Publications by authors named "Lucy F Stead"

24 Publications

  • Page 1 of 1

Patient-derived organoids and orthotopic xenografts of primary and recurrent gliomas represent relevant patient avatars for precision oncology.

Acta Neuropathol 2020 12 3;140(6):919-949. Epub 2020 Oct 3.

NORLUX Neuro-Oncology Laboratory, Department of Oncology, Luxembourg Institute of Health, 84, Val Fleuri, 1526, Luxembourg, Luxembourg.

Patient-based cancer models are essential tools for studying tumor biology and for the assessment of drug responses in a translational context. We report the establishment a large cohort of unique organoids and patient-derived orthotopic xenografts (PDOX) of various glioma subtypes, including gliomas with mutations in IDH1, and paired longitudinal PDOX from primary and recurrent tumors of the same patient. We show that glioma PDOXs enable long-term propagation of patient tumors and represent clinically relevant patient avatars that retain histopathological, genetic, epigenetic, and transcriptomic features of parental tumors. We find no evidence of mouse-specific clonal evolution in glioma PDOXs. Our cohort captures individual molecular genotypes for precision medicine including mutations in IDH1, ATRX, TP53, MDM2/4, amplification of EGFR, PDGFRA, MET, CDK4/6, MDM2/4, and deletion of CDKN2A/B, PTCH, and PTEN. Matched longitudinal PDOX recapitulate the limited genetic evolution of gliomas observed in patients following treatment. At the histological level, we observe increased vascularization in the rat host as compared to mice. PDOX-derived standardized glioma organoids are amenable to high-throughput drug screens that can be validated in mice. We show clinically relevant responses to temozolomide (TMZ) and to targeted treatments, such as EGFR and CDK4/6 inhibitors in (epi)genetically defined subgroups, according to MGMT promoter and EGFR/CDK status, respectively. Dianhydrogalactitol (VAL-083), a promising bifunctional alkylating agent in the current clinical trial, displayed high therapeutic efficacy, and was able to overcome TMZ resistance in glioblastoma. Our work underscores the clinical relevance of glioma organoids and PDOX models for translational research and personalized treatment studies and represents a unique publicly available resource for precision oncology.
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http://dx.doi.org/10.1007/s00401-020-02226-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7666297PMC
December 2020

Identification of candidate mediators of chemoresponse in breast cancer through therapy-driven selection of somatic variants.

Breast Cancer Res Treat 2020 Oct 30;183(3):607-616. Epub 2020 Jul 30.

School of Medicine, University of Leeds, Leeds, UK.

Purpose: More than a third of primary breast cancer patients are treated with cytotoxic chemotherapy, typically without guidance from predictive markers. Increased use of neoadjuvant chemotherapy provides opportunities for identification of molecules associated with treatment response, by comparing matched tumour samples before and after therapy. Our hypothesis was that somatic variants of increased prevalence after therapy promote resistance, while variants with reduced prevalence cause sensitivity.

Methods: We performed systematic analyses of matched pairs of cancer exomes from primary oestrogen receptor-positive/HER2-negative breast cancers (n = 6) treated with neoadjuvant epirubicin/cyclophosphamide. We identified candidate genes as mediators of chemotherapy response by consistent subclonal changes in somatic variant prevalence through therapy, predicted variant impact on gene function, and enrichment of specific functional pathways. Influence of candidate genes on breast cancer outcome was tested using publicly available breast cancer expression data (n = 1903).

Results: We identified 14 genes as the strongest candidate mediators of chemoresponse: TCHH, MUC17, ARAP2, FLG2, ABL1, CENPF, COL6A3, DMBT1, ITGA7, PLXNA1, S100PBP, SYNE1, ZFHX4, and CACNA1C. Genes contained somatic variants showing prevalence changes in up to 4 patients, with up to 3 being predicted as damaging. Genes coding for extra-cellular matrix components or related signalling pathways were significantly over-represented among variants showing prevalence changes. Expression of 5 genes (TCHH, ABL1, CENPF, S100PBP, and ZFHX4) was significantly associated with patient survival.

Conclusions: Genomic analysis of paired pre- and post-therapy samples resulting from neoadjuvant therapy provides a powerful method for identification of mediators of response. Genes we identified should be assessed as predictive markers or targets in chemo-sensitization.
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http://dx.doi.org/10.1007/s10549-020-05836-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497675PMC
October 2020

EGFRvIII upregulates DNA mismatch repair resulting in increased temozolomide sensitivity of MGMT promoter methylated glioblastoma.

Oncogene 2020 04 17;39(15):3041-3055. Epub 2020 Feb 17.

