• Loss of regulators of vacuolar ATPase function and ceramide synthesis results in multidrug sensitivity in schizosaccharomyces pombe.

      Dawson, Keren; Toone, W Mark; Jones, Nic; Wilkinson, Caroline R M; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, United Kingdom. (2008-06)
      We undertook a screen to isolate determinants of drug resistance in fission yeast and identified two genes that, when mutated, result in sensitivity to a range of structurally unrelated compounds, some of them commonly used in the clinic. One gene, rav1, encodes the homologue of a budding yeast protein which regulates the assembly of the vacuolar ATPase. The second gene, lac1, encodes a homologue of genes that are required for ceramide synthesis. Both mutants are sensitive to the chemotherapeutic agent doxorubicin, and using the naturally fluorescent properties of this compound, we found that both rav1 and lac1 mutations result in an increased accumulation of the drug in cells. The multidrug-sensitive phenotype of rav1 mutants can be rescued by up-regulation of the lag1 gene which encodes a homologue of lac1, whereas overexpression of either lac1 or lag1 confers multidrug resistance on wild-type cells. These data suggest that changing the amount of ceramide synthase activity in cells can influence innate drug resistance. The function of Rav1 appears to be conserved, as we show that SpRav1 is part of a RAVE-like complex in fission yeast and that loss of rav1 results in defects in vacuolar (H(+))-ATPase activity. Thus, we conclude that loss of normal V-ATPase function results in an increased sensitivity of Schizosaccharomyces pombe cells to drugs. The rav1 and lac1 genes are conserved in both higher eukaryotes and various pathogenic fungi. Thus, our data could provide the basis for strategies to sensitize tumor cells or drug-resistant pathogenic fungi to drugs.
    • Making connections at DNA replication forks: Mrc1 takes the lead.

      Labib, Karim; Cancer Research UK, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. (2008-10-24)
      In a recent issue of Molecular Cell, Lou et al. (2008) demonstrate that the Mrc1 protein associates with the DNA polymerase that acts on the leading strand at replication forks, suggesting a potential mechanism that could help to preserve genome stability.
    • Methods comparison for high-resolution transcriptional analysis of archival material on Affymetrix Plus 2.0 and Exon 1.0 microarrays.

      Linton, Kim M; Hey, Yvonne; Dibben, Sian; Miller, Crispin J; Freemont, Anthony J; Radford, John A; Pepper, Stuart D; Cancer Research UK Department of Medical Oncology, The Christie NHS Foundation Trust, Manchester, UK. kim.linton@christie.nhs.uk (2009-07)
      Microarray gene expression profiling of formalin-fixed paraffin-embedded (FFPE) tissues is a new and evolving technique. This report compares transcript detection rates on Affymetrix U133 Plus 2.0 and Human Exon 1.0 ST GeneChips across several RNA extraction and target labeling protocols, using routinely collected archival FFPE samples. All RNA extraction protocols tested (Ambion-Optimum, Ambion-RecoverAll, and Qiagen-RNeasy FFPE) provided extracts suitable for microarray hybridization. Compared with Affymetrix One-Cycle labeled extracts, NuGEN system protocols utilizing oligo(dT) and random hexamer primers, and cDNA target preparations instead of cRNA, achieved percent present rates up to 55% on Plus 2.0 arrays. Based on two paired-sample analyses, at 90% specificity this equalled an average 30 percentage-point increase (from 50% to 80%) in FFPE transcript sensitivity relative to fresh frozen tissues, which we have assumed to have 100% sensitivity and specificity. The high content of Exon arrays, with multiple probe sets per exon, improved FFPE sensitivity to 92% at 96% specificity, corresponding to an absolute increase of ~600 genes over Plus 2.0 arrays. While larger series are needed to confirm high correspondence between fresh-frozen and FFPE expression patterns, these data suggest that both Plus 2.0 and Exon arrays are suitable platforms for FFPE microarray expression analyses.
    • Mitochondrial dynamics and apoptosis: a painful separation.

