De Salvo, G.L.
Del Bianco, P.
Minard-Colin, V.
Chisholm, J.
Jenney, M.
Guillen, G.
Devalck, C.
Van Rijn, R.
Shipley, J.
Orbach, D.
Kelsey, A.
Rogers, T.
Guerin, F.
Scarzello, G.
Ferrari, A.
Cesen Mazic, M.
Merks, J.H.
Bisogno, G.
European Pediatric Soft Tissue Sarcoma Study Group,
(2024). Reappraisal of prognostic factors used in the European Pediatric Soft Tissue Sarcoma Study Group RMS 2005 study for localized rhabdomyosarcoma to optimize risk stratification and generate a prognostic nomogram. Cancer,
.
show abstract
full text
BACKGROUND: The objective of this study was to investigate the role of clinical factors together with FOXO1 fusion status in patients with nonmetastatic rhabdomyosarcoma (RMS) to develop a predictive model for event-free survival and provide a rationale for risk stratification in future trials. METHODS: The authors used data from patients enrolled in the European Pediatric Soft Tissue Sarcoma Study Group (EpSSG) RMS 2005 study (EpSSG RMS 2005; EudraCT number 2005-000217-35). The following baseline variables were considered for the multivariable model: age at diagnosis, sex, histology, primary tumor site, Intergroup Rhabdomyosarcoma Studies group, tumor size, nodal status, and FOXO1 fusion status. Main effects and significant second-order interactions of candidate predictors were included in a multiple Cox proportional hazards regression model. A nomogram was generated for predicting 5-year event-free survival (EFS) probabilities. RESULTS: The EFS and overall survival rates at 5 years were 70.9% (95% confidence interval, 68.6%-73.1%) and 81.0% (95% confidence interval, 78.9%-82.8%), respectively. The multivariable model retained five prognostic factors, including age at diagnosis interacting with tumor size, tumor primary site, Intergroup Rhabdomyosarcoma Studies clinical group, and FOXO1 fusion status. Based on each patient's total score in the nomogram, patients were stratified into four groups. The 5-year EFS rates were 94.1%, 78.4%, 65.2%, and 52.1% in the low-risk, intermediate-risk, high-risk, and very-high-risk groups, respectively, and the corresponding 5-year overall survival rates were 97.2%, 91.5%, 74.3%, and 60.8%, respectively. CONCLUSIONS: The results presented here provide the rationale to modify the EpSSG stratification, with the most significant change represented by the replacement of histology with fusion status. This classification was adopted in the new international trial launched by the EpSSG..
Picher, E.A.
Wahajuddin, M.
Barth, S.
Chisholm, J.
Shipley, J.
Pors, K.
(2024). The Capacity of Drug-Metabolising Enzymes in Modulating the Therapeutic Efficacy of Drugs to Treat Rhabdomyosarcoma. Cancers (basel),
Vol.16
(5).
show abstract
full text
Rhabdomyosarcoma (RMS) is a rare soft tissue sarcoma (STS) that predominantly affects children and teenagers. It is the most common STS in children (40%) and accounts for 5-8% of total childhood malignancies. Apart from surgery and radiotherapy in eligible patients, standard chemotherapy is the only therapeutic option clinically available for RMS patients. While survival rates for this childhood cancer have considerably improved over the last few decades for low-risk and intermediate-risk cases, the mortality rate remains exceptionally high in high-risk RMS patients with recurrent and/or metastatic disease. The intensification of chemotherapeutic protocols in advanced-stage RMS has historically induced aggravated toxicity with only very modest therapeutic gain. In this review, we critically analyse what has been achieved so far in RMS therapy and provide insight into how a diverse group of drug-metabolising enzymes (DMEs) possess the capacity to modify the clinical efficacy of chemotherapy. We provide suggestions for new therapeutic strategies that exploit the presence of DMEs for prodrug activation, targeted chemotherapy that does not rely on DMEs, and RMS-molecular-subtype-targeted therapies that have the potential to enter clinical evaluation..
Chisholm, J.
Mandeville, H.
Adams, M.
Minard-Collin, V.
Rogers, T.
Kelsey, A.
Shipley, J.
van Rijn, R.R.
de Vries, I.
van Ewijk, R.
de Keizer, B.
Gatz, S.A.
Casanova, M.
Hjalgrim, L.L.
Firth, C.
Wheatley, K.
Kearns, P.
Liu, W.
Kirkham, A.
Rees, H.
Bisogno, G.
Wasti, A.
Wakeling, S.
Heenen, D.
Tweddle, D.A.
Merks, J.H.
Jenney, M.
(2024). Frontline and Relapsed Rhabdomyosarcoma (FAR-RMS) Clinical Trial: A Report from the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG). Cancers (basel),
Vol.16
(5).
show abstract
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The Frontline and Relapsed Rhabdomyosarcoma (FaR-RMS) clinical trial is an overarching, multinational study for children and adults with rhabdomyosarcoma (RMS). The trial, developed by the European Soft Tissue Sarcoma Study Group (EpSSG), incorporates multiple different research questions within a multistage design with a focus on (i) novel regimens for poor prognostic subgroups, (ii) optimal duration of maintenance chemotherapy, and (iii) optimal use of radiotherapy for local control and widespread metastatic disease. Additional sub-studies focusing on biological risk stratification, use of imaging modalities, including [18F]FDG PET-CT and diffusion-weighted MRI imaging (DWI) as prognostic markers, and impact of therapy on quality of life are described. This paper forms part of a Special Issue on rhabdomyosarcoma and outlines the study background, rationale for randomisations and sub-studies, design, and plans for utilisation and dissemination of results..
Evans, R.J.
Perkins, D.W.
Selfe, J.
Kelsey, A.
Birch, G.P.
Shipley, J.M.
Schipper, K.
Isacke, C.M.
(2023). Endo180 (MRC2) Antibody-Drug Conjugate for the Treatment of Sarcoma. Mol cancer ther,
Vol.22
(2),
pp. 240-253.
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Although the 5-year survival rates for sarcoma patients have improved, the proportion of patients relapsing after first-line treatment remains high, and the survival of patients with metastatic disease is dismal. Moreover, the extensive molecular heterogeneity of the multiple different sarcoma subtypes poses a substantial challenge to developing more personalized treatment strategies. From the IHC staining of a large set of 625 human soft-tissue sarcomas, we demonstrate strong tumor cell staining of the Endo180 (MRC2) receptor in a high proportion of samples, findings echoed in gene-expression data sets showing a significantly increased expression in both soft-tissue and bone sarcomas compared with normal tissue. Endo180 is a constitutively recycling transmembrane receptor and therefore an ideal target for an antibody-drug conjugate (ADC). An anti-Endo180 monoclonal antibody conjugated to the antimitotic agent, MMAE via a cleavable linker, is rapidly internalized into target cells and trafficked to the lysosome for degradation, causing cell death specifically in Endo180-expressing sarcoma cell lines. In a sarcoma tumor xenograft model, the Endo180-vc-MMAE ADC, but not an isotype-vc-MMAE control or the unconjugated Endo180 antibody, drives on-target cytotoxicity resulting in tumor regression and a significant impairment of metastatic colonization of the lungs, liver and lymph nodes. These data, together with the lack of a phenotype in mice with an Mrc2 genetic deletion, provide preclinical proof-of-principle evidence for the future development of an Endo180-ADC as a therapeutic strategy in a broad range of sarcoma subtypes and, importantly, with potential impact both on the primary tumor and in metastatic disease..
Lak, N.S.
van Zogchel, L.M.
Zappeij-Kannegieter, L.
Javadi, A.
van Paemel, R.
Vandeputte, C.
De Preter, K.
De Wilde, B.
Chicard, M.
Iddir, Y.
Schleiermacher, G.
Ruhen, O.
Shipley, J.
Fiocco, M.
Merks, J.H.
van Noesel, M.M.
van der Schoot, C.E.
Tytgat, G.A.
Stutterheim, J.
(2023). Cell-Free DNA as a Diagnostic and Prognostic Biomarker in Pediatric Rhabdomyosarcoma. Jco precis oncol,
Vol.7,
p. e2200113.
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PURPOSE: Total cell-free DNA (cfDNA) and tumor-derived cfDNA (ctDNA) can be used to study tumor-derived genetic aberrations. We analyzed the diagnostic and prognostic potential of cfDNA and ctDNA, obtained from pediatric patients with rhabdomyosarcoma. METHODS: cfDNA was isolated from diagnostic plasma samples from 57 patients enrolled in the EpSSG RMS2005 study. To study the diagnostic potential, shallow whole genome sequencing (shWGS) and cell-free reduced representation bisulphite sequencing (cfRRBS) were performed in a subset of samples and all samples were tested using droplet digital polymerase chain reaction to detect methylated RASSF1A (RASSF1A-M). Correlation with outcome was studied by combining cfDNA RASSF1A-M detection with analysis of our rhabdomyosarcoma-specific RNA panel in paired cellular blood and bone marrow fractions and survival analysis in 56 patients. RESULTS: At diagnosis, ctDNA was detected in 16 of 30 and 24 of 26 patients using shallow whole genome sequencing and cfRRBS, respectively. Furthermore, 21 of 25 samples were correctly classified as embryonal by cfRRBS. RASSF1A-M was detected in 21 of 57 patients. The presence of RASSF1A-M was significantly correlated with poor outcome (the 5-year event-free survival [EFS] rate was 46.2% for 21 RASSF1A-M‒positive patients, compared with 84.9% for 36 RASSF1A-M‒negative patients [P < .001]). RASSF1A-M positivity had the highest prognostic effect among patients with metastatic disease. Patients both negative for RASSF1A-M and the rhabdomyosarcoma-specific RNA panel (28 of 56 patients) had excellent outcome (5-year EFS 92.9%), while double-positive patients (11/56) had poor outcome (5-year EFS 13.6%, P < .001). CONCLUSION: Analyzing ctDNA at diagnosis using various techniques is feasible in pediatric rhabdomyosarcoma and has potential for clinical use. Measuring RASSF1A-M in plasma at initial diagnosis correlated significantly with outcome, particularly when combined with paired analysis of blood and bone marrow using a rhabdomyosarcoma-specific RNA panel..
Pomella, S.
Cassandri, M.
D'Archivio, L.
Porrazzo, A.
Cossetti, C.
Phelps, D.
Perrone, C.
Pezzella, M.
Cardinale, A.
Wachtel, M.
Aloisi, S.
Milewski, D.
Colletti, M.
Sreenivas, P.
Walters, Z.S.
Barillari, G.
Di Giannatale, A.
Milano, G.M.
De Stefanis, C.
Alaggio, R.
Rodriguez-Rodriguez, S.
Carlesso, N.
Vakoc, C.R.
Velardi, E.
Schafer, B.W.
Guccione, E.
Gatz, S.A.
Wasti, A.
Yohe, M.
Ignatius, M.
Quintarelli, C.
Shipley, J.
Miele, L.
Khan, J.
Houghton, P.J.
Marampon, F.
Gryder, B.E.
De Angelis, B.
Locatelli, F.
Rota, R.
(2023). MYOD-SKP2 axis boosts tumorigenesis in fusion negative rhabdomyosarcoma by preventing differentiation through p57Kip2 targeting. Nat commun,
Vol.14
(1),
p. 8373.
show abstract
full text
Rhabdomyosarcomas (RMS) are pediatric mesenchymal-derived malignancies encompassing PAX3/7-FOXO1 Fusion Positive (FP)-RMS, and Fusion Negative (FN)-RMS with frequent RAS pathway mutations. RMS express the master myogenic transcription factor MYOD that, whilst essential for survival, cannot support differentiation. Here we discover SKP2, an oncogenic E3-ubiquitin ligase, as a critical pro-tumorigenic driver in FN-RMS. We show that SKP2 is overexpressed in RMS through the binding of MYOD to an intronic enhancer. SKP2 in FN-RMS promotes cell cycle progression and prevents differentiation by directly targeting p27Kip1 and p57Kip2, respectively. SKP2 depletion unlocks a partly MYOD-dependent myogenic transcriptional program and strongly affects stemness and tumorigenic features and prevents in vivo tumor growth. These effects are mirrored by the investigational NEDDylation inhibitor MLN4924. Results demonstrate a crucial crosstalk between transcriptional and post-translational mechanisms through the MYOD-SKP2 axis that contributes to tumorigenesis in FN-RMS. Finally, NEDDylation inhibition is identified as a potential therapeutic vulnerability in FN-RMS..
O'Brien, E.
Tse, C.
Tracy, I.
Reddin, I.
Selfe, J.
Gibson, J.
Tapper, W.
Pengelly, R.J.
Gao, J.
Aladowicz, E.
Petts, G.
Thway, K.
Popov, S.
Kelsey, A.
Underwood, T.J.
Shipley, J.
Walters, Z.S.
(2023). Pharmacological EZH2 inhibition combined with retinoic acid treatment promotes differentiation and apoptosis in rhabdomyosarcoma cells. Clin epigenetics,
Vol.15
(1),
p. 167.
show abstract
BACKGROUND: Rhabdomyosarcomas (RMS) are predominantly paediatric sarcomas thought to originate from muscle precursor cells due to impaired myogenic differentiation. Despite intensive treatment, 5-year survival for patients with advanced disease remains low (< 30%), highlighting a need for novel therapies to improve outcomes. Differentiation therapeutics are agents that induce differentiation of cancer cells from malignant to benign. The histone methyltransferase, Enhancer of Zeste Homolog 2 (EZH2) suppresses normal skeletal muscle differentiation and is highly expressed in RMS tumours. RESULTS: We demonstrate combining inhibition of the epigenetic modulator EZH2 with the differentiating agent retinoic acid (RA) is more effective at reducing cell proliferation in RMS cell lines than single agents alone. In PAX3-FOXO1 positive RMS cells this is due to an RA-driven induction of the interferon pathway resulting in apoptosis. In fusion negative RMS, combination therapy led to an EZH2i-driven upregulation of myogenic signalling resulting in differentiation. In both subtypes, EZH2 is significantly associated with enrichment of trimethylated lysine 27 on histone 3 (H3K27me3) in genes that are downregulated in untreated RMS cells and upregulated with EZH2 inhibitor treatment. These results provide insight into the mechanism that drives the anti-cancer effect of the EZH2/RA single agent and combination treatment and indicate that the reduction of EZH2 activity combined with the induction of RA signalling represents a potential novel therapeutic strategy to treat both subtypes of RMS. CONCLUSIONS: The results of this study demonstrate the potential utility of combining EZH2 inhibitors with differentiation agents for the treatment of paediatric rhabdomyosarcomas. As EZH2 inhibitors are currently undergoing clinical trials for adult and paediatric solid tumours and retinoic acid differentiation agents are already in clinical use this presents a readily translatable potential therapeutic strategy. Moreover, as inhibition of EZH2 in the poor prognosis FPRMS subtype results in an inflammatory response, it is conceivable that this strategy may also synergise with immunotherapies for a more effective treatment in these patients..
Milton, C.I.
Selfe, J.
Aladowicz, E.
Man, S.Y.
Bernauer, C.
Missiaglia, E.
Walters, Z.S.
Gatz, S.A.
Kelsey, A.
Generali, M.
Box, G.
Valenti, M.
de Haven-Brandon, A.
Galiwango, D.
Hayes, A.
Clarke, M.
Izquierdo, E.
Gonzalez De Castro, D.
Raynaud, F.I.
Kirkin, V.
Shipley, J.M.
(2022). FGF7-FGFR2 autocrine signaling increases growth and chemoresistance of fusion-positive rhabdomyosarcomas. Mol oncol,
Vol.16
(6),
pp. 1272-1289.
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full text
Rhabdomyosarcomas are aggressive pediatric soft-tissue sarcomas and include high-risk PAX3-FOXO1 fusion-gene-positive cases. Fibroblast growth factor receptor 4 (FGFR4) is known to contribute to rhabdomyosarcoma progression; here, we sought to investigate the involvement and potential for therapeutic targeting of other FGFRs in this disease. Cell-based screening of FGFR inhibitors with potential for clinical repurposing (NVP-BGJ398, nintedanib, dovitinib, and ponatinib) revealed greater sensitivity of fusion-gene-positive versus fusion-gene-negative rhabdomyosarcoma cell lines and was shown to be correlated with high expression of FGFR2 and its specific ligand, FGF7. Furthermore, patient samples exhibit higher mRNA levels of FGFR2 and FGF7 in fusion-gene-positive versus fusion-gene-negative rhabdomyosarcomas. Sustained intracellular mitogen-activated protein kinase (MAPK) activity and FGF7 secretion into culture media during serum starvation of PAX3-FOXO1 rhabdomyosarcoma cells together with decreased cell viability after genetic silencing of FGFR2 or FGF7 was in keeping with a novel FGF7-FGFR2 autocrine loop. FGFR inhibition with NVP-BGJ398 reduced viability and was synergistic with SN38, the active metabolite of irinotecan. In vivo, NVP-BGJ398 abrogated xenograft growth and warrants further investigation in combination with irinotecan as a therapeutic strategy for fusion-gene-positive rhabdomyosarcomas..
Hettmer, S.
Linardic, C.M.
Kelsey, A.
Rudzinski, E.R.
Vokuhl, C.
Selfe, J.
Ruhen, O.
Shern, J.F.
Khan, J.
Kovach, A.R.
Lupo, P.J.
Gatz, S.A.
Schäfer, B.W.
Volchenboum, S.
Minard-Colin, V.
Koscielniak, E.
Hawkins, D.S.
Bisogno, G.
Sparber-Sauer, M.
Venkatramani, R.
Merks, J.H.
Shipley, J.
(2022). Molecular testing of rhabdomyosarcoma in clinical trials to improve risk stratification and outcome: A consensus view from European paediatric Soft tissue sarcoma Study Group, Children's Oncology Group and Cooperative Weichteilsarkom-Studiengruppe. Eur j cancer,
Vol.172,
pp. 367-386.
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full text
Rhabdomyosarcomas (RMSs) are the most common soft tissue sarcomas in children/adolescents less than 18 years of age with an annual incidence of 1-2/million. Inter/intra-tumour heterogeneity raise challenges in clinical, pathological and biological research studies. Risk stratification in European and North American clinical trials previously relied on clinico-pathological features, but now, incorporates PAX3/7-FOXO1-fusion gene status in the place of alveolar histology. International working groups propose a coordinated approach through the INternational Soft Tissue SaRcoma ConsorTium to evaluate the specific genetic abnormalities and generate and integrate molecular and clinical data related to patients with RMS across different trial settings. We review relevant data and present a consensus view on what molecular features should be assessed. In particular, we recommend the assessment of the MYOD1-LR122R mutation for risk escalation, as it has been associated with poor outcomes in spindle/sclerosing RMS and rare RMS with classic embryonal histopathology. The prospective analyses of rare fusion genes beyond PAX3/7-FOXO1 will generate new data linked to outcomes and assessment of TP53 mutations and CDK4 amplification may confirm their prognostic value. Pathogenic/likely pathogenic germline variants in TP53 and other cancer predisposition genes should also be assessed. DNA/RNA profiling of tumours at diagnosis/relapse and serial analyses of plasma samples is recommended where possible to validate potential molecular biomarkers, identify new biomarkers and assess how liquid biopsy analyses can have the greatest benefit. Together with the development of new molecularly-derived therapeutic strategies that we review, a synchronised international approach is expected to enhance progress towards improved treatment assignment, management and outcomes for patients with RMS..
Ferrari, A.
Gatz, S.A.
Minard-Colin, V.
Alaggio, R.
Hovsepyan, S.
Orbach, D.
Gasparini, P.
Defachelles, A.-.
Casanova, M.
Milano, G.M.
Chisholm, J.C.
Jenney, M.
Bisogno, G.
Rogers, T.
Mandeville, H.C.
Shipley, J.
Miah, A.B.
Merks, J.H.
van der Graaf, W.T.
(2022). Shedding a Light on the Challenges of Adolescents and Young Adults with Rhabdomyosarcoma. Cancers (basel),
Vol.14
(24).
show abstract
full text
Rhabdomyosarcoma (RMS) is a typical tumour of childhood but can occur at any age. Several studies have reported that adolescent and young adult (AYA) patients with RMS have poorer survival than do younger patients. This review discusses the specific challenges in AYA patients with pediatric-type RMS, exploring possible underlying factors which may influence different outcomes. Reasons for AYA survival gap are likely multifactorial, and might be related to differences in tumor biology and intrinsic aggressiveness, or differences in clinical management (that could include patient referral patterns, time to diagnosis, enrolment into clinical trials, the adequacy and intensity of treatment), as well as patient factors (including physiology and comorbidity that may influence treatment tolerability, drug pharmacokinetics and efficacy). However, improved survival has been reported in the most recent studies for AYA patients treated on pediatric RMS protocols. Different strategies may help to further improve outcome, such as supporting trans-age academic societies and national/international collaborations; developing specific clinical trials without upper age limit; defining integrated and comprehensive approach to AYA patients, including the genomic aspects; establishing multidisciplinary tumor boards with involvement of both pediatric and adult oncologists to discuss all pediatric-type RMS patients; developing dedicated projects with specific treatment recommendations and registry/database..
Ruhen, O.
Lak, N.S.
Stutterheim, J.
Danielli, S.G.
Chicard, M.
Iddir, Y.
Saint-Charles, A.
Di Paolo, V.
Tombolan, L.
Gatz, S.A.
Aladowicz, E.
Proszek, P.
Jamal, S.
Stankunaite, R.
Hughes, D.
Carter, P.
Izquierdo, E.
Wasti, A.
Chisholm, J.C.
George, S.L.
Pace, E.
Chesler, L.
Aerts, I.
Pierron, G.
Zaidi, S.
Delattre, O.
Surdez, D.
Kelsey, A.
Hubank, M.
Bonvini, P.
Bisogno, G.
Di Giannatale, A.
Schleiermacher, G.
Schäfer, B.W.
Tytgat, G.A.
Shipley, J.
(2022). Molecular Characterization of Circulating Tumor DNA in Pediatric Rhabdomyosarcoma: A Feasibility Study. Jco precis oncol,
Vol.6,
p. e2100534.
show abstract
full text
PURPOSE: Rhabdomyosarcomas (RMS) are rare neoplasms affecting children and young adults. Efforts to improve patient survival have been undermined by a lack of suitable disease markers. Plasma circulating tumor DNA (ctDNA) has shown promise as a potential minimally invasive biomarker and monitoring tool in other cancers; however, it remains underexplored in RMS. We aimed to determine the feasibility of identifying and quantifying ctDNA in plasma as a marker of disease burden and/or treatment response using blood samples from RMS mouse models and patients. METHODS: We established mouse models of RMS and applied quantitative polymerase chain reaction (PCR) and droplet digital PCR (ddPCR) to detect ctDNA within the mouse plasma. Potential driver mutations, copy-number alterations, and DNA breakpoints associated with PAX3/7-FOXO1 gene fusions were identified in the RMS samples collected at diagnosis. Patient-matched plasma samples collected from 28 patients with RMS before, during, and after treatment were analyzed for the presence of ctDNA via ddPCR, panel sequencing, and/or whole-exome sequencing. RESULTS: Human tumor-derived DNA was detectable in plasma samples from mouse models of RMS and correlated with tumor burden. In patients, ctDNA was detected in 14/18 pretreatment plasma samples with ddPCR and 7/7 cases assessed by sequencing. Levels of ctDNA at diagnosis were significantly higher in patients with unfavorable tumor sites, positive nodal status, and metastasis. In patients with serial plasma samples (n = 18), fluctuations in ctDNA levels corresponded to treatment response. CONCLUSION: Comprehensive ctDNA analysis combining high sensitivity and throughput can identify key molecular drivers in RMS models and patients, suggesting potential as a minimally invasive biomarker. Preclinical assessment of treatments using mouse models and further patient testing through prospective clinical trials are now warranted..
Zhang, P.
Kitchen-Smith, I.
Xiong, L.
Stracquadanio, G.
Brown, K.
Richter, P.H.
Wallace, M.D.
Bond, E.
Sahgal, N.
Moore, S.
Nornes, S.
De Val, S.
Surakhy, M.
Sims, D.
Wang, X.
Bell, D.A.
Zeron-Medina, J.
Jiang, Y.
Ryan, A.J.
Selfe, J.L.
Shipley, J.
Kar, S.
Pharoah, P.D.
Loveday, C.
Jansen, R.
Grochola, L.F.
Palles, C.
Protheroe, A.
Millar, V.
Ebner, D.V.
Pagadala, M.
Blagden, S.P.
Maughan, T.S.
Domingo, E.
Tomlinson, I.
Turnbull, C.
Carter, H.
Bond, G.L.
(2021). Germline and Somatic Genetic Variants in the p53 Pathway Interact to Affect Cancer Risk, Progression, and Drug Response. Cancer res,
Vol.81
(7),
pp. 1667-1680.
show abstract
full text
Insights into oncogenesis derived from cancer susceptibility loci (SNP) hold the potential to facilitate better cancer management and treatment through precision oncology. However, therapeutic insights have thus far been limited by our current lack of understanding regarding both interactions of these loci with somatic cancer driver mutations and their influence on tumorigenesis. For example, although both germline and somatic genetic variation to the p53 tumor suppressor pathway are known to promote tumorigenesis, little is known about the extent to which such variants cooperate to alter pathway activity. Here we hypothesize that cancer risk-associated germline variants interact with somatic TP53 mutational status to modify cancer risk, progression, and response to therapy. Focusing on a cancer risk SNP (rs78378222) with a well-documented ability to directly influence p53 activity as well as integration of germline datasets relating to cancer susceptibility with tumor data capturing somatically-acquired genetic variation provided supportive evidence for this hypothesis. Integration of germline and somatic genetic data enabled identification of a novel entry point for therapeutic manipulation of p53 activities. A cluster of cancer risk SNPs resulted in increased expression of prosurvival p53 target gene KITLG and attenuation of p53-mediated responses to genotoxic therapies, which were reversed by pharmacologic inhibition of the prosurvival c-KIT signal. Together, our results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and identify novel combinatorial therapies. SIGNIFICANCE: These results offer evidence of how cancer susceptibility SNPs can interact with cancer driver genes to affect cancer progression and present novel therapeutic targets..
Bernauer, C.
Man, Y.K.
Chisholm, J.C.
Lepicard, E.Y.
Robinson, S.P.
Shipley, J.M.
(2021). Hypoxia and its therapeutic possibilities in paediatric cancers. Br j cancer,
Vol.124
(3),
pp. 539-551.
show abstract
full text
In tumours, hypoxia-a condition in which the demand for oxygen is higher than its availability-is well known to be associated with reduced sensitivity to radiotherapy and chemotherapy, and with immunosuppression. The consequences of hypoxia on tumour biology and patient outcomes have therefore led to the investigation of strategies that can alleviate hypoxia in cancer cells, with the aim of sensitising cells to treatments. An alternative therapeutic approach involves the design of prodrugs that are activated by hypoxic cells. Increasing evidence indicates that hypoxia is not just clinically significant in adult cancers but also in paediatric cancers. We evaluate relevant methods to assess the levels and extent of hypoxia in childhood cancers, including novel imaging strategies such as oxygen-enhanced magnetic resonance imaging (MRI). Preclinical and clinical evidence largely supports the use of hypoxia-targeting drugs in children, and we describe the critical need to identify robust predictive biomarkers for the use of such drugs in future paediatric clinical trials. Ultimately, a more personalised approach to treatment that includes targeting hypoxic tumour cells might improve outcomes in subgroups of paediatric cancer patients..
Rudzinski, E.R.
Kelsey, A.
Vokuhl, C.
Linardic, C.M.
Shipley, J.
Hettmer, S.
Koscielniak, E.
Hawkins, D.S.
Bisogno, G.
(2021). Pathology of childhood rhabdomyosarcoma: A consensus opinion document from the Children's Oncology Group, European Paediatric Soft Tissue Sarcoma Study Group, and the Cooperative Weichteilsarkom Studiengruppe. Pediatric blood & cancer,
Vol.68
(3).
Walters, Z.S.
Aladowicz, E.
Villarejo-Balcells, B.
Nugent, G.
Selfe, J.L.
Eve, P.
Blagg, J.
Rossanese, O.
Shipley, J.
(2021). Role for the Histone Demethylase KDM4B in Rhabdomyosarcoma via CDK6 and CCNA2: Compensation by KDM4A and Apoptotic Response of Targeting Both KDM4B and KDM4A. Cancers (basel),
Vol.13
(7).
show abstract
full text
Histone demethylases are epigenetic modulators that play key roles in regulating gene expression related to many critical cellular functions and are emerging as promising therapeutic targets in a number of tumor types. We previously identified histone demethylase family members as overexpressed in the pediatric sarcoma, rhabdomyosarcoma. Here we show high sensitivity of rhabdomyosarcoma cells to a pan-histone demethylase inhibitor, JIB-04 and identify a key role for the histone demethylase KDM4B in rhabdomyosarcoma cell growth through an RNAi-screening approach. Decreasing KDM4B levels affected cell cycle progression and transcription of G1/S and G2/M checkpoint genes including CDK6 and CCNA2, which are bound by KDM4B in their promoter regions. However, after sustained knockdown of KDM4B, rhabdomyosarcoma cell growth recovered. We show that this can be attributed to acquired molecular compensation via recruitment of KDM4A to the promoter regions of CDK6 and CCNA2 that are otherwise bound by KDM4B. Furthermore, upfront silencing of both KDM4B and KDM4A led to RMS cell apoptosis, not seen by reducing either alone. To circumvent compensation and elicit stronger therapeutic responses, our study supports targeting histone demethylase sub-family proteins through selective poly-pharmacology as a therapeutic approach..
Glosli, H.
Bisogno, G.
Kelsey, A.
Chisholm, J.C.
Gaze, M.
Kolb, F.
McHugh, K.
Shipley, J.
Gallego, S.
Merks, J.H.
Smeele, L.E.
Mandeville, H.
Ferrari, A.
Minard-Colin, V.
Corradini, N.
Jenney, M.
Zanetti, I.
De Salvo, G.L.
Orbach, D.
EpSSG members,
(2021). Non-parameningeal head and neck rhabdomyosarcoma in children, adolescents, and young adults: Experience of the European paediatric Soft tissue sarcoma Study Group (EpSSG) - RMS2005 study. Eur j cancer,
Vol.151,
pp. 84-93.
show abstract
full text
BACKGROUND/OBJECTIVES: The primary aim of this study was to analyse and evaluate the impact of different local treatments on the pattern of relapse in children with primary head and neck non-parameningeal (HNnPM) rhabdomyosarcoma (RMS), treated in the European paediatric Soft tissue sarcoma Study Group (EpSSG) RMS2005 study. The secondary aim was to assess whether current risk stratification is valid for this specific site. DESIGN/METHODS: This study includes all patients with localised HNnPM RMS enrolled in the RMS2005 study between 2005 and 2016. Treatment comprised chemotherapy adapted to risk group, with local surgery and/or radiation therapy. The main outcome measures were event-free survival (EFS) and overall survival (OS). RESULTS: A total of 165 patients were identified; the median age was 6.4 years (range, 0.1-25). The most common tumour sites were cheek/chin (22%) and nasal ala/nasolabial fold (20%). Histology was unfavourable for 40%, and regional nodal involvement present in 26%. Local therapy included surgery (58%) and/or radiotherapy (72%) to primary tumour and/or regional lymph nodes. After a median follow-up of 66 months (range, 6-158), 42 patients experienced an event, and 17 are still alive. Tumour events were frequent in oral primary (36%), parotid site (26%), cheek/chin (24%), and nasal ala/nasolabial fold (24%) and included locoregional failure in 84% of cases. The 5-year EFS and OS were 75% (95% confidence interval [CI]: 67.3-81.2) and 84.9% (95% CI: 77.5-89.7), respectively. Favourable histology was associated with a better EFS (82.3% versus 64.6%; p = 0.02) and nodal spread with a worse OS (88.6% versus 76.1%; p = 0.04). Different sublocations within the HNnPM primary did not have significant impact on outcome. CONCLUSION: Locoregional relapse/progression is the main tumour failure event in this site. Despite frequent unfavourable risk factors, HNnPM RMS remains a favourable location in the context of a risk-adapted strategy..
Shern, J.F.
Selfe, J.
Izquierdo, E.
Patidar, R.
Chou, H.-.
Song, Y.K.
Yohe, M.E.
Sindiri, S.
Wei, J.
Wen, X.
Rudzinski, E.R.
Barkauskas, D.A.
Lo, T.
Hall, D.
Linardic, C.M.
Hughes, D.
Jamal, S.
Jenney, M.
Chisholm, J.
Brown, R.
Jones, K.
Hicks, B.
