• A non-functional retinoblastoma tumor suppressor (RB) pathway in premenopausal breast cancer is associated with resistance to tamoxifen.

      Lehn, S; Fernö, M; Jirström, K; Rydén, L; Landberg, Göran; Center for Molecular Pathology, Department of Laboratory Medicine, Lund University, Skåne University Hospital, Malmö, Sweden. (2011-03-15)
      The retinoblastoma tumor suppressor (RB) is important for retaining cell cycle control and loss of RB function is commonly observed in various malignancies. Experimental and animal studies have shown that RB knockdown in ER+ (estrogen receptor) cell lines and xenografts leads to resistance to tamoxifen, indicating that RB-inactivation could be linked to impaired response to specific cancer treatments. To address this issue, we utilized a unique randomized trial including 500 premenopausal breast cancer patients receiving either two years of adjuvant tamoxifen treatment or no treatment after surgery, and defined the tamoxifen response in RB-subgroups. Non-functional RB tumors were defined by lack of concordance between RB-phosphorylation and proliferation, in comparison to RB-functional tumors displaying comparable RB-phosphorylation and proliferation. In the ER+ tumors harboring a functional RB pathway (N=204), patients benefited from adjuvant tamoxifen with fewer breast cancer recurrences (HR=0.53, 95% CI 0.34-0.81, P=0.003). In the small subgroup of ER+ and RB non-functional tumors there was no benefit of tamoxifen (HR=2.28, 95% CI 0.51-10.3, P=0.28). In a multivariate analysis, the interaction between status of the RB pathway and treatment was significant (P=0.010), validating that despite being a small subgroup of ER+ breast cancer, RB functional status appears to be linked to response to tamoxifen treatment. These findings are in line with earlier experimental data altogether suggesting that analyses of RB status in breast cancer have the potential to be one among other future predictive factors that needs to be analyzed in order to successfully identify patients that will benefit from tamoxifen treatment.
    • Non-invasive in vivo imaging of hypoxia-inducible factor 1 function in the growth of colorectal liver metastases

      Gieling, R; Telfer, Brian A; Williams, Kaye J; University of Manchester (2016)
    • Non-invasive pulsed ultrasound quantification of the resolution of basal cell carcinomas after photodynamic therapy.

      Allan, Ernest; Pye, David A; Levine, Edward; Moore, James V; Dept of Clinical Oncology, Christie Hospital, Manchester, UK. (2002)
      The probability of local control of basal cell carcinomas (BCC) treated by photodynamic therapy (PDT) depends strongly on lesion thickness, thicker lesions often requiring two treatments. We examine the utility of 20 MHz pulsed ultrasound (US) for the non-invasive measurement of thickness and rate of regression after PDT treatment. PDT was by topically applied 20% aminolaevulinic acid, followed at 6 h by a standard 100 J/cm(2) of 630 nm light. Patients ( n=60) were selected as being difficult to treat with existing modalities for reasons of likely poor quality of healing or of cosmesis in this very largely elderly population. Ultrasound 'A' scans were made immediately before treatment, and at first and subsequent follow-ups. Parameters measured non-invasively for BCC, adjacent normal skin, and for fibroses after previous conventional therapies, were (a) thickness of skin or lesion, (b) linear density of ultrasound echoes and (c) linear density of high-amplitude echoes. Prior to treatment, median skin thickness (to the dermal/subcutaneous boundary) was 2.6 mm (range 1.2-5.7), fibroses 2.5 mm (1.4-5.6) and BCC 1.5 mm (0.5-4.4). Median linear density of echoes for normal skin, fibroses and BCC plus underlying tissue were 5.6, 5.5 and 4.5, respectively, the BCC values being significantly lower ( p=0.002). The corresponding medians for high-amplitude echoes were 1.9, 1.9 and 1.1 (skin or fibrosis versus BCC, p=0.001). Patients whose BCCs appeared clinically to be controlled at up to 220 days after a single treatment, all had values of ultrasound parameters corresponding to skin/fibrosis and were significantly different from measurements on the same site prior to treatment. Patients whose tumours appeared to be reverting to the original BCC ultrasound pattern were subsequently found to be recurring as judged clinically. Non-invasive pulsed ultrasound indicates that rates of resolution vary widely between BCC of similar initial thickness and that the probability of clearance of BCC by PDT is determined largely by the deepest, sometimes small, regions within a lesion, with the overall area being relatively unimportant.
    • Non-surgical treatment of cervical carcinoma.

