Publications by authors named "Garry Ashton"

22 Publications

  • Page 1 of 1

Brain microenvironment-driven resistance to immune and targeted therapies in acral melanoma.

ESMO Open 2020 08;5(4)

Molecular Oncology Group, CRUK Manchester Institute, The University of Manchester, Nether Alderley, Macclesfield, UK

Background: Combination treatments targeting the MEK-ERK pathway and checkpoint inhibitors have improved overall survival in melanoma. Resistance to treatment especially in the brain remains challenging, and rare disease subtypes such as acral melanoma are not typically included in trials. Here we present analyses from longitudinal sampling of a patient with metastatic acral melanoma that became resistant to successive immune and targeted therapies.

Methods: We performed whole-exome sequencing and RNA sequencing on an acral melanoma that progressed on successive immune (nivolumab) and targeted (dabrafenib) therapy in the brain to identify resistance mechanisms. In addition, we performed growth inhibition assays, reverse phase protein arrays and immunoblotting on patient-derived cell lines using dabrafenib in the presence or absence of cerebrospinal fluid (CSF) in vitro. Patient-derived xenografts were also developed to analyse response to dabrafenib.

Results: Immune escape following checkpoint blockade was not due to loss of tumour cell recognition by the immune system or low neoantigen burden, but was associated with distinct changes in the microenvironment. Similarly, resistance to targeted therapy was not associated with acquired mutations but upregulation of the AKT/phospho-inositide 3-kinase pathway in the presence of CSF.

Conclusion: Heterogeneous tumour interactions within the brain microenvironment enable progression on immune and targeted therapies and should be targeted in salvage treatments.
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http://dx.doi.org/10.1136/esmoopen-2020-000707DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7437885PMC
August 2020

Stroma remodeling and reduced cell division define durable response to PD-1 blockade in melanoma.

Nat Commun 2020 02 12;11(1):853. Epub 2020 Feb 12.

Molecular Oncology Group, Cancer Research UK Manchester Institute, The University of Manchester, Alderley Park, Manchester, UK.

Although immune checkpoint inhibitors (ICIs) have achieved unprecedented results in melanoma, the biological features of the durable responses initiated by these drugs remain unknown. Here we show the genetic and phenotypic changes induced by treatment with programmed cell death-1 (PD-1) blockade in a genetically engineered mouse model of melanoma driven by oncogenic BRAF. In this controlled system anti-PD-1 treatment yields responses in ~35% of the tumors, and prolongs survival in ~27% of the animals. We identify increased stroma remodeling and reduced expression of proliferation markers as features associated with prolonged response. These traits are corroborated in two independent early on-treatment anti-PD-1 melanoma patient cohorts. These insights into the biological responses of tumors to ICI provide a strategy for identification of durable response early during the course of treatment and could improve patient stratification for checkpoint inhibitory drugs.
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http://dx.doi.org/10.1038/s41467-020-14632-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015935PMC
February 2020

Oxygen-enhanced MRI Is Feasible, Repeatable, and Detects Radiotherapy-induced Change in Hypoxia in Xenograft Models and in Patients with Non-small Cell Lung Cancer.

Clin Cancer Res 2019 07 3;25(13):3818-3829. Epub 2019 May 3.

Division of Cancer Sciences, University of Manchester, Manchester, United Kingdom.

Purpose: Hypoxia is associated with poor prognosis and is predictive of poor response to cancer treatments, including radiotherapy. Developing noninvasive biomarkers that both detect hypoxia prior to treatment and track change in tumor hypoxia following treatment is required urgently.

Experimental Design: We evaluated the ability of oxygen-enhanced MRI (OE-MRI) to map and quantify therapy-induced changes in tumor hypoxia by measuring oxygen-refractory signals in perfused tissue (perfused Oxy-R). Clinical first-in-human study in patients with non-small cell lung cancer (NSCLC) was performed alongside preclinical experiments in two xenograft tumors (Calu6 NSCLC model and U87 glioma model).

