• How liquid biopsies can change clinical practice in oncology

      Siravegna, G; Mussolin, B; Venesio, T; Marsoni, S; Seoane, J; Dive, Caroline; Papadopoulos, N; Kopetz, S; Corcoran, RB; Siu, LL; et al. (2019)
      Cell-free DNA fragments are shed into the bloodstream by tumor cells. The analysis of circulating tumor DNA (ctDNA), commonly known as liquid biopsy, can be exploited for a variety of clinical applications. ctDNA is being used to genotype solid cancers non-invasively, to track tumor dynamics and to detect the emergence of drug resistance. In a few settings, liquid biopsies have already entered clinical practice. For example, ctDNA is used to guide treatment in a subset of lung cancers. In this review, we discuss how recent improvements in the sensitivity and accuracy of ctDNA analyses have led to unprecedented advances in this research field. We further consider what is required for the routine deployment of liquid biopsies in the clinical diagnostic space. We pinpoint technical hurdles that liquid biopsies have yet to overcome, including pre-analytical and analytical challenges. We foresee how liquid biopsies will transform clinical practice: by complementing (or replacing) imaging to monitor treatment response and by detecting minimal residual disease after surgery with curative intent.
    • How many cancer cases and deaths are potentially preventable? Estimates for Australia in 2013.

      Wilson, L; Antonsson, A; Green, Adèle C; Jordan, S; Kendall, B; Nagle, C; Neale, R; Olsen, C; Webb, P; Whiteman, D; et al. (2017-10-06)
      Cancer is a leading cause of disease burden in Australia, particularly fatal burden, accounting for an estimated thirty percent of deaths. Many cancers develop because of exposure to lifestyle and environmental factors that are potentially modifiable. We aimed to quantify the proportions and numbers of cancer deaths and cases in Australia in 2013 attributable to 20 modifiable factors in eight broad groupings that are established causes of cancer, namely: tobacco smoke (smoking and second-hand), dietary factors (low intake of fruit, non-starchy vegetables and dietary fibre; and high intake of red and processed meat), overweight/obesity, alcohol, physical inactivity, solar ultraviolet radiation, infections (seven agents), and reproductive factors (lack of breastfeeding, menopausal hormone therapy use, combined oral contraceptive use). We estimated population attributable fractions (PAF) using standard formulae incorporating exposure prevalence and relative risk data. Of all cancer deaths in Australia in 2013, approximately 38% overall (males 41%, females 34%) could be attributed to the factors assessed; the corresponding PAF for cancer cases was 33% (males 34%, females 32%). Tobacco smoke was the leading cause of cancer deaths and cases, with PAFs of 23 and 13%, respectively, followed by dietary factors (5% deaths/5% cases), overweight/obesity (5%/4%) and infections (5%/3%). Cancer sites with the highest numbers of potentially preventable deaths/cases were lung (n = 6,776/9,272), colorectum (n = 1,974/7,380) and cutaneous melanoma (n = 1,390/7,918). We estimate that about 16,700 cancer deaths and 41,200 cancer cases could be prevented in Australia each year if people's exposures to 20 causal factors were aligned with levels recommended to minimise cancer risk.
    • How many diseases are colorectal cancer?

      Greystoke, Alastair; Mullamitha, Saifee A; Department of Medical Oncology, Christie NHS Foundation Trust, Manchester M20 4BX, UK ; School of Cancer and Imaging Sciences, University of Manchester, Manchester M13 9PL, UK. (2012)
      The development of personalised therapy and mechanism-targeted agents in oncology mandates the identification of the patient populations most likely to benefit from therapy. This paper discusses the increasing evidence as to the heterogeneity of the group of diseases called colorectal cancer. Differences in the aetiology and epidemiology of proximal and distal cancers are reflected in different clinical behaviour, histopathology, and molecular characteristics of these tumours. This may impact response both to standard cytotoxic therapies and mechanism-targeted agents. This disease heterogeneity leads to challenges in the design of clinical trials to assess novel therapies in the treatment of "colorectal cancer."
    • How many melanomas might be prevented if more people applied sunscreen regularly?

