• Identification of gene transcript signatures predictive for estrogen receptor and lymph node status using a stepwise forward selection artificial neural network modelling approach.

      Lancashire, Lee J; Rees, Robert C; Ball, Graham R; Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, United Kingdom. LLancashire@picr.man.ac.uk (2008-06)
      OBJECTIVE: The advent of microarrays has attracted considerable interest from biologists due to the potential for high throughput analysis of hundreds of thousands of gene transcripts. Subsequent analysis of the data may identify specific features which correspond to characteristics of interest within the population, for example, analysis of gene expression profiles in cancer patients to identify molecular signatures corresponding with prognostic outcome. These high throughput technologies have resulted in an unprecedented rate of data generation, often of high complexity, highlighting the need for novel data analysis methodologies that will cope with data of this nature. METHODS: Stepwise methods using artificial neural networks (ANNs) have been developed to identify an optimal subset of predictive gene transcripts from highly dimensional microarray data. Here these methods have been applied to a gene microarray dataset to identify and validate gene signatures corresponding with estrogen receptor and lymph node status in breast cancer. RESULTS: Many gene transcripts were identified whose expression could differentiate patients to very high accuracies based upon firstly whether they were positive or negative for estrogen receptor, and secondly whether metastasis to the axillary lymph node had occurred. A number of these genes had been previously reported to have a role in cancer. Significantly fewer genes were used compared to other previous studies. The models using the optimal gene subsets were internally validated using an extensive random sample cross-validation procedure and externally validated using a follow up dataset from a different cohort of patients on a newer array chip containing the same and additional probe sets. Here, the models retained high accuracies, emphasising the potential power of this approach in analysing complex systems. These findings show how the proposed method allows for the rapid analysis and subsequent detailed interrogation of gene expression signatures to provide a further understanding of the underlying molecular mechanisms that could be important in determining novel prognostic markers associated with cancer.
    • Immune evasion mechanisms in colorectal cancer liver metastasis patients vaccinated with TroVax (MVA-5T4).

      Elkord, Eyad; Dangoor, Adam; Burt, Deborah J; Southgate, Thomas D; Daayana, Sai; Harrop, Richard; Drijfhout, Jan W; Sherlock, David J; Hawkins, Robert E; Stern, Peter L; et al. (2009-02-17)
      We have recently reported the results of a phase II trial in which two TroVax [modified vaccinia ankara (MVA) encoding the tumour antigen 5T4] vaccinations were given to patients both pre- and post-surgical resection of liver metastases secondary to colorectal cancer (CRC). 5T4-specific cellular responses were assessed at the entry and 2 weeks after each vaccination by proliferation of fresh lymphocytes and ELISA for antibody responses; 18 from the 19 CRC patients mounted a 5T4-specific cellular and/or humoral response. Here, we present a comparison of individual and between patient responses over the course of the treatments using cryopreserved peripheral blood mononuclear cells (PBMC) samples from the baseline until after the fourth vaccination at 14 weeks. Assays used were proliferation assay with 5T4-Fc fusion protein, overlapping 32mer 5T4 peptides, MVA-LacZ and MVA-5T4 infected autologous monocytes. Responses to 5T4 protein or one or more peptide pools were pre-existing in 12/20 patients and subsequently 10 and 12 patients showed boosted and/or de novo responses, respectively. Cumulatively, 13/20 patients showed proliferative responses by week 14. We also assessed the levels of systemic T regulatory cells, plasma cytokine levels, phenotype of tumour-infiltrating lymphocytes including T regulatory cells and tumour HLA class I loss of expression. More than half of the patients showed phenotypes consistent with relative immune suppression and/or escape highlighting the complexity of positive and negative factors challenging any simple correlation with clinical outcome.
    • In silico screening and biological evaluation of inhibitors of Src-SH3 domain interaction with a proline-rich ligand.

      Atatreh, Noor; Stojkoski, Cvetan; Smith, Phillippa; Booker, Grant W; Dive, Caroline; Frenkel, A David; Freeman, Sally; Bryce, Richard A; School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Manchester M13 9PT, UK. (2008-02-01)
      Src signalling and transduction are directly involved in cell growth, cell cycle, malignant transformation and cell migration, providing therapeutic opportunities through inhibition of Src. Here we report virtual screening for novel compounds that inhibit the Src-SH3 protein-protein interaction with a proline-rich peptide ligand. Computational docking of the ZINC compound database was performed using GOLD. Top-scoring compounds were assayed using a fluorescence polarization-based assay. A benzoquinoline derivative showed micromolar inhibition of binding between Src-SH3 and the proline-rich peptide. Several analogues were subsequently assayed showing the requirement of a linker between the benzoquinoline and phenyl rings, and electron donating substituents on the phenyl ring.
    • Inn1 couples contraction of the actomyosin ring to membrane ingression during cytokinesis in budding yeast.