Results: MRI perfused Oxy-R tumor fraction measurement of hypoxia was validated with tissue pathology in both xenograft models. Calu6 and U87 experiments showed that MRI perfused Oxy-R tumor volume was reduced relative to control following single fraction 10-Gy radiation and fractionated chemoradiotherapy ( < 0.001) due to both improved perfusion and reduced oxygen consumption rate. Next, evaluation of 23 patients with NSCLC showed that OE-MRI was clinically feasible and that tumor perfused Oxy-R volume is repeatable [interclass correlation coefficient: 0.961 (95% CI, 0.858-0.990); coefficient of variation: 25.880%]. Group-wise perfused Oxy-R volume was reduced at 14 days following start of radiotherapy ( = 0.015). OE-MRI detected between-subject variation in hypoxia modification in both xenograft and patient tumors.

Conclusions: These findings support applying OE-MRI biomarkers to monitor hypoxia modification, to stratify patients in clinical trials of hypoxia-modifying therapies, to identify patients with hypoxic tumors that may fail treatment with immunotherapy, and to guide adaptive radiotherapy by mapping regional hypoxia.
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http://dx.doi.org/10.1158/1078-0432.CCR-18-3932DOI Listing
July 2019

Single-Cell Analysis Identifies LY6D as a Marker Linking Castration-Resistant Prostate Luminal Cells to Prostate Progenitors and Cancer.

Cell Rep 2018 12;25(12):3504-3518.e6

Prostate Oncobiology, Cancer Research UK Manchester Institute, The University of Manchester, Alderley Park SK10 4TG, UK; Belfast-Manchester Movember Centre of Excellence, Cancer Research UK Manchester Institute, The University of Manchester, Alderley Park SK10 4TG, UK. Electronic address:

The exact identity of castrate-resistant (CR) cells and their relation to CR prostate cancer (CRPC) is unresolved. We use single-cell gene profiling to analyze the molecular heterogeneity in basal and luminal compartments. Within the luminal compartment, we identify a subset of cells intrinsically resistant to castration with a bi-lineage gene expression pattern. We discover LY6D as a marker of CR prostate progenitors with multipotent differentiation and enriched organoid-forming capacity. Lineage tracing further reveals that LY6D CR luminal cells can produce LY6D luminal cells. In contrast, in luminal cells lacking PTEN, LY6D cells predominantly give rise to LY6D tumor cells, contributing to high-grade PIN lesions. Gene expression analyses in patients' biopsies indicate that LY6D expression correlates with early disease progression, including progression to CRPC. Our studies thus identify a subpopulation of luminal progenitors characterized by LY6D expression and intrinsic castration resistance. LY6D may serve as a prognostic maker for advanced prostate cancer.
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http://dx.doi.org/10.1016/j.celrep.2018.11.069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315111PMC
December 2018

Signaling pathway screening platforms are an efficient approach to identify therapeutic targets in cancers that lack known driver mutations: a case report for a cancer of unknown primary origin.

NPJ Genom Med 2018 20;3:15. Epub 2018 Jun 20.

1Signalling Networks in Cancer Group, Cancer Research UK, Manchester Institute, University of Manchester, Manchester, M20 4BX UK.

Precision medicine aims to tailor cancer therapies to target specific tumor-promoting aberrations. For tumors that lack actionable drivers, which occurs frequently in the clinic, extensive molecular characterization and pre-clinical drug efficacy studies will be required. A cell line maintained at low passage and a patient- derived xenograft model (PDX) were generated using a fresh biopsy from a patient with a poorly-differentiated neuroendocrine tumor of unknown primary origin. Next-generation sequencing, high throughput signaling network analysis, and drug efficacy trials were then conducted to identify actionable targets for therapeutic intervention. No actionable mutations were identified after whole exome sequencing of the patient's DNA. However, whole genome sequencing revealed amplification of the 3q and 5p chromosomal arms, that include the and genes, respectively. We then conducted pathway analysis, which revealed activation of the AKT pathway. Based on this analysis, efficacy of PIK3CA and AKT inhibitors were evaluated in the tumor biopsy-derived cell culture and PDX, and response to the AKT inhibitor AZD5363 was observed both in vitro and in vivo indicating the patient would benefit from targeted therapies directed against the serine/threonine kinase AKT. In conclusion, our study demonstrates that high throughput signaling pathway analysis will significantly aid in identifying actionable alterations in rare tumors and guide patient stratification into early-phase clinical trials.
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http://dx.doi.org/10.1038/s41525-018-0055-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6010465PMC
June 2018