      Sproston, Anthony R; Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Christie Hospital, Manchester. (2010-04-09)
      Since the discovery of radium in 1898, radiotherapy has been used in the treatment of cervical cancer, and it remains the mainstay of treatment today. Chemotherapy is in its infancy and has as yet been unable to improve long-term survival, despite encouraging response.
    • Noncoding mutations target cis-regulatory elements of the FOXA1 plexus in prostate cancer

      Zhou, S; Hawley, JR; Soares, F; Grillo, G; Teng, M; Madani, Tonekaboni SA; Hua, JT; Kron, KJ; Mazrooei, P; Ahmed, M; et al. (2020)
      Prostate cancer is the second most commonly diagnosed malignancy among men worldwide. Recurrently mutated in primary and metastatic prostate tumors, FOXA1 encodes a pioneer transcription factor involved in disease onset and progression through both androgen receptor-dependent and androgen receptor-independent mechanisms. Despite its oncogenic properties however, the regulation of FOXA1 expression remains unknown. Here, we identify a set of six cis-regulatory elements in the FOXA1 regulatory plexus harboring somatic single-nucleotide variants in primary prostate tumors. We find that deletion and repression of these cis-regulatory elements significantly decreases FOXA1 expression and prostate cancer cell growth. Six of the ten single-nucleotide variants mapping to FOXA1 regulatory plexus significantly alter the transactivation potential of cis-regulatory elements by modulating the binding of transcription factors. Collectively, our results identify cis-regulatory elements within the FOXA1 plexus mutated in primary prostate tumors as potential targets for therapeutic intervention.
    • Noninvasive tumor hypoxia measurement using magnetic resonance imaging in murine U87 glioma xenografts and in patients with glioblastoma.

      Linnik, I V; Scott, M L J; Holliday, K F; Woodhouse, N l; Waterton, J C; O'Connor, James P B; Barjat, H; Liess, C; Ulloa, J; Young, H; et al. (2014-05)
      There is a clinical need for noninvasive, nonionizing imaging biomarkers of tumor hypoxia and oxygenation. We evaluated the relationship of T1 -weighted oxygen-enhanced magnetic resonance imaging (OE-MRI) measurements to histopathology measurements of tumor hypoxia in a murine glioma xenograft and demonstrated technique translation in human glioblastoma multiforme.
    • Nonmyeloablative transplantation with or without alemtuzumab: comparison between 2 prospective studies in patients with lymphoproliferative disorders.

      Pérez-Simón, José A; Kottaridis, Panagiotis D; Martino, Rodrigo; Craddock, Charles; Caballero, Dolores; Chopra, Rajesh; García-Conde, Javier; Milligan, Donald W; Schey, Stephen; Urbano-Ispizua, Alvaro; et al. (2002-11-01)
      Although nonmyeloablative conditioning regimen transplantations (NMTs) induce engraftment of allogeneic stem cells with a low spectrum of toxicity, graft-versus-host disease (GVHD) remains a significant cause of morbidity and mortality. In vivo T-cell depletion, using alemtuzumab, has been shown to reduce the incidence of GVHD. However, this type of maneuver, although reducing GVHD, may have an adverse impact on disease response, because NMTs exhibit their antitumor activity by relying on a graft-versus-malignancy effect. To explore the efficacy of alemtuzumab compared with methotrexate (MTX) for GVHD prophylaxis, we have compared the results in 129 recipients of a sibling NMT enrolled in 2 prospective studies for chronic lymphoproliferative disorders. Both NMTs were based on the same combination of fludarabine and melphalan, but the United Kingdom regimen (group A) used cyclosporin A plus alemtuzumab, whereas the Spanish regimen (group B) used cyclosporin A plus MTX for GVHD prophylaxis. Patients receiving alemtuzumab had a higher incidence of cytomegalovirus (CMV) reactivation (85% versus 24%, P <.001) and a significantly lower incidence of acute GVHD (21.7% versus 45.1%, P =.006) and chronic GVHD (5% versus 66.7%, P <.001). Twenty-one percent of patients in group A and 67.5% in group B had complete or partial responses 3 months after transplantation (P <.001). Eighteen patients in group A received donor lymphocyte infusions (DLIs) to achieve disease control. At last follow-up there was no difference in disease status between the groups with 71% versus 67.5% (P =.43) of patients showing complete or partial responses in groups A and B, respectively. No significant differences were observed in event-free or overall survival between the 2 groups. In conclusion, alemtuzumab significantly reduced GVHD but its use was associated with a higher incidence of CMV reactivation. Patients receiving alemtuzumab often required DLIs to achieve similar tumor control but the incidence of GVHD was not significantly increased after DLI.
    • Normal breast tissue implanted into athymic nude mice identifies biomarkers of the effects of human pregnancy levels of estrogen.