      Olsen, C M; Wilson, L F; Green, Adèle C; Biswas, N; Loyalka, J; Whiteman, D C; Population Health Department, QIMR Berghofer Medical Research Institute, 300 Herston Road, Herston, Queensland, 4006, Australia (2018-01)
      Ultraviolet radiation causes cutaneous melanoma. Sunscreen prevents sunburn and protects skin cells against mutations. High-quality epidemiological studies suggest regular sunscreen use prevents melanoma.
    • How participants in cancer trials are chosen: ethics and conflicting interests.

      Jayson, Gordon C; Harris, John; Cancer Research UK, University of Manchester, Department of Medical Oncology, Christie Hospital, Withington, Manchester M20 4BX, UK. Gordon.Jayson@christie-tr.nwest.nhs.uk (2006-04)
      The development of new drugs for cancer is extremely complex and expensive, and poses ethical problems. In this article we will review issues in clinical trials for cancer drugs that will cast new light on the doctor-patient relationship and their interaction with industry, the health service, academic and administrative organizations. We show that the Declaration of Helsinki cannot be applied to cancer trials as it is currently written, that patients do not and perhaps cannot give fully informed consent to participate, and that the results of clinical trials do not translate into daily practice in a way that patients might expect.
    • How will haematologists use proteomics?

      Unwin, Richard D; Whetton, Anthony D; Stem Cell and Leukaemia Proteomics Laboratory, Faculty of Medical and Human Sciences, University of Manchester, Christie Hospital, Kinnaird House, Kinnaird Road, Withington, Manchester, UK M20 4QL. runwin@manchester.ac.uk (2007-11)
      Proteomics technologies are emerging as a useful tool in the identification of disease biomarkers, and in defining and characterising both normal physiological and disease processes. Many cellular changes in protein expression in response to an external stimulus or mutation can only be characterised at the proteome level. In these cases protein expression is often controlled by altered rates of translation and/or degradation, making proteomics an important tool in the analysis of biological systems. In the leukaemias, post-translational modification of proteins (e.g. phosphorylation, acetylation) plays a key role in the molecular pathology of the disease: such modifications can now be detected with novel proteomic methods. In a clinical setting, serum remains a relatively un-mined source of information for prognosis and response to therapy. This protein rich fluid represents an opportunity for proteomics research to benefit hematologists and others. In this review, we discuss the technologies available for the study of the proteome that offer realistic opportunities in haematology.
    • The Hox-2.4 gene is not involved in the generation of IL-3 dependent multipotent FDCP-mix cell lines.

      Just, Ursula; Kan, On; Fennelly, Jan; Dexter, T Michael; Spooncer, Elaine; Department of Experimental Haematology, Paterson Institute for Cancer Research, Manchester, UK. (1995)
      The establishment of IL-3-dependent multipotent progenitor cell lines from Hox-2.4-expressing bone marrow cells suggests that homeobox genes may contribute to immortalization of early myeloid cells. A survey of 20 independently derived multipotent IL-3-dependent cell lines established from either src-virus-infected long-term bone marrow cultures (FDCP-mix) or Multi-CSF-virus (M3MuV)-infected bone marrow revealed that Hox-2.4 was not expressed in any of these cell lines. In addition DNA rearrangements were not observed. We conclude that activation of Hox-2.4 is not an obligatory event in the immortalization of early myeloid cells.
    • The HOXB7 protein renders breast cancer cells resistant to tamoxifen through activation of the EGFR pathway.

      Jin, K; Kong, X; Shah, T; Penet, M; Wildes, F; Sgroi, D; Ma, X; Huang, Y; Kallioniemi, A; Landberg, Göran; et al. (2012-02-21)
      Multiple factors including long-term treatment with tamoxifen are involved in the development of selective estrogen receptor (ER) modulator resistance in ERα-positive breast cancer. Many underlying molecular events that confer resistance are known but a unifying theme is yet to be revealed. In this report, we provide evidence that HOXB7 overexpression renders MCF-7 cells resistant to tamoxifen via cross-talk between receptor tyrosine kinases and ERα signaling. HOXB7 is an ERα-responsive gene. Extended treatment of MCF-7 cells with tamoxifen resulted in progressively increasing levels of HOXB7 expression, along with EGFR and EGFR ligands. Up-regulation of EGFR occurs through direct binding of HOXB7 to the EGFR promoter, enhancing transcriptional activity. Finally, higher expression levels of HOXB7 in the tumor significantly correlated with poorer disease-free survival in ERα-positive patients with breast cancer on adjuvant tamoxifen monotherapy. These studies suggest that HOXB7 acts as a key regulator, orchestrating a major group of target molecules in the oncogenic hierarchy. Functional antagonism of HOXB7 could circumvent tamoxifen resistance.
    • HPV 16 infection and progression of cervical intra-epithelial neoplasia: analysis of HLA polymorphism and HPV 16 E6 sequence variants.