Mapping Hypoxia in Renal Carcinoma with Oxygen-enhanced MRI: Comparison with Intrinsic Susceptibility MRI and Pathology.

Radiology 2018 09 5;288(3):739-747. Epub 2018 Jun 5.

From the Centre for Imaging Sciences (R.A.L., J.H.N., Y.W., S.C., K.F.H., H.L., D.J.M., G.J.M.P., J.C.W.) and Division of Cancer Sciences (J.I., C.M.L.W., N.W.C., J.P.B.O.), University of Manchester, Manchester, England; Division of Radiotherapy and Imaging, The Institute of Cancer Research, London, England (Y.J., J.K.R.B., S.P.R.); Department of Pathology, Central Manchester University Hospitals NHS Foundation Trust, Manchester, England (G.N.B.); Department of Histology, CRUK Manchester Institute, Manchester, England (G.A.); Tumour Biology Team, The Breast Cancer Now Toby Robins Research Centre, The Institute of Cancer Research, London, England (A.R.R.); Department of Urology, Salford Royal Hospitals NHS Foundation Trust, Salford, England (S.M., N.W.C.); Bioxydyn Ltd, Manchester, England (G.J.M.P., J.C.W.); and Department of Radiology, The Christie NHS Foundation Trust, Manchester, England (J.P.B.O.).

Purpose To cross-validate T1-weighted oxygen-enhanced (OE) MRI measurements of tumor hypoxia with intrinsic susceptibility MRI measurements and to demonstrate the feasibility of translation of the technique for patients. Materials and Methods Preclinical studies in nine 786-0-R renal cell carcinoma (RCC) xenografts and prospective clinical studies in eight patients with RCC were performed. Longitudinal relaxation rate changes (∆R1) after 100% oxygen inhalation were quantified, reflecting the paramagnetic effect on tissue protons because of the presence of molecular oxygen. Native transverse relaxation rate (R2*) and oxygen-induced R2* change (∆R2*) were measured, reflecting presence of deoxygenated hemoglobin molecules. Median and voxel-wise values of ∆R1 were compared with values of R2* and ∆R2*. Tumor regions with dynamic contrast agent-enhanced MRI perfusion, refractory to signal change at OE MRI (referred to as perfused Oxy-R), were distinguished from perfused oxygen-enhancing (perfused Oxy-E) and nonperfused regions. R2* and ∆R2* values in each tumor subregion were compared by using one-way analysis of variance. Results Tumor-wise and voxel-wise ∆R1 and ∆R2* comparisons did not show correlative relationships. In xenografts, parcellation analysis revealed that perfused Oxy-R regions had faster native R2* (102.4 sec vs 81.7 sec) and greater negative ∆R2* (-22.9 sec vs -5.4 sec), compared with perfused Oxy-E and nonperfused subregions (all P < .001), respectively. Similar findings were present in human tumors (P < .001). Further, perfused Oxy-R helped identify tumor hypoxia, measured at pathologic analysis, in both xenografts (P = .002) and human tumors (P = .003). Conclusion Intrinsic susceptibility biomarkers provide cross validation of the OE MRI biomarker perfused Oxy-R. Consistent relationship to pathologic analyses was found in xenografts and human tumors, demonstrating biomarker translation. Published under a CC BY 4.0 license. Online supplemental material is available for this article.
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http://dx.doi.org/10.1148/radiol.2018171531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122194PMC
September 2018

PD-L1 expression and presence of TILs in small intestinal neuroendocrine tumours.

