diagnostic performance of WGS as the standalone method was remarkable and allowed detection of all clinically relevant genomic events in the diagnostic setting of B-cell ALL.

Diagnostic Yield From a Nationwide Implementation of Precision Medicine for all Children With Cancer.

PURPOSE: Several studies have indicated that broad genomic characterization of childhood cancer provides diagnostically and/or therapeutically relevant information in selected high-risk cases. However, the extent to which such characterization offers clinically actionable data in a prospective broadly inclusive setting remains largely unexplored. METHODS: We implemented prospective whole-genome sequencing (WGS) of tumor and germline, complemented by whole-transcriptome sequencing (RNA-Seq) for all children diagnosed with a primary or relapsed solid malignancy in Sweden. Multidisciplinary molecular tumor boards were set up to integrate genomic data in the clinical decision process along with a medicolegal framework enabling secondary use of sequencing data for research purposes. RESULTS: During the study's first 14 months, 118 solid tumors from 117 patients were subjected to WGS, with complementary RNA-Seq for fusion gene detection in 52 tumors. There was no significant geographic bias in patient enrollment, and the included tumor types reflected the annual national incidence of pediatric solid tumor types. Of the 112 tumors with somatic mutations, 106 (95%) exhibited alterations with a clear clinical correlation. In 46 of 118 tumors (39%), sequencing only corroborated histopathological diagnoses, while in 59 cases (50%), it contributed to additional subclassification or detection of prognostic markers. Potential treatment targets were found in 31 patients (26%), most commonly ALK mutations/fusions (n = 4), RAS/RAF/MEK/ERK pathway mutations (n = 14), FGFR1 mutations/fusions (n = 5), IDH1 mutations (n = 2), and NTRK2 gene fusions (n = 2). In one patient, the tumor diagnosis was revised based on sequencing. Clinically relevant germline variants were detected in 8 of 94 patients (8.5%). CONCLUSION: Up-front, large-scale genomic characterization of pediatric solid malignancies provides diagnostically valuable data in the majority of patients also in a largely unselected cohort.

The complement receptor C3AR constitutes a novel therapeutic target in NPM1-mutated AML.

Mutated nucleophosmin 1 (NPM1) is the most common genetic alteration in acute myeloid leukemia (AML), found in ∼30% of cases. Although mutations in this gene are considered favorable according to current risk stratification guidelines, a large fraction of patients will experience relapse, demonstrating the urgent need for new treatment options. Therefore, we aimed to identify cell surface proteins specifically expressed on NPM1-mutated AML cells, allowing for potential targeting with antibody-based therapies. Herein, we report on an arrayed flow cytometry-based screen directed to 362 cell surface markers. In comparing the cell surface expression on NPM1-mutated AML cells with primitive (CD34+ CD38-) normal bone marrow cells, we identified the complement receptor C3AR as being specifically expressed in NPM1-mutated AML. By flow cytometry and single-cell RNA sequencing, we further show that normal hematopoietic stem and progenitor cells lack detectable C3AR gene and protein expression, making it particularly suitable as a target for antibody therapy. We also demonstrate that C3AR in combination with GPR56 distinguishes the leukemic stem cells (LSCs) in NPM1-mutated AML from the normal hematopoietic stem cells, defining the LSC population, as shown by transplantation into immunodeficient mice. Mechanistically, the stimulation of C3AR-expressing cells with C3a, the ligand of C3AR, leads to the activation of ERK1/2 and increased survival of AML cells, suggesting that this is an important signaling axis in this subtype of AML. Finally, we show that antibodies directed against C3AR efficiently elicit natural killer cell-mediated killing of primary AML cells ex vivo, highlighting C3AR as a candidate therapeutic target in NPM1-mutated AML.

A Study Protocol for Validation and Implementation of Whole-Genome and -Transcriptome Sequencing as a Comprehensive Precision Diagnostic Test in Acute Leukemias.

Background: Whole-genome sequencing (WGS) and whole-transcriptome sequencing (WTS), with the ability to provide comprehensive genomic information, have become the focal point of research interest as novel techniques that can support precision diagnostics in routine clinical care of patients with various cancer types, including hematological malignancies. This national multi-center study, led by Genomic Medicine Sweden, aims to evaluate whether combined application of WGS and WTS (WGTS) is technically feasible and can be implemented as an efficient diagnostic tool in patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). In addition to clinical impact assessment, a health-economic evaluation of such strategy will be performed. Methods and Analysis: The study comprises four phases (i.e., retrospective, prospective, real-time validation, and follow-up) including approximately 700 adult and pediatric Swedish AML and ALL patients. Results of WGS for tumor (90×) and normal/germline (30×) samples as well as WTS for tumors only will be compared to current standard of care diagnostics. Primary study endpoints are diagnostic efficiency and improved diagnostic yield. Secondary endpoints are technical and clinical feasibility for routine implementation, clinical utility, and health-economic impact. Discussion: Data from this national multi-center study will be used to evaluate clinical performance of the integrated WGTS diagnostic workflow compared with standard of care. The study will also elucidate clinical and health-economic impacts of a combined WGTS strategy when implemented in routine clinical care. Clinical Trial Registration: [https://doi.org/10.1186/ISRCTN66987142], identifier [ISRCTN66987142].