      Bontkes, H J; Van Duin, M; De Gruijl, T D; Duggan-Keen, Margaret F; Walboomers, J M; Stukart, M J; Verheijen, R H; Helmerhorst, T J; Meijer, C J; Scheper, R J; et al. (1998-10-05)
      High-risk human papillomavirus (HPV) infection plays an important role in cervical intra-epithelial neoplasia (CIN), but HPV infection alone is not sufficient for progression to cervical cancer. Several lines of evidence suggest that cellular immune surveillance is important in the control of HPV infection and the development of CIN. The presentation to T cells of target viral peptides in the context of HLA molecules is influenced by the genetic polymorphisms of both HPV and HLA and thereby influences the host immune response and clinical outcome of HPV infection. HLA class I and II polymorphism in susceptibility for HPV 16 infection, development and progression of CIN was analyzed in a group of 118 patients participating in a prospective study of women with initial abnormal cytology. Patients were stratified according to HPV status and course of the disease. HLA-B*44 frequency was increased in the small group of patients with a lesion that showed clinical progression during follow-up [OR = 9.0 (4.6-17.5), p = 0.007]. HLA-DRB1*07 frequency was increased among HPV 16-positive patients compared with patients who were negative for all HPV types [OR = 5.9 (3.0-11.3), p = 0.02]. Our results are consistent with the immunogenetic factors associated with disease progression being different from those associated with susceptibility to HPV 16 infection. Sequencing of the HPV 16 E6 and E7 open reading frames of a subset of these patients (n = 40) showed the frequency of HPV 16 variants to be similar to other studies. However, there was no significant correlation between variant incidence and disease progression or viral persistence and no significant correlation with any HLA allele. It appears that multiple HLA types can influence HPV 16-associated cervical dysplasia but the role of HPV 16 variants in disease progression and susceptibility in relation to HLA polymorphism remains unclear.
    • HPV-related oropharyngeal cancer in the United Kingdom: an evolution in understanding of disease etiology.

      Schache, A; Powell, N; Cuschieri, K; Robinson, M; Leary, S; Mehanna, H; Rapozo, D; Long, A; Cubie, H; Junor, E; et al. (2016-08-28)
      A rising incidence of oropharyngeal squamous cell carcinoma (OPSCC) incidence has occurred throughout the developed world, where it has been attributed to an increasing impact of human papillomavirus (HPV) on disease etiology. This report presents the findings of a multicenter cross-sectional retrospective study aimed at determining the proportion of HPV-positive and HPV-negative OPSCC within the United Kingdom (UK). Archival tumor tissue blocks from 1602 patients previously diagnosed with OPSCC (2002-2011) were collated from 11 centers. HPV status was determined with 3 validated commercial tests to provide valid data for 1474 cases in total. Corresponding national incidence data from the same decade were obtained from UK Cancer registries.The overall proportion of HPV+ OPSCC between 2002-2011 was 51.8% (95% CI:49.3, 54.4) and this remained unchanged throughout the decade (unadjusted risk ratio:1.00 (95% CI:0.99, 1.02). However, over the same period, the incidence of OPSCC in the broader UK population underwent a 2-fold increase (age standardised rate (ASR) 2002:2.1 (95% CI:1.9, 2.2); 2011:4.1(95% CI:4.0, 4.3)). Although the number of OPSCC diagnosed within the UK from 2002-2011 nearly doubled, the proportion of HPV+ cases remained static at ~50%. Our results argue that the rapidly increasing incidence of OPSCC in the UK cannot be solely attributable to the influence of HPV. The parallel increase in HPV+ and HPV- cases we documented warrants further investigation, so that appropriate future prevention strategies for both types of disease can be implemented.
    • The HPV16 E6 and E7 proteins and the radiation resistance of cervical carcinoma.