Oncotarget 2018 Mar 12;9(19):14922-14938. Epub 2018 Feb 12.

Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester, UK.

Background: The extent of resistance to immune surveillance in patients with well-differentiated (Wd) (grade 1/2) small-intestinal neuroendocrine tumours (Si-NETs) is unknown.

Methods: Patients diagnosed with Wd Si-NETs (excluding appendix, which are considered to have a different biology to other midgut NETs) were eligible. Tumoural programmed death (PD)-ligand(L) 1 (PD-L1)/PD-L2/PD-1 and tumour infiltrating lymphocytes (TILs) [presence and phenotype] were analysed in archival tissue by immunohistochemistry (IHC); reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used for confirmation of IHC results.

Results: Of 109 patients screened, 62 were eligible: 54.8% were male; median age was 63.7 years (95%-CI 59.7-67.2); disease stage II: 4.8%, III: 40.3% and IV: 54.8%; 41.9% were functional. Analysed samples (67.1% from primary tumours, 32.9% from metastases) were of grade 1 (67.1%) or 2 (32.86%) with a median Ki-67 of 2%. From the total of 62 eligible patients, 70 and 63 samples were suitable for IHC and RT-qPCR analysis, respectively. PD-L1 expression within tumour cells and TILs were identified in 12.8% and 24.3% of samples respectively; 30% of samples showed PD-L1 expression within tumour cells and/or TILs. PD-1 was present in TILs in 22.8% of samples. Majority of samples showed significant presence of CD4 (focal 42.86%; moderate 2.86%) and CD8 (focal 92.86%; moderate 4.29%) TILs. IHC findings were confirmed with RT-qPCR; which showed higher expression levels of PD-L1 (p-value 0.007) and PD-1 (p-value 0.001) in samples positive for IHC compared to negative-IHC.

Conclusions: Thirty-percent of patients express PD-L1 within tumour cells and/or TILs. Identification of presence of TILs was also significant and warrant the investigation of immunotherapy in this setting.
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http://dx.doi.org/10.18632/oncotarget.24464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5871087PMC
March 2018

TIAM1 Antagonizes TAZ/YAP Both in the Destruction Complex in the Cytoplasm and in the Nucleus to Inhibit Invasion of Intestinal Epithelial Cells.

Cancer Cell 2017 05 13;31(5):621-634.e6. Epub 2017 Apr 13.

Cell Signalling Group, Cancer Research UK Manchester Institute, The University of Manchester, Manchester M20 4BX, UK. Electronic address:

Aberrant WNT signaling drives colorectal cancer (CRC). Here, we identify TIAM1 as a critical antagonist of CRC progression through inhibiting TAZ and YAP, effectors of WNT signaling. We demonstrate that TIAM1 shuttles between the cytoplasm and nucleus antagonizing TAZ/YAP by distinct mechanisms in the two compartments. In the cytoplasm, TIAM1 localizes to the destruction complex and promotes TAZ degradation by enhancing its interaction with βTrCP. Nuclear TIAM1 suppresses TAZ/YAP interaction with TEADs, inhibiting expression of TAZ/YAP target genes implicated in epithelial-mesenchymal transition, cell migration, and invasion, and consequently suppresses CRC cell migration and invasion. Importantly, high nuclear TIAM1 in clinical specimens associates with increased CRC patient survival. Together, our findings suggest that in CRC TIAM1 suppresses tumor progression by regulating YAP/TAZ activity.
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http://dx.doi.org/10.1016/j.ccell.2017.03.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5425402PMC
May 2017

A Biospecimen Proficiency Testing Program for Biobank Accreditation: Four Years of Experience.

Biopreserv Biobank 2016 Oct 19;14(5):429-439. Epub 2016 May 19.