Laboratory of Radiobiology & Experimental Radiation Oncology, Hubertus Wald Tumorzentrum - University Cancer Center Hamburg, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

The oncogene epidermal growth factor receptor variant III (EGFRvIII) is frequently expressed in glioblastomas (GBM) but its impact on therapy response is still under controversial debate. Here we wanted to test if EGFRvIII influences the sensitivity towards the alkylating agent temozolomide (TMZ). Therefore, we retrospectively analyzed the survival of 336 GBM patients, demonstrating that under standard treatment, which includes TMZ, EGFRvIII expression is associated with prolonged survival, but only in patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated tumors. Using isogenic GBM cell lines with endogenous EGFRvIII expression we could demonstrate that EGFRvIII increases TMZ sensitivity and results in enhanced numbers of DNA double-strand breaks and a pronounced S/G2-phase arrest after TMZ treatment. We observed a higher expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cells and patient tumor samples, which was most pronounced for MSH2 and MSH6. EGFRvIII-specific knockdown reduced MMR protein expression thereby increasing TMZ resistance. Subsequent functional kinome profiling revealed an increased activation of p38- and ERK1/2-dependent signaling in EGFRvIII expressing cells, which regulates MMR protein expression downstream of EGFRvIII. In summary, our results demonstrate that the oncoprotein EGFRvIII sensitizes a fraction of GBM to current standard of care treatment through the upregulation of DNA MMR.
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http://dx.doi.org/10.1038/s41388-020-1208-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7142016PMC
April 2020

Genomic and Expression Analyses Define MUC17 and PCNX1 as Predictors of Chemotherapy Response in Breast Cancer.

Mol Cancer Ther 2020 03 26;19(3):945-955. Epub 2019 Dec 26.

School of Medicine, University of Leeds, Leeds, United Kingdom.

Poor-prognosis breast cancers are treated with cytotoxic chemotherapy, but often without any guidance from therapy predictive markers because universally accepted markers are not currently available. Treatment failure, in the form of recurrences, is relatively common. We aimed to identify chemotherapy predictive markers and resistance pathways in breast cancer. Our hypothesis was that tumor cells remaining after neoadjuvant chemotherapy (NAC) contain somatic variants causing therapy resistance, while variants present pre-NAC but lost post-NAC cause sensitivity. Whole-exome sequencing was performed on matched pre- and post-NAC cancer cells, which were isolated by laser microdissection, from 6 cancer cases, and somatic variants selected for or against by NAC were identified. Somatic variant diversity was significantly reduced after therapy ( < 0.05). MUC17 variants were identified in 3 tumors and were selected against by NAC in each case, while PCNX1 variants were identified in 2 tumors and were selected for in both cases, implicating the function of these genes in defining chemoresponse. knockdown of MUC17 or PCNX1 was associated with significantly increased or decreased chemotherapy sensitivity, respectively ( < 0.05), further supporting their roles in chemotherapy response. Expression was tested for predictive value in two independent cohorts of chemotherapy-treated breast cancers ( = 53, = 303). Kaplan-Meier analyses revealed that low MUC17 expression was significantly associated with longer survival after chemotherapy, whereas low PCNX1 was significantly associated with reduced survival. We concluded that therapy-driven selection of somatic variants allows identification of chemotherapy response genes. With respect to MUC17 and PCNX1, therapy-driven selection acting on somatic variants, knockdown data concerning drug sensitivity, and survival analysis of expression levels in patient cohorts all define the genes as mediators of and predictive markers for chemotherapy response in breast cancer.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0940DOI Listing
March 2020

Longitudinal molecular trajectories of diffuse glioma in adults.

Nature 2019 12 20;576(7785):112-120. Epub 2019 Nov 20.

The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA.

The evolutionary processes that drive universal therapeutic resistance in adult patients with diffuse glioma remain unclear. Here we analysed temporally separated DNA-sequencing data and matched clinical annotation from 222 adult patients with glioma. By analysing mutations and copy numbers across the three major subtypes of diffuse glioma, we found that driver genes detected at the initial stage of disease were retained at recurrence, whereas there was little evidence of recurrence-specific gene alterations. Treatment with alkylating agents resulted in a hypermutator phenotype at different rates across the glioma subtypes, and hypermutation was not associated with differences in overall survival. Acquired aneuploidy was frequently detected in recurrent gliomas and was characterized by IDH mutation but without co-deletion of chromosome arms 1p/19q, and further converged with acquired alterations in the cell cycle and poor outcomes. The clonal architecture of each tumour remained similar over time, but the presence of subclonal selection was associated with decreased survival. Finally, there were no differences in the levels of immunoediting between initial and recurrent gliomas. Collectively, our results suggest that the strongest selective pressures occur during early glioma development and that current therapies shape this evolution in a largely stochastic manner.
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http://dx.doi.org/10.1038/s41586-019-1775-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6897368PMC
December 2019

Chemically induced neurite-like outgrowth reveals a multicellular network function in patient-derived glioblastoma cells.