      James, Dominic I; Martinou, Jean-Claude; Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. (2008-09)
      Reporting in Molecular Cell, Sheridan et al. (2008) and Breckenridge et al. (2008) show that mitochondrial fragmentation is not required to induce cell death. Meanwhile, Yamaguchi et al. show that proapoptotic Bcl-2 family members promote cytochrome c mobilization through Opa1-mediated cristae remodeling. Therefore, the connection between mitochondrial structure and apoptosis is more complex than previously imagined.
    • Multiple pathways differentially regulate global oxidative stress responses in fission yeast.

      Chen, Dongrong; Wilkinson, Caroline R M; Watt, Stephen; Penkett, Christopher J; Toone, W Mark; Jones, Nic; Bähler, Jürg; Cancer Research UK Fission Yeast Functional Genomics Group, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, United Kingdom. (2008-01)
      Cellular protection against oxidative damage is relevant to ageing and numerous diseases. We analyzed the diversity of genome-wide gene expression programs and their regulation in response to various types and doses of oxidants in Schizosaccharomyces pombe. A small core gene set, regulated by the AP-1-like factor Pap1p and the two-component regulator Prr1p, was universally induced irrespective of oxidant and dose. Strong oxidative stresses led to a much larger transcriptional response. The mitogen-activated protein kinase (MAPK) Sty1p and the bZIP factor Atf1p were critical for the response to hydrogen peroxide. A newly identified zinc-finger protein, Hsr1p, is uniquely regulated by all three major regulatory systems (Sty1p-Atf1p, Pap1p, and Prr1p) and in turn globally supports gene expression in response to hydrogen peroxide. Although the overall transcriptional responses to hydrogen peroxide and t-butylhydroperoxide were similar, to our surprise, Sty1p and Atf1p were less critical for the response to the latter. Instead, another MAPK, Pmk1p, was involved in surviving this stress, although Pmk1p played only a minor role in regulating the transcriptional response. These data reveal a considerable plasticity and differential control of regulatory pathways in distinct oxidative stress conditions, providing both specificity and backup for protection from oxidative damage.
    • Multiplexed assays for detection of mutations in PIK3CA.

      Board, Ruth E; Thelwell, Nicola J; Ravetto, Paul F; Little, Stephen; Ranson, Malcolm R; Dive, Caroline; Hughes, Andrew; Whitcombe, David; Discovery Medicine, AstraZeneca Pharmaceuticals, Macclesfield, UK. ruth.board@astrazeneca.com (2008-04)
      BACKGROUND: Mutations in the PIK3CA gene (phosphoinositide-3-kinase, catalytic, alpha polypeptide) have recently been described in a number of cancers, and their detection is currently limited because of the low sensitivity of conventional sequencing techniques. METHODS: We combined Amplification Refractory Mutation System (ARMS; AstraZeneca) allele-specific PCR and Scorpions (DxS) to develop assays for tumor-borne PIK3CA mutations and used real-time PCR to develop high-throughput multiplexed assays for the most commonly reported PIK3CA mutants (H1047L, H1047R, E542K, E545K). RESULTS: These assays were more sensitive than sequencing and could detect 5 copies of mutant DNA in proportions as low as 0.1% of the total DNA. We assayed DNA extracted from human tumors and detected PIK3CA mutation frequencies of 10.2% in colorectal cancer, 38.7% in breast cancer, 1.9% in lung cancer, and 2.9% in melanoma. In contrast, sequencing detected only 53% of the mutations detected by our assay. CONCLUSIONS: Multiplexed assays, which can easily be applied to clinical samples, have been developed for the detection of PIK3CA mutations.
    • Mutant CEBPA: priming stem cells for myeloid leukemogenesis.

      Somervaille, Tim C P; Cleary, M L; Cancer Research UK Leukaemia Biology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, M20 4BX, UK. (2009-11-06)
    • An MVA-based vaccine targeting the oncofetal antigen 5T4 in patients undergoing surgical resection of colorectal cancer liver metastases.