Angelini, P.
George, S.
Chesler, L.
Hubank, M.
Kelsey, A.
Gatz, S.A.
Skapek, S.X.
Hawkins, D.S.
Shipley, J.M.
Khan, J.
(2021). Genomic Classification and Clinical Outcome in Rhabdomyosarcoma: A Report From an International Consortium. J clin oncol,
Vol.39
(26),
pp. 2859-2871.
show abstract
full text
PURPOSE: Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. Despite aggressive therapy, the 5-year survival rate for patients with metastatic or recurrent disease remains poor, and beyond PAX-FOXO1 fusion status, no genomic markers are available for risk stratification. We present an international consortium study designed to determine the incidence of driver mutations and their association with clinical outcome. PATIENTS AND METHODS: Tumor samples collected from patients enrolled on Children's Oncology Group trials (1998-2017) and UK patients enrolled on malignant mesenchymal tumor and RMS2005 (1995-2016) trials were subjected to custom-capture sequencing. Mutations, indels, gene deletions, and amplifications were identified, and survival analysis was performed. RESULTS: DNA from 641 patients was suitable for analyses. A median of one mutation was found per tumor. In FOXO1 fusion-negative cases, mutation of any RAS pathway member was found in > 50% of cases, and 21% had no putative driver mutation identified. BCOR (15%), NF1 (15%), and TP53 (13%) mutations were found at a higher incidence than previously reported and TP53 mutations were associated with worse outcomes in both fusion-negative and FOXO1 fusion-positive cases. Interestingly, mutations in RAS isoforms predominated in infants < 1 year (64% of cases). Mutation of MYOD1 was associated with histologic patterns beyond those previously described, older age, head and neck primary site, and a dismal survival. Finally, we provide a searchable companion database (ClinOmics), containing all genomic variants, and clinical annotation including survival data. CONCLUSION: This is the largest genomic characterization of clinically annotated rhabdomyosarcoma tumors to date and provides prognostic genetic features that refine risk stratification and will be incorporated into prospective trials..
Loveday, C.
Litchfield, K.
Proszek, P.Z.
Cornish, A.J.
Santo, F.
Levy, M.
Macintyre, G.
Holryod, A.
Broderick, P.
Dudakia, D.
Benton, B.
Bakir, M.A.
Hiley, C.
Grist, E.
Swanton, C.
Huddart, R.
Powles, T.
Chowdhury, S.
Shipley, J.
O'Connor, S.
Brenton, J.D.
Reid, A.
de Castro, D.G.
Houlston, R.S.
Turnbull, C.
(2020). Genomic landscape of platinum resistant and sensitive testicular cancers. Nat commun,
Vol.11
(1),
p. 2189.
show abstract
full text
While most testicular germ cell tumours (TGCTs) exhibit exquisite sensitivity to platinum chemotherapy, ~10% are platinum resistant. To gain insight into the underlying mechanisms, we undertake whole exome sequencing and copy number analysis in 40 tumours from 26 cases with platinum-resistant TGCT, and combine this with published genomic data on an additional 624 TGCTs. We integrate analyses for driver mutations, mutational burden, global, arm-level and focal copy number (CN) events, and SNV and CN signatures. Albeit preliminary and observational in nature, these analyses provide support for a possible mechanistic link between early driver mutations in RAS and KIT and the widespread copy number events by which TGCT is characterised..
Loktev, A.
Shipley, J.M.
(2020). Desmoplastic small round cell tumor (DSRCT): emerging therapeutic targets and future directions for potential therapies. Expert opin ther targets,
Vol.24
(4),
pp. 281-285.
full text
van Erp, A.E.
van Houdt, L.
Hillebrandt-Roeffen, M.H.
van Bree, N.F.
Flucke, U.E.
Mentzel, T.
Shipley, J.
Desar, I.M.
Fleuren, E.D.
Versleijen-Jonkers, Y.M.
van der Graaf, W.T.
(2020). Olaparib and temozolomide in desmoplastic small round cell tumors: a promising combination in vitro and in vivo. J cancer res clin oncol,
Vol.146
(7),
pp. 1659-1670.
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full text
PURPOSE: Desmoplastic small round cell tumors (DSRCTs) are highly malignant and very rare soft tissue sarcomas with a high unmet need for new therapeutic options. Therefore, we examined poly(ADP-ribose) polymerase 1 (PARP1) and Schlafen-11 (SLFN11) expression in DSRCT tumor tissue and the combination of PARP inhibitor olaparib with the alkylating agent temozolomide (TMZ) in a preclinical DSRCT model. METHODS: PARP1 and SLFN11 have been described as predictive biomarkers for response to PARP inhibition. Expression of PARP1 and SLFN11 was assessed in 16 and 12 DSRCT tumor tissue samples, respectively. Effects of single-agent olaparib, and olaparib and TMZ combination treatment were examined using the preclinical JN-DSRCT-1 model. In vitro, single-agent and combination treatment effects on cell viability, the cell cycle, DNA damage and apoptosis were examined. Olaparib and TMZ combination treatment was also assessed in vivo. RESULTS: PARP1 and SLFN11 expression was observed in 100% and 92% of DSRCT tumor tissues, respectively. Olaparib treatment reduced cell viability and cell migration in a dose-dependent manner in vitro. Drug synergy between olaparib and TMZ was observed in vitro and in vivo. Combination treatment led to a cell-cycle arrest and induction of DNA damage and apoptosis, even when combined at low dosages. CONCLUSION: We show high PARP1 and SLFN11 expression in DSRCT tumor material and antitumor effects following olaparib and TMZ combination treatment in a preclinical DSRCT model. This suggests that olaparib and TMZ combination treatment could be a potential treatment option for DSRCTs..
Figeac, N.
Mohamed, A.D.
Sun, C.
Schönfelder, M.
Matallanas, D.
Garcia-Munoz, A.
Missiaglia, E.
Collie-Duguid, E.
De Mello, V.
Pobbati, A.V.
Pruller, J.
Jaka, O.
Harridge, S.D.
Hong, W.
Shipley, J.
Vargesson, N.
Zammit, P.S.
Wackerhage, H.
(2019). VGLL3 operates via TEAD1, TEAD3 and TEAD4 to influence myogenesis in skeletal muscle. J cell sci,
Vol.132
(13).
show abstract
full text
VGLL proteins are transcriptional co-factors that bind TEAD family transcription factors to regulate events ranging from wing development in fly, to muscle fibre composition and immune function in mice. Here, we characterise Vgll3 in skeletal muscle. We found that mouse Vgll3 was expressed at low levels in healthy muscle but that its levels increased during hypertrophy or regeneration; in humans, VGLL3 was highly expressed in tissues from patients with various muscle diseases, such as in dystrophic muscle and alveolar rhabdomyosarcoma. Interaction proteomics revealed that VGLL3 bound TEAD1, TEAD3 and TEAD4 in myoblasts and/or myotubes. However, there was no interaction with proteins from major regulatory systems such as the Hippo kinase cascade, unlike what is found for the TEAD co-factors YAP (encoded by YAP1) and TAZ (encoded by WWTR1). Vgll3 overexpression reduced the activity of the Hippo negative-feedback loop, affecting expression of muscle-regulating genes including Myf5, Pitx2 and Pitx3, and genes encoding certain Wnts and IGFBPs. VGLL3 mainly repressed gene expression, regulating similar genes to those regulated by YAP and TAZ. siRNA-mediated Vgll3 knockdown suppressed myoblast proliferation, whereas Vgll3 overexpression strongly promoted myogenic differentiation. However, skeletal muscle was overtly normal in Vgll3-null mice, presumably due to feedback signalling and/or redundancy. This work identifies VGLL3 as a transcriptional co-factor operating with the Hippo signal transduction network to control myogenesis..
Selfe, J.
Shipley, J.M.
(2019). IGF signalling in germ cells and testicular germ cell tumours: roles and therapeutic approaches. Andrology,
Vol.7
(4),
pp. 536-544.
show abstract
full text
The insulin-like growth factor (IGF) axis plays key roles in normal tissue growth and development as well as in the progression of several tumour types and their subsequent growth and progression to a metastatic phenotype. This review explores the role of IGF system in normal germ cell development and function in addition to examining the evidence for deregulation of IGF signalling in cancer, with particular relevance to evidence supporting a role in testicular germ cell tumours (TGCTs). Despite the clear preclinical rationale for targeting the IGF axis in cancer, there has been a lack of progress in identifying which patients may benefit from such therapy. Future employment of agents targeting the IGF pathway is expected to concentrate on their use in combination with other treatments to prevent resistance and exploit their potential as chemo- and radiosensitizers..
Romo-Morales, A.
Aladowicz, E.
Blagg, J.
Gatz, S.A.
Shipley, J.M.
(2019). Catalytic inhibition of KDM1A in Ewing sarcoma is insufficient as a therapeutic strategy. Pediatr blood cancer,
Vol.66
(9),
p. e27888.
show abstract
full text
BACKGROUND: Ewing sarcoma and desmoplastic small round cell tumors (DSRCT) are rare and clinically aggressive sarcomas usually characterized by oncogenic fusion proteins involving EWS. Emerging studies of Ewing sarcoma have demonstrated EWS-FLI1-driven chromatin remodeling as a key aspect of tumorigenicity. In particular, the lysine-specific demethylase KDM1A/LSD1 is linked to transcriptional regulation of target genes orchestrated by the EWS portion of the fusion protein interacting with repressive chromatin-remodeling complexes. Consistent with this model, depletion of KDM1A supports it is a molecular therapeutic target in Ewing sarcoma cells, but effective drugs need to be identified. PROCEDURE: A comprehensive phenotypic analysis of the effects of catalytic KDM1A inhibitors ORY-1001 and GSK2879552, including clinically relevant doses, was carried out in 2D and 3D spheroid models of Ewing sarcoma and DSRCT. RESULTS: Catalytic inhibition of KDM1A did not affect cell viability in 2D and 3D assays and had no impact on invasion in a 3D assay. CONCLUSIONS: Overall, evidence presented here does not support inhibition of KDM1A catalytic demethylase activity as an effective therapeutic strategy for Ewing sarcoma or DSRCT. However, roles of KDM1A beyond its demethylase activity should be considered for these sarcomas..
George, S.L.
Izquierdo, E.
Campbell, J.
Koutroumanidou, E.
Proszek, P.
Jamal, S.
Hughes, D.
Yuan, L.
Marshall, L.V.
Carceller, F.
Chisholm, J.C.
Vaidya, S.
Mandeville, H.
Angelini, P.
Wasti, A.
Bexelius, T.
Thway, K.
Gatz, S.A.
Clarke, M.
Al-Lazikani, B.
Barone, G.
Anderson, J.
Tweddle, D.A.
Gonzalez, D.
Walker, B.A.
Barton, J.
Depani, S.
Eze, J.
Ahmed, S.W.
Moreno, L.
Pearson, A.
Shipley, J.
Jones, C.
Hargrave, D.
Jacques, T.S.
Hubank, M.
Chesler, L.
(2019). A tailored molecular profiling programme for children with cancer to identify clinically actionable genetic alterations. Eur j cancer,
Vol.121,
pp. 224-235.
show abstract
full text
BACKGROUND: For children with cancer, the clinical integration of precision medicine to enable predictive biomarker-based therapeutic stratification is urgently needed. METHODS: We have developed a hybrid-capture next-generation sequencing (NGS) panel, specifically designed to detect genetic alterations in paediatric solid tumours, which gives reliable results from as little as 50Â ng of DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. In this study, we offered an NGS panel, with clinical reporting via a molecular tumour board for children with solid tumours. Furthermore, for a cohort of 12 patients, we used a circulating tumour DNA (ctDNA)-specific panel to sequence ctDNA from matched plasma samples and compared plasma and tumour findings. RESULTS: A total of 255 samples were submitted from 223 patients for the NGS panel. Using FFPE tissue, 82% of all submitted samples passed quality control for clinical reporting. At least one genetic alteration was detected in 70% of sequenced samples. The overall detection rate of clinically actionable alterations, defined by modified OncoKB criteria, for all sequenced samples was 51%. A total of 8 patients were sequenced at different stages of treatment. In 6 of these, there were differences in the genetic alterations detected between time points. Sequencing of matched ctDNA in a cohort of extracranial paediatric solid tumours also identified a high detection rate of somatic alterations in plasma. CONCLUSION: We demonstrate that tailored clinical molecular profiling of both tumour DNA and plasma-derived ctDNA is feasible for children with solid tumours. Furthermore, we show that a targeted NGS panel-based approach can identify actionable genetic alterations in a high proportion of patients..
Gatz, S.A.
Aladowicz, E.
Casanova, M.
Chisholm, J.C.
Kearns, P.R.
Fulda, S.
Geoerger, B.
Schäfer, B.W.
Shipley, J.M.
(2019). A Perspective on Polo-Like Kinase-1 Inhibition for the Treatment of Rhabdomyosarcomas. Front oncol,
Vol.9,
p. 1271.
show abstract
full text
Rhabdomyosarcomas are the most common pediatric soft tissue sarcoma and are a major cause of death from cancer in young patients requiring new treatment options to improve outcomes. High-risk patients include those with metastatic or relapsed disease and tumors with PAX3-FOXO1 fusion genes that encode a potent transcription factor that drives tumourigenesis through transcriptional reprogramming. Polo-Like Kinase-1 (PLK1) is a serine/threonine kinase that phosphorylates a wide range of target substrates and alters their activity. PLK1 functions as a pleiotropic master regulator of mitosis and regulates DNA replication after stress. Taken together with high levels of expression that correlate with poor outcomes in many cancers, including rhabdomyosarcomas, it is an attractive therapeutic target. This is supported in rhabdomyosarcoma models by characterization of molecular and phenotypic effects of reducing and inhibiting PLK1, including changes to the PAX3-FOXO1 fusion protein. However, as tumor re-growth has been observed, combination strategies are required. Here we review preclinical evidence and consider biological rationale for PLK1 inhibition in combination with drugs that promote apoptosis, interfere with activity of PAX3-FOXO1 and are synergistic with microtubule-destabilizing drugs such as vincristine. The preclinical effects of low doses of the PLK1 inhibitor volasertib in combination with vincristine, which is widely used in rhabdomyosarcoma treatment, show particular promise in light of recent clinical data in the pediatric setting that support achievable volasertib doses predicted to be effective. Further development of novel therapeutic strategies including PLK1 inhibition may ultimately benefit young patients with rhabdomyosarcoma and other cancers..
Hale, R.
Sandakly, S.
Shipley, J.
Walters, Z.
(2019). Epigenetic Targets in Synovial Sarcoma: A Mini-Review. Front oncol,
Vol.9,
p. 1078.
show abstract
Synovial Sarcomas (SS) are a type of Soft Tissue Sarcoma (STS) and represent 8-10% of all STS cases. Although SS can arise at any age, it typically affects younger individuals aged 15-35 and is therefore part of both pediatric and adult clinical practices. SS occurs primarily in the limbs, often near joints, but can present anywhere. It is characterized by the recurrent pathognomonic chromosomal translocation t(X;18)(p11.2;q11.2) that most frequently fuses SSX1 or SSX2 genes with SS18. This leads to the expression of the SS18-SSX fusion protein, which causes disturbances in several interacting multiprotein complexes such as the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex, also known as the BAF complex and the Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2). Furthermore, this promotes widespread epigenetic rewiring, leading to aberrant gene expression that drives the pathogenesis of SS. Good prognoses are characterized predominantly by small tumor size and young patient age. Whereas, high tumor grade and an increased genomic complexity of the tumor constitute poor prognostic factors. The current therapeutic strategy relies on chemotherapy and radiotherapy, the latter of which can lead to chronic side effects for pediatric patients. We will focus on the known roles of SWI/SNF, PRC1, and PRC2 as the main effectors of the SS18-SSX-mediated genome modifications and we present existing biological rationale for potential therapeutic targets and treatment strategies..
Gallego, S.
Zanetti, I.
Orbach, D.
Ranchère, D.
Shipley, J.
Zin, A.
Bergeron, C.
de Salvo, G.L.
Chisholm, J.
Ferrari, A.
Jenney, M.
Mandeville, H.C.
Rogers, T.
Merks, J.H.
Mudry, P.
Glosli, H.
Milano, G.M.
Ferman, S.
Bisogno, G.
European Paediatric Soft Tissue Sarcoma Study Group (EpSSG),
(2018). Fusion status in patients with lymph node-positive (N1) alveolar rhabdomyosarcoma is a powerful predictor of prognosis: Experience of the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG). Cancer,
Vol.124
(15),
pp. 3201-3209.
show abstract
BACKGROUND: Alveolar rhabdomyosarcoma (aRMS) with lymph node involvement (N1 classification) accounts for up to 10% of all cases of RMS. The prognosis is poor, and is comparable to that of distant metastatic disease. In the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) RMS2005 protocol, patients with a histologic diagnosis of aRMS/N1 received intensified chemotherapy with systematic locoregional treatment. METHODS: Patients with aRMS/N1 were enrolled prospectively after primary surgery/biopsy and fusion status was assessed in tumor samples. All patients received 9 cycles of induction chemotherapy and 6 months of maintenance therapy. Local treatment included radiotherapy to the primary site and lymph nodes with or without secondary surgical resection. RESULTS: A total of 103 patients were enrolled. The clinical characteristics of the patients were predominantly unfavorable: 90% had macroscopic residual disease after initial surgery/biopsy, 63% had locally invasive tumors, 77% had a tumor measuring >5 cm, and 81% had disease at unfavorable sites. Fusion genes involving forkhead box protein O1 (FOXO1) were detected in 56 of 84 patients. Events occurred in 52 patients: 43 developed disease recurrence, 7 had disease that was refractory to treatment, and 2 patients developed second neoplasms. On univariate analysis, unfavorable disease site, tumor invasiveness, Intergroup Rhabdomyosarcoma Study group III, and fusion-positive status correlated with worse prognosis. The 5-year event-free survival rate of patients with fusion-positive tumors was 43% compared with 74% in patients with fusion-negative tumors (P = .01). On multivariate analysis, fusion positivity and tumor invasiveness proved to be unfavorable prognostic markers. CONCLUSIONS: Fusion status and tumor invasiveness appear to have a strong impact on prognosis in patients with aRMS/N1. Fusion status will be used to stratify these patients in the next EpSSG RMS study, and treatment will be intensified in patients with fusion-positive tumors. Cancer 2018. © 2018 American Cancer Society..
Selfe, J.
Goddard, N.C.
McIntyre, A.
Taylor, K.R.
Renshaw, J.
Popov, S.D.
Thway, K.
Summersgill, B.
Huddart, R.A.
Gilbert, D.C.
Shipley, J.M.
(2018). IGF1R signalling in testicular germ cell tumour cells impacts on cell survival and acquired cisplatin resistance. J pathol,
Vol.244
(2),
pp. 242-253.
show abstract
full text
Testicular germ cell tumours (TGCTs) are the most frequent malignancy and cause of death from solid tumours in the 20- to 40-year age group. Although most cases show sensitivity to cis-platinum-based chemotherapy, this is associated with long-term toxicities and chemo-resistance. Roles for receptor tyrosine kinases other than KIT are largely unknown in TGCT. We therefore conducted a phosphoproteomic screen and identified the insulin growth factor receptor-1 (IGF1R) as both highly expressed and activated in TGCT cell lines representing the nonseminomatous subtype. IGF1R was also frequently expressed in tumour samples from patients with nonseminomas. Functional analysis of cell line models showed that long-term shRNA-mediated IGF1R silencing leads to apoptosis and complete ablation of nonseminoma cells with active IGF1R signalling. Cell lines with high levels of IGF1R activity also showed reduced AKT signalling in response to decreased IGF1R expression as well as sensitivity to the small-molecule IGF1R inhibitor NVP-AEW541. These results were in contrast to those in the seminoma cell line TCAM2 that lacked IGF1R signalling via AKT and was one of the two cell lines least sensitive to the IGF1R inhibitor. The dependence on IGF1R activity in the majority of nonseminomas parallels the known role of IGF signalling in the proliferation, migration, and survival of primordial germ cells, the putative cell of origin for TGCT. Upregulation of IGF1R expression and signalling was also found to contribute to acquired cisplatin resistance in an in vitro nonseminoma model, providing a rationale for targeting IGF1R in cisplatin-resistant disease. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland..
Bisogno, G.
Jenney, M.
Bergeron, C.
Gallego Melcón, S.
Ferrari, A.
Oberlin, O.
Carli, M.
Stevens, M.
Kelsey, A.
De Paoli, A.
Gaze, M.N.
Martelli, H.
Devalck, C.
Merks, J.H.
Ben-Arush, M.
Glosli, H.
Chisholm, J.
Orbach, D.
Minard-Colin, V.
De Salvo, G.L.
European paediatric Soft tissue sarcoma Study Group,
(2018). Addition of dose-intensified doxorubicin to standard chemotherapy for rhabdomyosarcoma (EpSSG RMS 2005): a multicentre, open-label, randomised controlled, phase 3 trial. Lancet oncol,
Vol.19
(8),
pp. 1061-1071.
show abstract
full text
BACKGROUND: Rhabdomyosarcoma is an aggressive tumour that can develop in almost any part of the body. Doxorubicin is an effective drug against rhabdomyosarcoma, but its role in combination with an established multidrug regimen remains controversial. Therefore, we aimed to evaluate the possible benefit of early dose intensification with doxorubicin in patients with non-metastatic rhabdomyosarcoma. METHODS: We did a multicentre, open-label, randomised controlled, phase 3 trial involving 108 hospitals from 14 countries. We included patients older than 6 months but younger than 21 years with a pathologically proven diagnosis of rhabdomyosarcoma. We assigned each patient to a specific subgroup according to the EpSSG stratification system. Those with embryonal rhabdomyosarcoma incompletely resected and localised at unfavourable sites with or without nodal involvement, or those with alveolar rhabdomyosarcoma without nodal involvement were considered at high risk of relapse. These high-risk patients were randomly assigned (1:1) to receive either nine cycles of IVA (ifosfamide 3 g/m2 given as a 3-h intravenous infusion on days 1 and 2, vincristine 1·5 mg/m2 weekly during the first 7 weeks then only on day 1 of each cycle [given as a single intravenous injection], and dactinomycin 1·5 mg/m2 on day 1 given as a single intravenous injection) or four cycles of IVA with doxorubicin 30 mg/m2 given as a 4-h intravenous infusion on days 1 and 2 followed by five cycles of IVA. The interval between cycles was 3 weeks. Randomisation was done using a web-based system and was stratified (block sizes of four) by enrolling country and risk subgroup. Neither investigators nor patients were masked to treatment allocation. The primary endpoint was 3-year event-free survival assessed by the investigator at each centre in the intention-to-treat population. Patients who received at least one dose of study treatment were considered in the safety analysis. In agreement with the independent data monitoring committee, the study was closed to patient entry on Dec 16, 2013, after futility analysis. This trial is registered with EudraCT, number 2005-000217-35, and is currently in follow-up. FINDINGS: Between Oct 1, 2005, and Dec 16, 2013, 484 patients were randomly assigned to receive each chemotherapy regimen (242 in the IVA group and 242 in the IVA plus doxorubicin group). Median follow-up was 63·9 months (IQR 44·6-78·9). The 3-year event-free survival was 67·5% (95% CI 61·2-73·1) in the IVA plus doxorubicin group and 63·3% (56·8-69·0) in the IVA group (hazard ratio 0·87, 95% CI 0·65-1·16; p=0·33). Grade 3-4 leucopenia (232 [93%] of 249 patients in the IVA plus doxorubicin group vs 194 [85%] of 227 in the IVA group; p=0·0061), anaemia (195 [78%] vs 111 [49%]; p<0·0001), thrombocytopenia (168 [67%] vs 59 [26%]; p<0.0001), and gastrointestinal adverse events (78 [31%] vs 19 [8%]; p<0·0001) were significantly more common in the IVA plus doxorubicin group than in the IVA group. Grade 3-5 infections (198 [79%] vs 128 [56%]; p<0·0001) were also significantly more common in the IVA plus doxorubicin group than in the IVA group, in which one patient had grade 5 infection. Two treatment-related deaths were reported (one patient developed septic shock and one affected by Goldenhar syndrome developed intractable seizures) in the IVA plus doxorubicin group, both occurring after the first cycle of treatment, and none were reported in the IVA group. INTERPRETATIONS: The addition of dose-intensified doxorubicin to standard IVA chemotherapy did not show a significant improvement in the outcome of patients with high-risk non-metastatic rhabdomyosarcoma. Therefore, the IVA chemotherapy regimen should remain the standard of care for patients with localised rhabdomyosarcoma in Europe. FUNDING: Fondazione Città della Speranza, Italy, and the Association Léon Berard Enfant Cancéreux, France..
Holme, H.
Gulati, A.
Brough, R.
Fleuren, E.D.
Bajrami, I.
Campbell, J.
Chong, I.Y.
Costa-Cabral, S.
Elliott, R.
Fenton, T.
Frankum, J.
Jones, S.E.
Menon, M.
Miller, R.
Pemberton, H.N.
Postel-Vinay, S.
Rafiq, R.
Selfe, J.L.
von Kriegsheim, A.
Munoz, A.G.
Rodriguez, J.
Shipley, J.
van der Graaf, W.T.
Williamson, C.T.
Ryan, C.J.
Pettitt, S.
Ashworth, A.
Strauss, S.J.
Lord, C.J.
(2018). Chemosensitivity profiling of osteosarcoma tumour cell lines identifies a model of BRCAness. Sci rep,
Vol.8
(1),
p. 10614.
show abstract
full text
Osteosarcoma (OS) is an aggressive sarcoma, where novel treatment approaches are required. Genomic studies suggest that a subset of OS, including OS tumour cell lines (TCLs), exhibit genomic loss of heterozygosity (LOH) patterns reminiscent of BRCA1 or BRCA2 mutant tumours. This raises the possibility that PARP inhibitors (PARPi), used to treat BRCA1/2 mutant cancers, could be used to target OS. Using high-throughput drug sensitivity screening we generated chemosensitivity profiles for 79 small molecule inhibitors, including three clinical PARPi. Drug screening was performed in 88 tumour cell lines, including 18 OS TCLs. This identified known sensitivity effects in OS TCLs, such as sensitivity to FGFR inhibitors. When compared to BRCA1/2 mutant TCLs, OS TCLs, with the exception of LM7, were PARPi resistant, including those with previously determined BRCAness LoH profiles. Post-screen validation experiments confirmed PARPi sensitivity in LM7 cells as well as a defect in the ability to form nuclear RAD51 foci in response to DNA damage. LM7 provides one OS model for the study of PARPi sensitivity through a potential defect in RAD51-mediated DNA repair. The drug sensitivity dataset we generated in 88 TCLs could also serve as a resource for the study of drug sensitivity effects in OS..
O'Brien, E.M.
Selfe, J.L.
Martins, A.S.
Walters, Z.S.
Shipley, J.M.
(2018). The long non-coding RNA MYCNOS-01 regulates MYCN protein levels and affects growth of MYCN-amplified rhabdomyosarcoma and neuroblastoma cells. Bmc cancer,
Vol.18
(1),
p. 217.
show abstract
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BACKGROUND: MYCN is amplified in small cell lung cancers and several pediatric tumors, including alveolar rhabdomyosarcomas and neuroblastomas. MYCN protein is known to play a key oncogenic role in both alveolar rhabdomyosarcomas and neuroblastomas. MYCN opposite strand (MYCNOS) is a gene located on the antisense strand to MYCN that encodes alternatively spliced transcripts, two of which (MYCNOS-01 and MYCNOS-02) are known to be expressed in neuroblastoma and small cell lung cancer with reciprocal regulation between MYCNOS-02 and MYCN reported for neuroblastomas. We sought to determine a functional role for MYCNOS-01 in alveolar rhabdomyosarcoma and neuroblastoma cells and identify any associated regulatory effects between MYCN and MYCNOS-01. METHODS: MYCNOS-01, MYCNOS-02 and MYCN expression levels were assessed in alveolar rhabdomyosarcoma and neuroblastoma cell lines and tumor samples from patients using Affymetrix microarray data and quantitative RT-PCR. Following MYCNOS-01 or MYCN siRNA knockdown and MYCNOS-01 overexpression, transcript levels were assayed by quantitative RT-PCR and MYCN protein expression assessed by Western blot and immunofluorescence. Additionally, effects on cell growth, apoptosis and cell cycle profiles were determined by a metabolic assay, caspase activity and flow cytometry, respectively. RESULTS: MYCNOS-01 transcript levels were generally higher in NB and RMS tumor samples and cell lines with MYCN genomic amplification. RNA interference of MYCNOS-01 expression did not alter MYCN transcript levels but decreased MYCN protein levels. Conversely, MYCN reduction increased MYCNOS-01 transcript levels, creating a negative feedback loop on MYCN protein levels. Reduction of MYCNOS-01 or MYCN expression decreased cell growth in MYCN-amplified alveolar rhabdomyosarcoma and neuroblastoma cell lines. This is consistent with MYCNOS-01-mediated regulation of MYCN contributing to the phenotype observed. CONCLUSIONS: An alternative transcript of MYCNOS, MYCNOS-01, post-transcriptionally regulates MYCN levels and affects growth in MYCN-amplified rhabdomyosarcoma and neuroblastoma cells..
Litchfield, K.
Levy, M.
Orlando, G.
Loveday, C.
Law, P.J.
Migliorini, G.
Holroyd, A.
Broderick, P.
Karlsson, R.
Haugen, T.B.
Kristiansen, W.
Nsengimana, J.
Fenwick, K.
Assiotis, I.
Kote-Jarai, Z.
Dunning, A.M.
Muir, K.
Peto, J.
Eeles, R.
Easton, D.F.
Dudakia, D.
Orr, N.
Pashayan, N.
UK Testicular Cancer Collaboration,
PRACTICAL Consortium,
Bishop, D.T.
Reid, A.
Huddart, R.A.
Shipley, J.
Grotmol, T.
Wiklund, F.
Houlston, R.S.
Turnbull, C.
(2017). Identification of 19 new risk loci and potential regulatory mechanisms influencing susceptibility to testicular germ cell tumor. Nat genet,
Vol.49
(7),
pp. 1133-1140.
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Genome-wide association studies (GWAS) have transformed understanding of susceptibility to testicular germ cell tumors (TGCTs), but much of the heritability remains unexplained. Here we report a new GWAS, a meta-analysis with previous GWAS and a replication series, totaling 7,319 TGCT cases and 23,082 controls. We identify 19 new TGCT risk loci, roughly doubling the number of known TGCT risk loci to 44. By performing in situ Hi-C in TGCT cells, we provide evidence for a network of physical interactions among all 44 TGCT risk SNPs and candidate causal genes. Our findings implicate widespread disruption of developmental transcriptional regulators as a basis of TGCT susceptibility, consistent with failed primordial germ cell differentiation as an initiating step in oncogenesis. Defective microtubule assembly and dysregulation of KIT-MAPK signaling also feature as recurrently disrupted pathways. Our findings support a polygenic model of risk and provide insight into the biological basis of TGCT..
Selfe, J.
Olmos, D.
Al-Saadi, R.
Thway, K.
Chisholm, J.
Kelsey, A.
Shipley, J.
(2017). Impact of fusion gene status versus histology on risk-stratification for rhabdomyosarcoma: Retrospective analyses of patients on UK trials. Pediatr blood cancer,
Vol.64
(7).
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BACKGROUND: Long-term toxicities from current treatments are a major issue in paediatric cancer. Previous studies, including our own, have shown prognostic value for the presence of PAX3/7-FOXO1 fusion genes in rhabdomyosarcoma (RMS). It is proposed to introduce PAX3/7-FOXO1 positivity as a component of risk stratification, rather than alveolar histology, in future clinical trials. PROCEDURE: To assess the potential impact of this reclassification, we have determined the changes to risk category assignment of 210 histologically reviewed patients treated in the UK from previous malignant mesenchymal tumour clinical trials for non-metastatic RMS based on identification of PAX3/7-FOXO1 by fluorescence in situ hybridisation and/or reverse transcription PCR. RESULTS: Using fusion gene positivity in the current risk stratification would reassign 7% of patients to different European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) risk groups. The next European trial would have 80% power to detect differences in event-free survival of 15% over 10 years and 20% over 5 years in reassigned patients. This would decrease treatment for over a quarter of patients with alveolar histology tumours that lack PAX3/7-FOXO1. CONCLUSIONS: Fusion gene status used in stratification may result in significant numbers of patients benefitting from lower treatment-associated toxicity. Prospective testing to show this reassignment maintains current survival rates is now required and is shown to be feasible based on estimated recruitment to a future EpSSG trial. Together with developing novel therapeutic strategies for patients identified as higher risk, this may ultimately improve the outcome and quality of life for patients with RMS..
van Erp, A.E.