J Cell Sci 2019 10 10;132(19). Epub 2019 Oct 10.

School of Medicine, University of Leeds, Leeds, LS2 9JT, UK

Tumor stem cells and malignant multicellular networks have been separately implicated in the therapeutic resistance of glioblastoma multiforme (GBM), the most aggressive type of brain cancer in adults. Here, we show that small-molecule inhibition of RHO-associated serine/threonine kinase proteins (ROCKi) significantly promoted the outgrowth of neurite-like cell projections in cultures of heterogeneous patient-derived GBM stem-like cells. These projections formed -induced cellular network (iNet) 'webs', which regressed after withdrawal of ROCKi. Connected cells within the iNet web exhibited long range Ca signal transmission, and significant lysosomal and mitochondrial trafficking. In contrast to their less-connected vehicle control counterparts, iNet cells remained viable and proliferative after high-dose radiation. These findings demonstrate a link between ROCKi-regulated cell projection dynamics and the formation of radiation-resistant multicellular networks. Our study identifies means to reversibly induce iNet webs , and may thereby accelerate future studies into the biology of GBM cellular networks.
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http://dx.doi.org/10.1242/jcs.228452DOI Listing
October 2019

Doomed from the TERT? A Two-Stage Model of Tumorigenesis in IDH-Wild-Type Glioblastoma.

Cancer Cell 2019 04;35(4):542-544

The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032, USA. Electronic address:

Using longitudinal molecular profiling, Körber et al. propose in this issue of Cancer Cell that IDH-wild-type glioblastomas initiate years pre-diagnosis with chromosome-level alterations that drive cell proliferation but require survival-promoting mutations, commonly in the TERT promoter, to form a detectable tumor. Multiple subclones drive disease progression, creating a therapeutic challenge.
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http://dx.doi.org/10.1016/j.ccell.2019.03.009DOI Listing
April 2019

Simulation of heterogeneous tumour genomes with HeteroGenesis and in silico whole exome sequencing.

Bioinformatics 2019 08;35(16):2850-2852

Leeds Institute of Medical Research at St James's, St James's University Hospital, Leeds, UK.

Summary: Tumour evolution results in progressive cancer phenotypes such as metastatic spread and treatment resistance. To better treat cancers, we must characterize tumour evolution and the genetic events that confer progressive phenotypes. This is facilitated by high coverage genome or exome sequencing. However, the best approach by which, or indeed whether, these data can be used to accurately model and interpret underlying evolutionary dynamics is yet to be confirmed. Establishing this requires sequencing data from appropriately heterogeneous tumours in which the exact trajectory and combination of events occurring throughout its evolution are known. We therefore developed HeteroGenesis: a tool to generate realistically evolved tumour genomes, which can be sequenced using weighted-Wessim (w-Wessim), an in silico exome sequencing tool that we have adapted from previous methods. HeteroGenesis simulates more complex and realistic heterogeneous tumour genomes than existing methods, can model different evolutionary dynamics, and enables the creation of multi-region and longitudinal data.

Availability And Implementation: HeteroGenesis and w-Wessim are freely available under the GNU General Public Licence from https://github.com/GeorgetteTanner, implemented in Python and supported on linux and MS Windows.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty1063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6691334PMC
August 2019

Intravenous delivery of oncolytic reovirus to brain tumor patients immunologically primes for subsequent checkpoint blockade.

Sci Transl Med 2018 01;10(422)

Institute of Cancer Research, 123 Old Brompton Road, London SW7 3RP, UK.

Immune checkpoint inhibitors, including those targeting programmed cell death protein 1 (PD-1), are reshaping cancer therapeutic strategies. Evidence suggests, however, that tumor response and patient survival are determined by tumor programmed death ligand 1 (PD-L1) expression. We hypothesized that preconditioning of the tumor immune microenvironment using targeted, virus-mediated interferon (IFN) stimulation would up-regulate tumor PD-L1 protein expression and increase cytotoxic T cell infiltration, improving the efficacy of subsequent checkpoint blockade. Oncolytic viruses (OVs) represent a promising form of cancer immunotherapy. For brain tumors, almost all studies to date have used direct intralesional injection of OV, because of the largely untested belief that intravenous administration will not deliver virus to this site. We show, in a window-of-opportunity clinical study, that intravenous infusion of oncolytic human (referred to herein as reovirus) leads to infection of tumor cells subsequently resected as part of standard clinical care, both in high-grade glioma and in brain metastases, and increases cytotoxic T cell tumor infiltration relative to patients not treated with virus. We further show that reovirus up-regulates IFN-regulated gene expression, as well as the PD-1/PD-L1 axis in tumors, via an IFN-mediated mechanism. Finally, we show that addition of PD-1 blockade to reovirus enhances systemic therapy in a preclinical glioma model. These results support the development of combined systemic immunovirotherapy strategies for the treatment of both primary and secondary tumors in the brain.
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http://dx.doi.org/10.1126/scitranslmed.aam7577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6276984PMC
January 2018

How to analyse the spatiotemporal tumour samples needed to investigate cancer evolution: A case study using paired primary and recurrent glioblastoma.