      Hampson, Lynne; El Hady, El Said Abd; Moore, James V; Kitchener, Henry C; Hampson, Ian N; University of Manchester Academic Unit of Obstetrics and Gynaecology, St. Mary's Hospital, Manchester M13 OJH, UK. (2001-06)
    • An HTS-compatible HTRF assay measuring the glycohydrolase activity of human PARG.

      Stowell, Alexandra I J; James, Dominic I; Waddell, Ian D; Bennett, N; Truman, C; Hardern, I; Ogilvie, Donald J; Cancer Research UK Manchester Institute Drug Discovery Unit, University of Manchester, Manchester, (2016-03-29)
      Poly(ADP-ribose)(PAR) polymers are transient post-translational modifications, and their formation is catalyzed by poly(ADP-ribose) polymerase (PARP) enzymes. A number of PARP inhibitors are in advanced clinical development for BRCA-mutated breast cancer, and olaparib has recently been approved for BRCA-mutant ovarian cancer; however, there has already been evidence of developed resistance mechanisms. Poly(ADP-ribose) glycohydrolase (PARG) catalyzes the hydrolysis of the endo- and exo-glycosidic bonds within the PAR polymers. As an alternative strategy, PARG is a potentially attractive therapeutic target. There is only one PARG gene, compared with 17 known PARP family members, and therefore a PARG inhibitor may have wider application with fewer compensatory mechanisms. Prior to the initiation of this project, there were no known existing cell-permeable small molecule PARG inhibitors for use as tool compounds to assess these hypotheses, and no suitable high-throughput screening (HTS)-compatible biochemical assays available to identify start points for a drug discovery project. The development of this newly-described high-throughput homogeneous time-resolved fluorescence (HTRF) assay has allowed HTS to proceed, and from this, the identification and advancement of multiple validated series of tool compounds for PARG inhibition.
    • Human breast cell proliferation and its relationship to steroid receptor expression.

      Clarke, Robert B; Breast Biology Group, Clinical Research Department, Christie Hospital, Manchester, UK. (2004-06)
      The steroidal regulation of proliferation and differentiation in the rodent mammary gland is well described, but how ovarian hormones regulate these processes in the human remains poorly understood. To investigate this, we developed the athymic nude mouse model in which intact normal human breast tissue is grafted subcutaneously and treated with estrogen and/or progesterone at human physiological serum levels. We demonstrated, first, that estrogen and not progesterone is the major epithelial cell mitogen in the adult non-pregnant, non-lactating breast, second, that estrogen induces progesterone receptor (PR) expression and, third, that PR expression is maximally induced at low estrogen concentrations while a higher amount of estrogen was required to induce proliferation. These data raised the question of whether one cell type possessed differential responses to high and low estrogen concentrations or whether PR expression and proliferation occurred in two cell populations. Using double-label immunofluorescence, we demonstrated that steroid receptor expression and cell proliferation (Ki67 antigen) occurred in separate cell populations in normal human breast epithelium, and that cells expressing the estrogen receptor-alpha (ERalpha) invariably contained the PR. We also found that this dissociation between steroid receptor expression and cell proliferation in normal epithelium was disrupted at an early stage in breast tumor formation. Recent findings presented herein support the proposal that some ERalpha/PR-positive epithelial cells are quiescent breast stem cells that act as 'steroid hormone sensors'. Such hormone sensor cells are likely to secrete positive or negative paracrine/juxtacrine factors dependent on the prevailing estrogen or progesterone concentration to influence the proliferative activity of adjacent ERalpha/PR-negative epithelial cells.
    • Human breast epithelial stem cells and their regulation.

      Kalirai, Helen; Clarke, Robert B; Breast Biology Group, Division of Cancer Studies, University of Manchester, Christie Hospital, Wilmslow Road, Manchester, M20 4BX, UK. (2006-01)
      This review summarizes the current evidence for the existence of human breast stem cells and the pathways involved in their regulation, and discusses how the disruption of these pathways may result in the generation of a population of cells with the capacity for unlimited self-renewal. Relevant data from mouse model systems are also discussed where appropriate. By understanding the molecular pathways that regulate self-renewal of normal mammary stem cells, it may be possible to target the activation of these pathways in breast tumours.
    • Human bronchoalveolar macrophage cytotoxicity for cultured human lung-tumour cells.