Versleijen-Jonkers, Y.M.
Hillebrandt-Roeffen, M.H.
van Houdt, L.
Gorris, M.A.
van Dam, L.S.
Mentzel, T.
Weidema, M.E.
Savci-Heijink, C.D.
Desar, I.M.
Merks, H.H.
van Noesel, M.M.
Shipley, J.
van der Graaf, W.T.
Flucke, U.E.
Meyer-Wentrup, F.A.
(2017). Expression and clinical association of programmed cell death-1, programmed death-ligand-1 and CD8+ lymphocytes in primary sarcomas is subtype dependent. Oncotarget,
.
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In order to explore the potential of immune checkpoint blockade in sarcoma, we investigated expression and clinical relevance of programmed cell death-1 (PD-1), programmed death ligand-1 (PD-L1) and CD8 in tumors of 208 sarcoma patients. Primary untreated osteosarcoma (n = 46), Ewing sarcoma (n = 32), alveolar rhabdomyosarcoma (n = 20), embryonal rhabdomyosarcoma (n = 77), synovial sarcoma (n = 22) and desmoplastic small round cell tumors (DSRCT) (n = 11) were examined immunohistochemically. PD-L1 expression was predominantly detected in alveolar and embryonal rhabdomyosarcomas (15% and 16%, respectively). In the alveolar subtype PD-L1 expression was associated with better overall, event-free and metastases-free survival. PD-1 expression on lymphocytes was predominantly seen in synovial sarcomas (18%). High levels of CD8+ lymphocytes were predominantly detected in osteosarcomas (35%) and associated with worse event-free survival in synovial sarcomas. Ewing sarcoma and DSRCTs showed PD-1 on tumor cells instead of on tumor infiltrating lymphocytes. Overall, expression and clinical associations were found to be subtype dependent. For the first time PD-1 expression on Ewing sarcoma (19%) and DSRCT (82%) tumor cells was described..
Jones, S.E.
Fleuren, E.D.
Frankum, J.
Konde, A.
Williamson, C.T.
Krastev, D.B.
Pemberton, H.N.
Campbell, J.
Gulati, A.
Elliott, R.
Menon, M.
Selfe, J.L.
Brough, R.
Pettitt, S.J.
Niedzwiedz, W.
van der Graaf, W.T.
Shipley, J.
Ashworth, A.
Lord, C.J.
(2017). ATR Is a Therapeutic Target in Synovial Sarcoma. Cancer res,
Vol.77
(24),
pp. 7014-7026.
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Synovial sarcoma (SS) is an aggressive soft-tissue malignancy characterized by expression of SS18-SSX fusions, where treatment options are limited. To identify therapeutically actionable genetic dependencies in SS, we performed a series of parallel, high-throughput small interfering RNA (siRNA) screens and compared genetic dependencies in SS tumor cells with those in >130 non-SS tumor cell lines. This approach revealed a reliance of SS tumor cells upon the DNA damage response serine/threonine protein kinase ATR. Clinical ATR inhibitors (ATRi) elicited a synthetic lethal effect in SS tumor cells and impaired growth of SS patient-derived xenografts. Oncogenic SS18-SSX family fusion genes are known to alter the composition of the BAF chromatin-remodeling complex, causing ejection and degradation of wild-type SS18 and the tumor suppressor SMARCB1. Expression of oncogenic SS18-SSX fusion proteins caused profound ATRi sensitivity and a reduction in SS18 and SMARCB1 protein levels, but an SSX18-SSX1 Δ71-78 fusion containing a C-terminal deletion did not. ATRi sensitivity in SS was characterized by an increase in biomarkers of replication fork stress (increased γH2AX, decreased replication fork speed, and increased R-loops), an apoptotic response, and a dependence upon cyclin E expression. Combinations of cisplatin or PARP inhibitors enhanced the antitumor cell effect of ATRi, suggesting that either single-agent ATRi or combination therapy involving ATRi might be further assessed as candidate approaches for SS treatment. Cancer Res; 77(24); 7014-26. ©2017 AACR..
Izquierdo, E.
Yuan, L.
George, S.
Hubank, M.
Jones, C.
Proszek, P.
Shipley, J.
Gatz, S.A.
Stinson, C.
Moore, A.S.
Clifford, S.C.
Hicks, D.
Lindsey, J.C.
Hill, R.M.
Jacques, T.S.
Chalker, J.
Thway, K.
O'Connor, S.
Marshall, L.
Moreno, L.
Pearson, A.
Chesler, L.
Walker, B.A.
De Castro, D.G.
(2017). Development of a targeted sequencing approach to identify prognostic, predictive and diagnostic markers in paediatric solid tumours. Oncotarget,
Vol.8
(67),
pp. 112036-112050.
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The implementation of personalised medicine in childhood cancers has been limited by a lack of clinically validated multi-target sequencing approaches specific for paediatric solid tumours. In order to support innovative clinical trials in high-risk patients with unmet need, we have developed a clinically relevant targeted sequencing panel spanning 311 kb and comprising 78 genes involved in childhood cancers. A total of 132 samples were used for the validation of the panel, including Horizon Discovery cell blends (n=4), cell lines (n=15), formalin-fixed paraffin embedded (FFPE, n=83) and fresh frozen tissue (FF, n=30) patient samples. Cell blends containing known single nucleotide variants (SNVs, n=528) and small insertion-deletions (indels n=108) were used to define panel sensitivities of ≥98% for SNVs and ≥83% for indels [95% CI] and panel specificity of ≥98% [95% CI] for SNVs. FFPE samples performed comparably to FF samples (n=15 paired). Of 95 well-characterised genetic abnormalities in 33 clinical specimens and 13 cell lines (including SNVs, indels, amplifications, rearrangements and chromosome losses), 94 (98.9%) were detected by our approach. We have validated a robust and practical methodology to guide clinical management of children with solid tumours based on their molecular profiles. Our work demonstrates the value of targeted gene sequencing in the development of precision medicine strategies in paediatric oncology..
Missiaglia, E.
Shepherd, C.J.
Aladowicz, E.
Olmos, D.
Selfe, J.
Pierron, G.
Delattre, O.
Walters, Z.
Shipley, J.
(2017). MicroRNA and gene co-expression networks characterize biological and clinical behavior of rhabdomyosarcomas. Cancer lett,
Vol.385,
pp. 251-260.
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Rhabdomyosarcomas (RMS) in children and adolescents are heterogeneous sarcomas broadly defined by skeletal muscle features and the presence/absence of PAX3/7-FOXO1 fusion genes. MicroRNAs are small non-coding RNAs that regulate gene expression in a cell context specific manner. Sequencing analyses of microRNAs in 64 RMS revealed expression patterns separating skeletal muscle, fusion gene positive and negative RMS. Integration with parallel gene expression data assigned biological functions to 12 co-expression networks/modules that reassuringly included myogenic roles strongly correlated with microRNAs known in myogenesis and RMS development. Modules also correlated with clinical outcome and fusion status. Regulation of microRNAs by the fusion protein was demonstrated after PAX3-FOXO1 reduction, exemplified by miR-9-5p. MiR-9-5p levels correlated with poor outcome, even within fusion gene positive RMS, and were higher in metastatic versus non-metastatic disease. MiR-9-5p reduction inhibited RMS cell migration. Our findings reveal microRNAs in a regulatory framework of biological and clinical significance in RMS..
Gilbert, D.C.
Al-Saadi, R.
Thway, K.
Chandler, I.
Berney, D.
Gabe, R.
Stenning, S.P.
Sweet, J.
Huddart, R.
Shipley, J.M.
(2016). Defining a New Prognostic Index for Stage I Nonseminomatous Germ Cell Tumors Using CXCL12 Expression and Proportion of Embryonal Carcinoma. Clin cancer res,
Vol.22
(5),
pp. 1265-1273.
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PURPOSE: Up to 50% of patients diagnosed with stage I nonseminomatous germ cell tumors (NSGCTs) harbor occult metastases. Patients are managed by surveillance with chemotherapy at relapse or adjuvant treatment up front. Late toxicities from chemotherapy are increasingly recognized. Based on a potential biologic role in germ cells/tumors and pilot data, our aim was to evaluate tumor expression of the chemokine CXCL12 alongside previously proposed markers as clinically useful biomarkers of relapse. EXPERIMENTAL DESIGN: Immunohistochemistry for tumor expression of CXCL12 was assessed as a biomarker of relapse alongside vascular invasion, histology (percentage embryonal carcinoma), and MIB1 staining for proliferation in formalin-fixed paraffin-embedded orchidectomy samples from patients enrolled in the Medical Research Council's TE08/22 prospective trials of surveillance in stage I NSGCTs. RESULTS: TE08/TE22 trial patients had a 76.4% 2-year relapse-free rate, and both CXCL12 expression and percentage embryonal carcinoma provided prognostic value independently of vascular invasion (stratified log rank test P = 0.006 for both). There was no additional prognostic value for MIB1 staining. A model using CXCL12, percentage embryonal carcinoma, and VI defines three prognostic groups that were independently validated. CONCLUSIONS: CXCL12 and percentage embryonal carcinoma both stratify patients' relapse risk over and above vascular invasion alone. This is anticipated to improve the stratification of patients and identify high-risk cases to be considered for adjuvant therapy..
Litchfield, K.
Levy, M.
Huddart, R.A.
Shipley, J.
Turnbull, C.
(2016). The genomic landscape of testicular germ cell tumours: from susceptibility to treatment. Nat rev urol,
Vol.13
(7),
pp. 409-419.
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The genomic landscape of testicular germ cell tumour (TGCT) can be summarized using four overarching hypotheses. Firstly, TGCT risk is dominated by inherited genetic factors, which determine nearly half of all disease risk and are highly polygenic in nature. Secondly KIT-KITLG signalling is currently the major pathway that is implicated in TGCT formation, both as a predisposition risk factor and a somatic driver event. Results from genome-wide association studies have also consistently suggested that other closely related pathways involved in male germ cell development and sex determination are associated with TGCT risk. Thirdly, the method of disease formation is unique, with tumours universally stemming from a noninvasive precursor lesion, probably of fetal origin, which lies dormant through childhood into adolescence and then eventually begins malignant growth in early adulthood. Formation of a 12p isochromosome, a hallmark of TGCT observed in nearly all tumours, is likely to be a key triggering event for malignant transformation. Finally, TGCT have been shown to have a distinctive somatic mutational profile, with a low rate of point mutations contrasted with frequent large-scale chromosomal gains. These four hypotheses by no means constitute a complete model that explains TGCT tumorigenesis, but advances in genomic technologies have enabled considerable progress in describing and understanding the disease. Further advancing our understanding of the genomic basis of TGCT offers a clear opportunity for clinical benefit in terms of preventing invasive cancer arising in young men, decreasing the burden of chemotherapy-related survivorship issues and reducing mortality in the minority of patients who have treatment-refractory disease..
Thway, K.
Robertson, D.
Jones, R.L.
Selfe, J.
Shipley, J.
Fisher, C.
Isacke, C.M.
(2016). Endosialin expression in soft tissue sarcoma as a potential marker of undifferentiated mesenchymal cells. Br j cancer,
Vol.115
(4),
pp. 473-479.
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BACKGROUND: Soft tissue sarcomas are a group of neoplasms with differentiation towards mesenchymal tissue, many of which are aggressive and chemotherapy resistant. Histology and immunoprofiles often overlap with neoplasms of other lineages, and establishing an accurate histopathological diagnosis is crucial for correct management, and therapeutic stratification. The endosialin cell surface glycoprotein is predominantly expressed by stromal fibroblasts and pericytes in epithelial neoplasms; however, tumour cell expression has been reported in small series of sarcomas. METHODS: We assessed endosialin expression by immunohistochemistry in a large set of 514 human soft tissue sarcomas. RESULTS: Tumour cell endosialin expression was seen in 89% of undifferentiated pleomorphic sarcomas (104/117), 77% adult fibrosarcomas/spindle cell sarcomas (20/26), 62% synovial sarcomas (37/60), 51% leiomyosarcomas (94/185) and 31% rhabdomyosarcomas (39/126). CONCLUSIONS: Endosialin immunohistochemistry has potential to distinguish undifferentiated and poorly differentiated sarcomas from other poorly differentiated, non-mesenchymal neoplasms. A Phase II trial randomising patients with advanced sarcomas to receive chemotherapy with/without an endosialin therapeutic antibody has recently completed enrolment. Endosialin expression could be used to select patients for such clinical trials. Based on our results, patients with undifferentiated pleomorphic sarcoma may be particularly suitable for such a therapeutic approach..
Mohamed, A.
Sun, C.
De Mello, V.
Selfe, J.
Missiaglia, E.
Shipley, J.
Murray, G.I.
Zammit, P.S.
Wackerhage, H.
(2016). The Hippo effector TAZ (WWTR1) transforms myoblasts and TAZ abundance is associated with reduced survival in embryonal rhabdomyosarcoma. J pathol,
Vol.240
(1),
pp. 3-14.
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The Hippo effector YAP has recently been identified as a potent driver of embryonal rhabdomyosarcoma (ERMS). Most reports suggest that the YAP paralogue TAZ (gene symbol WWTR1) functions as YAP but, in skeletal muscle, TAZ has been reported to promote myogenic differentiation, whereas YAP inhibits it. Here, we investigated whether TAZ is also a rhabdomyosarcoma oncogene or whether TAZ acts as a YAP antagonist. Immunostaining of rhabdomyosarcoma tissue microarrays revealed that TAZ is significantly associated with poor survival in ERMS. In 12% of fusion gene-negative rhabdomyosarcomas, the TAZ locus is gained, which is correlated with increased expression. Constitutively active TAZ S89A significantly increased proliferation of C2C12 myoblasts and, importantly, colony formation on soft agar, suggesting transformation. However, TAZ then switches to enhance myogenic differentiation in C2C12 myoblasts, unlike YAP. Conversely, lentiviral shRNA-mediated TAZ knockdown in human ERMS cells reduced proliferation and anchorage-independent growth. While TAZ S89A or YAP1 S127A similarly activated the 8XGTIIC-Luc Hippo reporter, only YAP1 S127A activated the Brachyury (T-box) reporter. Consistent with its oncogene function, TAZ S89A induced expression of the ERMS cancer stem cell gene Myf5 and the serine biosynthesis pathway (Phgdh, Psat1, Psph) in C2C12 myoblasts. Thus, TAZ is associated with poor survival in ERMS and could act as an oncogene in rhabdomyosarcoma. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland..
Bavetsias, V.
Lanigan, R.M.
Ruda, G.F.
Atrash, B.
McLaughlin, M.G.
Tumber, A.
Mok, N.Y.
Le Bihan, Y.-.
Dempster, S.
Boxall, K.J.
Jeganathan, F.
Hatch, S.B.
Savitsky, P.
Velupillai, S.
Krojer, T.
England, K.S.
Sejberg, J.
Thai, C.
Donovan, A.
Pal, A.
Scozzafava, G.
Bennett, J.M.
Kawamura, A.
Johansson, C.
Szykowska, A.
Gileadi, C.
Burgess-Brown, N.A.
von Delft, F.
Oppermann, U.
Walters, Z.
Shipley, J.
Raynaud, F.I.
Westaway, S.M.
Prinjha, R.K.
Fedorov, O.
Burke, R.
Schofield, C.J.
Westwood, I.M.
Bountra, C.
Müller, S.
van Montfort, R.L.
Brennan, P.E.
Blagg, J.
(2016). 8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors. J med chem,
Vol.59
(4),
pp. 1388-1409.
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We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay..
Litchfield, K.
Shipley, J.
Turnbull, C.
(2015). Common variants identified in genome-wide association studies of testicular germ cell tumour: an update, biological insights and clinical application. Andrology,
Vol.3
(1),
pp. 34-46.
show abstract
Testicular germ cell tumour (TGCT) is the most common cause of cancer in young men (aged 15-45Â years) in many populations. Multiple genome-wide association studies (GWAS) of TGCT have now been conducted, yielding over 25 disease-associated single-nucleotide polymorphism (SNP)s at 19 independent loci. The genes at these loci have provided rich biological and genetic insight into possible mechanisms underlying testicular germ cell oncogenesis. In this review, we summarize these mechanisms which can be grouped into five distinct categories: KIT/KITLG signalling, other pathways of male germ cell development/differentiation, telomerase function, microtubule assembly and DNA damage repair. The TGCT risk markers identified through GWAS include individual SNPs carrying per allele odds ratios (OR) in excess of 2.5. These ORs are among the highest reported in GWAS of any cancer type, hence suggesting a potential clinical utility in risk determination. Here, we present analysis of such an approach, using polygenic risk scores to calculate the combined effect of all risk loci on overall TGCT risk and discuss how a potential screening strategy may fit within a broader clinical context..
Gatz, S.A.
Shipley, J.M.
(2015). Less Can Be More for Gene Dose and Drug Sensitivity. Clin cancer res,
Vol.21
(21),
pp. 4750-4752.
show abstract
CDK4 is preclinically validated as a therapeutic target in PAX3-FOXO1 fusion gene-positive rhabdomyosarcomas. Pharmacologic targeting showed sensitivity but, contrary to expectation, CDK4 genomic amplification and overexpression associated with 25% of cases that exhibited the lowest sensitivities. This emphasizes the importance of tumor-specific preclinical studies to define and understand drug sensitivity..
Hingorani, P.
Missiaglia, E.
Shipley, J.
Anderson, J.R.
Triche, T.J.
Delorenzi, M.
Gastier-Foster, J.
Wing, M.
Hawkins, D.S.
Skapek, S.X.
(2015). Clinical Application of Prognostic Gene Expression Signature in Fusion Gene-Negative Rhabdomyosarcoma: A Report from the Children's Oncology Group. Clin cancer res,
Vol.21
(20),
pp. 4733-4739.
show abstract
PURPOSE: Pediatric rhabdomyosarcoma (RMS) has two common histologic subtypes: embryonal (ERMS) and alveolar (ARMS). PAX-FOXO1 fusion gene status is a more reliable prognostic marker than alveolar histology, whereas fusion gene-negative (FN) ARMS patients are clinically similar to ERMS patients. A five-gene expression signature (MG5) previously identified two diverse risk groups within the fusion gene-negative RMS (FN-RMS) patients, but this has not been independently validated. The goal of this study was to test whether expression of the MG5 metagene, measured using a technical platform that can be applied to routine pathology material, would correlate with outcome in a new cohort of patients with FN-RMS. EXPERIMENTAL DESIGN: Cases were taken from the Children's Oncology Group (COG) D9803 study of children with intermediate-risk RMS, and gene expression profiling for the MG5 genes was performed using the nCounter assay. The MG5 score was correlated with clinical and pathologic characteristics as well as overall and event-free survival. RESULTS: MG5 standardized score showed no significant association with any of the available clinicopathologic variables. The MG5 signature score showed a significant correlation with overall (N = 57; HR, 7.3; 95% CI, 1.9-27.0; P = 0.003) and failure-free survival (N = 57; HR, 6.1; 95% CI, 1.9-19.7; P = 0.002). CONCLUSIONS: This represents the first, validated molecular prognostic signature for children with FN-RMS who otherwise have intermediate-risk disease. The capacity to measure the expression of a small number of genes in routine pathology material and apply a simple mathematical formula to calculate the MG5 metagene score provides a clear path toward better risk stratification in future prospective clinical trials..
Litchfield, K.
Mitchell, J.S.
Shipley, J.
Huddart, R.
Rajpert-De Meyts, E.
Skakkebæk, N.E.
Houlston, R.S.
Turnbull, C.
(2015). Polygenic susceptibility to testicular cancer: implications for personalised health care. Br j cancer,
Vol.113
(10),
pp. 1512-1518.
show abstract
BACKGROUND: The increasing incidence of testicular germ cell tumour (TGCT) combined with its strong heritable basis suggests that stratified screening for the early detection of TGCT may be clinically useful. We modelled the efficiency of such a personalised screening approach, based on genetic risk profiling in combination with other diagnostic tools. METHODS: We compared the number of cases potentially detectable in the population under a number of screening models. The polygenic risk scoring (PRS) model was assumed to have a log-normal relative risk distribution across the 19 currently known TGCT susceptibility variants. The diagnostic performance of testicular biopsy and non-invasive semen analysis was also assessed, within a simulated combined screening programme. RESULTS: The area under the curve for the TGCT PRS model was 0.72 with individuals in the top 1% of the PRS having a nine-fold increased TGCT risk compared with the population median. Results from population-screening simulations only achieved a maximal positive predictive value (PPV) of 60%, highlighting broader clinical factors that challenge such strategies, not least the rare nature of TGCT. In terms of future improvements, heritability estimates suggest that a significant number of additional genetic risk factors for TGCT remain to be discovered, identification of which would potentially yield improvement of the PPV to 80-90%. CONCLUSIONS: While personalised screening models may offer enhanced TGCT risk discrimination, presently the case for population-level testing is not compelling. However, future advances, such as more routine generation of whole genome data is likely to alter the landscape. More targeted screening programs may plausibly then offer clinical benefit, particularly given the significant survivorship issues associated with the successful treatment of TGCT..
Litchfield, K.
Summersgill, B.
Yost, S.
Sultana, R.
Labreche, K.
Dudakia, D.
Renwick, A.
Seal, S.
Al-Saadi, R.
Broderick, P.
Turner, N.C.
Houlston, R.S.
Huddart, R.
Shipley, J.
Turnbull, C.
(2015). Whole-exome sequencing reveals the mutational spectrum of testicular germ cell tumours. Nat commun,
Vol.6,
p. 5973.
show abstract
full text
Testicular germ cell tumours (TGCTs) are the most common cancer in young men. Here we perform whole-exome sequencing (WES) of 42 TGCTs to comprehensively study the cancer's mutational profile. The mutation rate is uniformly low in all of the tumours (mean 0.5 mutations per Mb) as compared with common cancers, consistent with the embryological origin of TGCT. In addition to expected copy number gain of chromosome 12p and mutation of KIT, we identify recurrent mutations in the tumour suppressor gene CDC27 (11.9%). Copy number analysis reveals recurring amplification of the spermatocyte development gene FSIP2 (15.3%) and a 0.4 Mb region at Xq28 (15.3%). Two treatment-refractory patients are shown to harbour XRCC2 mutations, a gene strongly implicated in defining cisplatin resistance. Our findings provide further insights into genes involved in the development and progression of TGCT..
Litchfield, K.
Holroyd, A.
Lloyd, A.
Broderick, P.
Nsengimana, J.
Eeles, R.
Easton, D.F.
Dudakia, D.
Bishop, D.T.
Reid, A.
Huddart, R.A.
Grotmol, T.
Wiklund, F.
Shipley, J.
Houlston, R.S.
Turnbull, C.
(2015). Identification of four new susceptibility loci for testicular germ cell tumour. Nat commun,
Vol.6,
p. 8690.
show abstract
Genome-wide association studies (GWAS) have identified multiple risk loci for testicular germ cell tumour (TGCT), revealing a polygenic model of disease susceptibility strongly influenced by common variation. To identify additional single-nucleotide polymorphisms (SNPs) associated with TGCT, we conducted a multistage GWAS with a combined data set of >25,000 individuals (6,059 cases and 19,094 controls). We identified new risk loci for TGCT at 3q23 (rs11705932, TFDP2, P=1.5 × 10(-9)), 11q14.1 (rs7107174, GAB2, P=9.7 × 10(-11)), 16p13.13 (rs4561483, GSPT1, P=1.6 × 10(-8)) and 16q24.2 (rs55637647, ZFPM1, P=3.4 × 10(-9)). We additionally present detailed functional analysis of these loci, identifying a statistically significant relationship between rs4561483 risk genotype and increased GSPT1 expression in TGCT patient samples. These findings provide additional support for a polygenic model of TGCT risk and further insight into the biological basis of disease development..
Collinson, K.
Murray, M.J.
Orsi, N.M.
Cummings, M.
Shipley, J.
Joffe, J.K.
Coleman, N.
Stark, D.
(2014). Age-Related Biological Features of Germ Cell Tumors. Genes chromosomes & cancer,
Vol.53
(3),
pp. 215-227.
Yeste-Velasco, M.
Mao, X.
Grose, R.
Kudahetti, S.C.
Lin, D.
Marzec, J.
Vasiljevic, N.
Chaplin, T.
Xue, L.
Xu, M.
Foster, J.M.
Karnam, S.S.
James, S.Y.
Chioni, A.-.
Gould, D.
Lorincz, A.T.
Oliver, R.T.
Chelala, C.
Thomas, G.M.
Shipley, J.M.
Mather, S.J.
Berney, D.M.
Young, B.D.
Lu, Y.-.
(2014). Identification of ZDHHC14 as a novel human tumour suppressor gene. Journal of pathology,
Vol.232
(5),
pp. 566-577.
Ciarapica, R.
De Salvo, M.
Carcarino, E.
Bracaglia, G.
Adesso, L.
Leoncini, P.P.
Dall'Agnese, A.
Walters, Z.S.
Verginelli, F.
De Sio, L.
Boldrini, R.
Inserra, A.
Bisogno, G.
Rosolen, A.
Alaggio, R.
Ferrari, A.
Collini, P.
Locatelli, M.
Stifani, S.
Screpanti, I.
Rutella, S.
Yu, Q.
Marquez, V.E.
Shipley, J.
Valente, S.
Mai, A.
Miele, L.
Puri, P.L.
Locatelli, F.
Palacios, D.
Rota, R.
(2014). The Polycomb group (PcG) protein EZH2 supports the survival of PAX3-FOXO1 alveolar rhabdomyosarcoma by repressing FBXO32 (Atrogin1/MAFbx). Oncogene,
Vol.33
(32),
pp. 4173-4184.
show abstract
The Polycomb group (PcG) proteins regulate stem cell differentiation via the repression of gene transcription, and their deregulation has been widely implicated in cancer development. The PcG protein Enhancer of Zeste Homolog 2 (EZH2) works as a catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) by methylating lysine 27 on histone H3 (H3K27me3), a hallmark of PRC2-mediated gene repression. In skeletal muscle progenitors, EZH2 prevents an unscheduled differentiation by repressing muscle-specific gene expression and is downregulated during the course of differentiation. In rhabdomyosarcoma (RMS), a pediatric soft-tissue sarcoma thought to arise from myogenic precursors, EZH2 is abnormally expressed and its downregulation in vitro leads to muscle-like differentiation of RMS cells of the embryonal variant. However, the role of EZH2 in the clinically aggressive subgroup of alveolar RMS, characterized by the expression of PAX3-FOXO1 oncoprotein, remains unknown. We show here that EZH2 depletion in these cells leads to programmed cell death. Transcriptional derepression of F-box protein 32 (FBXO32) (Atrogin1/MAFbx), a gene associated with muscle homeostasis, was evidenced in PAX3-FOXO1 RMS cells silenced for EZH2. This phenomenon was associated with reduced EZH2 occupancy and H3K27me3 levels at the FBXO32 promoter. Simultaneous knockdown of FBXO32 and EZH2 in PAX3-FOXO1 RMS cells impaired the pro-apoptotic response, whereas the overexpression of FBXO32 facilitated programmed cell death in EZH2-depleted cells. Pharmacological inhibition of EZH2 by either 3-Deazaneplanocin A or a catalytic EZH2 inhibitor mirrored the phenotypic and molecular effects of EZH2 knockdown in vitro and prevented tumor growth in vivo. Collectively, these results indicate that EZH2 is a key factor in the proliferation and survival of PAX3-FOXO1 alveolar RMS cells working, at least in part, by repressing FBXO32. They also suggest that the reducing activity of EZH2 could represent a novel adjuvant strategy to eradicate high-risk PAX3-FOXO1 alveolar RMS..
Tremblay, A.M.
Missiaglia, E.
Galli, G.G.
Hettmer, S.
Urcia, R.
Carrara, M.
Judson, R.N.
Thway, K.
Nadal, G.
Selfe, J.L.
Murray, G.
Calogero, R.A.
De Bari, C.
Zammit, P.S.
Delorenzi, M.
Wagers, A.J.
Shipley, J.
Wackerhage, H.
Camargo, F.D.
(2014). The Hippo transducer YAP1 transforms activated satellite cells and is a potent effector of embryonal rhabdomyosarcoma formation. Cancer cell,
Vol.26
(2),
pp. 273-287.
show abstract
full text
The role of the Hippo pathway effector YAP1 in soft tissue sarcomas is poorly defined. Here we report that YAP1 activity is elevated in human embryonal rhabdomyosarcoma (ERMS). In mice, sustained YAP1 hyperactivity in activated, but not quiescent, satellite cells induces ERMS with high penetrance and short latency. Via its transcriptional program with TEAD1, YAP1 directly regulates several major hallmarks of ERMS. YAP1-TEAD1 upregulate pro-proliferative and oncogenic genes and maintain the ERMS differentiation block by interfering with MYOD1 and MEF2 pro-differentiation activities. Normalization of YAP1 expression reduces tumor burden in human ERMS xenografts and allows YAP1-driven ERMS to differentiate in situ. Collectively, our results identify YAP1 as a potent ERMS oncogenic driver and a promising target for differentiation therapy..
Hettmer, S.
Li, Z.
Billin, A.N.
Barr, F.G.
Cornelison, D.D.
Ehrlich, A.R.
Guttridge, D.C.
Hayes-Jordan, A.
Helman, L.J.
Houghton, P.J.
Khan, J.
Langenau, D.M.
Linardic, C.M.
Pal, R.
Partridge, T.A.
Pavlath, G.K.
Rota, R.
Schaefer, B.W.
Shipley, J.
Stillman, B.
Wexler, L.H.
Wagers, A.J.
Keller, C.
(2014). Rhabdomyosarcoma: Current Challenges and Their Implications for Developing Therapies. Cold spring harbor perspectives in biology,
Vol.6
(11).
Walters, Z.S.
Villarejo-Balcells, B.
Olmos, D.
Buist, T.W.
Missiaglia, E.
Allen, R.
Al-Lazikani, B.
Garrett, M.D.
Blagg, J.
Shipley, J.
(2014). JARID2 is a direct target of the PAX3-FOXO1 fusion protein and inhibits myogenic differentiation of rhabdomyosarcoma cells. Oncogene,
Vol.33
(9),
pp. 1148-1157.
show abstract
Rhabdomyosarcomas (RMS) are the most frequent soft-tissue sarcoma in children and characteristically show features of developing skeletal muscle. The alveolar subtype is frequently associated with a PAX3-FOXO1 fusion protein that is known to contribute to the undifferentiated myogenic phenotype of RMS cells. Histone methylation of lysine residues controls developmental processes in both normal and malignant cell contexts. Here we show that JARID2, which encodes a protein known to recruit various complexes with histone-methylating activity to their target genes, is significantly overexpressed in RMS with PAX3-FOXO1 compared with the fusion gene-negative RMS (t-test; P < 0.0001). Multivariate analyses showed that higher JARID2 levels are also associated with metastases at diagnosis, independent of fusion gene status and RMS subtype (n = 120; P = 0.039). JARID2 levels were altered by silencing or overexpressing PAX3-FOXO1 in RMS cell lines with and without the fusion gene, respectively. Consistent with this, we demonstrated that JARID2 is a direct transcriptional target of the PAX3-FOXO1 fusion protein. Silencing JARID2 resulted in reduced cell proliferation coupled with myogenic differentiation, including increased expression of Myogenin (MYOG) and Myosin Light Chain (MYL1) in RMS cell lines representative of both the alveolar and embryonal subtypes. Induced myogenic differentiation was associated with a decrease in JARID2 levels and this phenotype could be rescued by overexpressing JARID2. Furthermore, we that showed JARID2 binds to and alters the methylation status of histone H3 lysine 27 in the promoter regions of MYOG and MYL1 and that the interaction of JARID2 at these promoters is dependent on EED, a core component of the polycomb repressive complex 2 (PRC2). Therefore, JARID2 is a downstream effector of PAX3-FOXO1 that maintains an undifferentiated myogenic phenotype that is characteristic of RMS. JARID2 and other components of PRC2 may represent novel therapeutic targets for treating RMS patients..
Ehnman, M.
Missiaglia, E.
Folestad, E.
Selfe, J.
Strell, C.
Thway, K.
Brodin, B.
Pietras, K.
Shipley, J.
Östman, A.
Eriksson, U.