Int J Cancer 2018 04 14;142(8):1620-1626. Epub 2017 Dec 14.

Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, LS9 7TF, United Kingdom.

Many traits of cancer progression (e.g., development of metastases or resistance to therapy) are facilitated by tumour evolution: Darwinian selection of subclones with distinct genotypes or phenotypes that enable such progression. Characterising these subclones provide an opportunity to develop drugs to better target their specific properties but requires the accurate identification of somatic mutations shared across multiple spatiotemporal tumours from the same patient. Current best practices for calling somatic mutations are optimised for single samples, and risk being too conservative to identify shared mutations with low prevalence in some samples. We reasoned that datasets from multiple matched tumours can be used for mutual validation and thus propose an adapted two-stage approach: (1) low-stringency mutation calling to identify mutations shared across samples irrespective of the weight of evidence in a single sample; (2) high-stringency mutation calling to further characterise mutations present in a single sample. We applied our approach to three-independent cohorts of paired primary and recurrent glioblastoma tumours, two of which have previously been analysed using existing approaches, and found that it significantly increased the amount of biologically relevant shared somatic mutations identified. We also found that duplicate removal was detrimental when identifying shared somatic mutations. Our approach is also applicable when multiple datasets e.g. DNA and RNA are available for the same tumour.
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http://dx.doi.org/10.1002/ijc.31184DOI Listing
April 2018

Genomic Subtypes of Non-invasive Bladder Cancer with Distinct Metabolic Profile and Female Gender Bias in KDM6A Mutation Frequency.

Cancer Cell 2017 11;32(5):701-715.e7

Section of Molecular Oncology, Leeds Institute of Cancer and Pathology, St James's University Hospital, Beckett Street, Leeds LS9 7TF, UK. Electronic address:

Bladder cancer incurs a higher lifetime treatment cost than other cancers due to frequent recurrence of non-invasive disease. Improved prognostic biomarkers and localized therapy are needed for this large patient group. We defined two major genomic subtypes of primary stage Ta tumors. One of these was characterized by loss of 9q including TSC1, increased KI67 labeling index, upregulated glycolysis, DNA repair, mTORC1 signaling, features of the unfolded protein response, and altered cholesterol homeostasis. Comparison with muscle-invasive bladder cancer mutation profiles revealed lower overall mutation rates and more frequent mutations in RHOB and chromatin modifier genes. More mutations in the histone lysine demethylase KDM6A were present in non-invasive tumors from females than males.
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http://dx.doi.org/10.1016/j.ccell.2017.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5774674PMC
November 2017

Upregulated WEE1 protects endothelial cells of colorectal cancer liver metastases.

Oncotarget 2017 Jun;8(26):42288-42299

School of Medicine, University of Leeds, Leeds LS2 9JT, UK.

Surgical resection of colorectal cancer liver metastases (CLM) can be curative, yet 80% of patients are unsuitable for this treatment. As angiogenesis is a determinant of CLM progression we isolated endothelial cells from CLM and sought a mechanism which is upregulated, essential for angiogenic properties of these cells and relevant to emerging therapeutic options. Matched CLM endothelial cells (CLMECs) and endothelial cells of normal adjacent liver (LiECs) were superficially similar but transcriptome sequencing revealed molecular differences, one of which was unexpected upregulation and functional significance of the checkpoint kinase WEE1. Western blotting confirmed that WEE1 protein was upregulated in CLMECs. Knockdown of WEE1 by targeted short interfering RNA or the WEE1 inhibitor AZD1775 suppressed proliferation and migration of CLMECs. Investigation of the underlying mechanism suggested induction of double-stranded DNA breaks due to nucleotide shortage which then led to caspase 3-dependent apoptosis. The implication for CLMEC tube formation was striking with AZD1775 inhibiting tube branch points by 83%. WEE1 inhibitors might therefore be a therapeutic option for CLM and could be considered more broadly as anti-angiogenic agents in cancer treatment.
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http://dx.doi.org/10.18632/oncotarget.15039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522067PMC
June 2017

RAD51 Is a Selective DNA Repair Target to Radiosensitize Glioma Stem Cells.