      Swinburne, S; Moore, Michael; Cole, P; Host Defence Unit, Deaprtment of Medicine, Cardiothoraci Institute, Brompton Hospital, Fulham Road, London SW3 6HP. (1982-10)
      Human bronchoalveolar macrophages were separated from other free lung cells by density sedimentation on Percoll gradients. They were then tested for cytotoxicity against the human lung adenocarcinoma cell line A549, using a Selenomethionine-75 post-labelling assay. The cytotoxicity of the macrophages increased as the effector:target cell ratio was increased, approaching 100% at 20:1. There was no significant difference in the cytotoxicity of macrophages isolated from the lungs of bronchial-carcinoma or non-carcinoma patients. The highly cytotoxic nature of the macrophages was not due to selection of a more potent cytotoxic subpopulation of macrophages on the Percoll gradient, nor to a generally elevated activation of the macrophages due to the pathological conditions in the patients' lungs. An attempt to determine whether low concentrations of macrophages could potentiate target-cell growth proved negative. Cytotoxicity of macrophages for cultured lung target cells was not restricted to A549 cells and is not in accordance with the view that defective bronchoalveolar macrophage cytotoxicity contributes to the emergence of bronchial neoplasia.
    • Human colorectal mucosal O6-alkylguanine DNA-alkyltransferase activity and DNA-N7-methylguanine levels in colorectal adenoma cases and matched referents.

      Lees, Nicholas P; Harrison, Kathryn L; Hall, C Nicholas; Margison, Geoffrey P; Povey, Andrew C; Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, Christie Hospital, Manchester, UK. (2007-03)
      BACKGROUND AND AIMS: O(6)-alkylguanine DNA-alkyltransferase (MGMT) provides protection against alkylating agent-induced GC-->AT transition mutations. Such mutations are frequently seen in the KRAS oncogene of large colorectal adenomas, but whether adenoma or mutational risk in humans is influenced by MGMT activity and alkylating agent exposure is unclear. Hence, MGMT activity and, as an indicator of alkylating agent exposure, DNA-N7-methylguanine (N7-MeG) levels were determined in the normal tissue of patients with and without adenomas. METHODS: Biopsy specimens of normal colorectal mucosa were collected during colonoscopy from 85 patients with histologically proved colorectal adenomas (cases) and from 85 patients free of gastrointestinal neoplasia (referents) matched by age, sex and biopsy location. MGMT activity and N7-MeG levels were measured in colorectal tissue extracts and DNA, respectively. RESULTS: MGMT activity was higher in the normal mucosa of cases than in referents (6.65+/-3.03 vs 5.61+/-2.74 fmol/micro g DNA, p = 0.01). On stratification of cases, MGMT activity was found to be considerably greater in the normal mucosa of cases with large adenomas (p = 0.003) and slightly higher in cases with a GC-->AT transition mutation in the K-ras gene (p = 0.03). Elevated MGMT levels were associated with an increased risk of adenoma (OR 1.17, 95% CI 1.03 to 1.33 per unit increase in activity). Detectable levels of N7-MeG were found in DNA from 89% of cases and 93% of referents, with levels ranging from <0.1 to 7.7 micro mol/mol dG. Cases and referents had similar DNA-N7-MeG levels. CONCLUSIONS: Human exposure to methylating agents is widespread. MGMT activity is increased in the normal mucosa of patients with adenomas.
    • Human cord blood: a source of transplantable stem cells?

      Hows, Jill M; Marsh, Judith C W; Bradley, B A; Luft, Thomas; Coutinho, Lucia H; Testa, Nydia G; Dexter, T Michael; Kay Kendall Laboratory, Paterson Institute, Cancer Research Campaign, Manchester, UK. (1992)
      In a preliminary study we have shown that optimally collected human umbilical cord (HUC) blood cells grow significantly better in long term cultures (LTC) than normal adult marrow cells (NBM) p = 0.0007. The LTC findings are supported by the observation in clonogenic assay that a similar number of GM-CFC colonies can be grown from HUC blood and NBM mononuclear cells (MNC). Also there is a trend towards a higher proportion of primitive erythroid (BFU-E) and primitive megakaryocyte colonies (MK-CFC containing greater than 20 cells) in HUC blood compared with NBM. We suggest that further work on the feasibility of a HLA typed, cryopreserved HUC blood bank as a source of unrelated haemopoietic 'stem' cells for clinical transplantation is indicated.
    • The human cytomegalovirus immediate-early promoter is transcriptionally active in undifferentiated mouse embryonic stem cells.