1 Integrated Biobank of Luxembourg , Luxembourg, Luxembourg .

Biobanks produce and distribute biospecimens, ensuring their fitness for purpose and accurately qualifying them before distribution. In their efforts toward professionalization, biobanks can nowadays seek certification or accreditation. One of the requirements of these standards is regular participation in Proficiency Testing (PT) programs. An international PT program has been developed and provided to biobanks and other laboratories that perform specific tests to qualify different types of biospecimens. This PT program includes biospecimen testing schemes, as well as biospecimen processing interlaboratory exercises. This PT program supports the development of biobank quality assurance by providing the possibility to assess biobank laboratory performance and useful insights into biobank laboratory method performance characteristics and thus fulfill the demands from accreditation authorities.
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http://dx.doi.org/10.1089/bio.2015.0108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445200PMC
October 2016

Paradox-breaking RAF inhibitors that also target SRC are effective in drug-resistant BRAF mutant melanoma.

Cancer Cell 2015 Jan 11;27(1):85-96. Epub 2014 Dec 11.

Cancer Research UK Cancer Therapeutics Unit, The Institute of Cancer Research, London SM2 5NG, UK. Electronic address:

BRAF and MEK inhibitors are effective in BRAF mutant melanoma, but most patients eventually relapse with acquired resistance, and others present intrinsic resistance to these drugs. Resistance is often mediated by pathway reactivation through receptor tyrosine kinase (RTK)/SRC-family kinase (SFK) signaling or mutant NRAS, which drive paradoxical reactivation of the pathway. We describe pan-RAF inhibitors (CCT196969, CCT241161) that also inhibit SFKs. These compounds do not drive paradoxical pathway activation and inhibit MEK/ERK in BRAF and NRAS mutant melanoma. They inhibit melanoma cells and patient-derived xenografts that are resistant to BRAF and BRAF/MEK inhibitors. Thus, paradox-breaking pan-RAF inhibitors that also inhibit SFKs could provide first-line treatment for BRAF and NRAS mutant melanomas and second-line treatment for patients who develop resistance.
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http://dx.doi.org/10.1016/j.ccell.2014.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297292PMC
January 2015

Differential role of Th1 and Th2 cytokines in autotoxicity driven by CD19-specific second-generation chimeric antigen receptor T cells in a mouse model.

J Immunol 2014 Apr 12;192(8):3654-65. Epub 2014 Mar 12.

Clinical and Experimental Immunotherapy Group, Department of Medical Oncology, Institute of Cancer Sciences, The University of Manchester, Manchester, M20 4BX, United Kingdom;

T cells engrafted with chimeric AgRs (CAR) are showing exciting potential for targeting B cell malignancies in early-phase clinical trials. To determine whether the second-generation CAR was essential for optimal antitumor activity, two CD28-based CAR constructs targeting CD19 were tested for their ability to redirect mouse T cell function against established B cell lymphoma in a BALB/c syngeneic model system. T cells armed with either CAR eliminated A20 B cell lymphoma in vivo; however, one construct induced a T cell dose-dependent acute toxicity associated with a raised serum Th1 type cytokine profile on transfer into preconditioned mice. Moreover, a chronic toxicity manifested as granuloma-like formation in spleen, liver, and lymph nodes was observed in animals receiving T cells bearing either CD28 CAR, albeit with different kinetics dependent upon the specific receptor used. This phenotype was associated with an expansion of CD4+ CAR+ T cells and CD11b+ Gr-1(+) myeloid cells and increased serum Th2-type cytokines, including IL-10 and IL-13. Mouse T cells engrafted with a first-generation CAR failed to develop such autotoxicity, whereas toxicity was not apparent when T cells bearing the same receptors were transferred into C57BL/6 or C3H animals. In summary, the adoptive transfer of second-generation CD19-specific CAR T cells can result in a cell dose-dependent acute toxicity, whereas the prolonged secretion of high levels of Th2 cytokines from these CAR T cells in vivo drives a granulomatous reaction resulting in chronic toxicity. Strategies that prevent a prolonged Th2-cytokine biased CAR T cell response are clearly warranted.
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http://dx.doi.org/10.4049/jimmunol.1302148DOI Listing
April 2014

PLK1 and YY1 interaction in follicular lymphoma is associated with unfavourable outcome.