Stem Cell Reports 2017 01;8(1):125-139

Leeds Institute of Cancer and Pathology, University of Leeds, St James's University Hospital, Beckett Street, Leeds LS9 7TF, UK. Electronic address:

Patients with glioblastoma die from local relapse despite surgery and high-dose radiotherapy. Resistance to radiotherapy is thought to be due to efficient DNA double-strand break (DSB) repair in stem-like cells able to survive DNA damage and repopulate the tumor. We used clinical samples and patient-derived glioblastoma stem cells (GSCs) to confirm that the DSB repair protein RAD51 is highly expressed in GSCs, which are reliant on RAD51-dependent DSB repair after radiation. RAD51 expression and RAD51 foci numbers fall when these cells move toward astrocytic differentiation. In GSCs, the small-molecule RAD51 inhibitors RI-1 and B02 prevent RAD51 focus formation, reduce DNA DSB repair, and cause significant radiosensitization. We further demonstrate that treatment with these agents combined with radiation promotes loss of stem cells defined by SOX2 expression. This indicates that RAD51-dependent repair represents an effective and specific target in GSCs.
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http://dx.doi.org/10.1016/j.stemcr.2016.12.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5233453PMC
January 2017

A Case-Matched Gender Comparison Transcriptomic Screen Identifies eIF4E and eIF5 as Potential Prognostic Markers in Male Breast Cancer.

Clin Cancer Res 2017 May 16;23(10):2575-2583. Epub 2016 Dec 16.

Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom.

Breast cancer affects both genders, but is understudied in men. Although still rare, male breast cancer (MBC) is being diagnosed more frequently. Treatments are wholly informed by clinical studies conducted in women, based on assumptions that underlying biology is similar. A transcriptomic investigation of male and female breast cancer was performed, confirming transcriptomic data Biomarkers were immunohistochemically assessed in 697 MBCs ( = 477, training; = 220, validation set) and quantified in pre- and posttreatment samples from an MBC patient receiving everolimus and PI3K/mTOR inhibitor. Gender-specific gene expression patterns were identified. eIF transcripts were upregulated in MBC. eIF4E and eIF5 were negatively prognostic for overall survival alone (log-rank = 0.013; HR = 1.77, 1.12-2.8 and = 0.035; HR = 1.68, 1.03-2.74, respectively), or when coexpressed ( = 0.01; HR = 2.66, 1.26-5.63), confirmed in the validation set. This remained upon multivariate Cox regression analysis [eIF4E = 0.016; HR = 2.38 (1.18-4.8), eIF5 = 0.022; HR = 2.55 (1.14-5.7); coexpression = 0.001; HR = 7.04 (2.22-22.26)]. Marked reduction in eIF4E and eIF5 expression was seen post BEZ235/everolimus, with extended survival. Translational initiation pathway inhibition could be of clinical utility in MBC patients overexpressing eIF4E and eIF5. With mTOR inhibitors that target this pathway now in the clinic, these biomarkers may represent new targets for therapeutic intervention, although further independent validation is required. .
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http://dx.doi.org/10.1158/1078-0432.CCR-16-1952DOI Listing
May 2017

Exploring the surfaceome of Ewing sarcoma identifies a new and unique therapeutic target.

Proc Natl Acad Sci U S A 2016 Mar 15;113(13):3603-8. Epub 2016 Mar 15.

Medical Research Council Molecular Haematology Unit, Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DS, United Kingdom;

The cell surface proteome of tumors mediates the interface between the transformed cells and the general microenvironment, including interactions with stromal cells in the tumor niche and immune cells such as T cells. In addition, the cell surface proteome of individual cancers defines biomarkers for that tumor type and potential proteins that can be the target of antibody-mediated therapy. We have used next-generation deep RNA sequencing (RNA-seq) coupled to an in-house database of genes encoding cell surface proteins (herein referred to as the surfaceome) as a tool to define a cell surface proteome of Ewing sarcoma compared with progenitor mesenchymal stem cells. This subtractive RNA-seq analysis revealed a specific surfaceome of Ewing and showed unexpectedly that the leucine-rich repeat and Ig domain protein 1 (LINGO1) is expressed in over 90% of Ewing sarcoma tumors, but not expressed in any other somatic tissue apart from the brain. We found that the LINGO1 protein acts as a gateway protein internalizing into the tumor cells when engaged by antibody and can carry antibody conjugated with drugs to kill Ewing sarcoma cells. Therefore, LINGO1 is a new, unique, and specific biomarker and drug target for the treatment of Ewing sarcoma.
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http://dx.doi.org/10.1073/pnas.1521251113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822608PMC
March 2016

Elucidating drivers of oral epithelial dysplasia formation and malignant transformation to cancer using RNAseq.

Oncotarget 2015 Nov;6(37):40186-201

Precancer Genomics, Leeds Institute of Cancer and Pathology, Wellcome Trust Brenner Building, St James's University Hospital, Leeds, LS9 7TF, UK.