(2013). Distinct effects of ligand-induced PDGFRα and PDGFRβ signaling in the human rhabdomyosarcoma tumor cell and stroma cell compartments. Cancer res,
Vol.73
(7),
pp. 2139-2149.
show abstract
Platelet-derived growth factor receptors (PDGFR) α and β have been suggested as potential targets for treatment of rhabdomyosarcoma, the most common soft tissue sarcoma in children. This study identifies biologic activities linked to PDGF signaling in rhabdomyosarcoma models and human sample collections. Analysis of gene expression profiles of 101 primary human rhabdomyosarcomas revealed elevated PDGF-C and -D expression in all subtypes, with PDGF-D as the solely overexpressed PDGFRβ ligand. By immunohistochemistry, PDGF-CC, PDGF-DD, and PDGFRα were found in tumor cells, whereas PDGFRβ was primarily detected in vascular stroma. These results are concordant with the biologic processes and pathways identified by data mining. While PDGF-CC/PDGFRα signaling associated with genes involved in the reactivation of developmental programs, PDGF-DD/PDGFRβ signaling related to wound healing and leukocyte differentiation. Clinicopathologic correlations further identified associations between PDGFRβ in vascular stroma and the alveolar subtype and with presence of metastases. Functional validation of our findings was carried out in molecularly distinct model systems, where therapeutic targeting reduced tumor burden in a PDGFR-dependent manner with effects on cell proliferation, vessel density, and macrophage infiltration. The PDGFR-selective inhibitor CP-673,451 regulated cell proliferation through mechanisms involving reduced phosphorylation of GSK-3α and GSK-3β. Additional tissue culture studies showed a PDGFR-dependent regulation of rhabdosphere formation/cancer cell stemness, differentiation, senescence, and apoptosis. In summary, the study shows a clinically relevant distinction in PDGF signaling in human rhabdomyosarcoma and also suggests continued exploration of the influence of stromal PDGFRs on sarcoma progression..
Wickramasinghe, C.M.
Domaschenz, R.
Amagase, Y.
Williamson, D.
Missiaglia, E.
Shipley, J.
Murai, K.
Jones, P.H.
(2013). HES6 enhances the motility of alveolar rhabdomyosarcoma cells. Experimental cell research,
Vol.319
(1),
pp. 103-112.
Renshaw, J.
Taylor, K.R.
Bishop, R.
Valenti, M.
De Haven Brandon, A.
Gowan, S.
Eccles, S.A.
Ruddle, R.
Johnson, L.D.
Raynaud, F.I.
Selfe, J.
Thway, K.
Pietsch, T.
Pearson, A.D.
Shipley, J.
(2013). Dual blockade of the PI3K/AKT/mTOR (AZD8055) and RAS/MEK/ERK (AZD6244) pathways synergistically inhibits rhabdomyosarcoma cell growth in vitro and in vivo. Clin cancer res,
.
show abstract
full text
PURPOSE:
To provide rationale for using PI3K and/or MAPK pathway inhibitors to treat rhabdomyosarcomas (RMS), a major cause of pediatric/adolescent cancer deaths.
EXPERIMENTAL DESIGN:
The prevalence of PI3K/MAPK pathway activation in RMS clinical samples was assessed using immunohistochemistry. Compensatory signaling and crosstalk between PI3K/MAPK pathways was determined in RMS cell lines following p110α shRNA-mediated depletion. Pharmacological inhibition of reprogrammed signaling in stable p110α knockdown lines was used to determine the target-inhibition profile inducing maximal growth inhibition. The in vitro and in vivo efficacy of inhibitors of TORC1/2(AZD8055), MEK(AZD6244) and P13K/mTOR(NVP-BEZ235) were evaluated alone and in pair-wise combinations.
RESULTS:
PI3K pathway activation was seen in 82.5% rhabdomyosarcomas with co-activated MAPK in 36% and 46% of alveolar and embryonal sub-types respectively. p110α knockdown in cell lines over the short and long term was associated with compensatory expression of other p110 isoforms, activation of the MAPK pathway and cross-talk to reactivate the PI3K pathway. Combinations of PI3K pathway and MEK inhibitors synergistically inhibited cell growth in vitro. Treatment of RD cells with AZD8055 plus AZD6244 blocked reciprocal pathway activation, as evidenced by reduced AKT/ERK/S6 phosphorylation. In vivo, the synergistic effect on growth and changes in pharmacodynamic biomarkers was recapitulated using the AZD8055/AZD6244 combination but not NVP-BEZ235/AZD6244. Pharmacokinetic analysis provided evidence of drug-drug interaction with both combinations.
CONCLUSIONS:
Dual PI3K/MAPK pathway activation and compensatory signaling in both rhabdomyosarcoma subtypes predicts a lack of clinical efficacy for single agents targeting either pathway, supporting a therapeutic strategy combining a TORC1/2 with a MEK inhibitor..
Ruark, E.
Seal, S.
McDonald, H.
Zhang, F.
Elliot, A.
Lau, K.
Perdeaux, E.
Rapley, E.
Eeles, R.
Peto, J.
Kote-Jarai, Z.
Muir, K.
Nsengimana, J.
Shipley, J.
UK Testicular Cancer Collaboration (UKTCC),
Bishop, D.T.
Stratton, M.R.
Easton, D.F.
Huddart, R.A.
Rahman, N.
Turnbull, C.
(2013). Identification of nine new susceptibility loci for testicular cancer, including variants near DAZL and PRDM14. Nat genet,
Vol.45
(6),
pp. 686-689.
show abstract
full text
Testicular germ cell tumor (TGCT) is the most common cancer in young men and is notable for its high familial risks. So far, six loci associated with TGCT have been reported. From genome-wide association study (GWAS) analysis of 307,291 SNPs in 986 TGCT cases and 4,946 controls, we selected for follow-up 694 SNPs, which we genotyped in a further 1,064 TGCT cases and 10,082 controls from the UK. We identified SNPs at nine new loci (1q22, 1q24.1, 3p24.3, 4q24, 5q31.1, 8q13.3, 16q12.1, 17q22 and 21q22.3) showing association with TGCT (P < 5 × 10(-8)), which together account for an additional 4-6% of the familial risk of TGCT. The loci include genes plausibly related to TGCT development. PRDM14, at 8q13.3, is essential for early germ cell specification, and DAZL, at 3p24.3, is required for the regulation of germ cell development. Furthermore, PITX1, at 5q31.1, regulates TERT expression and is the third TGCT-associated locus implicated in telomerase regulation..
Tonelli, R.
McIntyre, A.
Camerin, C.
Walters, Z.S.
Di Leo, K.
Selfe, J.
Purgato, S.
Missiaglia, E.
Tortori, A.
Renshaw, J.
Astolfi, A.
Taylor, K.R.
Serravalle, S.
Bishop, R.
Nanni, C.
Valentijn, L.J.
Faccini, A.
Leuschner, I.
Formica, S.
Reis-Filho, J.S.
Ambrosini, V.
Thway, K.
Franzoni, M.
Summersgill, B.
Marchelli, R.
Hrelia, P.
Cantelli-Forti, G.
Fanti, S.
Corradini, R.
Pession, A.
Shipley, J.
(2012). Antitumor activity of sustained N-myc reduction in rhabdomyosarcomas and transcriptional block by antigene therapy. Clin cancer res,
Vol.18
(3),
pp. 796-807.
show abstract
PURPOSE: Rhabdomyosarcomas are a major cause of cancer death in children, described with MYCN amplification and, in the alveolar subtype, transcription driven by the PAX3-FOXO1 fusion protein. Our aim was to determine the prevalence of N-Myc protein expression and the potential therapeutic effects of reducing expression in rhabdomyosarcomas, including use of an antigene strategy that inhibits transcription. EXPERIMENTAL DESIGN: Protein expression was assessed by immunohistochemistry. MYCN expression was reduced in representative cell lines by RNA interference and an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide. Associated gene expression changes, cell viability, and apoptosis were analyzed in vitro. As a paradigm for antigene therapy, the effects of systemic treatment of mice with rhabdomyosarcoma cell line xenografts were determined. RESULTS: High N-Myc levels were significantly associated with genomic amplification, presence of the PAX3/7-FOXO1 fusion genes, and proliferative capacity. Sustained reduction of N-Myc levels in all rhabdomyosarcoma cell lines that express the protein decreased cell proliferation and increased apoptosis. Positive feedback was shown to regulate PAX3-FOXO1 and N-Myc levels in the alveolar subtype that critically decrease PAX3-FOXO1 levels on reducing N-Myc. Pharmacologic systemic administration of the antigene PNA can eliminate alveolar rhabdomyosarcoma xenografts in mice, without relapse or toxicity. CONCLUSION: N-Myc, with its restricted expression in non-fetal tissues, is a therapeutic target to treat rhabdomyosarcomas, and blocking gene transcription using antigene oligonucleotide strategies has therapeutic potential in the treatment of cancer and other diseases that has not been previously realized in vivo..
Thway, K.
Olmos, D.
Shah, C.
Flora, R.
Shipley, J.
Fisher, C.
(2012). Oncocytic adrenal cortical carcinosarcoma with pleomorphic rhabdomyosarcomatous metastases. Am j surg pathol,
Vol.36
(3),
pp. 470-477.
show abstract
Adrenal cortical carcinosarcoma is a rare variant of adrenal cortical carcinoma. Sarcomatous change in adrenal cortical carcinomas is exceptionally rare, with only 9 cases previously described. Adrenal cortical carcinosarcomas tend to be aggressive tumors, with locoregional recurrence and rapid metastases from the sarcomatoid component. We describe what seems to be the first case of sarcoma arising in oncocytic adrenal cortical carcinoma. The sarcomatous component here was pleomorphic rhabdomyosarcoma. This occurred in a 45-year-old man who had nodal and pulmonary metastases of the rhabdomyosarcomatous component at presentation and who died of progressive disease 11 months later. Here, we discuss the clinical, radiologic, and pathologic findings and review the literature on adrenal cortical carcinosarcomas..
Thway, K.
Selfe, J.
Shipley, J.
(2012). Immunohistochemical detection of glypican-5 in paraffin-embedded material: an optimized method for a novel research antibody. Appl immunohistochem mol morphol,
Vol.20
(2),
pp. 189-195.
show abstract
Glypican-5 (GPC5) is a cell surface heparan sulfate proteoglycan and 1 of 6 closely related members of the glypican family in mammals. Glypicans are predominantly expressed during development in cell-specific and tissue-specific contexts, and the expression of some is linked to developmental disorders and several visceral malignancies. We have previously shown that the region of amplification at 13q31.3 in a subset of rhabdomyosarcomas contains the GPC5 locus, and by copy number and gene expression analyses, that GPC5 is consistently expressed and upregulated in amplified tumors. As the immunohistochemical profile of GPC5 is untested, our aim was to optimize a commercially available anti-human GPC5 antibody for immunohistochemical use in formalin-fixed and paraffin-embedded (FFPE) tissue. Quantitative real-time polymerase chain reaction analyses of normal tissue samples indicated that the brain and testis highly expressed GPC5. High protein expression in these tissues and a cell line constructed to overexpress GPC5 were demonstrated by Western blotting. These normal tissues and the isogenic cell line were FFPE, and immunohistochemical expression of GPC5 was assessed using different methods of antigen retrieval, detection, and primary antibody concentration. The optimum conditions for detection were by heat-induced antigen retrieval, in sodium citrate buffer at pH 6. Enzyme-mediated retrieval did not produce effective detection, producing weaker, less well-localized GPC5 expression. We demonstrate that anti-human GPC5 antibody is amenable to use in FFPE tissue and with the optimized protocol we describe shows specific cellular localization and good staining intensity with minimal background staining..
Williamson, D.
Missiaglia, E.
Chisholm, J.
Shipley, J.
(2012). Inconvenience of convenience cohorts--letter. Cancer epidemiol biomarkers prev,
Vol.21
(8),
p. 1388.
Missiaglia, E.
Williamson, D.
Chisholm, J.
Wirapati, P.
Pierron, G.
Petel, F.
Concordet, J.-.
Thway, K.
Oberlin, O.
Pritchard-Jones, K.
Delattre, O.
Delorenzi, M.
Shipley, J.
(2012). Questionable Universal Validity of PAX3/FOXO1 Fusion Gene Status As Molecular Marker for Improvement of Risk Stratification in Rhabdomyosarcoma Therapy Reply. Journal of clinical oncology,
Vol.30
(32),
pp. 4040-4041.
Missiaglia, E.
Williamson, D.
Chisholm, J.
Wirapati, P.
Pierron, G.
Petel, F.
Concordet, J.-.
Thway, K.
Oberlin, O.
Pritchard-Jones, K.
Delattre, O.
Delorenzi, M.
Shipley, J.
(2012). PAX3/FOXO1 fusion gene status is the key prognostic molecular marker in rhabdomyosarcoma and significantly improves current risk stratification. J clin oncol,
Vol.30
(14),
pp. 1670-1677.
show abstract
PURPOSE: To improve the risk stratification of patients with rhabdomyosarcoma (RMS) through the use of clinical and molecular biologic data. PATIENTS AND METHODS: Two independent data sets of gene-expression profiling for 124 and 101 patients with RMS were used to derive prognostic gene signatures by using a meta-analysis. These and a previously published metagene signature were evaluated by using cross validation analyses. A combined clinical and molecular risk-stratification scheme that incorporated the PAX3/FOXO1 fusion gene status was derived from 287 patients with RMS and evaluated. RESULTS: We showed that our prognostic gene-expression signature and the one previously published performed well with reproducible and significant effects. However, their effect was reduced when cross validated or tested in independent data and did not add new prognostic information over the fusion gene status, which is simpler to assay. Among nonmetastatic patients, patients who were PAX3/FOXO1 positive had a significantly poorer outcome compared with both alveolar-negative and PAX7/FOXO1-positive patients. Furthermore, a new clinicomolecular risk score that incorporated fusion gene status (negative and PAX3/FOXO1 and PAX7/FOXO1 positive), Intergroup Rhabdomyosarcoma Study TNM stage, and age showed a significant increase in performance over the current risk-stratification scheme. CONCLUSION: Gene signatures can improve current stratification of patients with RMS but will require complex assays to be developed and extensive validation before clinical application. A significant majority of their prognostic value was encapsulated by the fusion gene status. A continuous risk score derived from the combination of clinical parameters with the presence or absence of PAX3/FOXO1 represents a robust approach to improving current risk-adapted therapy for RMS..
Yeste-Velasco, M.
Mao, X.
Grose, R.
Kudahetti, S.C.
Lin, D.
Chaplin, T.
Xue, L.
Xu, M.
Foster, J.M.
James, S.
Chioni, A.M.
Jeetle, S.
Vasiljevic, N.A.
Marzec, J.
Gould, D.
Jones, L.
Lorincz, A.T.
Oliver, R.T.
Mather, S.J.
Shipley, J.M.
Berney, D.M.
Young, B.D.
Lu, Y.-.
(2012). Identification of ZDHHC14 as a novel tumor suppressor gene commonly downregulated in human cancers. Cancer research,
Vol.72.
Gilbert, D.
Rapley, E.
Shipley, J.
(2011). Testicular germ cell tumours: predisposition genes and the male germ cell niche. Nat rev cancer,
Vol.11
(4),
pp. 278-288.
show abstract
Testicular germ cell tumours (TGCTs) of adults and adolescents are putatively derived from primordial germ cells or gonocytes. Recently reported genome-wide association studies implicate six gene loci that predispose to TGCT development. Remarkably, the functions of proteins encoded by genes within these regions bridge our understanding between the pathways involved in primordial germ cell physiology, male germ cell development and the molecular pathology of TGCTs. Furthermore, this improved understanding of the mechanisms underlying TGCT development and dissemination has clinical relevance for the management of patients with these tumours..
Thway, K.
Selfe, J.
Missiaglia, E.
Fisher, C.
Shipley, J.
(2011). Glypican-3 is expressed in rhabdomyosarcomas but not adult spindle cell and pleomorphic sarcomas. J clin pathol,
Vol.64
(7),
pp. 587-591.
show abstract
AIMS: Glypican-3 (GPC3) is a membrane-bound heparan sulphate proteoglycan. Glypicans are predominantly expressed during development in cell- and tissue-specific manners and act as co-receptors for many heparin-binding growth factors, playing important roles in cell growth and differentiation. GPC3 expression has been linked to several visceral malignancies, but its role in sarcomas is unexplored. Rhabdomyosarcomas (RMS), the most common paediatric soft tissue sarcomas (STS), are aggressive tumours that frequently show histological overlap with other STS. Immunohistochemistry was performed on tissue microarrays to study GPC3 expression in paediatric RMS and a variety of the more common spindle cell and pleomorphic adult sarcomas. METHODS: 642 tumour samples encompassing alveolar and embryonal RMS and adult STS (fibrosarcoma, synovial sarcoma, leiomyosarcoma and undifferentiated pleomorphic sarcoma) were stained with anti- GPC3, and percentage expression in tumour cells was scored on a scale of 0 to +3. RESULTS: GPC3 immunostaining was positive (+1 to +3 expression) in 34.7% (74/213) of RMS samples (15/60 alveolar, 54/143 embryonal, 5 RMS not otherwise specified), but negative or negligible in all other sarcoma samples. CONCLUSIONS: This is the first study of GPC3 in a large series of sarcomas, and highlights expression of GPC3 in RMS but not other STS. As glypicans are rarely expressed in mature tissue, GPC3 may be a therapeutic target in RMS, and as the protein can be shed from the cell surface, it may have future application as a biomarker for this disease..
Gilbert, D.C.
Chandler, I.
Summersgill, B.
McIntyre, A.
Missiaglia, E.
Goddard, N.C.
Huddart, R.A.
Shipley, J.
(2011). Genomic gain and over expression of CCL2 correlate with vascular invasion in stage I non-seminomatous testicular germ-cell tumours. Int j androl,
Vol.34
(4 Pt 2),
pp. e114-e121.
show abstract
Testicular germ-cell tumours (TGCT) are the most frequent solid tumour to affect young Caucasian adult males and have increased in incidence over recent decades. In clinical stage I non-seminomas, (NSGCT) histological vascular invasion (VI) is a prognostic factor for metastatic relapse. Using array comparative genomic hybridization, we have previously shown that the presence of VI is associated with gain of a region at 17q12, containing a cluster of genes encoding inflammatory cytokines. We here confirm this finding using fluorescence in situ hybridization (FISH) demonstrating gain in 12 out of 42 (29%) assessable samples. Interrogation of previously published expression microarray data suggests that of the genes contained within this region, CCL2 [monocyte chemoattractant protein 1 (MCP1)] is frequently overexpressed in TGCT. Immunohistochemistry confirms this finding in a collection of 67 clinical stage I NSGCT, demonstrating an association with the presence of VI (p=0.049) that was not seen with VEGF-A, MMP2 or MMP9, although all were frequently expressed. This work gives further insight into the mechanisms involved in invasion in this tumour type, which may ultimately have implications for the management of patients with stage I disease..
Zibat, A.
Missiaglia, E.
Rosenberger, A.
Pritchard-Jones, K.
Shipley, J.
Hahn, H.
Fulda, S.
(2011). Aberrant activation of hedgehog signaling confers a poor prognosis in embryonal and fusion gene negative alveolar rhabdomyosarcoma. Cancer research,
Vol.71.
Martins, A.S.
Olmos, D.
Missiaglia, E.
Shipley, J.
(2011). Targeting the insulin-like growth factor pathway in rhabdomyosarcomas: rationale and future perspectives. Sarcoma,
Vol.2011,
p. 209736.
show abstract
Rhabdomyosarcomas (RMS) are a heterogeneous group of tumors that share features of skeletal myogenesis and represent the most common pediatric soft tissue sarcoma. Even though significant advances have been achieved in RMS treatment, prognosis remains very poor for many patients. Several elements of the Insulin-like Growth Factor (IGF) pathway are involved in sarcomas, including RMS. The IGF2 ligand is highly expressed in most, if not all, RMS, and frequent overexpression of the receptor IGF1R is also found. This is confirmed here through mining expression profiling data of a large series of RMS samples. IGF signaling is implicated in the genesis, growth, proliferation, and metastasis of RMS. Blockade of this pathway is therefore a potential therapeutic strategy for the treatment of RMS. In this paper we examine the biological rationale for targeting the IGF pathway in RMS as well as the current associated preclinical and clinical experience..
Williamson, D.
Missiaglia, E.
de Reyniès, A.
Pierron, G.
Thuille, B.
Palenzuela, G.
Thway, K.
Orbach, D.
Laé, M.
Fréneaux, P.
Pritchard-Jones, K.
Oberlin, O.
Shipley, J.
Delattre, O.
(2010). Fusion gene-negative alveolar rhabdomyosarcoma is clinically and molecularly indistinguishable from embryonal rhabdomyosarcoma. J clin oncol,
Vol.28
(13),
pp. 2151-2158.
show abstract
PURPOSE: To determine whether the clinical and molecular biologic characteristics of the alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS) subtypes have relevance independent of the presence or absence of the PAX/FOXO1 fusion gene. PATIENTS AND METHODS: The fusion gene status of 210 histopathologically reviewed, clinically annotated rhabdomyosarcoma samples was determined by reverse transcriptase polymerase chain reaction. Kaplan-Meier analysis was used to assess event-free survival and overall survival in fusion gene-negative ARMS (ARMSn; n = 39), fusion gene-positive ARMS (ARMSp; n = 94), and ERMS (n = 77). A total of 101 RMS samples were also profiled for whole-genome expression, and 128 were profiled for genomic copy number imbalances. Profiling data were analyzed by supervised and unsupervised methods to compare features related to histopathology and fusion gene status. Results were also projected by meta-analysis techniques across three separate publically available data sets. RESULTS: Overall and event-free survival, frequency of metastases, and distribution of site at initial presentation were not significantly different between ARMSn and ERMS. Consistent with this, analysis of gene expression signatures could not reproducibly distinguish ARMSn from ERMS whereas fusion gene-positive cases were distinct. ARMSn and ERMS frequently show whole-chromosome copy number changes, notably gain of chromosome 8 with associated high levels of expression of genes from this chromosome. CONCLUSION: The clinical behavior and molecular characteristics of alveolar cases without a fusion gene are indistinguishable from embryonal cases and significantly different from fusion-positive alveolar cases. This implies that fusion gene status irrespective of histology is a critical factor in risk stratification of RMS..
Santarius, T.
Shipley, J.
Brewer, D.
Stratton, M.R.
Cooper, C.S.
(2010). EPIGENETICS AND GENETICS A census of amplified and overexpressed human cancer genes. Nature reviews cancer,
Vol.10
(1),
pp. 59-6.
Rao, P.K.
Missiaglia, E.
Shields, L.
Hyde, G.
Yuan, B.
Shepherd, C.J.
Shipley, J.
Lodish, H.F.
(2010). Distinct roles for miR-1 and miR-133a in the proliferation and differentiation of rhabdomyosarcoma cells. Faseb journal,
Vol.24
(9),
pp. 3427-11.
Goddard, N.C.
McIntyre, A.
Gilbert, D.
Kitazawa, S.
Shipley, J.
(2010). No Evidence for V600E BRAF Mutation in the Seminoma Cell Line TCam-2. Genes chromosomes & cancer,
Vol.49
(10),
pp. 963-4.
Cadalbert, L.C.
Sloss, C.M.
Cunningham, M.R.
Al-Mutairi, M.
McIntire, A.
Shipley, J.
Plevin, R.
(2010). Differential regulation of MAP kinase activation by a novel splice variant of human MAP kinase phosphatase-2. Cellular signalling,
Vol.22
(3),
pp. 357-9.
Zibat, A.
Missiaglia, E.
Rosenberger, A.
Pritchard-Jones, K.
Shipley, J.
Hahn, H.
Fulda, S.
(2010). Activation of the hedgehog pathway confers a poor prognosis in embryonal and fusion gene-negative alveolar rhabdomyosarcoma. Oncogene,
Vol.29
(48),
pp. 6323-8.
Noel, E.E.
Yeste-Velasco, M.
Mao, X.
Perry, J.
Kudahetti, S.C.
Li, N.F.
Sharp, S.
Chaplin, T.
Xue, L.
McIntyre, A.
Shan, L.
Powles, T.
Oliver, R.T.
Young, B.D.
Shipley, J.
Berney, D.M.
Joel, S.P.
Lu, Y.-.
(2010). The Association of CCND1 Overexpression and Cisplatin Resistance in Testicular Germ Cell Tumors and Other Cancers. American journal of pathology,
Vol.176
(6),
pp. 2607-9.
Missiaglia, E.
Shepherd, C.J.
Patel, S.
Thway, K.
Pierron, G.
Pritchard-Jones, K.
Renard, M.
Sciot, R.
Rao, P.
Oberlin, O.
Delattre, O.
Shipley, J.
(2010). MicroRNA-206 expression levels correlate with clinical behaviour of rhabdomyosarcomas. Br j cancer,
Vol.102
(12),
pp. 1769-1777.
show abstract
BACKGROUND: Rhabdomyosarcomas (RMSs) are primarily paediatric sarcomas that resemble developing skeletal muscle. Our aim was to determine the effects of microRNAs (miRNA) that have been implicated in muscle development on the clinical behaviour of RMSs. METHODS: Expression levels of miR-1, miR-206, miR-133a and miR-133b were quantified by RT-PCR in 163 primary paediatric RMSs, plus control tissues, and correlated with clinico-pathological features. Correlations with parallel gene expression profiling data for 84 samples were used to identify pathways associated with miR-206. Synthetic miR-206 was transfected into RMS cell lines and phenotypic responses assessed. RESULTS: Muscle-specific miRNAs levels were lower in RMSs compared with skeletal muscle but generally higher than in other normal tissues. Low miR-206 expression correlated with poor overall survival and was an independent predictor of shorter survival in metastatic embryonal and alveolar cases without PAX3/7-FOXO1 fusion genes. Low miR-206 expression also significantly correlated with high SIOP stage and the presence of metastases at diagnosis. High miR-206 expression strongly correlated with genes linked to muscle differentiation and low expression was associated with genes linked to MAPkinase and NFKappaB pathway activation. Increasing miR-206 expression in cell lines inhibited cell growth and migration and induced apoptosis that was associated with myogenic differentiation in some, but not all, cell lines. CONCLUSION: miR-206 contributes to the clinical behaviour of RMSs and the pleiotropic effects of miR-206 supports therapeutic potential..
Gilbert, D.C.
McIntyre, A.
Summersgill, B.
Missiaglia, E.
Goddard, N.C.
Chandler, I.
Huddart, R.A.
Shipley, J.
(2010). Minimum regions of genomic imbalance in stage I testicular embryonal carcinoma and association of 22q loss with relapse. Genes chromosomes cancer,
.
show abstract
Testicular germ cell tumors (TGCT) are the most frequent solid tumor to affect young adult males and are histologically divided into seminomas and nonseminomas (NS). NS comprise undifferentiated embryonal carcinoma (EC) and differentiated tumors with embryonic (teratoma) or extra-embryonic (choriocarcinoma, yolk sac tumor) features. In contrast to other subtypes, EC have uniform cellular morphology and lack normal cell infiltrates, ideal for nucleic acid profiling. EC are under-represented in previous studies due to their relative rarity. To gain insights into NS tumorigenesis, metastatic dissemination and potential markers of relapse, a full tiling path BAC platform was used to obtain array comparative genomic hybridization (aCGH) profiles from 32 formalin fixed paraffin embedded stage I EC samples from patients with follow-up data. In addition to identifying regions previously described in TGCT, novel minimum overlapping regions of gain at 6p21.33, 10q11.21, and 22q13.32 and loss at 22q12.2 were defined and confirmed by fluorescence in situ hybridization analyses. Specifically, the region at 6p21.33 included OCT3/4, the expression of which is involved in the maintenance of pluripotency and the 10q11.21 region contains the gene encoding the RAS activating factor RASGEF1A, the expression of which was demonstrably increased in RNA extracted from these samples. The region of loss at 22q12.2 was more frequently seen in tumors that relapsed and protein expression of genes from 22q12.2 included PIK3IP1, a negative regulator of PI3 kinase signaling was reduced. These data support the role for genes involved in pluripotency and RAS/PI3K signaling in EC development and progression. © 2010 Wiley-Liss, Inc..
Shipley, J.
(2010). Janet Shipley Case study. New scientist,
Vol.206
(2756),
pp. 51-1.
Summersgill, B.M.
Shipley, J.M.
(2010). Fluorescence in situ hybridization analysis of formalin fixed paraffin embedded tissues, including tissue microarrays. Methods mol biol,
Vol.659,
pp. 51-70.
show abstract
Formalin fixed paraffin embedded (FFPE) material is frequently the most convenient readily available source of diseased tissue, including tumors. Multiple cores of FFPE material are being used increasingly to construct tissue microarrays (TMAs) that enable simultaneous analyses of many archival samples. Fluorescence in situ hybridization (FISH) is an important approach to analyze FFPE material for specific genetic aberrations that may be associated with tumor types or subtypes, cellular morphology, and disease prognosis. Annealing, or hybridization of labeled nucleic acid sequences, or probes, to detect and locate one or more complementary nucleic acid sequences within fixed tissue sections allows the detection of structural (translocation/inversion) and numerical (deletion/gain) aberrations and their localization within tissues. The robust protocols described include probe preparation, hybridization, and detection and take 2-3 days to complete. A protocol is also described for the stripping of probes for repeat FISH in order to maximize the use of scarce tissue resources..
Gilbert, D.C.
Chandler, I.
McIntyre, A.
Goddard, N.C.
Gabe, R.
Huddart, R.A.
Shipley, J.
(2009). Clinical and biological significance of CXCL12 and CXCR4 expression in adult testes and germ cell tumours of adults and adolescents. J pathol,
Vol.217
(1),
pp. 94-102.
show abstract
Interaction between the chemokine CXCL12 (SDF1) and the G-protein coupled receptor CXCR4 is responsible for the maintenance of adult stem cell niches and is known to play an important role in utero in the migration of primordial germ cells. We demonstrate expression of CXCL12 by Sertoli cells and confirm CXCR4 expression by the germ cell population of the adult human testes. CXCR4 is also known to mediate organ-specific patterns of metastases in a range of common cancers. We identify consistent expression of CXCR4 mRNA and protein in testicular germ cell tumours (TGCT) that accounts for their patterns of relapse in sites of known CXCL12 expression. Extragonadal primary germ cell tumours express CXCR4 and their sites of occurrence are coincident with areas of known CXCL12 expression in utero. We show that CXCL12 stimulates the invasive migration of a TGCT cell line in vitro in a CXCR4-dependent fashion and activates ERK. Furthermore, we demonstrate that expression of CXCL12 in stage I non-seminomas is significantly associated with organ-confined disease post-orchidectomy and reduced risk of relapse (p = 0.003). This may be through the loss of CXCL12 gradients that might otherwise attract cells away from the primary tumour. We propose CXCL12 expression as a potential predictor of subsequent relapse that could lead to avoiding unnecessary treatment and associated late toxicities. Our observations support a role for CXCL12/CXCR4 in the adult germ cell population and demonstrate pathological function in germ cell tumour development and metastasis that may have clinical utility..
Missiaglia, E.
Selfe, J.
Hamdi, M.
Williamson, D.
Schaaf, G.
Fang, C.
Koster, J.
Summersgill, B.
Messahel, B.
Versteeg, R.
Pritchard-Jones, K.
Kool, M.
Shipley, J.
(2009). Genomic imbalances in rhabdomyosarcoma cell lines affect expression of genes frequently altered in primary tumors: an approach to identify candidate genes involved in tumor development. Genes chromosomes cancer,
Vol.48
(6),
pp. 455-467.
show abstract
Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue sarcomas. They resemble developing skeletal muscle and are histologically divided into two main subtypes; alveolar and embryonal RMS. Characteristic genomic aberrations, including the PAX3- and PAX7-FOXO1 fusion genes in alveolar cases, have led to increased understanding of their molecular biology. Here, we determined the effect of genomic copy number on gene expression levels through array comparative genomic hybridization (CGH) analysis of 13 RMS cell lines, confirmed by multiplex ligation-dependent probe amplification copy number analyses, combined with their corresponding expression profiles. Genes altered at the transcriptional level by genomic imbalances were identified and the effect on expression was proportional to the level of genomic imbalance. Extrapolating to a public expression profiling dataset for 132 primary RMS identified features common to the cell lines and primary samples and associations with subtypes and fusion gene status. Genes identified such as CDK4 and MYCN are known to be amplified, overexpressed, and involved in RMS tumorigenesis. Of the many genes identified, those with likely functional relevance included CENPF, DTL, MYC, EYA2, and FGFR1. Copy number and expression of FGFR1 was validated in additional primary material and found amplified in 6 out of 196 cases and overexpressed relative to skeletal muscle and myoblasts, with significantly higher expression levels in the embryonal compared with alveolar subtypes. This illustrates the ability to identify genes of potential significance in tumor development through combining genomic and transcriptomic profiles from representative cell lines with publicly available expression profiling data from primary tumors..