J Clin Pathol 2013 Sep 11;66(9):764-7. Epub 2013 Jun 11.

The Medical School, The University of Manchester, Manchester, Greater Manchester, UK.

Aims: Ying Yang 1 (YY1) is a transcription factor involved in both proliferation and apoptosis. It is prognostic in follicular lymphoma (FL), increased protein levels being associated with favourable outcome. PLK1 is a critical regulator of mitosis, playing a role in spindle formation and in regulation of the G2/M cell cycle checkpoint. PLK1 phosphorylates YY1 at the G2/M checkpoint with activation of YY1 and resultant progression from G2 into mitosis.

Methods: This study aims to investigate possible molecular coexpression and interaction of YY1 with PLK1 in FL using Duolink II in situ proximity ligation assay (PLA) in 51 FL samples in a tissue microarray.

Results: Positive PLA signals were present at variable frequency and Kaplan-Meier analysis showed association of signal frequency above the median with unfavourable outcome (p=0.0270). PLA signals were localised to the nuclear edge, with only one signal per cell, suggesting PLK1 and YY1 coexpression at the centrosome. In a minority of cells, two very close PLA signals were present in a single cell, and occasionally, there was a strong ring of semi-confluent fluorescent PLA signals round the nucleus of non-dividing cells, while rarely events were observed in the cytoplasm surrounding dividing cells.

Conclusions: The results confirm association of YY1 and PLK1 with outcome in FL and suggest coexpression at the centrosome. Given the reported interaction of YY1 with PLK1 at the centriole and promotion of cell division at the G2/M checkpoint, the results would concord with the known association of higher proliferation with poor outcome in FL.
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http://dx.doi.org/10.1136/jclinpath-2013-201461DOI Listing
September 2013

Standard preanalytical coding for biospecimens: review and implementation of the Sample PREanalytical Code (SPREC).

Biopreserv Biobank 2012 Aug;10(4):366-74

1 Integrated Biobank of Luxembourg , Luxembourg .

The first version of the Standard PREanalytical Code (SPREC) was developed in 2009 by the International Society for Biological and Environmental Repositories (ISBER) Biospecimen Science Working Group to facilitate documentation and communication of the most important preanalytical quality parameters of different types of biospecimens used for research. This same Working Group has now updated the SPREC to version 2.0, presented here, so that it contains more options to allow for recent technological developments. Existing elements have been fine tuned. An interface to the Biospecimen Reporting for Improved Study Quality (BRISQ) has been defined, and informatics solutions for SPREC implementation have been developed. A glossary with SPREC-related definitions has also been added.
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http://dx.doi.org/10.1089/bio.2012.0012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6463986PMC
August 2012

RAC2, AEP, and ICAM1 expression are associated with CNS disease in a mouse model of pre-B childhood acute lymphoblastic leukemia.

Blood 2011 Jul 23;118(3):638-49. Epub 2011 May 23.

Cancer Research UK Children's Cancer Group, Manchester Academic Health Science Centre, School of Cancer & Enabling Sciences, University of Manchester, Manchester, United Kingdom.

We developed a murine model of CNS disease to obtain a better understanding of the pathogenesis of CNS involvement in pre-B-cell acute lymphoblastic leukemia (ALL). Semiquantitative proteomic discovery-based approaches identified unique expression of asparaginyl endopeptidase (AEP), intercellular adhesion molecule 1 (ICAM1), and ras-related C3 botulinum toxin substrate 2 (RAC2), among others, in an invasive pre-B-cell line that produced CNS leukemia in NOD-SCID mice. Targeting RAC2 significantly inhibited in vitro invasion and delayed disease onset in mice. Induced expression of RAC2 in cell lines with low/absent expression of AEP and ICAM1 did not result in an invasive phenotype or murine CNS disease. Flow cytometric analysis identified an enriched population of blast cells expressing ICAM1/lymphocyte function associated antigen-1 (LFA-1)/CD70 in the CD10(+)/CD19(+) fraction of bone marrow aspirates obtained from relapsed compared with normal controls and those with primary disease. CD10(+)/CD19(+) fractions obtained from relapsed patients also express RAC2 and give rise to CNS disease in mice. Our data suggest that combinations of processes are involved in the pathogenesis of CNS disease in pre-B-cell ALL, support a model in which CNS disease occurs as a result of external invasion, and suggest that targeting the processes of adhesion and invasion unique to pre-B cells may prevent recurrences within the CNS.
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http://dx.doi.org/10.1182/blood-2010-09-307330DOI Listing
July 2011