Oral squamous cell carcinoma (OSCC) is a prevalent cancer with poor prognosis. Most OSCC progresses via a non-malignant stage called dysplasia. Effective treatment of dysplasia prior to potential malignant transformation is an unmet clinical need. To identify markers of early disease, we performed RNA sequencing of 19 matched HPV negative patient trios: normal oral mucosa, dysplasia and associated OSCC. We performed differential gene expression, principal component and correlated gene network analysis using these data. We found differences in the immune cell signatures present at different disease stages and were able to distinguish early events in pathogenesis, such as upregulation of many HOX genes, from later events, such as down-regulation of adherens junctions. We herein highlight novel coding and non-coding candidates for involvement in oral dysplasia development and malignant transformation, and speculate on how our findings may guide further translational research into the treatment of oral dysplasia.
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http://dx.doi.org/10.18632/oncotarget.5529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741888PMC
November 2015

Down-Regulation of miR-92 in Breast Epithelial Cells and in Normal but Not Tumour Fibroblasts Contributes to Breast Carcinogenesis.

PLoS One 2015 5;10(10):e0139698. Epub 2015 Oct 5.

Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, United Kingdom.

Background: MicroRNA (miR) expression is commonly dysregulated in many cancers, including breast. MiR-92 is one of six miRs encoded by the miR-17-92 cluster, one of the best-characterised oncogenic miR clusters. We examined expression of miR-92 in the breast epithelium and stroma during breast cancer progression. We also investigated the role of miR-92 in fibroblasts in vitro and showed that down-regulation in normal fibroblasts enhances the invasion of breast cancer epithelial cells.

Methodology/principal Findings: We used laser microdissection (LMD) to isolate epithelial cells from matched normal, DCIS and invasive tissue from 9 breast cancer patients and analysed miR-92 expression by qRT-PCR. Expression of ERβ1, a direct miR-92 target, was concurrently analysed for each case by immunohistochemistry. LMD was also used to isolate matched normal (NFs) and cancer-associated fibroblasts (CAFs) from 14 further cases. Effects of miR-92 inhibition in fibroblasts on epithelial cell invasion in vitro was examined using a Matrigel™ assay. miR-92 levels decreased in microdissected epithelial cells during breast cancer progression with highest levels in normal breast epithelium, decreasing in DCIS (p<0.01) and being lowest in invasive breast tissue (p<0.01). This was accompanied by a shift in cell localisation of ERβ1 from nuclear expression in normal breast epithelium to increased cytoplasmic expression during progression to DCIS (p = 0.0078) and invasive breast cancer (p = 0.031). ERβ1 immunoreactivity was also seen in stromal fibroblasts in tissues. Where miR-92 expression was low in microdissected NFs this increased in matched CAFs; a trend also seen in cultured primary fibroblasts. Down-regulation of miR-92 levels in NFs but not CAFs enhanced invasion of both MCF-7 and MDA-MB-231 breast cancer epithelial cells.

Conclusions: miR-92 is gradually lost in breast epithelial cells during cancer progression correlating with a shift in ERβ1 immunoreactivity from nuclei to the cytoplasm. Our data support a functional role in fibroblasts where modification of miR-92 expression can influence the invasive capacity of breast cancer epithelial cells. However in silico analysis suggests that ERβ1 may not be the most important miR-92 target in breast cancer.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0139698PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4593575PMC
June 2016

Using common variants to indicate cancer genes.

Int J Cancer 2015 Jan 14;136(1):241-5. Epub 2014 May 14.

Leeds Institute of Cancer and Pathology, University of Leeds, St James's University Hospital, Leeds, United Kingdom.

The catalogue of tumour-specific somatic mutations (SMs) is growing rapidly owing to the advent of next-generation sequencing. Identifying those mutations responsible for the development and progression of the disease, so-called driver mutations, will increase our understanding of carcinogenesis and provide candidates for targeted therapeutics. The phenotypic consequence(s) of driver mutations cause them to be selected for within the tumour environment, such that many approaches aimed at distinguishing drivers are based on finding significantly somatically mutated genes. Currently, these methods are designed to analyse, or be specifically applied to, nonsynonymous mutations: those that alter an encoded protein. However, growing evidence suggests the involvement of noncoding transcripts in carcinogenesis, mutations in which may also be disease-driving. We wished to test the hypothesis that common DNA variation rates within humans can be used as a baseline from which to score the rate of SMs, irrespective of coding capacity. We preliminarily tested this by applying it to a dataset of 159,498 SMs and using the results to rank genes. This resulted in significant enrichment of known cancer genes, indicating that the approach has merit. As additional data from cancer sequencing studies are made publicly available, this approach can be refined and applied to specific cancer subtypes. We named this preliminary version of our approach PRISMAD (polymorphism rates indicate somatic mutations as drivers) and have made it publicly accessible, with scripts, via a link at www.precancer.leeds.ac.uk/software-and-datasets.
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http://dx.doi.org/10.1002/ijc.28951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4277321PMC
January 2015

An integrated inspection of the somatic mutations in a lung squamous cell carcinoma using next-generation sequencing.