      Elkord, Eyad; Dangoor, Adam; Drury, Noel L; Harrop, Richard; Burt, Deborah J; Drijfhout, Jan W; Hamer, Caroline; Andrews, Danielle; Naylor, Stuart; Sherlock, David J; et al. (2009-03-17)
      We investigated the use of a therapeutic vaccine, TroVax in patients undergoing surgical resection of colorectal cancer liver metastases. Systemic immunity generated by vaccination before and after resection of metastases was measured in addition to assessing safety and analyzing the function and phenotype of tumor-associated lymphocytes. Twenty patients were scheduled to receive 2 TroVax vaccinations at 2-week intervals preoperatively and 2 postoperatively; if immune responses were detected, 2 further vaccinations were offered. Blood was taken at trial entry and 2 weeks after each vaccination; tumor biopsies were collected at surgery. 5T4-specific cellular responses were assessed by lymphocyte proliferation and enzyme-linked immunosorbent spot, with antibody responses by enzyme-linked immunosorbent assay. Immunohistochemistry characterized the phenotype of tumor-infiltrating lymphocytes. Seventeen of 19 colorectal cancer patients showed 5T4 expression in the liver metastases or surrounding stroma and 18 mounted a 5T4-specific cellular and/or humoral response. In patients who received at least 4 vaccinations and potentially curative surgery (n=15), those with above median 5T4-specific proliferative responses or T-cell infiltration into the resected tumor showed significantly longer survival compared with those with below median responses. Seven of 8 patients who had preexisting proliferative responses to 5T4 were longer-term survivors; these patients showed significantly higher proliferative responses after vaccination than those who subsequently died. These data suggest that the magnitude of 5T4 proliferative responses and the density of CD3 cells in colorectal cancer liver metastases are associated with longer survival. These observations warrant more studies to identify the precise underlying mechanisms.
    • Novel therapeutic strategies by regulatory T cells in allergy.

      Elkord, Eyad; Immunology Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK. eelkord@picr.man.ac.uk (2008)
      Natural CD4+CD25+FoxP3+ regulatory T cells (Treg) actively suppress physiological and pathological responses, therefore playing a critical role in controlling peripheral tolerance to self antigens and maintaining immune homeostasis. In normal individuals, natural Treg and interleukin- 10-secreting Treg are able to suppress Th2 responses to allergens, whereas lower levels of Treg or defect in their functionality have been described as potential mechanisms for inducing allergic diseases. In animal models, adoptive transfer of CD4+CD25+Treg has been shown as a promising strategy for preventing or treating allergic disorders. Recent studies show that induction of Treg activity is associated with suppression of allergic responses in allergic patients treated with specific immunotherapy. Herein, I review the potential of Treg as exciting targets for developing new immunotherapeutic strategies for treating allergic diseases.
    • O(6)-methylguanine-DNA methyltransferase depletion and DNA damage in patients with melanoma treated with temozolomide alone or with lomeguatrib.

      Watson, Amanda J; Middleton, Mark R; McGown, Gail; Thorncroft, Mary R; Ranson, Malcolm R; Hersey, Peter; McArthur, Grant A; Davis, Ian D; Thomson, D; Beith, Jane; et al. (2009-04-21)
      We evaluated the pharmacodynamic effects of the O(6)-methylguanine-DNA methyltransferase (MGMT) inactivator lomeguatrib (LM) on patients with melanoma in two clinical trials. Patients received temozolomide (TMZ) for 5 days either alone or with LM for 5, 10 or 14 days. Peripheral blood mononuclear cells (PBMCs) were isolated before treatment and during cycle 1. Where available, tumour biopsies were obtained after the last drug dose in cycle 1. Samples were assayed for MGMT activity, total MGMT protein, and O(6)-methylguanine (O(6)-meG) and N7-methylguanine levels in DNA. MGMT was completely inactivated in PBMC from patients receiving LM, but detectable in those on TMZ alone. Tumours biopsied on the last day of treatment showed complete inactivation of MGMT but there was recovery of activity in tumours sampled later. Significantly more O(6)-meG was present in the PBMC DNA of LM/TMZ patients than those on TMZ alone. LM/TMZ leads to greater MGMT inactivation, and higher levels of O(6)-meG than TMZ alone. Early recovery of MGMT activity in tumours suggested that more protracted dosing with LM is required. Extended dosing of LM completely inactivated PBMC MGMT, and resulted in persistent levels of O(6)-meG in PBMC DNA during treatment.
    • O6-Methylguanine-DNA methyltransferase inactivation and chemotherapy.