Loss of ATF2 function leads to cranial motoneuron degeneration during embryonic mouse development.

PLoS One 2011 Apr 21;6(4):e19090. Epub 2011 Apr 21.

Cell Regulation Department, Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom.

The AP-1 family transcription factor ATF2 is essential for development and tissue maintenance in mammals. In particular, ATF2 is highly expressed and activated in the brain and previous studies using mouse knockouts have confirmed its requirement in the cerebellum as well as in vestibular sense organs. Here we present the analysis of the requirement for ATF2 in CNS development in mouse embryos, specifically in the brainstem. We discovered that neuron-specific inactivation of ATF2 leads to significant loss of motoneurons of the hypoglossal, abducens and facial nuclei. While the generation of ATF2 mutant motoneurons appears normal during early development, they undergo caspase-dependent and independent cell death during later embryonic and foetal stages. The loss of these motoneurons correlates with increased levels of stress activated MAP kinases, JNK and p38, as well as aberrant accumulation of phosphorylated neurofilament proteins, NF-H and NF-M, known substrates for these kinases. This, together with other neuropathological phenotypes, including aberrant vacuolisation and lipid accumulation, indicates that deficiency in ATF2 leads to neurodegeneration of subsets of somatic and visceral motoneurons of the brainstem. It also confirms that ATF2 has a critical role in limiting the activities of stress kinases JNK and p38 which are potent inducers of cell death in the CNS.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0019090PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3080913PMC
April 2011

Standard preanalytical coding for biospecimens: defining the sample PREanalytical code.

Cancer Epidemiol Biomarkers Prev 2010 Apr 23;19(4):1004-11. Epub 2010 Mar 23.

Integrated Biobank of Luxembourg, 6 rue Nicolas Ernest Barblé, L-1210 Luxembourg.

Background: Management and traceability of biospecimen preanalytical variations are necessary to provide effective and efficient interconnectivity and interoperability between Biobanks.

Methods: Therefore, the International Society for Biological and Environmental Repositories Biospecimen Science Working Group developed a "Standard PREanalytical Code" (SPREC) that identifies the main preanalytical factors of clinical fluid and solid biospecimens and their simple derivatives.

Results: The SPREC is easy to implement and can be integrated into Biobank quality management systems and databases. It can also be extended to nonhuman biorepository areas. Its flexibility allows integration of new novel technological developments in future versions. SPREC version 01 is presented in this article.

Conclusions And Impact: Implementation of the SPREC is expected to facilitate and consolidate international multicenter biomarker identification research and biospecimen research in the clinical Biobank environment.
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http://dx.doi.org/10.1158/1055-9965.EPI-09-1268DOI Listing
April 2010

Eradication of established B-cell lymphoma by CD19-specific murine T cells is dependent on host lymphopenic environment and can be mediated by CD4+ and CD8+ T cells.

J Immunother 2009 Apr;32(3):207-18

Department of Medical Oncology, Paterson Institute for Cancer Research, Wilmslow Road, Manchester, M20 4BX, UK.