      Blance, Rognvald N; Sims, Andrew H; Anderson, Elizabeth; Howell, Anthony; Clarke, Robert B; Breast Biology Group, School of Cancer and Imaging Sciences, Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom. (2009-03)
      We have generated a novel model system for the study of estrogen intervention in normal breast tissue. Nulliparous human breast tissue was implanted into immunocompromised nude mice and treated with high-dose estrogen to simulate the effects of pregnancy. Treatment of mice with human mid-pregnancy levels of 17beta-estradiol for a period of 4 weeks was followed by 4 weeks of withdrawal to mimic involution. Gene expression in the xenograft tissue was then analyzed by real-time reverse transcription-PCR to identify differences between treated and control tissues. Ten genes previously identified as altered by pregnancy in rodent models were found to be differentially expressed in human breast tissue with a > or =1.8-fold up-regulation of CDC42, TGFbeta3, DCN, KRT14, LTF, and AREG and a > or =0.7-fold down-regulation of STAT1, CTGF, IGF1, and VAMP1. Immunohistochemical analysis of archival paraffin-embedded adult premenopausal human breast tissue specimens identified a significantly lower level of expression of STAT1 (P < 0.05, Mann-Whitney U test) in parous compared with age-matched nulliparous tissue (median of 24% compared with 42% epithelial cells positive). We conclude that many of the pregnancy-induced breast cancer-protective changes observed in rodent models also occur in human breast tissue following intervention using human pregnancy levels of estrogen and that STAT1 expression is a potential biomarker of parity-induced breast cancer protection in the human breast.
    • Normal human breast xenografts activate N-nitrosodimethylamine: identification of potential target cells for an environmental nitrosamine.

      Zaidi, S N; Laidlaw, Ian; Howell, Anthony; Potten, Christopher S; Cooper, Donald P; O'Connor, Peter J; Department of Carcinogenesis, Christie Hospital (NHS) Trust, Manchester, UK. (1992-07)
      Normal human breast tissue maintained as xenografts in female Balb/c (nu/nu) athymic mice is capable of metabolising N-nitrosodimethylamine (NDMA) to active intermediates that will react with DNA. Administration of NDMA to mice with slow-release implants of 17 beta-oestradiol which provide human physiological (luteal phase) circulating oestrogen levels and increase cell proliferation in the xenograft (Laidlaw et al., 1992), leads to an apparent increase in the extent of reaction with DNA compared to controls without oestrogen implants. In mice with oestrogen implants, measurements of the amounts of the promutagenic lesion, O6-methyl-2'-deoxyguanosine formed in DNA clearly indicated a dose related increase in the extent of reaction. Detection of O6-methyl-2'-deoxyguanosine using immunohistochemical procedures revealed that the nuclei of cells of the glandular epithelium, supportive tissue and adipose tissue, in decreasing order of prevalence, were positively stained for the presence of this DNA lesion. Epithelial cells, which are the putative target cells for carcinogenesis in the breast, are therefore prone to promutagenic damage as a result of exposure to an environmental nitrosamine.
    • Not all perlecans are created equal: interactions with fibroblast growth factor (FGF) 2 and FGF receptors.