      Sanchez-Diaz, Alberto; Marchesi, Vanessa; Murray, Stephen M; Jones, Richard C; Pereira, Gislene; Edmondson, Ricky D; Allen, Terence D; Labib, Karim; Cancer Research U.K., Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK. (2008-04)
      By rapidly depleting each of the essential budding yeast proteins of unknown function, we identified a novel factor that we call Inn1, which associates with the contractile actomyosin ring at the end of mitosis and is needed for cytokinesis. We show that Inn1 has a C2 domain at the amino terminus of the protein that is required for ingression of the plasma membrane, whereas the remainder of the protein recruits Inn1 to the actomyosin ring. The lethal effects of deleting the INN1 gene can be suppressed by artificial fusion of the C2 domain to other components of the actomyosin ring, restoring membrane ingression on contraction of the actomyosin ring. Our data indicate that recruitment of the C2 domain of Inn1 to the contractile actomyosin ring is crucial for ingression of the plasma membrane during cytokinesis in budding yeast.
    • Int6/eIF3e promotes general translation and Atf1 abundance to modulate Sty1 MAPK-dependent stress response in fission yeast.

      Udagawa, T; Nemoto, N; Wilkinson, Caroline R M; Narashimhan, J; Jiang, L; Watt, S; Zook, A; Jones, Nic; Wek, R; Bähler, J; et al. (2008-08-08)
      int-6 is one of the frequent integration sites for mouse mammary tumor viruses. Although its product is the e-subunit of translation initiation factor eIF3, other evidence indicates that it interacts with proteasomes or other proteins to regulate protein stability. Here we report that the fission yeast int6(+) is required for overcoming stress imposed by histidine starvation, using the drug 3-aminotriazole (3AT). Microarray and complementary Northern studies using wild-type, int6Delta or gcn2Delta mutants indicate that 3AT-treated wild-type yeast induces core environmental stress response (CESR) genes in addition to typical general amino acid control (GAAC) genes whose transcription depends on the eIF2 kinase, Gcn2. In agreement with this, Sty1 MAPK and its target transcription factor Atf1, which signal the CESR, are required for overcoming 3AT-induced starvation. We find that Int6 is required for maintaining the basal level of Atf1 and for rapid transcriptional activation of the CESR on 3AT-insult. Pulse labeling experiments indicate that int6Delta significantly slows down de novo protein synthesis. Moreover, Atf1 protein half-life was reduced in int6Delta cells. These effects would account for the compromised Atf1 activity on 3AT-induced stress. Thus, the robust protein synthesis promoted by intact eIF3 appears to be a part of the requisites for sound Sty1 MAPK-dependent signaling governed by the activity of the Atf1 transcription factor.
    • An introduction to artificial neural networks in bioinformatics--application to complex microarray and mass spectrometry datasets in cancer studies.

      Lancashire, Lee J; Lemetre, Christophe; Ball, Graham R; Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, UK. llancashire@picr.man.ac.uk (2009-05)
      Applications of genomic and proteomic technologies have seen a major increase, resulting in an explosion in the amount of highly dimensional and complex data being generated. Subsequently this has increased the effort by the bioinformatics community to develop novel computational approaches that allow for meaningful information to be extracted. This information must be of biological relevance and thus correlate to disease phenotypes of interest. Artificial neural networks are a form of machine learning from the field of artificial intelligence with proven pattern recognition capabilities and have been utilized in many areas of bioinformatics. This is due to their ability to cope with highly dimensional complex datasets such as those developed by protein mass spectrometry and DNA microarray experiments. As such, neural networks have been applied to problems such as disease classification and identification of biomarkers. This review introduces and describes the concepts related to neural networks, the advantages and caveats to their use, examples of their applications in mass spectrometry and microarray research (with a particular focus on cancer studies), and illustrations from recent literature showing where neural networks have performed well in comparison to other machine learning methods. This should form the necessary background knowledge and information enabling researchers with an interest in these methodologies, but not necessarily from a machine learning background, to apply the concepts to their own datasets, thus maximizing the information gain from these complex biological systems.
    • 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.