PLoS One 2013 11;8(11):e78823. Epub 2013 Nov 11.

Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, West Yorkshire, United Kingdom.

Squamous cell carcinoma (SCC) of the lung kills over 350,000 people annually worldwide, and is the main lung cancer histotype with no targeted treatments. High-coverage whole-genome sequencing of the other main subtypes, small-cell and adenocarcinoma, gave insights into carcinogenic mechanisms and disease etiology. The genomic complexity within the lung SCC subtype, as revealed by The Cancer Genome Atlas, means this subtype is likely to benefit from a more integrated approach in which the transcriptional consequences of somatic mutations are simultaneously inspected. Here we present such an approach: the integrated analysis of deep sequencing data from both the whole genome and whole transcriptome (coding and non-coding) of LUDLU-1, a SCC lung cell line. Our results show that LUDLU-1 lacks the mutational signature that has been previously associated with tobacco exposure in other lung cancer subtypes, and suggests that DNA-repair efficiency is adversely affected; LUDLU-1 contains somatic mutations in TP53 and BRCA2, allelic imbalance in the expression of two cancer-associated BRCA1 germline polymorphisms and reduced transcription of a potentially endogenous PARP2 inhibitor. Functional assays were performed and compared with a control lung cancer cell line. LUDLU-1 did not exhibit radiosensitisation or an increase in sensitivity to PARP inhibitors. However, LUDLU-1 did exhibit small but significant differences with respect to cisplatin sensitivity. Our research shows how integrated analyses of high-throughput data can generate hypotheses to be tested in the lab.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078823PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3823931PMC
August 2014

Accurately identifying low-allelic fraction variants in single samples with next-generation sequencing: applications in tumor subclone resolution.

Hum Mutat 2013 Oct 11;34(10):1432-8. Epub 2013 Jul 11.

Leeds Institute of Cancer and Pathology, St James's University Hospital, University of Leeds, Leeds, West Yorkshire, LS9 7TF, England.

Current methods for resolving genetically distinct subclones in tumor samples require somatic mutations to be clustered by allelic frequencies, which are determined by applying a variant calling program to next-generation sequencing data. Such programs were developed to accurately distinguish true polymorphisms and somatic mutations from the artifactual nonreference alleles introduced during library preparation and sequencing. However, numerous variant callers exist with no clear indication of the best performer for subclonal analysis, in which the accuracy of the assigned variant frequency is as important as correctly indicating whether the variant is present or not. Furthermore, sequencing depth (the number of times that a genomic position is sequenced) affects the ability to detect low-allelic fraction variants and accurately assign their allele frequencies. We created two synthetic sequencing datasets, and sequenced real KRAS amplicons, with variants spiked in at specific ratios, to assess which caller performs best in terms of both variant detection and assignment of allelic frequencies. We also assessed the sequencing depths required to detect low-allelic fraction variants. We found that VarScan2 performed best overall with sequencing depths of 100×, 250×, 500×, and 1,000× required to accurately identify variants present at 10%, 5%, 2.5%, and 1%, respectively.
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http://dx.doi.org/10.1002/humu.22365DOI Listing
October 2013

The transcriptional consequences of somatic amplifications, deletions, and rearrangements in a human lung squamous cell carcinoma.

Neoplasia 2012 Nov;14(11):1075-86

Leeds Institute of Molecular Medicine, University of Leeds, Leeds, United Kingdom.

Lung cancer causes more deaths, worldwide, than any other cancer. Several histologic subtypes exist. Currently, there is a dearth of targeted therapies for treating one of the main subtypes: squamous cell carcinoma (SCC). As for many cancers, lung SCC karyotypes are often highly anomalous owing to large somatic structural variants, some of which are seen repeatedly in lung SCC, indicating a potential causal association for genes therein. We chose to characterize a lung SCC genome to unprecedented detail and integrate our findings with the concurrently characterized transcriptome. We aimed to ascertain how somatic structural changes affected gene expression within the cell in ways that could confer a pathogenic phenotype. We sequenced the genomes of a lung SCC cell line (LUDLU-1) and its matched lymphocyte cell line (AGLCL) to more than 50x coverage. We also sequenced the transcriptomes of LUDLU-1 and a normal bronchial epithelium cell line (LIMM-NBE1), resulting in more than 600 million aligned reads per sample, including both coding and non-coding RNA (ncRNA), in a strand-directional manner. We also captured small RNA (<30 bp). We discovered significant, but weak, correlations between copy number and expression for protein-coding genes, antisense transcripts, long intergenic ncRNA, and microRNA (miRNA). We found that miRNA undergo the largest change in overall expression pattern between the normal bronchial epithelium and the tumor cell line. We found evidence of transcription across the novel genomic sequence created from six somatic structural variants. For each part of our integrated analysis, we highlight candidate genes that have undergone the largest expression changes.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3514744PMC
http://dx.doi.org/10.1593/neo.121380DOI Listing
November 2012

Mutation detection by clonal sequencing of PCR amplicons and grouped read typing is applicable to clinical diagnostics.