Stoop, H.
Honecker, F.
Geijn, G.J.
Gillis, A.J.
Cools, M.C.
de Boer, M.
Bokemeyer, C.
Wolffenbuttel, K.P.
Drop, S.L.
de Krijger, R.R.
Dennis, N.
Summersgill, B.
McIntyre, A.
Shipley, J.
Oosterhuis, J.W.
Looijenga, L.H.
(2008). Stem cell factor as a novel diagnostic marker for early malignant germ cells. Journal of pathology,
Vol.216
(1),
pp. 43-12.
Knight, J.F.
Shepherd, C.J.
Rizzo, S.
Brewer, D.
Jhavar, S.
Dodson, A.R.
Cooper, C.S.
Eeles, R.
Falconer, A.
Kovacs, G.
Garrett, M.D.
Norman, A.R.
Shipley, J.
Hudson, D.L.
(2008). TEAD1 and c-Cbl are novel prostate basal cell markers that correlate with poor clinical outcome in prostate cancer. Br j cancer,
Vol.99
(11),
pp. 1849-1858.
show abstract
Prostate cancer is the most frequently diagnosed male cancer, and its clinical outcome is difficult to predict. The disease may involve the inappropriate expression of genes that normally control the proliferation of epithelial cells in the basal layer and their differentiation into luminal cells. Our aim was to identify novel basal cell markers and assess their prognostic and functional significance in prostate cancer. RNA from basal and luminal cells isolated from benign tissue by immunoguided laser-capture microdissection was subjected to expression profiling. We identified 112 and 267 genes defining basal and luminal populations, respectively. The transcription factor TEAD1 and the ubiquitin ligase c-Cbl were identified as novel basal cell markers. Knockdown of either marker using siRNA in prostate cell lines led to decreased cell growth in PC3 and disrupted acinar formation in a 3D culture system of RWPE1. Analyses of prostate cancer tissue microarray staining established that increased protein levels of either marker were associated with decreased patient survival independent of other clinicopathological metrics. These data are consistent with basal features impacting on the development and clinical course of prostate cancers..
McIntyre, A.
Gilbert, D.
Goddard, N.
Looijenga, L.
Shipley, J.
(2008). Genes, chromosomes and the development of testicular germ cell tumors of adolescents and adults. Genes chromosomes cancer,
Vol.47
(7),
pp. 547-557.
show abstract
Testicular germ cell tumors (TGCTs) of adults and adolescents are thought to be derived from primordial germ cells or gonocytes. TGCTs develop postpuberty from precursor lesions known as intratubular germ cell neoplasia undifferentiated. The tumors can be divided into two groups based on their histology and clinical behavior; seminomas resemble primordial germ cells or gonocytes and nonseminomas resemble embryonic or extraembryonic tissues at various stages of differentiation. The most undifferentiated form of nonseminoma, embryonal carcinoma, resembles embryonic stem cells in terms of morphology and expression profiling, both mRNAs and microRNAs. Evidence supports both environmental factors and genetic predisposition underlying the development of TGCTs. Various models of development have been proposed and are discussed. In TGCTs, gain of material from the short arm of chromosome 12 is invariable: genes from this region include the proto-oncogene KRAS, which has activating mutations in approximately 10% of tumors or is frequently overexpressed. A number of different approaches to increase the understanding of the development and progression of TGCTs have highlighted the involvement of KIT, RAS/RAF/MAPK, STAT, and PI3K/AKT signaling. We review the role of these signaling pathways in this process and the potential influence of environmental factors in the development of TGCTs..
Morgenstern, D.A.
Rees, H.
Sebire, N.J.
Shipley, J.
Anderson, J.
(2008). Rhabdomyosarcoma subtyping by immunohistochemical assessment of myogenin: Tissue array study and review of the literature. Pathology & oncology research,
Vol.14
(3),
pp. 233-6.
Canzonetta, C.
Mulligan, C.
Deutsch, S.
Ruf, S.
O'Doherty, A.
Lyle, R.
Borel, C.
Lin-Marq, N.
Delom, F.
Groet, J.
Schnappauf, F.
De Vita, S.
Averill, S.
Priestley, J.V.
Martin, J.E.
Shipley, J.
Denyer, G.
Epstein, C.J.
Fillat, C.
Estivill, X.
Tybulewicz, V.L.
Fisher, E.M.
Antonarakis, S.E.
Nizetic, D.
(2008). DYRK1A-dosage imbalance perturbs NRSF/REST levels, deregulating pluripotency and embryonic stem cell fate in Down syndrome. American journal of human genetics,
Vol.83
(3),
pp. 388-13.
Mao, X.
Young, B.D.
Chaplin, T.
Shipley, J.
Lu, Y.-.
(2008). Subtle genomic alterations and genomic instability revealed in diploid cancer cell lines. Cancer letters,
Vol.267
(1),
pp. 49-6.
Summersgill, B.
Clark, J.
Shipley, J.
(2008). Fluorescence and chromogenic in situ hybridization to detect genetic aberrations in formalin-fixed paraffin embedded material, including tissue microarrays. Nat protoc,
Vol.3
(2),
pp. 220-234.
show abstract
Screening for specific genetic aberrations by fluorescence and chromogenic in situ hybridization (fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH)) can reveal associations with tumor types or subtypes, cellular morphology and clinical behavior. FISH and CISH methodologies are based on the specific annealing (hybridization) of labeled genomic sequences (probes) to complementary nucleic acids within fixed cells to allow their detection, quantification and spatial localization. Formalin-fixed paraffin embedded (FFPE) material is the most widely available source of tumor samples. Increasingly, tissue microarrays (TMAs) consisting of multiple cores of FFPE material are being used to enable simultaneous analyses of many archival samples. Here we describe robust protocols for the FISH and CISH analyses of genetic aberrations in FFPE tissue, including TMAs. Protocols include probe preparation, hybridization and detection. Steps are described to reduce background fluorescence and strip probes for repeat FISH analyses to maximize the use of tissue resources. The basic protocol takes 2-3 d to complete..
Williamson, D.
Selfe, J.
Gordon, T.
Lu, Y.-.
Pritchard-Jones, K.
Murai, K.
Jones, P.
Workman, P.
Shipley, J.
(2007). Role for amplification and expression of glypican-5 in rhabdomyosarcoma. Cancer res,
Vol.67
(1),
pp. 57-65.
show abstract
Overexpression of genes, through genomic amplification and other mechanisms, can critically affect the behavior of tumor cells. Genomic amplification of the 13q31-32 region is reported in many tumors, including rhabdomyosarcomas that are primarily pediatric sarcomas resembling developing skeletal muscle. The minimum overlapping region of amplification at 13q31-32 in rhabdomyosarcomas was defined as containing two genes: Glypican-5 (GPC5) encoding a cell surface proteoglycan and C13orf25 encompassing the miR-17-92 micro-RNA cluster. Genomic copy number and gene expression analyses of rhabdomyosarcomas indicated that GPC5 was the only gene consistently expressed and up-regulated in all cases with amplification. Constitutive overexpression and knockdown of GPC5 expression in rhabdomyosarcoma cell lines increased and decreased cell proliferation, respectively. A correlation between expression levels of nascent pre-rRNA and GPC5 (P = 0.001), but not a C13orf25 transcript containing miR-17-92, in primary samples supports an association of GPC5 with proliferative capacity in vivo. We show that GPC5 increases proliferation through potentiating the action of the growth factors fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and Wnt1A. GPC5 enhanced the intracellular signaling of FGF2 and HGF and altered the cellular distribution of FGF2. The mesoderm-inducing effect of FGF2 and FGF4 in Xenopus blastocysts was also enhanced. Our data are consistent with a role of GPC5, in the context of sarcomagenesis, in enhancing FGF signaling that leads to mesodermal cell proliferation without induction of myogenic differentiation. Furthermore, the properties of GPC5 make it an attractive target for therapeutic intervention in rhabdomyosarcomas and other tumors that amplify and/or overexpress the gene..
Zhou, Y.P.
Ye, H.T.
Martin-Subero, J.I.
Gesk, S.
Hamoudi, R.
Lu, Y.J.
Wang, R.
Shipley, J.
Siebert, R.
Isaacson, P.G.
Dogan, A.
Du, M.Q.
(2007). The pattern of genomic gains in salivary gland MALT lymphomas. Haematol-hematol j,
Vol.92
(7),
pp. 921-927.
show abstract
Background and Objectives Salivary gland mucosa-associated lymphoid tissue (MALT) lymphomas typically lack chromosomal translocations and the molecular genetics underlying their development is unknown. The aim of this study was to investigate chromosomal changes in these lymphomas.Design and Methods We performed comparative genomic hybridisation using DNA samples extracted from microdissected tumour cells in 19 salivary gland MALT lymphomas. Recurrent chromosomal changes were further verified by interphase fluorescence in situ hybridization (FISH).Results Chromosomal gains were much more common than losses. Recurrent gains were found at 1p32-ter (42%), 9q33-34 (84%), 11q11-13 (42%), 17 (58%) and 18q21-22 (42%). Among these, the recurrent gains at 9q34, 11q11-13 and 18q21 were nearly the exclusive gain of the corresponding chromosome. Notably, chromosomal gains at 9q34, 11q13 and 18q21 were frequently concurrent, with 12/19 cases affecting at least two of the three loci. The genomic gains at these chromosomal regions were further confirmed by interphase FISH with probes targeting the TRAF2 and CARD9 (9q34), RELA and CCND1 (11q13), and MALT1 (18q21) loci.Interpretation and Conclusions Salivary gland MALT lymphomas show a conserved pattern of chromosomal gains, which appear to target genes positively modulating cell survival and proliferation..
Goddard, N.C.
McIntyre, A.
Summersgill, B.
Gilbert, D.
Kitazawa, S.
Shipley, J.
(2007). KIT and RAS signalling pathways in testicular germ cell tumours: new data and a review of the literature. Int j androl,
Vol.30
(4),
pp. 337-348.
show abstract
Testicular germ cell tumours (TGCTs) are the leading cause of cancer deaths in young male Caucasians. Identifying changes in DNA copy number can pinpoint genes involved in tumour development. We defined the smallest overlapping regions of imbalance in TGCTs using array comparative genomic hybridization analysis. Novel regions, or regions which refined those previously reported, were identified. The expression profile of genes from 12p, which is invariably gained in TGCTs, and amplicons defined at 12p11.2-12.1 and 4q12, suggest KRAS and KIT involvement in TGCT and seminoma development, respectively. Amplification of these genes was not found in intratubular germ cell neoplasia adjacent to invasive disease showing these changes, suggesting their involvement in tumour progression. Activating mutations of RAS genes (KRAS or NRAS) and overexpression of KRAS were mutually exclusive events. These, correlations between the expression levels of KIT, KRAS and GRB7 (which encodes an adapter molecule known to interact with the KIT tyrosine kinase receptor) and other reported evidence reviewed here, are consistent with a role for activation of KIT and RAS signalling in TGCT development. In order to assess a role for KIT in seminomas, we modulated the level of KIT expression in TCam-2, a seminoma cell line. The likely seminomatous origin of this cell line was supported by demonstrating KIT and OCT3/4 overexpression and gain of 12p material. Reducing the expression of KIT in TCam-2 through RNA inhibition resulted in decreased cell viability. Further understanding of KIT and RAS signalling in TGCTs may lead to novel therapeutic approaches for these tumours..
Slater, O.
Shipley, J.
(2007). Clinical relevance of molecular genetics to paediatric sarcomas. J clin pathol,
Vol.60
(11),
pp. 1187-1194.
show abstract
full text
The application of cytogenetic and molecular genetic analyses to paediatric sarcomas has identified a number of characteristic changes associated with types and subtypes of sarcomas. This has led to increased understanding of the underlying molecular biology of some sarcomas and provided an important adjunct to standard morphological and immunohistochemical diagnoses. Characteristic genetic abnormalities, particularly specific chromosome translocations and associated fusion genes, have diagnostic and in some cases prognostic value. There is also the potential to detect micrometastastic disease. Fusion genes are most readily detected by fluorescence in situ hybridisation and reverse transcription-PCR technologies. The expression profiles of tumours with specific fusion genes are characteristically similar and the molecular signatures of sarcomas are also proving to be of diagnostic and prognostic value. Furthermore, fusion genes and other emerging molecular events associated with sarcomas represent potential targets for novel therapeutic approaches which are desperately required to improve the outcome of children with certain categories of sarcoma, including rhabdomyosarcomas and the Ewing's family of tumours. Increased understanding of the molecular biology of sarcomas is leading towards more effective treatments which may complement or be less toxic than conventional radiotherapy and cytotoxic chemotherapy. Here we review paediatric sarcomas that have associated molecular genetic changes which can increase diagnostic and prognostic accuracy and impact on clinical management..
McIntyre, A.
Summersgill, B.
Lu, Y.J.
Missiaglia, E.
Kitazawa, S.
Oosterhuis, J.W.
Looijenga, L.H.
Shipley, J.
(2007). Genomic copy number and expression patterns in testicular germ cell tumours. Br j cancer,
Vol.97
(12),
pp. 1707-1712.
show abstract
Testicular germ cell tumours of adults and adolescents (TGCT) include seminomas (SE) and nonseminomas (NS), with spermatocytic seminomas (SSE) representing a distinct entity in older men. SE and NS have gain of 12p material in all cases, whereas SSE are associated with overrepresentation of chromosome 9. Here, we compare at the chromosomal level, copy number imbalances with global expression changes, identified by comparative expressed sequence hybridisation analyses, in seven SE, one combined tumour, seven NS and seven cell lines. Positive correlations were found consistent with copy number as a main driver of expression change, despite reported differences in methylation status in SE and NS. Analysis of chromosomal copy number and expression data could not distinguish between SE and NS, in-keeping with a similar genetic pathogenesis. However, increased expression from 4q22, 5q23.2 and 9p21 distinguished SSE from SE and NS and decreased copy number and expression from 2q36-q37 and 6q24 was a specific feature of NS-derived cell lines. Our analysis also highlights 19 regions with both copy number and expression imbalances in greater than 40% of cases. Mining available expression array data identified genes from these regions as candidates for involvement in TGCT development. Supplementary data is available at http://www.crukdmf.icr.ac.uk/array/array.html..
Mensah, A.
Mulligan, C.
Linehan, J.
Ruf, S.
O'Doherty, A.
Grygalewicz, B.
Shipley, J.
Groet, J.
Tybulewicz, V.
Fisher, E.
Brandner, S.
Nizetic, D.
(2007). An additional human chromosome 21 causes suppression of neural fate of pluripotent mouse embryonic stem cells in a teratoma model. Bmc developmental biology,
Vol.7,
p. 10.
Rees, H.
Williamson, D.
Papanastasiou, A.
Jina, N.
Nabarro, S.
Shipley, J.
Anderson, J.
(2006). The MET receptor tyrosine kinase contributes to invasive tumour growth in rhabdomyosarcomas. Growth factors,
Vol.24
(3),
pp. 197-12.
Ma, J.
Gao, M.
Lu, Y.
Feng, X.
Zhang, J.
Lin, D.
Xiao, T.
Hu, Z.
Yuan, J.
Su, K.
Shipley, J.
Xue, J.
Gao, Y.
(2006). Gain of 1q25-32, 12q23-24 3, and 17q 12-22 facilitates tumorigenesis and progression of human squamous cell lung cancer. Journal of pathology,
Vol.210
(2),
pp. 205-9.
Anderson, J.
Gibson, S.
Williamson, D.
Rampling, D.
Austin, C.
Shipley, J.
Sebire, N.
Brock, P.
(2006). Rapid and accurate determination of MYCN copy number and 1p deletion in neuroblastoma by quantitative PCR. Pediatric blood & cancer,
Vol.46
(7),
pp. 820-5.
Looijenga, L.H.
Hersmus, R.
Gillis, A.J.
Pfundt, R.
Stoop, H.J.
van Gurp, R.J.
Veltman, J.
Beverloo, H.B.
van Drunen, E.
van Kessel, A.G.
Pera, R.R.
Schneider, D.T.
Summersgill, B.
Shipley, J.
McIntyre, A.
van der Spek, P.
Schoenmakers, E.
Oosterhuis, J.W.
(2006). Genomic and expression profiling of human spermatocytic seminomas:: Primary spermatocyte as tumorigenic precursor and DMRT1 as candidate chromosome 9 gene. Cancer research,
Vol.66
(1),
pp. 290-13.
Strefford, J.C.
Lane, T.M.
Hill, A.
LeRoux, L.
Foot, N.J.
Shipley, J.
Oliver, R.T.
Lu, Y.J.
Young, B.D.
(2006). Molecular characterisation of the t(1;15)(p22;q22) translocation in the prostate cancer cell line LNCaP. Cytogenetic and genome research,
Vol.112
(1-2),
pp. 45-8.
Horwich, A.
Shipley, J.
Huddart, R.
(2006). Testicular germ-cell cancer. Lancet,
Vol.367
(9512),
pp. 754-765.
show abstract
Testicular germ-cell tumours (TGCTs) represent the model of a curable malignancy; sensitive tumour markers, accurate prognostic classification, logical series of management trials, and high cure rates in both seminomas and non-seminomas have enabled a framework of effective cancer therapy. Understanding the molecular biology of TGCT could help improve treatment of other cancers. The typical presentation in young adults means that issues of long-term toxicity become especially important in judging appropriate management. A focus of recent developments has been to tailor aggressiveness of treatment to the severity of the prognosis. Recent changes affect the most common subtypes and include the reduction of chemotherapy for patients who have metastastic non-seminomas and a good prognosis, and alternatives to adjuvant radiotherapy in stage I seminomas. We summarise advances in the understanding and management of TGCT during the past decade..
Zhou, Y.P.
Ye, H.T.
Martin-Subero, J.I.
Hamoudi, R.
Lu, Y.J.
Wang, R.
Siebert, R.
Shipley, J.
Isaacson, P.G.
Dogan, A.
Du, M.Q.
(2006). Distinct comparative genomic hybridisation profiles in gastric mucosa-associated lymphoid tissue lymphomas with and without t(11;18)(q21;q21). British journal of haematology,
Vol.133
(1),
pp. 35-8.
Hanks, S.
Coleman, K.
Summersgill, B.
Messahel, B.
Williamson, D.
Pritchard-Jones, K.
Strefford, J.
Swansbury, J.
Plaja, A.
Shipley, J.
Rahman, N.
(2006). Comparative genomic hybridization and BUB1B mutation analyses in childhood cancers associated with mosaic variegated aneuploidy syndrome. Cancer lett,
Vol.239
(2),
pp. 234-238.
show abstract
We previously demonstrated that constitutional BUB1B mutations cause mosaic variegated aneuploidy, a condition characterized by constitutional aneuploidies and childhood cancer predisposition. To further investigate the role of BUB1B in cancer predisposition we performed comparative genomic hybridization analysis in an embryonal rhabdomyosarcoma from an MVA case with biallelic BUB1B mutations, revealing aneuploidies typical of sporadic E-RMS, with gain of chromosomes 3, 8, 13 and loss of chromosomes 9, 14, X. To investigate whether somatic BUB1B mutations occur in sporadic childhood cancers we screened 30 Wilms tumours, 10 acute lymphoblastic leukemias, nine rhabdomyosarcomas and 11 rhabdomyosarcoma cell lines for BUB1B mutations. We identified seven exonic and six intronic variants. Six of the exonic variants were synonymous and one resulted in a non-synonymous conservative missense alteration that was also present in a control. These data suggest that the genetic progression in rhabdomyosarcoma from MVA and non-MVA cases may be similar, but that somatic BUB1B mutations are unlikely to be common in sporadic childhood cancers known to be associated with MVA..
Barker, K.T.
Spendlove, H.E.
Banu, N.S.
Bridge, J.A.
Fisher, C.
Shipley, J.
Garrett, M.
Manyonda, I.
Houlston, R.S.
(2006). No evidence for epigenetic inactivation of fumarate hydratase in leiomyomas and leiomyosarcomas. Cancer lett,
Vol.235
(1),
pp. 136-140.
show abstract
Germline mutations in Fumarate Hydratase (FH) cause the development of leiomyomas and leiomyosarcomas in the syndromes Multiple Cutaneous and Uterine Leiomyomata (MCUL1) and Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC). There is little evidence, however, that FH mutation plays a role in the development of sporadic leiomyomas or leiomyosarcomas. Such observations do not, however, exclude a role for FH in tumour development outside the context of MCUL1/HLRCC, as it is possible that FH expression could be silenced by epigenetic mechanisms. To explore this possibility we have developed a highly specific antibody to FH and analysed a series of forty-five fresh-frozen uterine leiomyomas and nine leiomyosarcomas for FH expression..
Williamson, D.
Lu, Y.-.
Fang, C.
Pritchard-Jones, K.
Shipley, J.
(2006). Nascent pre-rRNA overexpression correlates with an adverse prognosis in alveolar rhabdomyosarcoma. Genes chromosomes cancer,
Vol.45
(9),
pp. 839-845.
show abstract
Comparative expressed sequence hybridization (CESH) is an expression profiling technique which identifies chromosomal regions corresponding to differential gene expression. Here, we observe that various tumor samples including rhabdomyosarcoma show very prominent staining on the short arms of the acrocentric chromosomes suggesting an increase in expression of ribosomal RNA synthesized from the repetitive rDNA of the nucleolar organizer regions located on these chromosomes. Survival analysis showed a correlation with overexpression from this region and a poor prognosis in rhabdomyosarcoma. This phenomenon was studied in an extended set of rhabdomyosarcoma tumor samples using quantitative real-time reverse transcriptase-PCR to quantify levels of pre-rRNA (precursor ribosomal RNA). It was demonstrated first that the strong CESH signals did correspond to a marked increase in pre-rRNA expression and second that high pre-rRNA expression correlated with an adverse prognosis in alveolar subtype rhabdomyosarcoma. In addition, we demonstrate that pre-rRNA expression is significantly correlated with tumor stage. We conclude that measuring expression of pre-rRNA by real-time PCR is a useful prognostic marker in alveolar rhabdomyosarcoma. Furthermore, given that we have observed similar rDNA staining in all cancer types that we have studied by CESH, we propose that pre-rRNA overexpression is a general phenomenon in cancer and that our real-time PCR assay may be applicable as a prognostic marker in other tumor types..
Shipley, J.
(2006). Putting the colours into chromogenic in situ hybridization (CISH). J pathol,
Vol.210
(1),
pp. 1-2.
show abstract
Recurrent genomic alterations are the hallmarks of particular cancers. Application of molecular cytogenetic technologies to tumour material in order to detect these alterations has become important for molecular diagnostics and research. A dual-colour chromogenic in situ hybridization (dc-CISH) method described recently in the Journal of Pathology has the advantage of visualizing two probes simultaneously with the ability to discern morphological features. In addition, the bright field microscopy required is readily accessible to many laboratories. The approach has been validated by comparison of results with standard analyses for HER-2 amplification status in formalin-fixed, paraffin-embedded breast cancers and is applicable to the analysis of other clinically relevant genomic aberrations as well as of use in research investigations..
Cooper, C.S.
Nicholson, A.G.
Foster, C.
Dodson, A.
Edwards, S.
Fletcher, A.
Roe, T.
Clark, J.
Joshi, A.
Norman, A.
Feber, A.
Lin, D.
Gao, Y.
Shipley, J.
Cheng, S.-.
(2006). Nuclear overexpression of the E2F3 transcription factor in human lung cancer. Lung cancer,
Vol.54
(2),
pp. 155-162.
show abstract
BACKGROUND: The E2F3 transcription factor has an established role in controlling cell cycle progression. In previous studies we have provided evidence that nuclear E2F3 overexpression represents a mechanism that drives the development of human bladder cancer and that determines aggressiveness in human prostate cancer. We have proposed a model in which E2F3 overexpression co-operates with removal of the E2F inhibitor pRB to facilitate cancer development. Since small cell lung cancers (SCLC) have one of the highest reported frequencies of functional abnormalities in the pRB protein (90%) of any human cancer, we wish to assess to what extent E2F3 would be overexpressed in this and other classes of human lung cancer. METHODS: Immunohistochemical techniques were used to assess the E2F3 status in 428 samples of lung cancers, lung carcinoids, normal bronchial epithelium and normal lung tissue. RESULTS: E2F3 is overexpressed in 55-70% of squamous cell carcinomas and 79% of adenocarcinomas of the lung. In addition very high level expression of nuclear E2F3 is found in almost all small cell lung cancers analysed. When considered together with published data our observations indicate that co-operation between pRB functional knockouts and E2F3 overexpression may represent a mechanism of development of SCLC..
Kote-Jarai, Z.
Salmon, A.
Mengitsu, T.
Copeland, M.
Ardern-Jones, A.
Locke, I.
Shanley, S.
Summersgill, B.
Lu, Y.-.
Shipley, J.
Eeles, R.
(2006). Increased level of chromosomal damage after irradiation of lymphocytes from BRCA1 mutation carriers. Br j cancer,
Vol.94
(2),
pp. 308-310.
show abstract
Deleterious mutations in the BRCA1 gene predispose women to an increased risk of breast and ovarian cancer. Many functional studies have suggested that BRCA1 has a role in DNA damage repair and failure in the DNA damage response pathway often leads to the accumulation of chromosomal aberrations. Here, we have compared normal lymphocytes with those heterozygous for a BRCA1 mutation. Short-term cultures were irradiated (8Gy) using a high dose rate and subsequently metaphases were analysed by 24-colour chromosome painting (M-FISH). We scored the chromosomal rearrangements in the metaphases from five BRCA1 mutation carriers and from five noncarrier control samples 6 days after irradiation. A significantly higher level of chromosomal damage was detected in the lymphocytes heterozygous for BRCA1 mutations compared with normal controls; the average number of aberrations per mitosis was 3.48 compared with 1.62 in controls (P=0.0001). This provides new evidence that heterozygous mutation carriers have a different response to DNA damage compared with noncarriers and that BRCA1 has a role in DNA damage surveillance. Our finding has implications for treatment and screening of BRCA1 mutation carriers using modalities that involve irradiation..
Williamson, D.
Lu, Y.-.
Gordon, T.
Sciot, R.
Kelsey, A.
Fisher, C.
Poremba, C.
Anderson, J.
Pritchard-Jones, K.
Shipley, J.
(2005). Relationship between MYCN copy number and expression in rhabdomyosarcomas and correlation with adverse prognosis in the alveolar subtype. J clin oncol,
Vol.23
(4),
pp. 880-888.
show abstract
PURPOSE: Amplification of the transcription factor MYCN is an important molecular diagnostic tool in stratifying treatment for neuroblastoma. Increased copy number and overexpression of MYCN in the pediatric cancer rhabdomyosarcoma has been described in a number of small studies with conflicting conclusions about its association with clinicopathologic characteristics. We aimed to study the phenomenon in the largest series to date. PATIENTS AND METHODS: Using quantitative polymerase chain reaction, we measured MYCN copy number and expression levels in rhabdomyosarcoma samples from 113 and 92 individuals with a confirmed diagnosis of rhabdomyosarcoma, respectively. RESULTS: Increased copy number of MYCN was found to be a feature of both the embryonal and alveolar subtypes. The copy number and expression levels were significantly greater in the alveolar subtype, although the range of expression in both subtypes spanned several orders of magnitude. MYCN copy number showed a significant correlation with expression in the alveolar subtype; this relationship between copy number and expression could be modeled as a logarithmic function. It is notable that relatively high expression frequently occurred in embryonal rhabdomyosarcoma without high copy number and that low expression was found in some cases with high copy number. In patients with alveolar rhabdomyosarcoma, overexpression (greater than median) or gain of genomic copies of MYCN were significantly associated with adverse outcome. CONCLUSION: MYCN deregulation is a feature of rhabdomyosarcoma tumorigenesis, defines groups of patients with a poor prognosis, and is a potential target for novel therapies..
Shipley, J.
(2005). Identification and significance of amplified and overexpressed genes in selected solid tumours. Chromosome research,
Vol.13,
pp. 2-1.
Williams, S.V.
Adams, J.
Coultar, J.
Summersgill, B.M.
Shipley, J.
Knowles, M.A.
(2005). Assessment by M-FISH of karyotypic complexity and cytogenetic evolution in bladder cancer in vitro. Genes chromosomes & cancer,
Vol.43
(4),
pp. 315-14.
Wilson, C.
Yang, J.S.
Strefford, O.C.
Summersgil, B.
Young, B.D.
Shipley, J.
Oliver, T.
Lu, Y.J.
(2005). Overexpression of genes on 16q associated with cisplatin resistance of testicular germ cell tumor cell lines. Genes chromosomes & cancer,
Vol.43
(2),
pp. 211-6.
McIntyre, A.
Summersgill, B.
Spendlove, H.E.
Huddart, R.
Houlston, R.
Shipley, J.
(2005). Activating mutations and/or expression levels of tyrosine kinase receptors GRB7, RAS, and BRAF in testicular germ cell tumors. Neoplasia,
Vol.7
(12),
pp. 1047-1052.
show abstract
full text
Amplification and/or overexpression of genes encoding tyrosine kinase receptors KIT and ERBB2 have been reported in testicular germ cell tumors (TGCTs). These receptors can bind the adaptor molecule GRB7 encoded by a gene adjacent to ERBB2 at 17q12, a region also frequently gained in TGCTs. GRB7 binding may be involved in the activation of RAS signaling and KRAS2 maps to 12p, which is constitutively gained in TGCT and lies within a minimum overlapping region of amplification at 12p11.2-12.1, a region we have previously defined. RAS proteins activate BRAF, and activating mutations of genes encoding these proteins have been described in various tumors. Here we determine the relationships between expression levels and activating mutations of these genes in a series of 65 primary TGCTs and 4 TCGT cell lines. High levels of expression and activating mutations in RAS were mutually exclusive events, and activating mutations in RAS were only identified in the seminoma subtype. Mutations in BRAF were not identified. Increased ERBB2 expression was associated with differentiated nonseminoma histology excised from lymph nodes postchemotherapy. Mutation, elevated expression, and correlations between expression levels of KRAS2, GRB7, and KIT are consistent with their involvement in the development of TGCTs..
McIntyre, A.
Summersgill, B.
Grygalewicz, B.
Gillis, A.J.
Stoop, J.
van Gurp, R.J.
Dennis, N.
Fisher, C.
Huddart, R.
Cooper, C.
Clark, J.
Oosterhuis, J.W.
Looijenga, L.H.
Shipley, J.
(2005). Amplification and overexpression of the KIT gene is associated with progression in the seminoma subtype of testicular germ cell tumors of adolescents and adults. Cancer res,
Vol.65
(18),
pp. 8085-8089.
show abstract
We have previously identified amplification at 4q12 in testicular germ cell tumors of adolescents and adults centered around the KIT gene encoding a tyrosine kinase transmembrane receptor. Analysis of primary testicular germ cell tumors totaling 190 cases revealed 21% of the seminoma subtype with an increased copy number of KIT whereas this change was rarely found in the nonseminomas. In most cases, gain of KIT did not include the immediately flanking noncoding DNA or the flanking genes KDR and PDGFRA. Increased copy number of KIT was not found in the putative precursor lesion, carcinoma in situ (CIS), adjacent to tumor with this change. KIT overexpression was found independent of gain and KIT immunostaining was stronger in selected cases with gain of KIT compared to those without. Taken together with activating mutations of KIT in exon 17 identified in 13% of seminomas, this suggests that the KIT gene product plays a role in the progression of CIS towards seminoma, the further understanding of which may lead to novel less toxic therapeutic approaches..
Lu, Y.J.
Yang, J.S.
Noel, E.
Skoulakis, S.
Chaplin, T.
Raghavan, M.
Purkis, T.
Mcintyre, A.
Kudahetti, S.C.
Naase, M.
Berney, D.
Shipley, J.
Oliver, R.T.
Young, B.D.
(2005). Association between large-scale genomic homozygosity without chromosomal loss and nonseminomatous germ cell tumor development. Cancer research,
Vol.65
(20),
pp. 9137-5.
Edwards, S.
Campbell, C.
Flohr, P.
Shipley, J.
Giddings, I.
Te-Poele, R.
Dodson, A.
Foster, C.
Clark, J.
Jhavar, S.
Kovacs, G.
Cooper, C.S.