      Verbeek, Barbara; Southgate, Thomas D; Gilham, David E; Margison, Geoffrey P; Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. (2008)
      INTRODUCTION: Alkylating agents are frequently used in the chemotherapy of many types of cancer. This group of drugs mediates cell death by damaging DNA and therefore, understandably, cellular DNA repair mechanisms can influence both their antitumour efficacy and their dose-limiting toxicities. SOURCES OF DATA: This review focuses on the mechanism of action of the DNA repair protein, O(6)-methylguanine-DNA methyltransferase (MGMT) and its exploitation in cancer therapy and reviews the current literature. AREAS OF AGREEMENT: MGMT can provide resistance to alkylating agents by DNA damage reversal. Inhibition of tumour MGMT by pseudosubstrates to overcome tumour resistance is under clinical evaluation. In addition, MGMT overexpression in haematopoietic stem cells has been shown in animal models to protect normal cells against the myelosuppressive effects of chemotherapy: this strategy has also entered clinical trials. AREAS OF CONTROVERSY: MGMT inhibitors enhance the myelotoxic effect of O(6)-alkylating drugs and therefore reduce the maximum-tolerated dose of these agents. Retroviral vectors used for chemoprotective gene therapy are associated with insertional mutagenesis and leukaemia development. GROWING POINTS: The results of ongoing preclinical and clinical research involving various aspects of MGMT modulation should provide new prospects for the treatment of glioma, melanoma and other cancer types. AREAS TIMELY FOR DEVELOPING RESEARCH: Tissue- and tumour-specific approaches to the modulation of MGMT together with other DNA repair functions and in combination with immuno- or radiotherapy are promising strategies to improve alkylating agent therapy.
    • Obesity and cancer: pathophysiological and biological mechanisms.

      Renehan, Andrew G; Roberts, Darren L; Dive, Caroline; Department of Surgery, School of Cancer and Imaging Sciences, University of Manchester, Christie Hospital NHS Foundation Trust, Wilmslow Road, Manchester, UK. arenehan@picr.man.ac.uk (2008-02)
      Excess body weight (overweight and obesity) is characterized by chronic hyperinsulinaemia and insulin resistance, and is implicated both in cancer risk and cancer mortality. The list of cancers at increased risk of development in an "obesogenic" environment include common adult cancers such as endometrium, post-menopausal breast, colon and kidney, but also less common malignancies such as leukaemia, multiple myeloma, and non-Hodgkin's lymphoma. The pathophysiological and biological mechanisms underpinning these associations are only starting to be understood. Insulin resistance is at the heart of many, but there are several other candidate systems including insulin-like growth factors, sex steroids, adipokines, obesity-related inflammatory markers, the nuclear factor kappa beta (NF-kappa B) system and oxidative stresses. With such as diversity of obesity-related cancers, it is unlikely that there is a "one system fits all" mechanism. While public health strategies to curb the spread of the obesity epidemic appear ineffective, there is a need to better understand the processes linking obesity and cancer as a pre-requisite to the development of new approaches to the prevention and treatment of obesity-related cancers.
    • Optimisation of circulating biomarkers of cell death for routine clinical use.

      Greystoke, Alastair; Cummings, Jeffrey; Ward, Timothy H; Simpson, Kathryn L; Renehan, Andrew G; Butt, Fouziah; Moore, David; Gietema, J; Blackhall, Fiona H; Ranson, Malcolm R; et al. (2008-05)
      BACKGROUND: M30 and M65 enzyme-linked immunosorbent assays detect circulating cytokeratin 18 fragments released during caspase-dependent or total cell death, respectively, and have potential as biomarkers in epithelial cancers. While these assays have been validated, their robustness for routine clinical use is unknown. PATIENTS AND METHODS: M30 and M65 were measured in matched serum and plasma samples from 31 lung cancer patients and 18 controls. RESULTS: Time allowable between sample acquisition and processing is critical for assays in clinical use. A 4-h delay in processing at room temperature increased M30 (P < 0.0001), an effect minimised by incubation on ice. M30 and M65 in serum were resistant to processing variations including delays. Serum and plasma measurements correlated well although M30 but not M65 was lower in serum (P < 0.0005). Less variation between duplicate assays was observed in serum. Prolonged storage (-80 degrees C) led to increased M30 (12%, 6 months; 34%, 1 year). Sample dilution in the supplied assay diluent proved non-linear, whereas dilution in donor serum or porcine plasma restored linearity up to a ratio of 1 : 6. CONCLUSION: We present recommendations that improve the reliability of these assays for clinical use and recommend serum as the preferred matrix with data more resistant to variations in collection.
    • Phase I trial of AEG35156 administered as a 7-day and 3-day continuous intravenous infusion in patients with advanced refractory cancer.