The asthma candidate gene NPSR1 mediates isoform specific downstream signalling.

BACKGROUND: Neuropeptide S Receptor 1 (NPSR1, GPRA, GPR154) was first identified as an asthma candidate gene through positional cloning and has since been replicated as an asthma and allergy susceptibility gene in several independent association studies. In humans, NPSR1 encodes two G protein-coupled receptor variants, NPSR1-A and NPSR1-B, with unique intracellular C-termini. Both isoforms show distinct expression pattern in asthmatic airways. Although NPSR1-A has been extensively studied, functional differences and properties of NPSR1-B have not yet been clearly examined. Our objective was to investigate downstream signalling properties of NPSR1-B and functional differences between NPSR1-A and NPSR1-B. METHODS: HEK-293 cells transiently overexpressing NPSR1-A or NPSR1-B were stimulated with the ligand neuropeptide S (NPS) and downstream signalling effects were monitored by genome-scale affymetrix expression-arrays. The results were verified by NPS concentration-response and time series analysis using qRT-PCR, cAMP and Ca²âº assays, and cAMP/PKA, MAPK/JNK and MAPK/ERK pathway specific reporter assays. RESULTS: NPSR1-B signalled through the same pathways and regulated the same genes as NPSR1-A, but NPSR1-B yielded lower induction on effector genes than NPSR1-A, with one notable exception, CD69, a marker of regulatory T cells. CONCLUSIONS: We conclude that NPSR1-B is regulating essentially identical set of genes as NPSR1-A, with few, but possibly important exceptions, and that NPSR1-A induces stronger signalling effects than NPSR1-B. Our findings suggest an isoform-specific link to pathogenetic processes in asthma and allergy.

Biological and genetic interaction between tenascin C and neuropeptide S receptor 1 in allergic diseases.

Neuropeptide S receptor 1 (NPSR1, GPRA 154, GPRA) has been verified as a susceptibility gene for asthma and related phenotypes. The ligand for NPSR1, Neuropeptide S (NPS), activates signalling through NPSR1 and microarray analysis has identified Tenascin C (TNC) as a target gene of NPS-NPSR1 signalling. TNC has previously been implicated as a risk gene for asthma. We aimed therefore to study the genetic association of TNC in asthma- and allergy-related disorders as well as the biological and genetic interactions between NPSR1 and TNC. Regulation of TNC was investigated using NPS stimulated NPSR1 transfected cells. We genotyped 12 TNC SNPs in the cross-sectional PARSIFAL study (3113 children) and performed single SNP association, haplotype association and TNC and NPSR1 gene-gene interaction analyses. Our experimental results show NPS-dependent upregulation of TNC-mRNA. The genotyping results indicate single SNP and haplotype associations for several SNPs in TNC with the most significant association to rhinoconjunctivitis for a haplotype, with a frequency of 29% in cases (P = 0.0005). In asthma and atopic sensitization significant gene-gene interactions were found between TNC and NPSR1 SNPs, indicating that depending on the NPSR1 genotype, TNC can be associated with either an increased or a decreased risk of disease. We conclude that variations in TNC modifies, not only risk for asthma, but also for rhinoconjunctivitis. Furthermore, we show epistasis based on both a direct suggested regulatory effect and a genetic interaction between NPSR1 and TNC. These results suggest merging of previously independent pathways of importance in the development of asthma- and allergy-related traits.

Clonal competition within complex evolutionary hierarchies shapes AML over time.

Clonal heterogeneity and evolution has major implications for disease progression and relapse in acute myeloid leukemia (AML). To model clonal dynamics in vivo, we serially transplanted 23 AML cases to immunodeficient mice and followed clonal composition for up to 15 months by whole-exome sequencing of 84 xenografts across two generations. We demonstrate vast changes in clonality that both progress and reverse over time, and define five patterns of clonal dynamics: Monoclonal, Stable, Loss, Expansion and Burst. We also show that subclonal expansion in vivo correlates with a more adverse prognosis. Furthermore, clonal expansion enabled detection of very rare clones with AML driver mutations that were undetectable by sequencing at diagnosis, demonstrating that the vast majority of AML cases harbor multiple clones already at diagnosis. Finally, the rise and fall of related clones enabled deconstruction of the complex evolutionary hierarchies of the clones that compete to shape AML over time.

Microenvironmental control of breast cancer subtype elicited through paracrine platelet-derived growth factor-CC signaling.

Breast tumors of the basal-like, hormone receptor-negative subtype remain an unmet clinical challenge, as there is high rate of recurrence and poor survival in patients following treatment. Coevolution of the malignant mammary epithelium and its underlying stroma instigates cancer-associated fibroblasts (CAFs) to support most, if not all, hallmarks of cancer progression. Here we delineate a previously unappreciated role for CAFs as determinants of the molecular subtype of breast cancer. We identified paracrine crosstalk between cancer cells expressing platelet-derived growth factor (PDGF)-CC and CAFs expressing the cognate receptors in human basal-like mammary carcinomas. Genetic or pharmacological intervention of PDGF-CC activity in mouse models of cancer resulted in conversion of basal-like breast cancers into a hormone receptor-positive state that enhanced sensitivity to endocrine therapy in previously resistant tumors. We conclude that specification of breast cancer to the basal-like subtype is under microenvironmental control and is therapeutically actionable.