B-cell malignancies seem to be particularly amenable to immunotherapy and as such make particularly attractive targets for adoptive T-cell therapy. Murine T cells gene-modified to express a chimeric immune receptor specific for CD19+ (aCD19z) efficiently kill CD19 B-cell lymphoma cells in vitro. aCD19z T cells also secrete high levels of interleukin-2 during culture with target cells in a CD86 independent manner. aCD19z T cells proved effective at eradicating established B-cell lymphoma in a syngeneic model system when combined with a lymphodepleting preconditioning regimen. In mice deficient of T, B, and natural killer cells (severe combined immunodeficient/Beige), aCD19z T cells efficiently eradicated long-term (13 d) established tumors with 100% of treated animals remaining tumor free for greater than 77 days. Although gene-modified CD4+ and CD8+ were both active in this setting, poor engraftment by CD8+ T cells coupled with the rigorous expansion of CD4+ cells in the Balb/c background suggests that CD4+ T cells may be playing a predominant role in lymphoma rejection in this model. Taken together, the therapeutic effectiveness of aCD19z T cells in this model supports a recently opened phase 1 trial of this receptor in non-Hodgkin lymphoma.
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http://dx.doi.org/10.1097/CJI.0b013e318194a921DOI Listing
April 2009

Eotaxin-2 and colorectal cancer: a potential target for immune therapy.

Clin Cancer Res 2007 Oct;13(19):5719-28

Cancer Research UK Department of Medical Oncology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, University of Manchester, United Kingdom.

Purpose: To study the production of chemokines by colorectal hepatic metastases.

Experimental Design: Biopsies of resected colorectal hepatic metastases and nonneoplastic adjacent liver tissue were screened for chemokines using protein arrays and results were confirmed by ELISA and immunohistochemistry.

Results: Two chemokines, eotaxin-2 and MCP-1, were found at elevated levels within the tumor biopsy compared with adjacent liver. The relative increase in expression from tumor was much higher for eotaxin-2 than MCP-1, with 10 of 25 donors having a >100-fold increase in expression compared with 0 of 24 donors for MCP-1. In a parallel analysis, eotaxin-2 was also found at elevated levels in the tumor region of primary colorectal cancer biopsies. Immunohistochemical staining indicated that carcinoembryonic antigen-positive tumor cells stained strongly for eotaxin-2, implicating these cells as the predominant source of the chemokine. In vitro studies confirmed that several colorectal tumor lines produce eotaxin-2 and that secretion of this chemokine could be depressed by IFN-gamma and enhanced by the Th2-type cytokines interleukin-4 and interleukin-13. Jurkat T cells were engineered to express the receptor for eotaxin-2 (CCR3). These cells effectively migrated in response to eotaxin-2 protein, suggesting that immune cells gene modified to express a chemokine receptor may have improved abilities to home to tumor.

Conclusions: Taken together, these observations confirm eotaxin-2 as a chemokine strongly associated with primary and metastatic tumors of colorectal origin. Furthermore, the importance of this result may be a useful tool in the development of targeted therapeutic approaches to colorectal tumors.
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http://dx.doi.org/10.1158/1078-0432.CCR-07-1145DOI Listing
October 2007

Feedback regulation of p38 activity via ATF2 is essential for survival of embryonic liver cells.

Genes Dev 2007 Aug;21(16):2069-82

Cell Regulation Department, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom.

The ATF2 transcription factor is phosphorylated by the stress-activated mitogen-activated protein kinases (MAPKs) JNK and p38. We show that this phosphorylation is essential for ATF2 function in vivo, since a mouse carrying mutations in the critical phosphorylation sites has a strong phenotype identical to that seen upon deletion of the DNA-binding domain. In addition, combining this mutant with a knockout of the ATF2 homolog, ATF7, results in embryonic lethality with severe abnormalities in the developing liver and heart. The mutant fetal liver is characterized by high levels of apoptosis in developing hepatocytes and haematopoietic cells. Furthermore, we observe a significant increase in active p38 due to loss of a negative feedback loop involving the ATF2-dependent transcriptional activation of MAPK phosphatases. In embryonic liver cells, this increase drives apoptosis, since it can be suppressed by chemical inhibition of p38. Our findings demonstrate the importance of finely regulating the activities of MAPKs during development.
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http://dx.doi.org/10.1101/gad.430207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1948861PMC
August 2007