      Dean, Emma J; Jodrell, Duncan; Connolly, Kate; Danson, Sarah; Jolivet, Jacques; Durkin, J; Morris, Stephen; Jowle, Debra; Ward, Timothy H; Cummings, Jeffrey; et al. (2009-04-01)
      PURPOSE: To establish the maximum-tolerated dose and evaluate tolerability, pharmacokinetics, pharmacodynamic effects, and antitumor activity of AEG35156, a second-generation antisense to X-linked inhibitor of apoptosis (XIAP) protein, in patients with advanced refractory malignant tumors. PATIENTS AND METHODS: This was a first-in-man, open-label, phase I dose-escalation study. AEG35156 was administered by continuous intravenous infusion over 7 days (7DI) or 3 days (3DI) of a 21-day treatment cycle. Dose escalation started at 48 mg/m(2)/d and continued until consistent dose-limiting toxicity (DLT) was observed. RESULTS: Thirty-eight patients were entered in seven cohorts. Grade 3 to 4 adverse events were uncommon and were predominantly abnormal laboratory values: elevated ALT, thrombocytopenia, and lymphopenia. DLTs comprised elevated hepatic enzymes, hypophosphatemia, and thrombocytopenia. The maximum-tolerated doses were defined as 125 mg/m(2)/d for the 7DI regimen and < or = 213 mg/m(2)/d for the 3DI schedule. AEG35156 area under the plasma concentration curve and peak plasma concentration increased proportionally with dose. Suppression of XIAP mRNA levels was maximal at 72 hours (mean suppression, 21%), and this coincided with a dramatic decrease in circulating tumor cells in a patient with non-Hodgkin's lymphoma. Two further patients had unconfirmed partial responses. Circulating biomarkers of cell death and apoptosis altered in association with drug infusion and toxicity. CONCLUSION: In this first-in-man study, AEG35156 was well tolerated, with predictable toxicities, pharmacokinetic properties, and clinical evidence of antitumor activity in patients with refractory lymphoma, melanoma, and breast cancer. Phase I/II trials of AEG35156 chemotherapy combinations are ongoing in patients with pancreatic, breast, non-small-cell lung cancer, acute myeloid leukemia, lymphoma, and solid tumors for which docetaxel is indicated.
    • A phase II trial of lomeguatrib and temozolomide in metastatic colorectal cancer.

      Khan, O A; Ranson, Malcolm R; Michael, M; Olver, I; Levitt, N C; Mortimer, Peter; Watson, Amanda J; Margison, Geoffrey P; Midgley, R; Middleton, Mark R; et al. (2008-05-20)
      To evaluate the tumour response to lomeguatrib and temozolomide (TMZ) administered for 5 consecutive days every 4 weeks in patients with metastatic colorectal carcinoma. Patients with stage IV metastatic colorectal carcinoma received lomeguatrib (40 mg) and TMZ (50-200 mg m(-2)) orally for 5 consecutive days every 4 weeks. Response was determined every two cycles. Pharmacokinetics of lomeguatrib and TMZ as well as their pharmacodynamic effects in peripheral blood mononuclear cells (PBMC) were determined. Nineteen patients received 49 cycles of treatments. Despite consistent depletion of O(6)-methylguanine-DNA methyltransferase in PBMC, none of the patients responded to treatment. Three patients had stable disease, one for the duration of the study, and no fall in carcinoembryonic antigen was observed in any patient. Median time to progression was 50 days. The commonest adverse effects were gastrointestinal and haematological and these were comparable to those of TMZ when given alone. This combination of lomeguatrib and TMZ is not efficacious in metastatic colorectal cancer. If further studies are to be performed, emerging data suggest that higher daily doses of lomeguatrib and a dosing period beyond that of TMZ should be evaluated.
    • Preclinical efficacy of the bioreductive alkylating agent RH1 against paediatric tumours.