(2005). Expression analysis onto microarrays of randomly selected cDNA clones highlights HOXB13 as a marker of human prostate cancer. Br j cancer,
Vol.92
(2),
pp. 376-381.
show abstract
In a strategy aimed at identifying novel markers of human prostate cancer, we performed expression analysis using microarrays of clones randomly selected from a cDNA library prepared from the LNCaP prostate cancer cell line. Comparisons of expression profiles in primary human prostate cancer, adjacent normal prostate tissue, and a selection of other (nonprostate) normal human tissues, led to the identification of a set of clones that were judged as the best candidate markers of normal and/or malignant prostate tissue. DNA sequencing of the selected clones revealed that they included 10 genes that had previously been established as prostate markers: NKX3.1, KLK2, KLK3 (PSA), FOLH1 (PSMA), STEAP2, PSGR, PRAC, RDH11, Prostein and FASN. Following analysis of the expression patterns of all selected and sequenced genes through interrogation of SAGE databases, a further three genes from our clone set, HOXB13, SPON2 and NCAM2, emerged as additional candidate markers of human prostate cancer. Quantitative RT-PCR demonstrated the specificity of expression of HOXB13 in prostate tissue and revealed its ubiquitous expression in a series of 37 primary prostate cancers and 20 normal prostates. These results demonstrate the utility of this expression-microarray approach in hunting for new markers of individual human cancer types..
Williamson, D.
Lu, Y.J.
McIntyre, A.
Summersgill, B.
Wang, R.B.
Selfe, J.
Cooper, C.
Clark, J.
Shipley, J.
(2004). Comparative genomic hybridisation and comparative expressed sequence hybridisation as complements to microarray analyses of tumours. Mutagenesis,
Vol.19
(6),
pp. 509-1.
Hearle, N.
Lucassen, A.
Wang, R.
Lim, W.
Ross, F.
Wheeler, R.
Moore, I.
Shipley, J.
Houlston, R.
(2004). Mapping of a translocation breakpoint in a Peutz-Jeghers hamartoma to the putative PJS locus at 19q13 4 and mutation analysis of candidate genes in polyp and STKII-negative PJS cases. Genes chromosomes & cancer,
Vol.41
(2),
pp. 163-7.
McIntyre, A.
Summersgill, B.
Jafer, O.
Rodriguez, S.
Zafarana, G.
Oosterhuis, J.W.
Gillis, A.J.
Looijenga, L.
Cooper, C.
Huddart, R.
Clark, J.
Shipley, J.
(2004). Defining minimum genomic regions of imbalance involved in testicular germ cell tumors of adolescents and adults through genome wide microarray analysis of cDNA clones. Oncogene,
Vol.23
(56),
pp. 9142-9147.
show abstract
Identifying changes in DNA copy number can pinpoint genes that may be involved in tumor development. Here we have defined the smallest overlapping regions of imbalance (SORI) in testicular germ cell tumors other than the 12p region, which has been previously investigated. Definition of the regions was achieved through comparative genomic hybridization (CGH) analysis of a 4559 cDNA clone microarray. A total of 14 SORI were identified, which involved at least five of the 11 samples analysed. Many of these refined regions were previously reported using chromosomal or allelic imbalance studies. The SORI included gain of material from the regions 4q12, 17q21.3, 22q11.23 and Xq22, and loss from 5q33, 11q12.1, 16q22.3 and 22q11. Comparison with parallel chromosomal CGH data supported involvement of most regions. The various SORI span between one and 20 genes and highlight potential oncogenes/tumor suppressor genes to be investigated further..
Lee, Y.-.
John, M.
Falconer, A.
Edwards, S.
Clark, J.
Flohr, P.
Roe, T.
Wang, R.
Shipley, J.
Grimer, R.J.
Mangham, D.C.
Thomas, J.M.
Fisher, C.
Judson, I.
Cooper, C.S.
(2004). A gene expression signature associated with metastatic outcome in human leiomyosarcomas. Cancer res,
Vol.64
(20),
pp. 7201-7204.
show abstract
Metastasis is a major factor associated with poor prognosis in cancer, but little is known of its molecular mechanisms. Although the clinical behavior of soft tissue sarcomas is highly variable, few reliable determinants of outcome have been identified. New markers that predict clinical outcome, in particular the ability of primary tumors to develop metastatic tumors, are urgently needed. Here, we have chosen leiomyosarcoma as a model for examining the relationship between gene expression profile and the development of metastasis in soft tissue sarcomas. Using cDNA microarray, we have identified a gene expression signature associated with metastasis in sarcoma that allowed prediction of the future development of metastases of primary tumors (Kaplan-Meier analysis P = 0.001). Our finding may aid the tailoring of therapy for individual sarcoma patients, where the aggressiveness of treatment is affected by the predicted outcome of disease..
Gordon, A.
McManus, A.
Anderson, J.
Fisher, C.
Abe, S.
Nojima, T.
Pritchard-Jones, K.
Shipley, J.
(2003). Chromosomal imbalances in pleomorphic rhabdomyosarcomas and identification of the alveolar rhabdomyosarcoma-associated PAX3-FOXO1A fusion gene in one case. Cancer genet cytogenet,
Vol.140
(1),
pp. 73-77.
show abstract
Rhabdomyosarcomas (RMS) are soft tissue sarcomas resembling developing skeletal muscle, and pleomorphic rhabdomyosarcomas (PRMS) are a rare nonpediatric entity. Little molecular cytogenetic information exists for PRMS, and their relationship to other subtypes of rhabdomyosarcoma and other sarcomas is unclear. Chromosomal imbalances were determined in seven well-characterized cases of PRMS using comparative genomic hybridization. The smallest overlapping regions of gain were 1p22 approximately p33 (71%), 7p (43%), 18/18q (43%), and 20/20p (43%), and the regions of loss were 10q23 (71%), 15q21 approximately q22 (57%), 3p, 5q32 approximately qter, and 13 (all 43%). Four of the seven cases had amplicons involving the regions 1p21 approximately p31, 1q21 approximately q25, 3p12, 3q26 approximately qtel, 4q28 approximately q31, 8q21 approximately q23/8q, and 22q. These regions are distinct from those frequently associated with the alveolar subtype, whereas the embryonal subtype without anaplasia is rarely associated with amplification events other than gain/amplification of 8q material. The regions of imbalance appeared more similar to those reported for malignant fibrous histiocytomas (MFH) and osteosarcomas, consistent with the suggestion that PRMS can be considered part of the spectrum of MFH. In addition, one of the cases classified as PRMS showed evidence for the presence of a PAX3-FOXO1A fusion gene, which is characteristic of the alveolar subtype of RMS..
Tiffin, N.
Williams, R.D.
Robertson, D.
Hill, S.
Shipley, J.
Pritchard-Jones, K.
(2003). WT1 expression does not disrupt myogenic differentiation in C2C12 murine myoblasts or in human rhabdomyosarcoma. Exp cell res,
Vol.287
(1),
pp. 155-165.
show abstract
WT1 encodes a tissue-specific transcription factor important in early mesenchymal differentiation. Altered expression or mutation of WT1 occurs in malignancies derived from such tissues. These include Wilms tumour, a paediatric kidney cancer that may show heterologous differentiation into primitive skeletal muscle, especially in tumours with WT1 mutation. A putative role for WT1 in inhibiting myogenesis has been suggested by transient transfection of C(2)C(12) myoblasts. However, using a more robust model of stable transfectants of C(2)C(12) expressing inducible WT1 isoforms, we found no inhibition of myogenic differentiation. We also investigated a possible role for WT1 in the disrupted myogenesis seen in rhabdomyosarcoma, a paediatric cancer resembling foetal skeletal muscle. WT1 expression levels measured by quantitative real-time reverse transcription polymerase chain reaction were low or absent in those tumours with a PAX-FKHR fusion gene characteristic of the alveolar subtype, and were higher in cases lacking these fusion genes. Overall, there was a weak positive correlation between expression of myogenic differentiation marker genes and WT1 levels. We conclude that expression of WT1 in C(2)C(12) cells and in rhabdomyosarcoma does not inhibit myogenic differentiation..
Osin, P.
Lu, Y.-.
Stone, J.
Crook, T.
Houlston, R.S.
Gasco, M.
Gusterson, B.A.
Shipley, J.
(2003). Distinct genetic and epigenetic changes in medullary breast cancer. Int j surg pathol,
Vol.11
(3),
pp. 153-158.
show abstract
Genetic instability resulting in chromosome aneuploidy or mismatch repair deficiency characterizes cancer. Medullary carcinoma (MC) of the breast is a specific form of breast cancer with unique clinical, epidemiologic, and prognostic features, suggesting distinctive tumorigenic pathways. To investigate the nature of the genetic changes associated with MC we analyzed a series of 22 tumors. Chromosomal imbalances were assessed by comparative genomic hybridization (CGH) and mismatch repair (MMR) deficiency tested for through assessment of microsatellite instability (MSI) and expression of MLH1 and MSH2 genes. MMR deficiency was detected in only a small proportion of cases. The chromosomal copy number changes showed some similarities to BRCA1-associated tumors. A high level of BRCA1 promoter hypermethylation was detected, suggesting a possible role of this gene in MC development..
Wang, R.
Titley, J.C.
Lu, Y.-.
Summersgill, B.M.
Bridge, J.A.
Fisher, C.
Shipley, J.
(2003). Loss of 13q14-q21 and gain of 5p14-pter in the progression of leiomyosarcoma. Mod pathol,
Vol.16
(8),
pp. 778-785.
show abstract
Leiomyosarcomas of soft tissues are an aggressive group of tumors with a high incidence of recurrence. Little is known about the molecular genetic changes associated with clinical outcome. Therefore, we studied 28 leiomyosarcoma samples of similar grade using comparative genomic hybridization and DNA flow cytometry and identified a difference in survival time associated with ploidy status and the number of chromosomal aberrations. The average survival time was shown to decrease with increase in chromosomal aberrations identified using comparative genomic hybridization. The average survival time was shorter in the near-tetraploid group than in the diploid and triploid group. Gain of 5p14-pter was significantly more common in near-tetraploid tumors. The survival time of patients with near-tetraploidy together with gain of 5p14-pter was reduced, and 50% died within the 1st year. Furthermore, loss of 13q14-q21 was significantly more frequent in the <5-year than in the >5-year survival group (P =.01). These results suggest that 13q14-q21 loss and 5p14-pter gain at diagnosis could be used to identify patients with leiomyosarcoma who are likely to have a shorter survival time and who might benefit from early treatment intensification..
Lu, Y.-.
Williamson, D.
Wang, R.
Summersgill, B.
Rodriguez, S.
Rogers, S.
Pritchard-Jones, K.
Campbell, C.
Shipley, J.
(2003). Expression profiling targeting chromosomes for tumor classification and prediction of clinical behavior. Genes chromosomes cancer,
Vol.38
(3),
pp. 207-214.
show abstract
Tumors are associated with altered or deregulated gene products that affect critical cellular functions. Here we assess the use of a global expression profiling technique that identifies chromosome regions corresponding to differential gene expression, termed comparative expressed sequence hybridization (CESH). CESH analysis was performed on a total of 104 tumors with a diagnosis of rhabdomyosarcoma, leiomyosarcoma, prostate cancer, and favorable-histology Wilms tumors. Through the use of the chromosome regions identified as variables, support vector machine analysis was applied to assess classification potential, and feature selection (recursive feature elimination) was used to identify the best discriminatory regions. We demonstrate that the CESH profiles have characteristic patterns in tumor groups and were also able to distinguish subgroups of rhabdomyosarcoma. The overall CESH profiles in favorable-histology Wilms tumors were found to correlate with subsequent clinical behavior. Classification by use of CESH profiles was shown to be similar in performance to previous microarray expression studies and highlighted regions for further investigation. We conclude that analysis of chromosomal expression profiles can group, subgroup, and even predict clinical behavior of tumors to a level of performance similar to that of microarray analysis. CESH is independent of selecting sequences for interrogation and is a simple, rapid, and widely accessible approach to identify clinically useful differential expression..
Tiffin, N.
Williams, R.D.
Shipley, J.
Pritchard-Jones, K.
(2003). PAX7 expression in embryonal rhabdomyosarcoma suggests an origin in muscle satellite cells. Br j cancer,
Vol.89
(2),
pp. 327-332.
show abstract
Rhabdomyosarcoma (RMS) is a common paediatric soft tissue sarcoma that resembles developing foetal skeletal muscle. Tumours of the alveolar subtype frequently harbour one of two characteristic translocations that juxtapose PAX3 or PAX7, and the forkhead-related gene FKHR (FOXO1A). The embryonal subtype of RMS is not generally associated with these fusion genes. Here, we have quantified the relative levels of chimaeric and wild-type PAX transcripts in various subtypes of RMS (n=34) in order to assess the relevance of wild-type PAX3 and PAX7 gene expression in these tumours. We found that upregulation of wild-type PAX3 is independent of the presence of either fusion gene and is unlikely to contribute to tumorigenesis. Most strikingly, upregulated PAX7 expression is almost entirely restricted to cases without PAX3-FKHR or PAX7-FKHR fusion genes and may contribute to tumorigenesis in the absence of chimaeric PAX transcription factors. Furthermore, as myogenic satellite cells are known to express PAX7, this pattern of PAX7 expression suggests this cell type as the origin of these tumours. This is corroborated by the detection of MET (c-met) expression, a marker for the myogenic satellite cell lineage, in all RMS samples expressing wild-type PAX7..
Rodriguez, S.
Jafer, O.
Goker, H.
Summersgill, B.M.
Zafarana, G.
Gillis, A.J.
van Gurp, R.J.
Oosterhuis, J.W.
Lu, Y.-.
Huddart, R.
Cooper, C.S.
Clark, J.
Looijenga, L.H.
Shipley, J.M.
(2003). Expression profile of genes from 12p in testicular germ cell tumors of adolescents and adults associated with i(12p) and amplification at 12p11 2-p12 1. Oncogene,
Vol.22
(12),
pp. 1880-1891.
show abstract
Gain of 12p material is invariably associated with testicular germ cell tumors (TGCTs) of adolescents and adults, most usually as an isochromosome 12p. We analyzed TGCTs with i(12p) using a global approach to expression profiling targeting chromosomes (comparative expressed sequence hybridization, CESH). This indicated overexpression of genes from 12p11.2-p12.1 relative to testis tissue and fibroblasts. The nonseminoma subtype showed higher levels of expression than seminomas. Notably, 12p11.2-p12.1 is amplified in about 10% of TGCTs and CESH analysis of such amplicon cases showed high levels of overexpression from this region. Microarray analysis, including cDNA clones representing most UniGene clusters from 12p11.2-p12.1, was applied to DNA and RNA from 5 TGCTs with amplification of 12p11.2-p12.1 and seven TGCTs with gain of the entire short arm of chromosome 12. Expression profiles were consistent with the CESH data and overexpression of EST595078, MRPS35 and LDHB at 12p11.2-p12.1 was detected in most TGCTs. High-level overexpression of BCAT1 was specific to nonseminomas and overexpression of genes such as CMAS, EKI1, KRAS2, SURB7 and various ESTs correlated with their amplification. Genes such as CCND2, GLU3, LRP6 and HPH1 at 12p13 were also overexpressed. The overexpressed sequences identified, particularly those in the region amplified, represent candidate genes for involvement in TGCT development..
Martin, A.J.
Summersgill, B.M.
Fisher, C.
Shipley, J.M.
Dean, A.F.
(2002). Chromosomal imbalances in meningeal solitary fibrous tumors. Cancer genetics and cytogenetics,
Vol.135
(2),
pp. 160-5.
Ladanyi, M.
Antonescu, C.R.
Leung, D.H.
Woodruff, J.M.
Kawai, A.
Healey, J.H.
Brennan, M.F.
Bridge, J.A.
Neff, J.R.
Barr, F.G.
Goldsmith, J.D.
Brooks, J.S.
Goldblum, J.R.
Ali, S.Z.
Shipley, J.
Cooper, C.S.
Fisher, C.
Skytting, B.
Larsson, O.
(2002). Impact of SYT-SSX fusion type on the clinical behavior of synovial sarcoma: A multi-institutional retrospective study of 243 patients. Cancer research,
Vol.62
(1),
pp. 135-6.
Cattaneo, E.
Roberts, I.
Shipley, J.
Gordon, A.
Nacheva, E.
Coleman, N.
(2002). Translocations involving chromosome 15 in embryonal rhabdomyosarcoma. Journal of pathology,
Vol.198,
pp. 26A-1.
Trupiano, J.K.
Rice, T.W.
Herzog, K.
Barr, F.G.
Shipley, J.
Fisher, C.
Goldblum, J.R.
(2002). Mediastinal synovial sarcoma: Report of two cases with molecular genetic analysis. Annals of thoracic surgery,
Vol.73
(2),
pp. 628-3.
Gruszka-Westwood, A.M.
Atkinson, S.
Summersgill, B.M.
Shipley, J.
Elnenaei, M.O.
Jain, P.
Hamoudi, R.A.
Kaeda, J.S.
Wotherspoon, A.C.
Matutes, E.
Catovsky, D.
(2002). Unusual case of leukemic mantle cell lymphoma with amplified CCNDI/IGH fusion gene. Genes chromosomes & cancer,
Vol.33
(2),
pp. 206-7.
Lu, Y.-.
Hing, S.
Williams, R.
Pinkerton, R.
Shipley, J.
Pritchard-Jones, K.
UK Children's Cancer Study Goup Wilms' tumor group,
(2002). Chromosome 1q expression profiling and relapse in Wilms' tumour. Lancet,
Vol.360
(9330),
pp. 385-386.
show abstract
Gene-expression patterns have proved useful for tumour classification and prediction of prognosis. We investigated a new gene-expression profiling method that targets individual chromosomes--comparative expressed sequence hybridisation (CESH)--to establish if any expression pattern was associated with relapse, and if this pattern could accurately predict outcome. We studied 18 cases of Wilms' tumour with favourable histological findings. Relative overexpression of genes on the long arm of chromosome 1 was shown in all tumours that subsequently relapsed but in none of those from patients in remission (odds ratio 357, 95% CI 6.4-19960). This finding shows the value of CESH analysis to rapidly screen for chromosomal regions that carry genes whose differential expression is important in establishment of tumour behaviour..
Clark, J.
Edwards, S.
John, M.
Flohr, P.
Gordon, T.
Maillard, K.
Giddings, I.
Brown, C.
Bagherzadeh, A.
Campbell, C.
Shipley, J.
Wooster, R.
Cooper, C.S.
(2002). Identification of amplified and expressed genes in breast cancer by comparative hybridization onto microarrays of randomly selected cDNA clones. Genes chromosomes cancer,
Vol.34
(1),
pp. 104-114.
show abstract
Microarray analysis using sets of known human genes provides a powerful platform for identifying candidate oncogenes involved in DNA amplification events but suffers from the disadvantage that information can be gained only on genes that have been preselected for inclusion on the array. To address this issue, we have performed comparative genome hybridization (CGH) and expression analyses on microarrays of clones, randomly selected from a cDNA library, prepared from a cancer containing the DNA amplicon under investigation. Application of this approach to the BT474 breast carcinoma cell line, which contains amplicons at 20q13, 17q11-21, and 17q22-23, identified 50 amplified and expressed genes, including genes from these regions previously proposed as candidate oncogenes. When considered together with data from microarray expression profiles and Northern analyses, we were able to propose five genes as new candidate oncogenes where amplification in breast cancer cell lines was consistently associated with higher levels of RNA expression. These included the HB01 histone acetyl transferase gene at 17q22-23 and the TRAP100 gene, which encodes a thyroid hormone receptor-associated protein coactivator, at 17q11-21. The results demonstrate the utility of this microarray-based CGH approach in hunting for candidate oncogenes within DNA amplicons..
Summersgill, B.
Thornton, P.
Atkinson, S.
Matutes, E.
Shipley, J.
Catovsky, D.
Houlston, R.S.
Yuille, M.R.
(2002). Chromosomal imbalances in familial chronic lymphocytic leukaemia: a comparative genomic hybridisation analysis. Leukemia,
Vol.16
(7),
pp. 1229-1232.
show abstract
A subset of B cell chronic lymphocytic leukaemia (CLL) is familial. Lack of large families makes it attractive to exploit methods in addition to genetic linkage analysis for the identification of a susceptibility locus. One strategy that can localise regions of the genome that may harbour tumour suppressor genes is to identify regions of chromosomal imbalance using comparative genomic hybridisation (CGH) analysis. We examined 24 familial CLL cases by CGH analysis. Losses that are documented as arising frequently in sporadic CLL were observed at a comparable frequency in familial CLL. However, gains and losses in two regions of the X chromosome - Xp11.2-p21 and Xq21-qter - appear more common in familial CLL than in sporadic CLL. This suggests these regions may harbour a susceptibility locus for CLL. There is also some evidence that chromosome regions 2p12-p14 and 4q11-q21 may harbour predisposition genes..
Barker, K.T.
Bevan, S.
Wang, R.
Lu, Y.J.
Flanagan, A.M.
Bridge, J.A.
Fisher, C.
Finlayson, C.J.
Shipley, J.
Houlston, R.S.
(2002). Low frequency of somatic mutations in the FH/multiple cutaneous leiomyomatosis gene in sporadic leiomyosarcomas and uterine leiomyomas. British journal of cancer,
Vol.87
(4),
pp. 446-3.
Barker, K.T.
Bevan, S.
Wang, R.
Lu, Y.-.
Flanagan, A.M.
Bridge, J.A.
Fisher, C.
Finlayson, C.J.
Shipley, J.
Houlston, R.S.
(2002). Low frequency of somatic mutations in the FH/multiple cutaneous leiomyomatosis gene in sporadic leiomyosarcomas and uterine leiomyomas. British journal of cancer,
Vol.87
(4),
pp. 446-448.
Xie, Y.T.
Skytting, B.
Nilsson, G.
Grimer, R.J.
Mangham, C.D.
Fisher, C.
Shipley, J.
Bjerkehagen, B.
Myklebost, O.
Larsson, O.
(2002). The SYT-SSX1 fusion type of synovial sarcoma is associated with increased expression of cyclin A and D1 : A link between t(X;18)(p11 2; q11 2) and the cell cycle machinery. Oncogene,
Vol.21
(37),
pp. 5791-6.
Davies, H.
Bignell, G.R.
Cox, C.
Stephens, P.
Edkins, S.
Clegg, S.
Teague, J.
Woffendin, H.
Garnett, M.J.
Bottomley, W.
Davis, N.
Dicks, E.
Ewing, R.
Floyd, Y.
Gray, K.
Hall, S.
Hawes, R.
Hughes, J.
Kosmidou, V.
Menzies, A.
Mould, C.
Parker, A.
Stevens, C.
Watt, S.
Hooper, S.
Wilson, R.
Jayatilake, H.
Gusterson, B.A.
Cooper, C.
Shipley, J.
Hargrave, D.
Pritchard-Jones, K.
Maitland, N.
Chenevix-Trench, G.
Riggins, G.J.
Bigner, D.D.
Palmieri, G.
Cossu, A.
Flanagan, A.
Nicholson, A.
Ho, J.W.
Leung, S.Y.
Yuen, S.T.
Weber, B.L.
Seigler, H.F.
Darrow, T.L.
Paterson, H.
Marais, R.
Marshall, C.J.
Wooster, R.
Stratton, M.R.
Futreal, P.A.
(2002). Mutations of the BRAF gene in human cancer. Nature,
Vol.417
(6892),
pp. 949-954.
show abstract
Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma..
Osin, P.
Lu, Y.J.
Bell, A.
Stone, J.
Houlston, R.S.
Gusterson, B.A.
Shipley, J.
(2001). Medullary carcinoma of breast shows a distinct pattern of chromosomal copy number chances, but not DNA mismatch repair deficit. Modern pathology,
Vol.14
(1),
pp. 33A-33A.
Osin, P.
Lu, Y.J.
Bell, A.
Stone, J.
Houlston, R.S.
Gusterson, B.A.
Shipley, J.
(2001). Medullary carcinoma of breast shows a distinct pattern of chromosomal copy number changes, but not DNA mismatch repair deficit. Laboratory investigation,
Vol.81
(1),
pp. 33A-33A.
Barker, K.
Bevan, S.
Wang, R.
Alam, A.
Tomlinson, I.
Shipley, J.
Houlston, R.
(2001). The role of MUCL1 in sporadic leiomyomas and leimyosarcomas. Journal of medical genetics,
Vol.38,
pp. S66-1.
Barker, K.
Martinez, A.
Wang, R.
Bevan, S.
Murday, V.
Shipley, J.
Houlston, R.
Harper, J.
(2001). PTEN mutations are uncommon in Proteus syndrome. Journal of medical genetics,
Vol.38
(7),
pp. 480-2.
full text
Wang, R.
Lu, Y.J.
Fisher, C.
Bridge, J.A.
Shipley, J.
(2001). Characterization of chromosome aberrations associated with soft-tissue leiomyosarcomas by twenty-four-color karyotyping and comparative genomic hybridization analysis. Genes chromosomes & cancer,
Vol.31
(1),
pp. 54-11.
Gordon, T.
McManus, A.
Anderson, J.
Min, T.
Swansbury, J.
Pritchard-Jones, K.
(2001). Cytogenetic abnormalities in 42 rhabdomyosarcoma: A United Kingdom cancer cytogenetics group study. Medical and pediatric oncology,
Vol.36
(2),
pp. 259-267.
Hing, S.
Lu, Y.J.
Summersgill, B.
King-Underwood, L.
Nicholson, J.
Grundy, P.
Grundy, R.
Gessler, M.
Shipley, J.
Pritchard-Jones, K.
(2001). Gain of 1q is associated with adverse outcome in favorable histology Wilms' tumors. Am j pathol,
Vol.158
(2),
pp. 393-398.
show abstract
Although several genes/genetic loci involved in the etiology of Wilms' tumor have been identified, little is known of the molecular changes associated with relapse. We therefore undertook an analysis by comparative genomic hybridization (CGH) of 58 tumor samples of favorable histology Wilms' tumor taken at initial diagnosis and/or relapse. Tumors with anaplastic histology were excluded as this is known to be associated with p53 mutation and a poor prognosis. A control group of 21 Wilms' tumors that did not relapse was also analyzed. The overall frequency of gains or losses of genetic material detected by CGH was similar in both groups (77% in relapsing tumors and 70% in the nonrelapse group) as was the median number of changes per tumor (relapse group: n = 4, range, 1 to 19; nonrelapse group: n = 3, range, 1 to 8). However, gain of 1q was significantly more frequent in the relapse series [27 of 46 (59%) versus 5 of 21 (24%), P: = 0.019]. In 12 matched tumor pairs, the CGH profiles, including 1q gain, were similar at diagnosis and relapse, with little evidence for further copy number changes being involved in clonal evolution. The results suggest that 1q gain at diagnosis could be used to identify patients with favorable histology Wilms' tumor at increased risk of relapse who might benefit from early treatment intensification..
Gordon, T.
McManus, A.
Anderson, J.
Min, T.
Swansbury, J.
Pritchard-Jones, K.
Shipley, J.
United kingdom Children's Cancer Study Group,
United Kingdom Cancer Cytogenetics Group,
(2001). Cytogenetic abnormalities in 42 rhabdomyosarcoma: a United Kingdom Cancer Cytogenetics Group Study. Med pediatr oncol,
Vol.36
(2),
pp. 259-267.
show abstract
BACKGROUND: Rhabdomyosarcomas are the most common type of pediatric soft tissue sarcoma. The cytogenetic literature on RMS is biased towards the less common alveolar subtype (ARMS), which is frequently associated with specific translocations and the PAX3/7-FKHR fusion genes. Relatively few karyotypes are reported for the embryonal subtype (ERMS). The aim of this study was to further cytogenetic knowledge of RMS subtypes. PROCEDURE: Representative examples of all karyotypes from UKCCG; member laboratories were reexamined and their histopathologies reviewed through the United Kingdom Children's Cancer Study (Group) (UKCCSG). Molecular evidence for the PAX3/7-FKHR fusion genes was available for five ERMS and seven ARMS cases and compiled with the karyotypes. RESULTS: Clonal chro mosome aberrations were characterized for 25 ERMS and 17 ARMS cases. Thirty-six percent of the ERMS cases involved translocation breakpoints in the 1p11-q11 region. Ten of the seventeen cases of ARMS showed cytogenetic evidence for the t(2;13)(q35;q14), consistent with molecular data available from four of these. Two further ARMS cases revealed a PAX3-FKHR and a variant PAX7-FKHR fusion gene product that were not detected cytogenetically. CONCLUSIONS: Many of the karyotypes from both subtypes were complex. The frequent involvement of the 1p11-1q11 region and gain of chromosomes 2, 8, 12, and 13 in ERMS may be functionally significant. There was no evidence for involvement of the PAX3/7-FKHR genes in ERMS, and cryptic involvement was found in some ARMS. There were no consistent chromosomal rearrangements associated with apparently translocation negative ARMS cases..
Anderson, J.
Gordon, T.
McManus, A.
Mapp, T.
Gould, S.
Kelsey, A.
McDowell, H.
Pinkerton, R.
Shipley, J.
Pritchard-Jones, K.
UK Children's Cancer Study Group (UKCCSG) and the UK Cancer Cytogenetics Group,
(2001). Detection of the PAX3-FKHR fusion gene in paediatric rhabdomyosarcoma: a reproducible predictor of outcome?. Br j cancer,
Vol.85
(6),
pp. 831-835.
show abstract
Rhabdomyosarcoma has 2 major histological subtypes, embryonal and alveolar. Alveolar histology is associated with the fusion genes PAX3-FKHR and PAX7-FKHR. Definition of alveolar has been complicated by changes in terminology and subjectivity. It is currently unclear whether adverse clinical behaviour is better predicted by the presence of these fusion genes or by alveolar histology. We have determined the presence of the PAX3/7-FKHR fusion genes in 91 primary rhabdomyosarcoma tumours using a combination of classical cytogenetics, FISH and RT-PCR, with a view to determining the clinical characteristics of tumours with and without the characteristic translocations. There were 37 patients with t(2;13)/PAX3-FKHR, 8 with t(1;13) PAX7-FKHR and 46 with neither translocation. One or other of the characteristic translocations was found in 31/38 (82%) of alveolar cases. Univariate survival analysis revealed the presence of the translocation t(2;13)/PAX3-FKHR to be an adverse prognostic factor. With the difficulties in morphological diagnosis of alveolar rhabdomyosarcoma on increasingly used small needle biopsy specimens, these data suggest that molecular analysis for PAX3-FKHR will be a clinically useful tool in treatment stratification in the future. This hypothesis requires testing in a prospective study. Variant t(1;13)/PAX7-FKHR appears biologically different, occurring in younger patients with more localised disease..
Summersgill, B.M.
Jafer, O.
Wang, R.
Goker, H.
Niculescu-Duvaz, I.
Huddart, R.
Shipley, J.
(2001). Definition of chromosome aberrations in testicular germ cell tumor cell lines by 24-color karyotyping and complementary molecular cytogenetic analyses. Cancer genet cytogenet,
Vol.128
(2),
pp. 120-129.
show abstract
Many of the reported karyotypes for adult testicular germ cell tumors (GCTs) are complex and incomplete, although the presence of an isochromosome 12p, i(12p), and gain of 12p material have consistently been found. Here, an accurate definition of the chromosome aberrations associated with four cell lines derived from GCTs (GCT27, H12.1, Tera1, and Tera2) has been produced using 24-color karyotyping by mulifluor in situ hybridization, comparative genomic hybridization analysis, and further fluorescence in situ hybridization analysis to confirm some chromosomal assignments and refine involvement of specific regions of 12p. There was karyotypic heterogeneity. Isochromosomes in addition to i(12p) were found, as were other rearrangements with breakpoints at or near centromeric regions. The most frequent non-centromeric breakpoints were at 1p31 approximately p32, 1p21 approximately p22, 11q13, and Xq22, although consistent partner chromosomes were not involved. One cell line (Tera1) showed a subtle dosage increase in the copy number of a 12p probe known to be within the smallest overlapping region of amplification that has been defined in a number of testicular GCTs with amplicons at 12p11 approximately p12. The chromosome rearrangements and associated imbalances may be significant in GCT progression and the characterized cell lines can be used to investigate these further..
Lu, Y.
Condie, A.
Bennett, J.D.
Fry, M.J.
Yuille, M.R.
Shipley, J.
(2001). Disruption of the ATM gene in breast cancer. Cancer genet cytogenet,
Vol.126
(2),
pp. 97-101.
show abstract
Mutations in the ATM gene, which maps to 11q22-23, cause the multisystem recessive syndrome ataxia-telangiectasia (AT). Breast cancer has been reported in AT patients and carriers. Sporadic breast cancer is associated with loss of heterozygosity at or in the region of ATM and chromosomal abnormalities involving 11q23. We have investigated the chromosomes, nuclei and released chromatin fibers from nine primary breast carcinoma and eight cell lines by fluorescence in situ hybridization with four fluorochrome-labeled cosmids spanning the ATM gene. The ATM gene was disrupted in one primary breast carcinoma and in the cell lines MDA-MB-231 and MCF-7. The role of these aberrations in breast carcinomas, which may lead to gene dosage or dominant negative effects on gene function, requires further investigation..