      Knox, Sarah; Merry, Catherine L R; Stringer, Sally E; Melrose, James; Whitelock, John; Commonwealth Scientific Industrial Research Organization (CSIRO) Molecular Science, North Ryde 2113, Australia. (2002-04-26)
      Human basement membrane heparan sulfate proteoglycan (HSPG) perlecan binds and activates fibroblast growth factor (FGF)-2 through its heparan sulfate (HS) chains. Here we show that perlecans immunopurified from three cellular sources possess different HS structures and subsequently different FGF-2 binding and activating capabilities. Perlecan isolated from human umbilical arterial endothelial cells (HUAEC) and a continuous endothelial cell line (C11 STH) bound similar amounts of FGF-2 either alone or complexed with FGFRalpha1-IIIc or FGFR3alpha-IIIc. Both perlecans stimulated the growth of BaF3 cell lines expressing FGFR1b/c; however, only HUAEC perlecan stimulated those cells expressing FGFR3c, suggesting that the source of perlecan confers FGF and FGFR binding specificity. Despite these differences in FGF-2 activation, the level of 2-O- and 6-O-sulfation was similar for both perlecans. Interestingly, perlecan isolated from a colon carcinoma cell line that was capable of binding FGF-2 was incapable of activating any BaF3 cell line unless the HS was removed from the protein core. The HS chains also exhibited greater bioactivity after digestion with heparinase III. Collectively, these data clearly demonstrate that the bioactivity of HS decorating a single PG is dependent on its cell source and that subtle changes in structure including secondary interactions have a profound effect on biological activity.
    • A novel approach to the analysis of SUMOylation with the independent use of trypsin and elastase digestion followed by database searching utilising consecutive residue addition to lysine.

      Chicooree, Navin; Griffiths, John R; Connolly, Yvonne; Tan, Chong-Teik; Malliri, Angeliki; Eyers, C; Smith, Duncan L; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK; School of Chemistry, University of Manchester, Brunswick Street, Manchester, M13 9SU, UK. (2013-01-15)
      Identification of sites of protein SUMOylation is of great importance due its functional diversity within the cell. To date, most approaches to this problem rely on site-directed mutagenesis and/or highly specialised mass spectrometry approaches. We present a novel alternative approach to the site mapping of SUMOylation using trypsin and elastase digestion, routine mass spectrometry and an unbiased isotag database searching strategy.
    • Novel asymmetric photosensitizers: an in vitro study.

      Haylett, Ann K; Forbes, E; MacLennan, A; Truscott, T G; Moore, James V; Department of Experimental Radiation Oncology, Paterson Institute for Cancer Research, Manchester, UK. (1996-08-02)
      A series of compounds based on an asymmetrical protoporphyrin molecule have been examined. The paired groups of sensitizers differed in terms of the presence or absence of a permanent positive charge, in the alkyl side chain length and in having either a primary or secondary amine substituent. The effects of these variables on drug uptake, partition coefficient and photodynamic cell kill were tested. Drug uptake and partition coefficient were shown to be correlated. Differences in gross uptake were found within paired groups of sensitizers although cell-associated uptake alone did not correlate with clonogenic cell survival. Of the compounds tested it was the sensitizers with alkyl side chains, rather than the permanently positively charged compounds, which resulted in the greatest degree of clonogenic cell kill.
    • Novel cell culture technique for primary ductal carcinoma in situ: role of Notch and epidermal growth factor receptor signaling pathways.