      Hussein, Deema; Holt, Sarah V; Brookes, K E; Klymenko, T; Adamski, J K; Hogg, Alison; Estlin, E J; Ward, Timothy H; Dive, Caroline; Makin, Guy W J; et al. (2009-07-07)
      BACKGROUND: Despite substantial improvements in childhood cancer survival, drug resistance remains problematic for several paediatric tumour types. The urgent need to access novel agents to treat drug-resistant disease should be expedited by pre-clinical evaluation of paediatric tumour models during the early stages of drug development in adult cancer patients. METHODS/RESULTS: The novel cytotoxic RH1 (2,5-diaziridinyl-3-[hydroxymethyl]-6-methyl-1,4-benzoquinone) is activated by the obligate two-electron reductase DT-diaphorase (DTD, widely expressed in adult tumour cells) to a potent DNA interstrand cross-linker. In acute viability assays against neuroblastoma, osteosarcoma, and Ewing's sarcoma cell lines RH1 IC(50) values ranged from 1-200 nM and drug potency correlated both with DTD levels and drug-induced apoptosis. However, synergy between RH1 and cisplatin or doxorubicin was only seen in low DTD expressing cell lines. In clonogenic assays RH1 IC(50) values ranged from 1.5-7.5 nM and drug potency did not correlate with DTD level. In A673 Ewing's sarcoma and 791T osteosarcoma tumour xenografts in mice RH1 induced apoptosis 24 h after a single bolus injection (0.4 mg/kg) and daily dosing for 5 days delayed tumour growth relative to control. CONCLUSION: The demonstration of RH1 efficacy against paediatric tumour cell lines, which was performed concurrently with the adult Phase 1 Trial, suggests that this agent may have clinical usefulness in childhood cancer.
    • Preclinical evaluation of M30 and M65 ELISAs as biomarkers of drug induced tumor cell death and antitumor activity.

      Cummings, Jeffrey; Hodgkinson, Cassandra L; Odedra, Rajesh; Sini, Patrizia; Heaton, Simon P; Mundt, Kirsten E; Ward, Timothy H; Wilkinson, Robert W; Growcott, Jim; Hughes, Andrew; et al. (2008-03)
      M30 and M65 are ELISAs that detect different circulating forms of cytokeratin 18. Using the aurora kinase inhibitor AZD1152 and the SW620 human colon cancer xenograft, experiments were conducted to qualify preclinically both assays as serologic biomarkers of cell death. Using two different apoptotic markers, the kinetics of cell death induced by AZD1152 was first characterized in vitro in three different cell lines and shown to peak 5 to 7 days after drug addition. Treatment of non-tumor-bearing rats with AZD1152 (25 mg/kg) produced no alterations in circulating baseline values of M30 and M65 antigens. In treated, tumor-bearing animals, M30 detected a 2- to 3-fold (P < 0.05) increase in plasma antigen levels by day 5 compared with controls. This correlated to a 3-fold increase in the number of apoptotic cells detected on day 5 in SW620 xenografts using immunohistochemistry. By contrast, M65 did not detect a drug-induced increase in circulating antigen levels at day 5. However, M65 plasma levels correlated to changes in tumor growth in control animals (r(2) = 0.93; P < 0.01) and also followed the magnitude of the temporal effect of AZD1152 on tumor growth. An intermediate but active dose of AZD1152 (12.5 mg/kg) produced a less significant increase in M30 plasma levels at day 5. It was also confirmed that the plasma profiles of M30 and M65 mirrored closely those measured in whole tumor lysates. We conclude that M30 is a pharmacodynamic biomarker of AZD1152-induced apoptosis in the SW620 xenograft model, whereas M65 is a biomarker of therapeutic response.
    • Predicting the myelotoxicity of chemotherapy: the use of pretreatment O6-methylguanine-DNA methyltransferase determination in peripheral blood mononuclear cells.