Summersgill, B.
Osin, P.
Lu, Y.J.
Huddart, R.
Shipley, J.
(2001). Chromosomal imbalances associated with carcinoma in situ and associated testicular germ cell tumours of adolescents and adults. Br j cancer,
Vol.85
(2),
pp. 213-220.
show abstract
Carcinoma in situ (CIS) or intratubular germ cell neoplasia is generally considered the precursor lesion of adult testicular germ cell tumours (TGCT). The chromosomal imbalances associated with CIS and the corresponding seminoma (SE) or nonseminoma (NS) have been determined by comparative genomic hybridization (CGH) analysis of microdissected material from seven cases. Significantly, the CIS showed no gain of 12p material whereas in the invasive components of all cases gain of 12p was found, in 2 cases associated with amplification of the 12p11.2-12.1 region. Interphase fluorescence in situ analysis was consistent with this and provided evidence for the i(12p) or 12p11.2-12.1 amplification in the SE and NS but not in the corresponding CIS. This suggests a role for these changes in progression of CIS to invasive testicular cancer or progression of the invasive disease. Other imbalances such as gain of material from chromosomes 1, 5, 7, 8, 12q and X and loss of material from chromosome 18 were frequently identified (> 40% of cases) in the CIS associated with both SE and NS as well as in the invasive components. Loss of material from chromosome 4 and 13 and gain of 2p were more frequently found in the invasive components. The results shed light on the genetic relationship between the non-invasive and invasive components of testicular cancer and the stage at which particular chromosomal changes may be important..
Lu, Y.J.
Williamson, D.
Clark, J.
Wang, R.
Tiffin, N.
Skelton, L.
Gordon, T.
Williams, R.
Allan, B.
Jackman, A.
Cooper, C.
Pritchard-Jones, K.
Shipley, J.
(2001). Comparative expressed sequence hybridization to chromosomes for tumor classification and identification of genomic regions of differential gene expression. Proc natl acad sci u s a,
Vol.98
(16),
pp. 9197-9202.
show abstract
full text
Altered expression of genes can have phenotypic consequences in cancer development and treatment, developmental abnormalities, and differentiation processes. Here we describe a rapid approach, termed comparative expressed sequence hybridization (CESH), which gives a genome-wide view of relative expression patterns within tissues according to chromosomal location. No prior knowledge of genes or cloning is required, and minimal amounts of tissue can be used. Expression profiles are achieved in a manner similar to the identification of chromosomal imbalances by comparative genomic hybridization analysis. The approach is demonstrated to indicate a chromosomal region that harbors overexpressed genes that may be associated with a drug-resistant phenotype. In addition, known and new regions of differential gene expression in both normal tissues and tumor samples from the soft tissue sarcoma group of rhabdomyosarcoma (RMS) are indicated. These regions included 2p24; overexpression of MYCN at 2p24 was confirmed by quantitative reverse transcription-PCR for all of the alveolar RMS cases and did not necessarily correspond to genomic amplification. Evidence including region specific microarray analysis indicated that overexpression of several genes from a region may be required for detection by CESH. This evidence is consistent with clusters of functionally related genes and mechanisms that affect the expression of a number of genes at a particular genomic location. The distinctive CESH profiles demonstrated in different subtypes of RMS show potential for tumor classification..
Roberts, I.
Gordon, A.
Wang, R.
Pritchard-Jones, K.
Shipley, J.
Coleman, N.
(2001). Molecular cytogenetic analysis consistently identifies translocations involving chromosomes 1, 2 and 15 in five embryonal rhabdomyosarcoma cell lines and a PAX-FOXO1A fusion gene negative alveolar rhabdomyosarcoma cell line. Cytogenet cell genet,
Vol.95
(3-4),
pp. 134-142.
show abstract
Rhabdomyosarcoma in children is a "small round blue cell tumour" that displays skeletal muscle differentiation. Two main histological variants are recognised, alveolar (ARMS) and embryonal (ERMS) rhabdomyosarcoma. Whereas consistent chromosome translocations characteristic of ARMS have been reported, no such cytogenetic abnormality has yet been described in ERMS. We have used multiple colour chromosome painting to obtain composite karyotypes for five ERMS cell lines and one PAX-FOXO1A fusion gene negative ARMS. The cell lines were assessed by spectral karyotyping (SKY), tailored multi-fluorophore fluorescence in situ hybridisation (M-FISH) using series of seven colour paint sets generated to examine specific abnormalities, and comparative genomic hybridisation (CGH). This approach enabled us to obtain karyotypes of the cell lines in greater detail than previously possible. Several recurring cytogenetic abnormalities were demonstrated, including translocations involving chromosomes 1 and 15 and chromosomes 2 and 15, in 4/6 and 2/6 cell lines respectively. All six cell lines demonstrated abnormalities of chromosome 15. Translocations between chromosomes 1 and 15 have previously been recorded in two primary cases of ERMS by conventional cytogenetics. Analysis of the translocation breakpoints may suggest mechanisms of ERMS tumourigenesis and may enable the development of novel approaches to the clinical management of this tumour..
Jones, C.
Foschini, M.P.
Chaggar, R.
Lu, Y.J.
Wells, D.
Shipley, J.M.
Eusebi, V.
Lakhani, S.R.
(2000). Comparative genomic hybridization analysis of myoepithelial carcinoma of the breast. Laboratory investigation,
Vol.80
(6),
pp. 831-6.
Roberts, I.
Gordon, T.
Shipley, J.
Coleman, N.
(2000). Consistent chromosomal translocations in embryonal rhabdomyosarcoma. Journal of pathology,
Vol.190,
pp. 44A-1.
Smedley, D.
Sidhar, S.
Birdsall, S.
Bennett, D.
Herlyn, M.
Cooper, C.
Shipley, J.
(2000). Characterization of chromosome 1 abnormalities in malignant melanomas. Genes chromosomes cancer,
Vol.28
(1),
pp. 121-125.
show abstract
Chromosome 1 abnormalities are the most commonly detected aberrations in many cancers including malignant melanomas. Specific breakpoints are reported for malignant melanomas throughout the chromosome but especially at 1p36 and at several sites throughout 1p22-q21. In addition, partial deletions and loss of heterozygosity have been found on 1p indicating the possible location of tumor suppressor genes. Here we have characterized the involvement of chromosome 1 in a series of seven malignant melanoma cell lines. Initial chromosome painting studies revealed that six of the cell lines had chromosome 1 rearrangements. Deletions involving 1p10-32, 1q11-44, and 1q25-44 were observed. The other rearrangement breakpoints included three in the 1q10-p11 region with the rest at 1p36, 1p34, 1p32, 1p31, 1p12-13, 1q21, and 1q23. The breaks at 1q10-p11 were investigated further using an alpha-satellite 1 centromere probe and yeast artificial chromosomes (YACs) from the region. Two of the 1q10-p11 breaks mapped in the centromeric region, while the others mapped to variable sites. This suggests that the role of these rearrangements in the pathogenesis of melanomas does not involve the alteration of specific oncogenes in the breakpoint region. During the YAC mapping a previously undetected, small (<1 Mbp) del(1)(p10p11) was identified. This deletion lies within minimal overlapping deleted regions reported in head and neck as well as breast carcinomas and it could therefore facilitate the isolation of a carcinoma-associated tumor suppressor gene..
Gordon, A.T.
Brinkschmidt, C.
Anderson, J.
Coleman, N.
Dockhorn-Dworniczak, B.
Pritchard-Jones, K.
Shipley, J.
(2000). A novel and consistent amplicon at 13q31 associated with alveolar rhabdomyosarcoma. Genes chromosomes cancer,
Vol.28
(2),
pp. 220-226.
show abstract
Rhabdomyosarcomas are the most common soft-tissue sarcoma found in children. The alveolar subtype is clinically more aggressive than the embryonal subtype. In addition to the presence of specific chromosome translocations and associated fusion gene products in a high proportion of the alveolar subtype, we previously showed that tumors with this histology frequently show evidence of genomic amplification. Here, we substantially extended the number of alveolar rhabdomyosarcoma samples examined by comparative genomic hybridization analysis. Regions of loss were noted, including the smallest overlapping regions corresponding to 16q, 17/17p, and 9q32-34, in 16%, 10%, and 10% of cases, respectively (44 primary samples/6 cell lines). Amplification or gain at 12q13-15 in the region of the MDM2/GLI1/SAS/CDK4 loci and 2p24 at the MYCN locus was found in 28% and 32% of cases, respectively. Single amplicons were found at locations that in other samples showed consistent gain, including the regions 5q15-23, 7q21-31, 11p11-14, 17q23-24, and 20q13, and amplification was found in two cases at 15q24-26. However, most striking was a novel region of amplification or gain at 13q31 in 19% of cases (51 primary samples/6 cell lines). This indicates that a gene or genes at 13q31 are significant in the development or progression of alveolar rhabdomyosarcoma..
Lu, Y.J.
Morris, J.S.
Edwards, P.A.
Shipley, J.
(2000). Evaluation of 24-color multifluor-fluorescence in-situ hybridization (M-FISH) karyotyping by comparison with reverse chromosome painting of the human breast cancer cell line T-47D. Chromosome res,
Vol.8
(2),
pp. 127-132.
show abstract
Multifluor-fluorescence in-situ hybridization (M-FISH) chromosome paints for all the chromosomes in the human complement labeled with different combinations of fluorochromes is a recent technological development enabling assignment of chromosomal material to rearranged chromosomes. Little data is available on the accuracy and limitations of the approach to the analysis of complex karyotypes, which are characteristic of many malignant diseases. Here we compare M-FISH analysis of the breast-cancer-derived cell line T-47D with a previous analysis by reverse chromosome painting analysis of flow-sorted chromosomes from the same material. This demonstrated a high degree of concordance. It also illustrated the limitations of M-FISH analysis, including difficulties identifying small regions of chromosomal material and intrachromosomal rearrangements. Confirmation of selected aberrations using less-complex mixtures of painting probes and further definition of abnormalities using single copy markers may be required. The detailed karyotype description possible by M-FISH analysis contrasts with the definition in the original G-banding analysis. This and the level of concordance with reverse FISH painting supports the utility of the approach in the definition of complex karyotypes..
Lu, Y.J.
Osin, P.
Lakhani, S.
Gusterson, B.
Shipley, J.
(1999). CGH analysis of lobular carcinoma in situ and atypical lobular hyperplasia. British journal of cancer,
Vol.80,
pp. 82-1.
Smedley, D.
Hamoudi, R.
Lu, Y.J.
Cooper, C.
Shipley, J.
(1999). Cloning and mapping of members of the MYM family. Genomics,
Vol.60
(2),
pp. 244-4.
Lu, Y.J.
Dong, X.Y.
Shipley, J.
Zhang, R.G.
Cheng, S.J.
(1999). Chromosome 3 imbalances are the most frequent aberration found in non-small cell lung carcinoma. Lung cancer,
Vol.23
(1),
pp. 61-6.
van de Rijn, M.
Barr, F.G.
Xiong, Q.B.
Hedges, M.
Shipley, J.
Fisher, C.
(1999). Poorly differentiated synovial sarcoma - An analysis of clinical, pathologic, and molecular genetic features. American journal of surgical pathology,
Vol.23
(1),
pp. 106-7.
Lu, Y.J.
Birdsall, S.
Summersgill, B.
Smedley, D.
Osin, P.
Fisher, C.
Shipley, J.
(1999). Dual colour fluorescence in situ hybridization to paraffin-embedded samples to deduce the presence of the der(X)t(X;18)(p11 2;q11 2) and involvement of either the SSX1 or SSX2 gene: a diagnostic and prognostic aid for synovial sarcoma. J pathol,
Vol.187
(4),
pp. 490-496.
show abstract
Identification of the t(X;18)(p11.2;q11.2) and the fusion gene products, SYT-SSX1 and SYT-SSX2, associated with a high proportion of synovial sarcomas, has been shown to be a useful diagnostic aid. This study demonstrates the application of dual colour fluorescence in situ hybridization to paraffin-embedded samples to deduce the presence of the derivative X chromosome and also the position of the breakpoint on chromosome X at either the SSX1 or the SSX2 gene. This used region specific markers from chromosomes X and 18 and an optimized protocol involving microwave exposure. Novel and rapid scoring criteria were validated which circumvented potential problems of nuclear truncation and defining cell boundaries. This involved blind analysis of two negative sarcoma samples and three synovial sarcomas in which corresponding frozen material had been previously shown to have the translocation involving different SSX genes. Six new cases diagnosed as synovial sarcoma were also analysed; two monophasic and two biphasic case were deduced to have a breakpoint in the SSX1 gene, one monophasic case an SSX2 breakpoint, and one case did not show rearrangement of the region. The ability to analyse formalin-fixed, paraffin-embedded samples in this way has practical implications for aiding the diagnosis of difficult cases, recently ascribed prognostic relevance, and allows further retrospective studies to be carried out. The methodology is also applicable to the identification of other tumour specific translocations in paraffin-embedded material..
Anderson, J.
Gordon, A.
McManus, A.
Shipley, J.
Pritchard-Jones, K.
(1999). Disruption of imprinted genes at chromosome region 11p15 5 in paediatric rhabdomyosarcoma. Neoplasia,
Vol.1
(4),
pp. 340-348.
show abstract
full text
Rhabdomyosarcomas are characterized by loss of heterozygosity (LOH) at chromosome region 11p15.5, a region known to contain several imprinted genes including insulin-like growth factor 2 (IGF2), H19, and p57(KIP2). We analyzed 48 primary tumour samples and found distinct genetic changes at 11p15.5 in alveolar and embryonal histological subtypes. LOH was a feature of embryonal tumours, but at a lower frequency than previous studies. Loss of imprinting (LOI) of the IGF2 gene was detected in 6 of 13 informative cases, all harbouring PAX3-FKHR or PAX7-FKHR fusion genes characteristic of alveolar histology. In contrast, H19 imprinting was maintained in 14 of 15 informative cases and the case with H19 LOI had maintenance of the IGF2 imprint indicating separate mechanisms controlling imprinting of IGF2 and H19. The adult promoter of IGF2, P1, was used in 5 of 14 tumours and its expression was unrelated to IGF2 imprinting status implying a further mechanism of altered IGF2 regulation. The putative tumour suppressor gene p57(KIP2) was expressed in 15 of 29 tumours and expression was unrelated to allele status. Moreover, in tumours with p57(KIP2) expression, there was no evidence for inactivating mutations, suggesting that p57(KIP2) is not a tumour suppressor in rhabdomyosarcoma..
Smedley, D.
Demiroglu, A.
Abdul-Rauf, M.
Heath, C.
Cooper, C.
Shipley, J.
Cross, N.C.
(1999). ZNF198-FGFR1 transforms Ba/F3 cells to growth factor independence and results in high level tyrosine phosphorylation of STATS 1 and 5. Neoplasia,
Vol.1
(4),
pp. 349-355.
show abstract
full text
The ZNF198- FGFR1 fusion gene arises as a result of the t(8;13)(p11;q12) in the 8p11 myeloproliferative syndrome. To determine the transforming properties of this chimeric protein we transfected ZNF198-FGFR1 into the interleukin (IL)-3 dependent cell line Ba/F3. Growth factor independent subclones were obtained in which ZNF198-FGFR1, STAT1, and STAT5 were constitutively tyrosine phosphorylated, as determined by immunoprecipitation and Western blot analysis. To test the hypothesis that constitutive activation of ZNF198-FGFR1 tyrosine kinase activity is a result of self-association of the fusion protein, we in vitro transcribed and translated ZNF198-FGFR1 and a derivative construct, ZNF198- FGFR1deltaC-myc, in which the C-terminal FGFR1 epitope was replaced by a c-myc tag. As expected, an anti-FGFR1 antibody immunoprecipitated ZNF198-FGFR1 but not ZNF198-FGFRdeltaC-myc. However when both products were translated together, both were coimmunoprecipitated by anti-FGFR1 antisera. Similar results were obtained by using an anti-myc antibody and demonstrated a physical interaction between the two proteins. Analysis of COS-7 cells transfected with ZNF198-FGFR1 demonstrated that the fusion gene, in contrast to normal FGFR1, is located in the cytoplasm. We conclude that ZNF198-FGFR1 is a cytoplasmic protein that self-associates and has constitutive transformation activity. These data suggest that ZNF198-FGFR1 plays a primary role in the pathogenesis of the t(8;13) myeloproliferative syndrome and is the first report to implicate STAT proteins in FGFR1-mediated signaling..
Anderson, J.
Gordon, A.
Pritchard-Jones, K.
Shipley, J.
(1999). Genes, chromosomes, and rhabdomyosarcoma. Genes chromosomes cancer,
Vol.26
(4),
pp. 275-285.
show abstract
Rhabdomyosarcomas are a heterogeneous group of malignant tumors and are the most common soft-tissue sarcoma of childhood. Rhabdomyosarcomas resemble developing skeletal muscle, notably in their expression of the MRF family of transcription factors and the PAX3 and PAX7 genes. These PAX genes are also involved through specific translocations, t(2;13)(q35;q14) and variant t(1;13)(p36;q14) in the alveolar subtype, which result in PAX3-FKHR and PAX7-FKHR fusion genes, respectively. The fusion genes are thought critically to affect downstream targets of PAX3 and PAX7 or possibly have novel targets. Similar downstream changes may also be involved in embryonal and fusion gene negative cases. Genomic amplification of such genes as MYCN, MDM2, CDK4, and PAX7-FKHR is a feature mainly of the alveolar subtype, while specific chromosomal gains, including chromosomes 2, 8, 12, and 13, are associated with the embryonal subtype. Loss of alleles and imprinting at 11p15.5 and disruption of genes such as IGF2, ATR, PTC, P16, and TP53 have also been implicated in rhabdomyosarcoma development. Whereas there is now a realistic possibility of cure in the majority of cases, there remains a subset that is resistant to multimodality therapy, including high-dose chemotherapy. Characterization of the defining molecular features of tumors that are likely to behave aggressively represents a particular challenge. Current research is leading toward a better understanding of rhabdomyosarcoma tumorigenesis, which may ultimately result in novel therapeutic strategies that increase the overall cure. Genes Chromosomes Cancer 26:275-285, 1999..
Anderson, J.
Gordon, A.
Pritchard-Jones, K.
Shipley, J.
(1999). Genes, chromosomes, and rhabdomyosarcoma. Genes, chromosomes and cancer,
Vol.26
(4),
pp. 275-285.
Birdsall, S.
Osin, P.
Lu, Y.J.
Fisher, C.
Shipley, J.
(1999). Synovial sarcoma specific translocation on associated with both epithelial and spindle cell components. International journal of cancer,
Vol.82
(4),
pp. 605-4.
Birdsall, S.H.
McManus, A.
Shipley, J.M.
Crolla, J.A.
(1998). Origin of the mar3 in the myeloid cell line HL-60 determined by fluorescence in situ hybridization. Cancer genetics and cytogenetics,
Vol.103
(1),
pp. 86-2.
Soundy, V.
Bailey, D.
Chaggar, R.
Lu, Y.J.
Osin, P.
Odel, C.
Ramachandra, S.
Shipley, J.M.
Lakhani, S.R.
(1998). Myoepithelial carcinoma of the breast A detailed analysis using immunohistochemistry, loss of heterozygosity and comparative genomic hybridisation. Journal of pathology,
Vol.184,
pp. 22A-1.
Smedley, D.
Somers, G.
Venter, D.
Chow, C.W.
Cooper, C.
Shipley, J.
(1998). Characterization of a t(8;13)(p11;q11-12) in an atypical myeloproliferative disorder. Genes chromosomes cancer,
Vol.21
(1),
pp. 70-73.
show abstract
Fourteen cases of an atypical myeloproliferative disorder associated with consistent translocations involving 8p11-12 have previously been described. A t(8;13)(p11;q11-12) was the most common, but variant t(8;9)(p11-12;q32-34) and t(6;8)(q27;p12) were also reported. Here we have used a series of yeast artificial chromosomes (YACs) derived from the 8p11 and 13q11-12 regions to analyse one of the t(8;13) cases by fluorescence in situ hybridization (FISH). YACs flanking the 13q11-12 breakpoint and spanning the 8p11 breakpoint have been isolated. These YACs will facilitate characterization of the genes involved in this rearrangement..
Osin, P.
Shipley, J.
Lui, Y.J.
Crook, T.
Gusterson, B.A.
(1998). Experimental pathology and breast cancer genetics: Looking at malignant and premalignant tissues using new technologies. European journal of cancer,
Vol.34,
pp. S96-1.
Osin, P.
Shipley, J.
Liu, Y.J.
Crook, T.
Gusterson, B.A.
(1998). Experimental pathology and breast cancer genetics: Looking at malignant and premalignant tissues using new technologies. European journal of cancer,
Vol.34,
pp. S3-1.
Osin, P.P.
Lu, Y.J.
Lakhani, S.
Pilotti, S.
Gusterson, B.A.
Shipley, J.
(1998). Gains and losses of genomic material in Atypical Lobular Hyperplasia (ALH) and Lobular Carcinoma In Situ (LCIS) of the breast suggest these lesions represent a similar stage of tumour progression. Journal of pathology,
Vol.184,
pp. 15A-1.
Shipley, J.
Fisher, C.
(1998). Chromosome translocations in sarcomas and the analysis of paraffin-embedded material. Journal of pathology,
Vol.184
(1),
pp. 1-3.
van de Rijn, M.
Barr, F.G.
Xiong, Q.B.
Hedges, M.
Shipley, J.
Fisher, C.
(1998). Poorly differentiated synovial sarcoma: an analysis of clinical, pathologic, and molecular genetic features. Laboratory investigation,
Vol.78
(1),
pp. 15A-1.
van de Rijn, M.
Barr, F.G.
Xiong, Q.B.
Hedges, M.
Shipley, J.
Fisher, C.
(1998). Poorly differentiated synovial sarcoma: an analysis of clinical, pathologic, and molecular genetic features. Modern pathology,
Vol.11
(1),
pp. 15A-1.
Smedley, D.
Somers, G.
Venter, D.
Chow, C.W.
Cooper, C.
Shipley, J.
(1998). Characterization of a t(8;13)(p11;q11-12) in an atypical myeloproliferative disorder. Genes, chromosomes and cancer,
Vol.21
(1),
pp. 70-73.
Smedley, D.
Hamoudi, R.
Clark, J.
Warren, W.
Abdul-Rauf, M.
Somers, G.
Venter, D.
Fagan, K.
Cooper, C.
Shipley, J.
(1998). The t(8;13)(p11;q11-12) rearrangement associated with an atypical myeloproliferative disorder fuses the fibroblast growth factor receptor 1 gene to a novel gene RAMP. Hum mol genet,
Vol.7
(4),
pp. 637-642.
show abstract
A recently described atypical myeloproliferative disorder is invariably associated with reciprocal translocations involving 8p11-12. The most common rearrangement is a t(8;13)(p11;q11-12). Here we determine that this translocation results in the fusion of the fibroblast growth factor receptor 1 gene (FGFR1), a member of the receptor tyrosine kinase family at 8p11, to a novel gene at 13q11-12 designated RAMP . The predicted RAMP protein exhibits strong homology to the product of a recently cloned candidate gene for X-linked mental retardation, DXS6673E . We also provide the first report of a novel, putative metal-binding motif, present as five tandem repeats in both RAMP and DXS6673E. RT-PCR detected only one of the two possible fusion transcripts, encoding a product in which the N-terminal 641 amino acids of RAMP become joined to the tyrosine kinase domain of FGFR1. Receptor tyrosine kinases are not commonly involved in the formation of tumour-specific fusion proteins. However, the previous reports of involvement of receptor tyrosine kinases in fusion proteins in non-Hodgkin's lymphoma, chronic myelomonocytic leukaemia and papillary thyroid carcinoma described similar rearrangements. By analogy with these, we propose that the RAMP-FGFR1 fusion product will contribute to progression of this myeloproliferative disorder by constitutive activation of tyrosine kinase function..
Summersgill, B.
Goker, H.
Osin, P.
Huddart, R.
Horwich, A.
Fisher, C.
Shipley, J.
(1998). Establishing germ cell origin of undifferentiated tumors by identifying gain of 12p material using comparative genomic hybridization analysis of paraffin-embedded samples. Diagn mol pathol,
Vol.7
(5),
pp. 260-266.
show abstract
An estimated 10% of adult cancer patients present with undifferentiated carcinoma. The diagnosis of germ cell tumor (GCT) in such patients can be difficult but has important implications for patient management. Male testicular GCT is characterized by an isochromosome 12p, i(12p), or additional 12p material, in some cases restricted to the 12p11.2-p12.1 region. A gain of 12p material can indicate that a tumor, which may not be present in the testis, is of germ cell origin. Formalin-fixed, paraffin-embedded samples are the most widely available material for diagnostic analysis and retrospective studies. We have compared the identification of 12p gain in snap-frozen samples with corresponding paraffin-embedded material from three clearly defined testicular GCTs using comparative genomic hybridization analysis. In this preliminary study, paraffin-embedded tumor samples of uncertain histogenesis from seven patients were then analyzed. Tumor samples from three of these patients showed a gain of 12p material, and in one patient, gain was restricted to the 12p11.2-p12 region. The clinical picture and response to therapy were generally consistent with the 12p status, though lack of 12p gain may not exclude a diagnosis of GCT..
Lu, Y.J.
Osin, P.
Lakhani, S.R.
Di Palma, S.
Gusterson, B.A.
Shipley, J.M.
(1998). Comparative genomic hybridization analysis of lobular carcinoma in situ and atypical lobular hyperplasia and potential roles for gains and losses of genetic material in breast neoplasia. Cancer res,
Vol.58
(20),
pp. 4721-4727.
show abstract
Lobular carcinoma in situ (LCIS) and atypical lobular hyperplasia (ALH) of the breast are cytologically similar breast lesions that reportedly carry different relative risks of subsequent development of invasive carcinoma. They are frequently multifocal and bilateral. We have identified the chromosomal copy number changes in 31 LCIS and 14 ALH lesions from 28 cases and also the 7 invasive carcinomas that subsequently developed in 6 of these cases. This was achieved by comparative genomic hybridization analysis of microdissected formalin-fixed, paraffin-embedded material. There was no significant difference between the aberrations found in the unilateral versus the bilateral cases of LCIS. Loss of material from 16p, 16q, 17p, and 22q and also gain of material from 6q were found at a similar high frequency in LCIS and ALH. Loss of these genomic regions may indicate the locations of genes that predispose to the development of the lesions, and the results are consistent with LCIS and ALH representing the same genetic stage of development. Comparison of the comparative genomic hybridization results from LCIS/ALH with those from ductal carcinoma in situ and invasive cancer showed some similarities at the chromosomal level, but it also showed significant differences, including gain of 1q and 8q and evidence for genomic amplification, which were not found in LCIS/ALH. A genetic model is postulated for the possible relationships between noninvasive lobular lesions and invasive breast carcinoma, delineating potential roles for specific chromosome copy number changes..
Rozycka, M.
Lu, Y.J.
Brown, R.A.
Lau, M.R.
Shipley, J.M.
Fry, M.J.
(1998). cDNA cloning of a third human C2-domain-containing class II phosphoinositide 3-kinase, PI3K-C2gamma, and chromosomal assignment of this gene (PIK3C2G) to 12p12. Genomics,
Vol.54
(3),
pp. 569-574.
show abstract
Phosphoinositide (PI) 3-kinases have been shown to have critical roles in signaling pathways that regulate proliferation, oncogenic transformation, cell survival, cell migration, and intracellular protein trafficking. We have previously used reverse-transcription polymerase chain reaction methods to identify novel PI 3-kinase isoforms in normal human breast and in lymph nodes containing metastatic breast cancer. Here we report the cDNA cloning of a Class II PI 3-kinase found in normal breast tissue. This gene (PIK3C2G) encodes the third distinct protein of the human Class II PI 3-kinase family, PI3K-C2gamma. PIK3C2G was mapped to chromosome 12 at 12p12 by fluorescence in situ hybridization..
Summersgill, B.
Goker, H.
Weber-Hall, S.
Huddart, R.
Horwich, A.
Shipley, J.
(1998). Molecular cytogenetic analysis of adult testicular germ cell tumours and identification of regions of consensus copy number change. Br j cancer,
Vol.77
(2),
pp. 305-313.
show abstract
full text
A series of adult testicular germ cell tumours consisting of eight seminomas, 14 non-seminomas (including two cell lines) and two combined tumours was analysed by comparative genomic hybridization and, in some cases, by interphase fluorescence in situ hybridization. The gain of 12p was identified in all cases and additional material from chromosomes 7 and 8 was found in over 70% of cases, in keeping with previous analyses. Other consistent regions of gain included 1q24-q31 (50%), 2p16-pter (41%), 2q22-q32 (45%) and Xq11-q21 (50%). The loss of 1p32-p36 (36%), 9q31-qter (36%), 11q14-qter (50%), 16p (36%) and 18p (45%) and the loss of material from chromosomes 4 and 5 (50% and 36% respectively) were also found in all histological subtypes. The loss of 1p material was confirmed in four cases by interphase FISH analysis and shown, with one exception, not to involve the loss of the D1Z2 locus at 1p36.3, which is commonly deleted in paediatric germ cell tumours. An association between gain of 6q21-q24 with cases resistant to chemotherapy (P < 0.01) was observed. In addition, loss of chromosome 19 and 22 material and gain of 5q14-q23, 6q21-q24 and 13q were found at a significantly lower frequency in seminoma than non-seminoma. These regions may contain genes involved in the divergent development of seminoma and non-seminoma..
Osin, P.
Shipley, J.
Lu, Y.J.
Crook, T.
Gusterson, B.A.
(1998). Experimental pathology and breast cancer genetics: new technologies. Recent results cancer res,
Vol.152,
pp. 35-48.
show abstract
The goal is to understand the critical events in tumour development and to apply this understanding to new approaches to diagnosis, prevention and treatment. It is clear that breast cancer is a heterogeneous disease at the molecular level, raising the possibility of a future functional classification based on mechanisms rather than morphology. These molecular phenotypes will also confer predictive value on the potential of the tumour to invade, metastasise and respond to or resist new therapeutic strategies. Studies of the genome in individuals are predicted also to enable the identification of polymorphisms that are associated with increased susceptibility to environmental factors, in addition to possibly explaining de novo variations in responses to drugs and radiation. The difficulty is how to identify which, of the approximately 30,000 genes expressed by a typical cancer cell alone or in combination, are the ones involved in these processes. The majority of breast cancers have such a multitude of molecular changes that it is difficult to distinguish between those that are critical to tumour progression and those that are epiphenomena of genetic instability and abnormalities in DNA repair. The identification of the earliest events in carcinogenesis must be the best hope, as it will then be possible to target the events that predispose to other secondary changes before they occur. Genomics and proteomics is the current hope to take us forward. This involves the application of a number of new technologies to facilitate the profiling of individual tumours, including laser-guided microdissection of microscopic lesions, comparative genomic hybridisation and loss of heterozygosity analysis of DNA using microarray technology to study DNA and expressed RNAs and protein profiling using 2D gel mass spectroscopy. With over 100,000 mRNAs and proteins to examine in complex tissues and in various combinations, there is obviously going to be a requirement for a large investment in computing power (bioinformatics) to facilitate the analysis of these data in relation to the clinical characteristics of the individual tumour and the patient..
Lu, Y.J.
Birdsall, S.
Osin, P.
Gusterson, B.
Shipley, J.
(1997). Phyllodes tumors of the breast analyzed by comparative genomic hybridization and association of increased 1q copy number with stromal overgrowth and recurrence. Genes chromosomes & cancer,
Vol.20
(3),
pp. 275-7.
Sheaff, M.
McManus, A.
Scheimberg, I.
Paris, A.
Shipley, J.
Baithun, S.
(1997). Primitive neuroectodermal tumor of the kidney confirmed by fluorescence in situ hybridization. American journal of surgical pathology,
Vol.21
(4),
pp. 461-8.
Brett, D.
Whitehouse, S.
Antonson, P.
Shipley, J.
Cooper, C.
Goodwin, G.