      Farnie, Gillian; Clarke, Robert B; Spence, Katherine; Pinnock, Natasha; Brennan, Keith; Anderson, Neil G; Bundred, Nigel J; Department of Surgery and Breast Biology Group, Division of Cancer Studies, Faculty of Medicine and Human Sciences, University of Manchester, Christie Hospital NHS Trust, Wilmslow Road, M20 9BX, Manchester, UK. (2007-04-18)
      BACKGROUND: The epidermal growth factor receptor (EGFR) and Notch signaling pathways have been implicated in self-renewal of normal breast stem cells. We investigated the involvement of these signaling pathways in ductal carcinoma in situ (DCIS) of the breast. METHODS: Samples of normal breast tissue (n = 15), pure DCIS tissue of varying grades (n = 35), and DCIS tissue surrounding an invasive cancer (n = 7) were used for nonadherent (i.e., mammosphere) culture. Mammosphere cultures were treated at day 0 with gefitinib (an EGFR inhibitor), DAPT (N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester) (a gamma-secretase inhibitor), or Notch 4-neutralizing antibody. Mammosphere-forming efficiency (MFE) was calculated by dividing the number of mammospheres of 60 microm or more formed by the number of single cells seeded and is expressed as a percentage. The Notch 1 intracellular domain (NICD) was detected immunohistochemically in paraffin-embedded DCIS tissue from 50 patients with at least 60 months of follow-up. All statistical tests were two-sided. RESULTS: DCIS had a greater MFE than normal breast tissue (1.5% versus 0.5%, difference = 1%, 95% confidence interval [CI] = 0.62% to 1.25%, P<.001). High-grade DCIS had a greater MFE than low-grade DCIS (1.6% versus 1.09%, difference = 0.51%, 95% CI = 0.07% to 0.94%, P = .01). The MFE of high-grade DCIS treated with gefitinib in the absence of exogenous EGF was lower than that of high-grade DCIS treated with mammosphere medium lacking gefitinib and exogenous EGF (0.56% versus 1.36%, difference 0.8%, 95% CI = 0.33% to 1.4%, P = .004). Increased Notch signaling as detected by NICD staining was associated with recurrence at 5 years (P = .012). DCIS MFE was reduced when Notch signaling was inhibited using either DAPT (0.89% versus 0.51%, difference = 0.38%, 95% CI = 0.2% to 0.6%, P<.001) or a Notch 4-neutralizing antibody (0.97% versus 0.2%, difference = 0.77%, 95% CI = 0.52% to 1.0%, P<.001). CONCLUSION: We describe a novel primary culture technique for DCIS. Inhibition of the EGFR or Notch signaling pathways reduced DCIS MFE.
    • Novel cytotoxic drugs: old challenges, new solutions.

      Ismael, Gustavo F V; Rosa, Daniela D; Mano, Max S; Awada, Ahmad; Department of Medical Oncology, Jules Bordet Institute, Université Libre de Bruxelles, Belgium. (2008-02)
      The discovery of cytotoxic agents was revolutionary for anticancer therapy in the last century, improving survival rates and the quality of life of patients with different types of tumours. However, the development of agents that combine efficacy, safety and convenience remains a great challenge, due to the narrow therapeutic index of some drugs, the fact that they may damage not only cancer cells, but also normal and healthy tissue and the occurrence of resistance, limiting anticancer efficacy. Novel cytotoxic agents have brought certain advantages over the conventional ones, such as shorter administration time, mechanisms to overcome drug resistance and lower incidence of adverse events. In this review we highlight the development of promising novel cytotoxic drugs that will hopefully offer not only gains in efficacy, but also in safety, tolerability and convenience in the treatment of patients with cancer.
    • A novel deletion within exon 6 of TP53 in a family with Li-Fraumeni-like syndrome, and LOH in a benign lesion from a mutation carrier.

      Varley, Jennifer; Thorncroft, Mary R; McGown, Gail; Tricker, Karen J; Birch, Jillian M; Evans, D Gareth R; CRC Department of Cancer Genetics, Christie Hospital, Manchester, U.K. (1996-08)
      We report here a family with some of the characteristics of Li-Fraumeni syndrome (Li-Fraumeni-like) in which there is a 2 base pair deletion within exon 6 of TP53 in two affected individuals. Of particular interest in this family is a study of loss of heterozygosity (LOH) of the TP53 gene, and the finding that there is LOH in all cancers available for study from mutation carriers, and additionally from a benign endometrial polyp from one of those patients. Two other family members, one with a rectal carcinoma aged 55, the other with two separate benign lesions under the age of 45, were both wild-type for the TP53 mutation.
    • A novel DNA damage recognition protein in Schizosaccharomyces pombe.