      Sabharwal, A; Waters, R; Danson, Sarah; Clamp, Andrew R; Lorigan, Paul C; Thatcher, Nick; Margison, Geoffrey P; Middleton, Mark R; Department of Medical Oncology, University of Oxford, Oxford, UK. (2011-12-21)
      To assess the value of pretreatment O-methylguanine-DNA methyltransferase (MGMT) expression in peripheral blood mononuclear cells (PBMCs) in predicting haematological toxicity with O-alkylating agent chemotherapy, we explored this relationship retrospectively in melanoma patients. Ninety-three patients treated with temozolomide or dacarbazine in four clinical trials were assessed, and a model of the interaction between MGMT expression and haematological toxicity was constructed. Nadir white-cell and platelet counts were related to, and hence could be predicted from, pretreatment MGMT. Leucopenia and thrombocytopenia were more prevalent amongst patients with low pretreatment MGMT, according to the highest grades of toxicity experienced and/or the dose intensity patients could sustain. Addition of interferon to chemotherapy or compression of the temozolomide schedule increased the toxicity. The model also predicts significant myelotoxicity where PBMC MGMT is inactivated, consistent with the experience in the clinic with lomeguatrib and O-benzylguanine. Determination of MGMT in PBMC can identify patients at greatest risk of toxicity or who are suitable for dose intensification.
    • Quantitative mass spectrometry-based techniques for clinical use: biomarker identification and quantification.

      Simpson, Kathryn L; Whetton, Anthony D; Dive, Caroline; Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, United Kingdom. KSimpson@PICR.man.ac.uk (2009-05-01)
      The potential for development of personalised medicine through the characterisation of novel biomarkers is an exciting prospect for improved patient care. Recent advances in mass spectrometric (MS) techniques, liquid phase analyte separation and bioinformatic tools for high throughput now mean that this goal may soon become a reality. However, there are challenges to be overcome for the identification and validation of robust biomarkers. Bio-fluids such as plasma and serum are a rich source of protein, many of which may reflect disease status, and due to the ease of sampling and handling, novel blood borne biomarkers are very much sought after. MS-based methods for high throughput protein identification and quantification are now available such that the issues arising from the huge dynamic range of proteins present in plasma may be overcome, allowing deep mining of the blood proteome to reveal novel biomarker signatures for clinical use. In addition, the development of sensitive MS-based methods for biomarker validation may bypass the bottleneck created by the need for generation and usage of reliable antibodies prior to large scale screening. In this review, we discuss the MS-based methods that are available for clinical proteomic analysis and highlight the progress made and future challenges faced in this cutting edge area of research.
    • Quantitative multiplexed quantum dot immunohistochemistry.

      Sweeney, Elizabeth; Ward, Timothy H; Gray, N; Womack, C; Jayson, Gordon C; Hughes, Andrew; Dive, Caroline; Byers, Richard J; Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, Wilmslow Road, Manchester, 420 4BX, UK. (2008-09-19)
      Quantum dots are photostable fluorescent semiconductor nanocrystals possessing wide excitation and bright narrow, symmetrical, emission spectra. These characteristics have engendered considerable interest in their application in multiplex immunohistochemistry for biomarker quantification and co-localisation in clinical samples. Robust quantitation allows biomarker validation, and there is growing need for multiplex staining due to limited quantity of clinical samples. Most reported multiplexed quantum dot staining used sequential methods that are laborious and impractical in a high-throughput setting. Problems associated with sequential multiplex staining have been investigated and a method developed using QDs conjugated to biotinylated primary antibodies, enabling simultaneous multiplex staining with three antibodies. CD34, Cytokeratin 18 and cleaved Caspase 3 were triplexed in tonsillar tissue using an 8h protocol, each localised to separate cellular compartments. This demonstrates utility of the method for biomarker measurement enabling rapid measurement of multiple co-localised biomarkers on single paraffin tissue sections, of importance for clinical trial studies.