(1997). The SYT protein involved in the t(X;18) synovial sarcoma translocation is a transcriptional activator localised in nuclear bodies. Hum mol genet,
Vol.6
(9),
pp. 1559-1564.
show abstract
The t(X;18)(p11.2;q11.2) translocation found in synovial sarcomas results in the fusion of the SYT gene on chromosome 18 to either of two closely related genes SSX1 and SSX2 on chromosome X. The resulting chimaeric genes express SYT-SSX1 or SYT-SSX2 fusion proteins in which the C-terminal amino acids of SYT are replaced by amino acids from the C-terminus of the SSX proteins. Using green fluorescent protein fusions we demonstrate that the SYT, SSX and the SYT-SSX proteins are nuclear proteins. We demonstrate that whilst the SSX1 protein has a uniform nuclear distribution the SYT protein has a speckled distribution in the cell nucleus, and this distribution is retained with the SYT-SSX2 fusion protein. Since the SYT speckles do not co-localise with PML-containing bodies (PODs) or spliceosomes it is possible that they represent a novel nuclear structure. Transfection of constructs expressing GAL4 fusion proteins demonstrate that the SYT domains present in the SYT-SSX fusion proteins can activate transcription of a luciferase reporter. It is proposed that the t(X;18) translocation results in the generation of an SYT-SSX transcriptional co-activator in which the addition of the C-terminal SSX domain to SYT provides a new interacting domain that redirects the SYT activation domain to different target promoters..
Clark, J.
Lu, Y.J.
Sidhar, S.K.
Parker, C.
Gill, S.
Smedley, D.
Hamoudi, R.
Linehan, W.M.
Shipley, J.
Cooper, C.S.
(1997). Fusion of splicing factor genes PSF and NonO (p54nrb) to the TFE3 gene in papillary renal cell carcinoma. Oncogene,
Vol.15
(18),
pp. 2233-2239.
show abstract
We demonstrate that the cytogenetically defined translocation t(X;1)(p11.2;p34) observed in papillary renal cell carcinomas results in the fusion of the splicing factor gene PSF located at 1p34 to the TFE3 helix-loop-helix transcription factor gene at Xp11.2. In addition we define an X chromosome inversion inv(X)(p11.2;q12) that results in the fusion of the NonO (p54nrb) gene to TFE3. NonO (p54nrb), the human homologue of the Drosophila gene NonAdiss which controls the male courtship song, is closely related to PSF and also believed to be involved in RNA splicing. In each case the rearrangement results in the fusion of almost the entire splicing factor protein to the TFE3 DNA-binding domain. These observations suggest the possibility of intriguing links between the processes of RNA splicing, DNA transcription and oncogenesis..
Lu, Y.
Birdsall, S.
Osin, P.
Gusterson, B.
Shipley, J.
(1997). Phyllodes tumors of the breast analyzed by comparative genomic hybridization and association of increased 1q copy number with stromal overgrowth and recurrence. Genes, chromosomes and cancer,
Vol.20
(3),
pp. 275-281.
Brown, R.A.
Ho, L.K.
Weber-Hall, S.J.
Shipley, J.M.
Fry, M.J.
(1997). Identification and cDNA cloning of a novel mammalian C2 domain-containing phosphoinositide 3-kinase, HsC2-PI3K. Biochem biophys res commun,
Vol.233
(2),
pp. 537-544.
show abstract
Phosphoinositide (PI) 3-kinases have been shown to have critical roles in signal transduction, cell transformation and intracellular protein trafficking. Reverse-transcription polymerase chain reaction methods, using degenerate primers derived from the lipid kinase consensus region, were utilised to identify PI 3-kinases in the normal human breast. Here we report the cDNA cloning of a novel human PI 3-kinase isoform, HsC2-PI3K. This PI 3-kinase is most closely related to the recently described C2 domain-containing family of PI 3-kinases which includes Drosophila PI3K_68D/cpk and murine cpk-m/p170. Sequence analysis suggests that HsC2-PI3K is a second distinct mammalian member of the C2 domain-containing PI 3-kinase family. Northern blot analysis of human tissues indicates that HsC2-PI3K is widely expressed. Fluorescence in situ hybridisation has mapped HsC2-PI3K to chromosome 1q32..
Mitchell, P.J.
Barker, K.T.
Shipley, J.
Crompton, M.R.
(1997). Characterisation and chromosome mapping of the human non receptor tyrosine kinase gene, brk. Oncogene,
Vol.15
(12),
pp. 1497-1502.
show abstract
The brk gene encodes a non-receptor protein tyrosine kinase that consists of single SH3, SH2 and catalytic domains. Although BRK shows strongest sequence similarity to members of the SRC family of PTKs, there are several key structural and regulatory differences that place it on its own amongst non-receptor PTKs. In this study we have isolated genomic DNA clones corresponding to the human brk locus and used these to determine the intron-exon structure of the brk gene. The genomic structure of brk consists of 8 exons, whose boundaries are distinct from other non-receptor PTK family members, again indicating a structural and functional divergence. Alternate splicing of the primary brk transcript generates a distinct mRNA which encodes a truncated protein consisting of an SH3 domain and a novel C-terminal proline rich sequence. Using an antiserum raised to the SH3 domain, we have demonstrated that the product of this alternate brk transcript is expressed in the human breast tumour cell line T-47D. We have previously reported that expression of a tumour derived brk cDNA in mouse embryonic fibroblasts and human mammary epithelial cells supports anchorage independent growth, and in the latter potentiates the mitogenic response to epidermal growth factor. The protein encoded by the genomic sequence derived from normal human tissue is identical to that encoded by the tumour derived cDNA, and therefore the altered growth regulation is not associated with mutations within brk. In addition, we have identified a 5' genomic region that has promoter activity. The brk gene has been assigned to chromosome 20q 13.3 [corrected] using fluorescence in situ hybridisation (FISH)..
Birdsall, S.H.
Stamps, A.C.
Gusterson, B.A.
Shipley, J.M.
Gill, S.E.
Cooper, C.S.
(1996). No rearrangement of the CHOP and TLS/FUS genes in two cases of phyllodes tumor of the breast. Cancer genetics and cytogenetics,
Vol.87
(1),
pp. 90-2.
Byrne, P.C.
Shipley, J.M.
Chave, K.J.
Sanders, P.G.
Snell, K.
(1996). Characterisation of a human serine hydroxymethyltransferase pseudogene and its localisation to 1p32 3-33. Human genetics,
Vol.97
(3),
pp. 340-5.
McManus, A.P.
Gusterson, B.A.
Pinkerton, C.R.
Shipley, J.M.
(1996). Specificity of the EWS/WT1 gene fusion for desmoplastic small round cell tumour - Reply. Journal of pathology,
Vol.180
(4),
pp. 462-1.
Summersgill, B.M.
Huddart, R.A.
Fisher, C.
Horwich, A.
Shipley, J.
(1996). Identification of isochromosome 12P and other chromosomal copy number changes in male germ cell tumours by comparative genomic hybridisation (CGH); A new diagnostic tool?. British journal of cancer,
Vol.74,
pp. 11-1.
Ramani, P.
Shipley, J.
(1996). Recent advances in the diagnosis, prognosis and classification of childhood solid tumours. British medical bulletin,
Vol.52
(4),
pp. 724-18.
McManus, A.P.
Gusterson, B.A.
Pinkerton, C.R.
Shipley, J.M.
(1996). The molecular pathology of small round-cell tumours--relevance to diagnosis, prognosis, and classification. J pathol,
Vol.178
(2),
pp. 116-121.
show abstract
Substantial improvements have been made in the treatment and survival of children with SRCT, resulting in an increased emphasis on precise histological diagnosis. Although diagnostic procedures such as electron microscopy and immunocytochemistry contribute in poorly differentiated cases, an accurate diagnosis can remain elusive in a proportion of SRCTs. The cytogenetic and molecular genetic abnormalities characteristic of the different SRCTs can now be consistently and rapidly identified from minimal quantities of tumour material, using the techniques of FISH and PCR. This, coupled with the identification of novel phenotypic characteristics, has had a major impact on SRCT diagnosis. The aim of a tumour classification is to identify disease entities which are biologically distinct and whose recognition is of clinical value. The recent advances described above demonstrate that the SRCTs are genotypically and phenotypically distinct tumour types and that the genetic abnormalities represent key alterations that influence both the morphology and the clinical behaviour of the tumour. This suggests that these advanced phenotypic and genotypic analyses should form an integral and complementary part of the laboratory assessment and clinical management of these forms of paediatric cancer..
Shipley, J.
Crew, J.
Birdsall, S.
Gill, S.
Clark, J.
Fisher, C.
Kelsey, A.
Nojima, T.
Sonobe, H.
Cooper, C.
Gusterson, B.
(1996). Interphase fluorescence in situ hybridization and reverse transcription polymerase chain reaction as a diagnostic aid for synovial sarcoma. Am j pathol,
Vol.148
(2),
pp. 559-567.
show abstract
Identification of the t(X;18)(p11.2;q11.2) that is associated with a high proportion of synovial sarcoma can be a useful diagnostic aid. The translocation results in fusion of the SYT gene on chromosome 18 to either the SSX1 or the SSX2 gene, two homologous genes within Xp11.2. Two-color interphase fluorescence in situ hybridization and reverse transcription polymerase chain reaction were assessed as approaches to identify the rearrangement in well characterized cases. The presence of the translocation, and the specific chromosome X gene disrupted, were inferred from the configuration of signals from chromosome-specific centromere probes, paints, and markers flanking each gene in preparations of interphase nuclei. Rearrangement was found in two cell lines and eight of nine tumor samples, including analysis of five touch imprints. This was consistent with cytogenetic data in four cases and reverse transcription polymerase chain reaction analysis using primers known to amplify both SYT-SSX1 and SYT-SSX2 transcripts. The transcripts were distinguished by restriction with LspI and SmaI. Contrary to previous suggestions, there was no obvious correlation between histological subtype and involvement of the SSX1 or SSX2 gene. These approaches could also be applied to the identification of tumor-free margins and metastatic disease..
Shipley, J.
Weber-Hall, S.
Birdsall, S.
(1996). Loss of the chromosomal region 5q11-q31 in the myeloid cell line HL-60: characterization by comparative genomic hybridization and fluorescence in situ hybridization. Genes chromosomes cancer,
Vol.15
(3),
pp. 182-186.
show abstract
Comparative genomic hybridization was used to identify the regions of genomic gain and loss in the myeloid cell line HL-60. These included amplification at 8q24 corresponding to previous reports of overrepresentation of the MYC gene; loss of material from the short arms of chromosomes 9 (9p21-p23), 10, and 17; loss of the chromosome regions 9q32-qter and 14q11-q24; and an extra copy of chromosome 18. Additionally, deletion of the 5q11-q31 region was noted and was associated with translocation of chromosome 5 material to chromosomes 16 and a dic(5;17)(q11;p11) chromosome (previously described as mar 3). Loss of chromosome 5 material in myeloid malignancies, including the M2 subtype from which HL-60 was derived, is usually associated with interstitial deletions of the long arm, including the critical 5q31 region, resulting in a 5q- chromosome. The HL-60 cell line may be a useful model to investigate the role of potential tumour suppressor genes associated with loss of 5q material in myeloid leukaemias..
McManus, A.P.
Min, T.
Swansbury, G.J.
Gusterson, B.A.
Pinkerton, C.R.
Shipley, J.M.
(1996). der(16)t(1;16)(q21;q13) as a secondary change in alveolar rhabdomyosarcoma A case report and review of the literature. Cancer genet cytogenet,
Vol.87
(2),
pp. 179-181.
show abstract
Alveolar rhabdomyosarcoma is an aggressive childhood tumor that exhibits muscle cell differentiation. Cytogenetically, it is characterized by t(2;13)(q35;q14); no consistent secondary abnormalities have been reported. Cytogenetic analysis of bone marrow in a case of alveolar rhabdomyosarcoma revealed t(2;13)(q35;q14) and der(16)t(1;16)(q21;q13). The present case and a review of the literature suggest that up to 11% of these tumors possess der(16)t(1;16)(q21;q13). This is similar to the incidence observed in the Ewing family of tumors, where unbalanced der(16)t(1;16) translocations, resulting in partial trisomy of 1q, are regarded as a consistent secondary cytogenetic change..
McManus, A.P.
O'Reilly, M.A.
Jones, K.P.
Gusterson, B.A.
Mitchell, C.D.
Pinkerton, C.R.
Shipley, J.M.
(1996). Interphase fluorescence in situ hybridization detection of t(2;13)(q35;q14) in alveolar rhabdomyosarcoma--a diagnostic tool in minimally invasive biopsies. J pathol,
Vol.178
(4),
pp. 410-414.
show abstract
The identification of t(2;13)(q35;q14) is a useful aid in the accurate diagnosis of rhabdomyosarcoma, distinguishing it from other small round cell tumours and supporting the distinction between alveolar and embryonal forms. Cytogenetic analysis is difficult and with the increased use of minimally invasive biopsy methods and primary chemotherapy, adequate tumour material is not always available. To overcome these difficulties, two-colour interphase fluorescence in situ hybridization (FISH) to detect t(2;13)(q35;q14) was developed and its utility in assessing minimally invasive biopsies was investigated. Two cosmid clones which mapped proximal or distal to the breakpoint region 13q14 and one yeast artificial chromosome clone that mapped distal to the 2q35 breakpoint were identified. In interphase cells containing t(2;13)(q35;q14), the configuration of cosmid and yeast artificial chromosome signals demonstrated the presence of the translocation. Five cases of rhabdomyosarcoma were analysed by interphase FISH. The t(2;13)(q35;q14) was detected in all four alveolar tumours and was confirmed by cytogenetics in two cases, but was absent in one embryonal tumour. This sensitive detection method is applicable to minimal amounts of fresh or frozen tumour..
McMANUS, A.P.
O'REILLY, M.-.
PRITCHARD JONES, K.
GUSTERSON, B.A.
MITCHELL, C.D.
PINKERTON, C.R.
SHIPLEY, J.M.
(1996). INTERPHASE FLUORESCENCEIN SITU HYBRIDIZATION DETECTION OF t(2;13)(q35;q14) IN ALVEOLAR RHABDOMYOSARCOMA—A DIAGNOSTIC TOOL IN MINIMALLY INVASIVE BIOPSIES. The journal of pathology,
Vol.178
(4),
pp. 410-414.
Weber-Hall, S.
McManus, A.
Anderson, J.
Nojima, T.
Abe, S.
Pritchard-Jones, K.
Shipley, J.
(1996). Novel formation and amplification of the PAX7-FKHR fusion gene in a case of alveolar rhabdomyosarcoma. Genes chromosomes cancer,
Vol.17
(1),
pp. 7-13.
show abstract
Alveolar rhabdomyosarcomas frequently exhibit specific translocations, resulting in the fusion of the FKHR gene at 13q14 with either the PAX3 or PAX7 gene at 2q35 and 1p36, respectively. Comparative genomic hybridization revealed amplification at 13q14 and 1p36, suggesting amplification of the PAX7-FKHR fusion gene in two cases of alveolar rhabdomyosarcoma. A PAX7-FKHR fusion transcript was demonstrated in both cases by reverse transcription-polymerase chain reaction followed by sequence analysis. In one case, amplification of the PAX7 gene and 3'-and 5'-FKHR gene sequences was demonstrated by using interphase fluorescence in situ hybridization on tumor imprints. The colocalization, variable copy number, and distribution of signals from the three cosmids was consistent with amplification of these sequences on double minutes, which were present cytogenetically. Chromatin release studies suggested that the amplified sequences correlated with amplification of the PAX7-FKHR fusion gene which resulted from the insertion of PAX7 sequences into the first intron of FKHR gene, in keeping with the absence of cytogenetic evidence for derivative chromosomes..
Sidhar, S.K.
Clark, J.
Gill, S.
Hamoudi, R.
Crew, A.J.
Gwilliam, R.
Ross, M.
Linehan, W.M.
Birdsall, S.
Shipley, J.
Cooper, C.S.
(1996). The t(X;l)(p11 2;q21 2) translocation in papillary renal cell carcinoma fuses a novel gene PRCC to the TFE3 transcription factor gene. Human molecular genetics,
Vol.5
(9),
pp. 1333-6.
Smith, P.D.
Barker, K.T.
Wang, J.
Lu, Y.J.
Shipley, J.
Crompton, M.R.
(1996). ICAAR, a novel member of a new family of transmembrane, tyrosine phosphatase-like proteins. Biochemical and biophysical research communications,
Vol.229
(2),
pp. 402-10.
Weber-Hall, S.
Anderson, J.
McManus, A.
Abe, S.
Nojima, T.
Pinkerton, R.
Pritchard-Jones, K.
Shipley, J.
(1996). Gains, losses, and amplification of genomic material in rhabdomyosarcoma analyzed by comparative genomic hybridization. Cancer res,
Vol.56
(14),
pp. 3220-3224.
show abstract
In this study, 10 embryonal and 14 alveolar rhabdomyosarcoma (RMS) tumor samples, including 4 cell lines derived from tumors of the alveolar subtype, were analyzed by comparative genomic hybridization. In the embryonal tumors, the gain of whole or most of various chromosomes, notably chromosomes 2 (60% of cases), 13 (60%), 12 (60%), 8 (60%), 7 (50%), 17 (40%), 18 (40%), and 19 (40%), and the loss of chromosomes 16 (40%), 10 (30%), 15 (20%), and 14 (20%) were found. One case showed evidence of genomic amplification at 12q13-15. In contrast, the alveolar tumors and cell lines showed consistent evidence of genomic amplification, with multiple amplicons in some cases. The amplicons were localized to l2q13-15 (50%), 2p24 (36%), 13q14 (14%), l3q32 (14%), 1q36 (14%), 1q21 (7%), and 8q13-21 (7%). Four cases had additional copies of chromosome 17 or l7q. These changes were in addition to the presence of fusion gene transcripts that are associated with translocations specific to alveolar RMS. The results show that distinct patterns of primarily gains of specific chromosomal material are associated with the embryonal subtype of RMS, and that genomic amplification seems to play an important role in the alveolar subtype. Notably, these distinct changes predominantly involved chromosomes 2, 12, and 13 in both subtypes..
Zani, V.J.
Asou, N.
Jadayel, D.
Heward, J.M.
Shipley, J.
Nacheva, E.
Takasuki, K.
Catovsky, D.
Dyer, M.J.
(1996). Molecular cloning of complex chromosomal translocation t(8;14;12)(q24 1;q32 3;q24 1) in a Burkitt lymphoma cell line defines a new gene (BCL7A) with homology to caldesmon. Blood,
Vol.87
(8),
pp. 3124-3134.
show abstract
Chromosome 12q24.1 is a recurrent breakpoint in high-grade B-cell non-Hodgkin lymphoma (B-NHL). To identify the genes involved at 12q24.1, molecular cloning of a three-way translocation t(8;14;12)(q24.1;q32.3;q24.1) in a Burkitt lymphoma cell line (Wien 133) was performed; all four translocation breakpoints were cloned and sequenced. Analysis of clones encompassing the der(12)(12;14)(q24.1;q32.3) breakpoint showed a CpG island from chromosome 12q24.1 juxtaposed in a tail-to-tail configuration with a productively rearranged Ig VH4-DH-JH5 gene. A total of 4.5 kb of genomic DNA including the CpG island was sequenced and analyzed using gene-identification programs; all three programs identified a potential 92-bp exon within the centromeric boundary of the CpG island. Using this as a probe, an RNA transcript of 3.8 kb, expressed at low levels in a wide variety of normal tissues, was detected. Overlapping cDNA clones were isolated and sequenced. The longest open-reading frame predicted a serine-rich protein of 231 amino acids. This protein, termed BCL7A, exhibited no recognizable protein motifs but showed homology with the actin-binding protein, caldesmon. In Wien 133, the BCL7A breakpoint occurred within the first intron and resulted in a MYC-BCL7A fusion transcript, with exon I of BCL7A being replaced by MYC exon I. The normal, untranslocated allele of BCL7A was also expressed without mutation. One of the 11 other B-NHL cell lines examined with 12q24.1 cytogenetic abnormalities, a mediastinal B-NHL cell line (Karpas 1106), showed biallelic rearrangement within the first intron of BCL7A, which was adjacent to the breakpoint observed in Wien 133. Disruption of the amino-terminus of BCL7A defines a new mechanism in the pathogenesis of a subset of high-grade B-NHL..
Clark, J.
Benjamin, H.
Gill, S.
Sidhar, S.
Goodwin, G.
Crew, J.
Gusterson, B.A.
Shipley, J.
Cooper, C.S.
(1996). Fusion of the EWS gene to CHN, a member of the steroid thyroid receptor gene superfamily, in a human myxoid chondrosarcoma. Oncogene,
Vol.12
(2),
pp. 229-7.
GILL, S.
MCMANUS, A.P.
CREW, A.J.
BENJAMIN, H.
SHEER, D.
GUSTERSON, B.A.
PINKERTON, C.R.
PATEL, K.
COOPER, C.S.
SHIPLEY, J.M.
(1995). FUSION OF THE EWS GENE TO A DNA SEGMENT FROM 9Q22-31 IN A HUMAN MYXOID CHONDROSARCOMA. Genes chromosomes & cancer,
Vol.12
(4),
pp. 307-4.
SMITH, A.L.
MITCHELL, P.J.
SHIPLEY, J.
ROGERS, M.
CROMPTON, M.R.
(1995). A NOVEL PROTEIN-TYROSINE-PHOSPHATASE IN HUMAN MAMMARY EPITHELIAL-CELLS. Journal of cellular biochemistry,
,
pp. 43-1.
COOPER, C.
CLARK, J.
ROCQUES, P.
CREW, J.
GILL, S.
SHIPLEY, J.
CHAN, A.
GUSTERSON, B.
(1995). CHARACTERIZATION OF NOVEL GENES SYT AND SSX INVOLVED IN THE T(X, 18)(P11 2, Q11 2) TRANSLOCATION FOUND IN HUMAN SYNOVIAL SARCOMA. Journal of cellular biochemistry,
,
pp. 50-1.
Birdsall, S.H.
Shipley, J.M.
Summersgill, B.M.
Black, A.J.
Jackson, P.
Kissin, M.W.
Gusterson, B.A.
(1995). Cytogenetic findings in a case of nodular fasciitis of the breast. Cancer genet cytogenet,
Vol.81
(2),
pp. 166-168.
show abstract
We report the cytogenetic findings in a case of nodular fasciitis of the breast. The abnormalities found in all 11 metaphases available for analysis were -2, -2, -13, der(15)t(2;15)(q31;q26), + der(?) t(?;2), + mar1, + mar2. Other consistent abnormalities were also identified. Fluorescence in situ hybridization (FISH) was used to confirm the origin of some of the chromosomes. A large acrocentric chromosome was confirmed to be derived from chromosome 15 with chromosome 2 material translocated onto the q arm. The metacentric der(?)t(?;2) was demonstrated to have part of chromosome 2 on the q arm. No other chromosome 2 material was found. Eight of 11 cells were tetraploid and had two copies of a del(6)(q16q24)..
McManus, A.P.
Gusterson, B.A.
Pinkerton, C.R.
Shipley, J.M.
(1995). Diagnosis of Ewing's sarcoma and related tumours by detection of chromosome 22q12 translocations using fluorescence in situ hybridization on tumour touch imprints. J pathol,
Vol.176
(2),
pp. 137-142.
show abstract
It is increasingly recognized that the identification of t(11;22)(q24;q12) is a useful aid in the accurate diagnosis of Ewing's sarcoma and related tumours. However, cytogenetic studies have a low success rate and adequate tumour is not always available. This study describes the use of fluorescence in situ hybridization (FISH) to detect translocations at 22q12, the site of the EWS gene involved in t(11;22)(q24;q12), on tumour touch imprints made from true cut core-needle biopsy and frozen tumour. Of the seven tumours analysed, five diagnosed as Ewing's sarcoma or primitive neuroectodermal tumour demonstrated chromosome translocation at 22q12. This is a rapid and reliable method to detect a diagnostically relevant chromosome translocation using minimal amounts of fresh or frozen tumour..
Shipley, J.
Williams, S.
O'Byrne, A.
Kearney, L.
Jones, T.
Young, B.
Dyer, M.
Catovsky, D.
Sheer, D.
Gusterson, B.
(1995). Characterization of a t(10;11)(p13-14;q14-21) in the monoblastic cell line U937. Genes chromosomes cancer,
Vol.13
(2),
pp. 138-142.
show abstract
Previous analysis of the monoblastic cell line U937 has shown that several sublines contain a rearranged chromosome arm 11q. In order to determine the true nature of the rearrangement, fluorescence in situ hybridization (FISH) was carried out with various combinations of single copy anonymous markers, clones containing genes, a chromosome 10 paint, and an 11 centromere specific sequence. The rearrangement was deduced to be a reciprocal translocation between chromosomes 10 and 11 described as t(10;11)(p13-14;q14-21). The breakpoint on chromosome 11 is telomeric to the INT2 gene and the pHS11 probe at 11q13, and centromeric to the marker D11S36 localized to 11q14.3-q22.1 and the MLL gene at 11q23. Similar translocations have been reported in various acute leukemias, principally of the monocytic lineage, and also in T-cell precursor acute lymphocytic leukemias. Further characterization of the genetic rearrangements in U937 may lead to the isolation of genes important in leukemogenesis and provide an in vitro system for their study..
Birdsall, S.H.
Summersgill, B.M.
Egan, M.
Fentiman, I.S.
Gusterson, B.A.
Shipley, J.M.
(1995). Additional copies of 1q in sequential samples from a phyllodes tumor of the breast. Cancer genet cytogenet,
Vol.83
(2),
pp. 111-114.
show abstract
Cytogenetic and fluorescent in situ hybridization (FISH) analysis has been performed on consecutive samples, taken 4 weeks apart, from a phyllodes breast tumor. This revealed the presence of two different chromosome 1 derivatives, namely a dic(1;10)(q10;q24) in the first sample and an i(1) (q10) in the second. In one cell out of 25 from the second sample both derivative chromosomes were seen. A chromosome 21 was lost in both samples. These results are consistent with phyllodes tumors having a clonal origin..
CREW, A.J.
CLARK, J.
FISHER, C.
GILL, S.
GRIMER, R.
MITCHELL, P.
CHAND, A.
SHIPLEY, J.
GUSTERSON, B.A.
COOPER, C.S.
(1995). FUSION OF SYT TO 2 GENES, SSX1 AND SSX2, ENCODING PROTEINS WITH HOMOLOGY TO THE KRUPPEL-ASSOCIATED BOX IN HUMAN SYNOVIAL SARCOMA. Embo journal,
Vol.14
(10),
pp. 2333-8.
full text
Smith, A.L.
Mitchell, P.J.
Shipley, J.
Gusterson, B.A.
Rogers, M.V.
Crompton, M.R.
(1995). Pez: a novel human cDNA encoding protein tyrosine phosphatase- and ezrin-like domains. Biochem biophys res commun,
Vol.209
(3),
pp. 959-965.
show abstract
We have isolated cDNAs from normal human breast tissue and breast tumour cells that encode a protein (pez) with features of a novel non-receptor tyrosine phosphatase possessing N-terminal sequence homology to the ezrin-band 4.1-merlin-radixin protein family. Northern blot analysis indicates that pez is expressed in a variety of human tissues including kidney, skeletal muscle, lung and placenta. Fluorescence in situ hybridization has mapped pez to chromosome 1 region q32.2-41. Sequence identity to a characterized polymorphic marker confirms this localization..
Shipley, J.M.
Birdsall, S.
Clark, J.
Crew, J.
Gill, S.
Linehan, M.
Gnarra, J.
Fisher, S.
Craig, I.W.
Cooper, C.S.
(1995). Mapping the X chromosome breakpoint in two papillary renal cell carcinoma cell lines with a t(X;1)(p11 2;q21 2) and the first report of a female case. Cytogenet cell genet,
Vol.71
(3),
pp. 280-284.
show abstract
A t(X;1)(p11.2;q21.2) has been reported in cases of papillary renal cell tumors arising in males. In this study two cell lines derived from this tumor type have been used to indicate the breakpoint region on the X chromosome. Both cell lines have the translocation in addition to other rearrangements and one is derived from the first female case to be reported with the t(X;1)(p11.2;q21.2). Fluorescence in situ hybridization (FISH) has been used to position YACs belonging to contigs in the Xp11.2 region relative to the breakpoint. When considered together with detailed mapping information from the Xp11.2 region the position of the breakpoint in both cell lines was suggested as follows: Xpter-->Xp11.23-OATL1-GATA1-WAS-TFE3-SY P-t(X;1)-DXS255-CLCN5-DXS146-OATL2- Xp11.22-->Xcen. The breakpoint was determined to lie in an uncloned region between SYP and a YAC called FTDM/1 which extends 1 Mb distal to DXS255. These results are contrary to the conclusion from previous FISH studies that the breakpoint was near the OATL2 locus, but are consistent with, and considerably refine, the position that had been established by molecular analysis..
CLARK, J.
CREW, A.
SHIPLEY, J.
GILL, S.
ROCQUES, P.
MONACO, A.
GUSTERSON, B.
COOPER, C.
(1994). SUBLOCALIZATION OF THE T(X-18)(P11 2 Q11 2) TRANSLOCATION BREAKPOINT IN SYNOVIAL SARCOMA. Cytogenetics and cell genetics,
Vol.65
(3),
pp. 164-1.
Shipley, J.M.
Clark, J.
Crew, A.J.
Birdsall, S.
Rocques, P.J.
Gill, S.
Chelly, J.
Monaco, A.P.
Abe, S.
Gusterson, B.A.
(1994). The t(X;18)(p11 2;q11 2) translocation found in human synovial sarcomas involves two distinct loci on the X chromosome. Oncogene,
Vol.9
(5),
pp. 1447-1453.
show abstract
A high proportion of synovial sarcomas contain the reciprocal translocation t(X;18)(p11.2;q11.2). We have previously localized the breakpoint on the X chromosome between the X chromosome marker DXS255 and an ornithine aminotransferase (OAT) pseudogene region designated OATL2. Subsequently by fluorescence in situ hybridization (FISH) we provided evidence that YACs corresponding to the OATL2 locus spanned the break-point. In order to confirm the position of this breakpoint cosmids corresponding to the OATL2 region were isolated. Most of these cosmids mapped to four cosmid contigs designated C1-C4. Analysis of two contigs, C1- and C4, using FISH established that in four of six synovial sarcomas examined the breakpoint occurs between these two contigs: C1 lies distal to the break-point while C4 is proximal. In contrast we provide evidence that the breakpoint in the remaining two tumours mapped to a second pseudogene region called OATL1 that is telomeric to the OATL2 locus. This heterogeneity of the breakpoint position on the X chromosome explains why in previous mapping studies there have been discrepancies between the results obtained by different laboratories..
Clark, J.
Rocques, P.J.
Crew, A.J.
Gill, S.
Shipley, J.
Chan, A.M.
Gusterson, B.A.
Cooper, C.S.
(1994). Identification of novel genes, SYT and SSX, involved in the t(X;18)(p11 2;q11 2) translocation found in human synovial sarcoma. Nat genet,
Vol.7
(4),
pp. 502-508.
show abstract
Human synovial sarcomas contain a recurrent and specific chromosomal translocation t(X;18)(p11.2;q11.2). By screening a synovial sarcoma cDNA library with a yeast artificial chromosome spanning the X chromosome breakpoint, we have identified a hybrid transcript that contains 5' sequences (designated SYT) mapping to chromosome 18 and 3' sequences (designated SSX) mapping to chromosome X. An SYT probe detected genomic rearrangements in 10/13 synovial sarcomas. Sequencing of cDNA clones shows that the normal SYT gene encodes a protein rich in glutamine, proline and glycine, and indicates that in synovial sarcoma rearrangement of the SYT gene results in the formation of an SYT-SSX fusion protein. Both SYT and SSX failed to exhibit significant homology to known gene sequences..
CREW, A.J.
CLARK, J.P.
SHIPLEY, J.M.
GILL, S.E.
ROCQUES, P.J.
MONACO, A.P.
GUSTERSON, B.A.
COOPER, C.S.
(1993). SUBLOCALIZATION OF THE T(X-18)(P11 2-Q11 2) TRANSLOCATION BREAKPOINT IN SYNOVIAL SARCOMA. American journal of human genetics,
Vol.53
(3),
pp. 1279-1.
Shipley, J.
Crew, J.
Gusterson, B.
(1993). The molecular biology of soft tissue sarcomas. Eur j cancer,
Vol.29A
(14),
pp. 2054-2058.
Karolak, M.
Tracy, I.
Shipley, J.
Walters, Z.S.
Targeting EZH2 for the treatment of soft tissue sarcomas. Journal of cancer metastasis and treatment,
.