Hum Mutat 2013 Jan 11;34(1):248-54. Epub 2012 Oct 11.

Leeds Institute of Molecular Medicine, Leeds University, Leeds, UK.

We describe a sensitive technique for mutation detection using clonal sequencing. We analyzed DNA extracted from 13 cancer cell lines and 35 tumor samples and applied a novel approach to identify disease-associated somatic mutations. By matching reads against an index of known variants, noise can be dramatically reduced, enabling the detection and quantification of those variants, even when they are present at less than 1% of the total sequenced population; this is comparable to, or better than, current diagnostic methods. Following the identification or exclusion of known variants, unmatched reads are grouped for BLAST searching to identify novel variants or contaminants. Known variants, novel variants, and contaminants were readily identified in tumor tissue using this approach. Our approach also enables an estimation of the per-base sequencing error rate, providing a confidence threshold for interpretation of the results in the clinic. This novel approach has immediate applicability to clinical testing for disease-associated genetic variants.
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http://dx.doi.org/10.1002/humu.22207DOI Listing
January 2013

Functional and developmental expression of a zebrafish Kir1.1 (ROMK) potassium channel homologue Kcnj1.

J Physiol 2011 Mar 24;589(Pt 6):1489-503. Epub 2011 Jan 24.

Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, UK.

The zebrafish, Danio rerio, is emerging as an important model organism for the pathophysiological study of some human kidney diseases, but the sites of expression and physiological roles of a number of protein orthologues in the zebrafish nephron remain mostly undefined. Here we show that a zebrafish potassium channel is orthologous to the mammalian kidney potassium channel, ROMK. The cDNA (kcnj1) encodes a protein (Kcnj1) that when expressed in Xenopus laevis oocytes displayed pH- and Ba2+-sensitive K+-selective currents, but unlike the mammalian channel, was completely insensitive to the peptide inhibitor tertiapin-Q. In the pronephros, kcnj1 transcript expression was restricted to a distal region and overlapped with that of sodium–chloride cotransporter Nkcc, chloride channel ClC-Ka, and ClC-Ka/b accessory subunit Barttin, indicating the location of the diluting segment. In a subpopulation of surface cells, kcnj1 was coexpressed with the a1a.4 isoform of the Na+/K+-ATPase, identifying these cells as potential K+ secretory cells in this epithelium. At later stages of development, kcnj1 appeared in cells of the developing gill that also expressed the a1a.4 subunit.Morpholino antisense-mediated knockdown of kcnj1 was accompanied by transient tachycardia followed by bradycardia, effects consistent with alterations in extracellular K+ concentration in the embryo.Our findings indicate that Kcnj1 is expressed in cells associated with osmoregulation and acts as a K+ efflux pathway that is important in maintaining extracellular levels of K+ in the developing embryo.
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http://dx.doi.org/10.1113/jphysiol.2010.200295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3082106PMC
March 2011

KvDB; mining and mapping sequence variants in voltage-gated potassium channels.

Hum Mutat 2010 Aug;31(8):908-17

University of Leeds, Woodhouse Lane, Leeds, LS2 9JT, UK.

We have created KvDB: a voltage-gated potassium (Kv) channel-specific database that houses natural and experimental variant data and includes highly curated multiple sequence alignments and additional analytical tools, such as structural variant mapping and transmembrane segment prediction. KvDB is available at www.bioinformatics.leeds.ac.uk/KvDB. Analyzing the characterized gene variants in terms of topological location revealed the following. The S4, S4-S5, S5, S5-S6, and S6 segments are most likely to house disease-causing variants. Neurological disorders are more likely to be caused by variants affecting voltage sensing, whereas cardiac disorders are more likely to be caused by variants in the pore. Long QT Syndrome 2 (LQT2) is more often caused by N-terminus variation, a region containing a domain that affects deactivation, suggesting a potential disease mechanism. Conversely, a higher proportion of LQT1-causing variants reside in S4-S5, suggesting communication of voltage-sensing to the pore as a disease mechanism. By structurally mapping functionally characterized variants, we also provide mechanistic insight into Kv channel function; identifying an intersubunit interaction that may be partly responsible for setting activation voltage. Investigating phenotypically characterized variants that map to the same position as functionally characterized ones indicates only weak association between locations that cause disease and those that alter electrophysiological properties.
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http://dx.doi.org/10.1002/humu.21295DOI Listing
August 2010