      Ward, Christopher M; Stern, Peter L; Immunology Group, Paterson Institute for Cancer Research (PICR), Christie Hospital NHS Trust, Manchester, United Kingdom. wardcm@hotmail.com (2002)
      It has been reported recently that the cytomegalovirus (CMV) immediate-early promoter is transcriptionally inactive in undifferentiated mouse embryonic stem (ES) cells. This result is surprising, since the CMV promoter is used to express transgenes in a variety of cell lines. We studied the expression of a human CMV-driven enhanced green fluorescent protein (EGFP) reporter gene (pEGFP-N1) in five undifferentiated mouse ES cell lines (BL/6III, D3, E14TG2a, MESC20, and 129) and found EGFP expression in all of these cell lines. Under optimal conditions, between 50%-80% transfection efficiencies could be achieved, and EGFP expression levels were maintained for at least 72 hours. Therefore, the human CMV promoter remains a useful system for transgene expression in undifferentiated ES cells.
    • Human cytomegalovirus infection upregulates the mitochondrial transcription and translation machineries.

      Karniely, S; Weekes, M; Antrobus, R; Rorbach, J; van Haute, L; Umrania, Y; Smith, Duncan L; Stanton, R; Minczuk, M; Lehner, P; et al. (2016)
      Infection with human cytomegalovirus (HCMV) profoundly affects cellular metabolism. Like in tumor cells, HCMV infection increases glycolysis, and glucose carbon is shifted from the mitochondrial tricarboxylic acid cycle to the biosynthesis of fatty acids. However, unlike in many tumor cells, where aerobic glycolysis is accompanied by suppression of mitochondrial oxidative phosphorylation, HCMV induces mitochondrial biogenesis and respiration. Here, we affinity purified mitochondria and used quantitative mass spectrometry to determine how the mitochondrial proteome changes upon HCMV infection. We found that the mitochondrial transcription and translation systems are induced early during the viral replication cycle. Specifically, proteins involved in biogenesis of the mitochondrial ribosome were highly upregulated by HCMV infection. Inhibition of mitochondrial translation with chloramphenicol or knockdown of HCMV-induced ribosome biogenesis factor MRM3 abolished the HCMV-mediated increase in mitochondrially encoded proteins and significantly impaired viral growth under bioenergetically restricting conditions. Our findings demonstrate how HCMV manipulates mitochondrial biogenesis to support its replication.
    • Human cytotoxic T lymphocytes stimulated by endogenously processed human papillomavirus type 11 E7 recognize a peptide containing a HLA-A2 (A*0201) motif.

      Tarpey, I; Stacey, Simon N; Hickling, J; Birley, H D; Renton, A; McIndoe, A; Davies, D H; Division of Life Sciences, King's College, London, U.K. (1994-02)
      Cytotoxic T lymphocytes (CTL) may play an important role in the control of human papillomavirus (HPV)-induced anogenital neoplasias, but have been difficult to study owing to the difficulty in obtaining sufficient quantities of infectious virus. To address this we have stimulated human HPV-specific CTL in vitro using low-density cells (LDC) from peripheral blood mononuclear cells (PBMC). Low-density cells were used to present synthetic peptides, or endogenously processed peptides expressed from recombinant vaccinia viruses, to high-density PBMC (predominantly lymphocytes) for 6 days. Cytotoxic T lymphocytes stimulated with endogenously processed HPV 11 E7 recognized the synthetic HLA-A2 (A*0201) motif-containing nonamer, 4-12. In reciprocal experiments, CTL stimulated with this peptide in vitro recognized targets expressing endogenously processed E7. The responses in each case were A2 restricted and peptide specific. Two additional A2 motif-containing nonamers from HPV 6b E7 (21-30 and 47-55) also elicited peptide-specific, A2-restricted CTL. The data illustrate the potential that in vitro stimulation with LDC has in understanding CTL responses to experimentally problematic viral systems such as HPV, and may offer a route to specific immunotherapy of HPV-associated lesions.