      Pearson, Steven J; Wharton, Stephen; Watson, Amanda J; Begum, Ghazala; Butt, Amna; Glynn, Nicola; Williams, David M; Shibata, Takayuki; Santibanez-Koref, Mauro F; Margison, Geoffrey P; et al. (2006)
      Toxic and mutagenic O6-alkylguanine adducts in DNA are repaired by O6-alkylguanine-DNA alkyltransferases (MGMT) by transfer of the alkyl group to a cysteine residue in the active site. Comparisons in silico of prokaryotes and lower eukaryotes reveal the presence of a group of proteins [alkyltransferase-like (ATL) proteins] showing amino acid sequence similarity to MGMT, but where the cysteine at the putative active site is replaced by tryptophan. To examine whether ATL proteins play a role in the biological effects of alkylating agents, we inactivated the gene, referred to as atl1+, in Schizosaccharomyces pombe, an organism that does not possess a functional MGMT homologue. The mutants are substantially more susceptible to the toxic effects of the methylating agents, N-methyl-N-nitrosourea, N-methyl-N'nitro-N-nitrosoguanidine and methyl methanesulfonate and longer chain alkylating agents including N-ethyl-N-nitrosourea, ethyl methanesulfonate, N-propyl-N-nitrosourea and N-butyl-N-nitrosourea. Purified Atl1 protein does not transfer methyl groups from O6-methylguanine in [3H]-methylated DNA but reversibly inhibits methyl transfer by human MGMT. Atl1 binds to short single-stranded oligonucleotides containing O6-methyl, -benzyl, -4-bromothenyl or -hydroxyethyl-guanine but does not remove the alkyl group or base and does not cleave the oligonucleotide in the region of the lesion. This suggests that Atl1 acts by binding to O6-alkylguanine lesions and signalling them for processing by other DNA repair pathways. This is the first report describing an activity that protects S.pombe against the toxic effects of O6-alkylguanine adducts and the biological function of a family of proteins that is widely found in prokaryotes and lower eukaryotes.
    • A novel dual function retrovirus expressing multidrug resistance 1 and O6-alkylguanine-DNA-alkyltransferase for engineering resistance of haemopoietic progenitor cells to multiple chemotherapeutic agents.

      Jelinek, J; Rafferty, Joseph A; Cmejla, R; Hildinger, M; Chinnasamy, Dhanalakshmi; Lashford, Linda S; Ostertag, W; Margison, Geoffrey P; Dexter, T Michael; Fairbairn, Leslie J; et al. (1999-08)
      Following transduction with a retrovirus (SF1MIH) expressing both the multidrug resistance 1 (MDR1) and O6-alkylguanine-DNA-alkyltransferase (ATase) proteins, human erythroleukaemic progenitor (K562) cells were isolated which were resistant to killing by the MDR1 substrate, colchicine. In colony-forming survival assays, K562-SF1MIH cells exhibited resistance to colchicine and doxorubicin, as well as to the O6-alkylating agents N-Methyl-N-nitrosourea (MNU) and temozolomide. Furthermore, the resistance to doxorubicin was abolished by preincubation with the MDR1 inhibitor verapamil while resistance to MNU was ablated by the specific ATase inactivator, O6-benzylguanine (O6-beG) confirming that resistance to doxorubicin and MNU was conferred by MDR1 and ATase, respectively. When K562-SF1MIH were exposed to combinations of colchicine and MNU or doxorubicin and temozolomide, simultaneous resistance to these agents was observed. Thus, transduction of K562 with SF1MIH conferred dual resistance to these cells. These data offer the prospect of designing vectors that will confer resistance to entire regimens of chemotherapy rather than just to individual components of such drug cocktails, thereby substantially increasing the efficacy of therapy. Furthermore, the use of such dual expression constructs is likely to be highly informative for the design of effective in vivo selection protocols, an issue likely to make a major impact in a clinical context in gene therapy in the near future.
    • Novel heparin/heparan sulfate mimics as inhibitors of HGF/SF-induced MET activation

      Raiber, Eun-Ang; Wilkinson, James A; Manetti, Fabrizio; Botta, Maurizio; Deakin, Jon A; Gallagher, John T; Lyon, Malcolm; Ducki, Sylvie W; Centre for Molecular Drug Design, Cockcroft Building, University of Salford, Salford, UK. (2007-11-15)
      The synthesis of simple, non-sugar glycosaminoglycan (GAG) mimics has been achieved and the analogues evaluated for their ability to inhibit the activation of the MET receptor by hepatocyte growth factor/scatter factor (HGF/SF).
    • Novel heterocyclic betaines relevant to the mechanism of tyrosinase-catalysed oxidation of phenols.

      Clews, John; Cooksey, Christopher J; Garratt, Peter; Land, Edward J; Ramsden, Christopher A; Riley, Patrick A; Department of Chemistry, Keele University, Keele, Staffordshire, UK ST5 5BG. (1998)