• Qualification of M30 and M65 ELISAs as surrogate biomarkers of cell death: long term antigen stability in cancer patient plasma

      Cummings, Jeffrey; Ranson, Malcolm R; Butt, Fouziah; Moore, David; Dive, Caroline; Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, England. jcummings@picr.man.ac.uk (2007-11)
      PURPOSE: M30 and M65 ELISAs are proposed as surrogate biomarkers of tumour cell death in patients and are being applied increasingly in the pharmacodynamic (PD) evaluation of anticancer drugs during clinical trials. In the absence of such data, we have studied the long-term stability of the antigens of both assays in plasma of cancer patients stored at -80 degrees C over 2 years. RESULTS: No evidence was detected of degradation in the M65 antigen. However, in a proportion of patients significant increases in levels of M30 antigen were detected CONCLUSION: Plasma samples for M65 analysis can be stored at -80 degrees C for 2 years; however, caution is recommended when considering long-term storage of samples for the M30 assay.
    • Quality of life (QoL) outcomes with futibatinib treatment in FOENIX-CCA2 - A phase II study in patients (pts) with intrahepatic cholangiocarcinoma (iCCA) harboring FGFR2 gene fusions/rearrangements

      Valle, Juan W; Hollebecque, A.; Furuse, J.; Goyal, L.; Meric-Bernstam, F.; Morlock, R.; He, Y.; Benhadji, K.; Bridgewater, J.; Division of Cancer Sciences, University of Manchester/The Christie NHS Foundation Trust, Manchester, (2020)
      Background: Cancer treatment can produce AEs that result in a reduced QoL. Futibatinib, a highly selective irreversible FGFR1e4 inhibitor, demonstrated an objective response rate (ORR) of 37.3% and median 8.3-mo duration of response in the interim analysis of FOENIX-CCA2, a mutlicenter phase II trial of pts with advanced, refractory iCCA harboring an FGFR2 fusion/rearrangement; grade 3 treatment-related AEs (TRAEs) occurred in 57% of pts (most commonly, hyperphosphatemia [26.9%]). Change in pt-reported outcomes (PROs) from baseline (BL) for the interim data of the phase II trial are presented here. Methods: Pts enrolled into FOENIX-CCA2 (NCT02052778), had locally advanced/ metastatic unresectable iCCA and received oral futibatinib 20 mg once daily (QD) until disease progression/intolerance. PRO measures included EORTC-QLQ-C30 (5 functional and 9 physical measures) and EQ-5D-3L (utility index and 5 dimensions: anxiety/ depression, mobility, pain/discomfort, self-care, and usual activity). PROs were collected at screening, cycles 2 and 4, every 3 cycles after cycle 4 and at the end of treatment. Change in mean score from BL was assessed using predefined clinically meaningful thresholds for each time point with 19 observations (through cycle 13). Results: Sixty-seven of 103 enrolled pts had 6 months of follow-up and 57 (85.1%) had PRO completion data at BL and 1 assessment. EORTC mean global health status score was high at BL (68.7) and maintained through cycle 13 (70.8), a trend observed across all EORTC measures. The only clinically meaningful changes ( 10-point changes) in this timeframe were for constipation symptoms at cycles 2 and 4 (worsened +12.4 and +10.7, respectively) and dyspnea at cycle 10 (improved -12.2). Mean EQ-5D-3L index scores improved from 70.9 at BL to 79.1 at cycle 13 (approximately 273 days on treatment). Conclusions: Overall, the interim PROs from FOENIX-CCA2 were encouraging. These data suggest that despite the occurrence of TRAEs, a 20-mg-QD dose of futibatinib in pts with iCCA provides a promising clinical response without adversely impacting QoL.
    • Quantifying antivascular effects of monoclonal antibodies to vascular endothelial growth factor: insights from imaging.

      O'Connor, James P B; Carano, Richard A D; Clamp, Andrew R; Ross, Jed; Ho, Calvin C K; Jackson, Alan; Parker, Geoff J M; Rose, Chris J; Peale, Franklin V; Friesenhahn, Michel; et al. (2009)
    • The quantitation and kinetics of unscheduled (repair) DNA synthesis in ultraviolet-irradiated human skin by automated image analysis.

      Chadwick, Caroline A; Diggle, S P; Young, A R; Potten, Christopher S; CRC Department of Epithelial Biology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, U.K. (1996-10)
      Grain counting by eye is a tedious and time-consuming technique but one with great potential in cell kinetics and for the study of DNA excision repair activity (unscheduled DNA synthesis or UDS). We have been investigating the levels of UDS in human skin sections exposed in situ to ultraviolet radiation using a short-term incubation in tritiated thymidine and autoradiography and the decline in UDS levels with time (repair kinetics). We have adapted an automated image analysis system automatically to assess the number of grains over epidermal cell nuclei in autoradiographs of sections of epidermis. An excellent correlation was observed between visual counting and machine measurement of the area (in pixels) occupied by silver grains. The levels of UDS declined with time as lesions are progressively repaired. The half time (+/- standard deviation) for the reduction in UDS is 7.25 +/- 0.18 h. The grain counts can be significantly increased by increasing the autoradiographic exposure, by increasing the concentration of tritiated thymidine and by increasing the incubation time.
    • Quantitation of extracts containing tumour angiogenesis factor (TAF) by radioimmunometric and radioimmunoassays.

      Schor, Ana M; Kumar, S; Phillips, P; Clinical Research Laboratories, Christie Hospital and Holt Radium Institute, Manchester, M20 9BX, England. (1980-06-15)
      An antiserum which is able to inhibit TAF-induced neovascularization in vivo (TAF antiserum) was used to develop two quantitative assays for TAF-containing tumour extracts (tumor TAF). 1) Radioimmunometric assay (RIMA): the IgG of the TAF antiserum was labelled with 125I. An excess of 125I-IgG was incubated with increasing concentrations of tumour TAF and the antigen-bound fraction was precipitated by addition of Clq. 2) Radioimmunoassay (RIA): an excess of iodinated antigen (tumour TAF) was incubated with TAF antiserum diluted so that binding in the absence of unlabelled antigen represented 70-80% of the maximum binding. When tumour TAF was added, competition between labelled and unlabelled antigen for the TAF antibody binding sites resulted in displacement of the former by increasing concentrations of the latter. A second antibody was used to precipitate the bound labelled antigen. Of the two assays, the RIMA was the more sensitive and, due to the lack of a purified antigen, allowed standardization of the results in a more accurate manner. Our data show that there was a good correlation between the ability of tumour TAF to induce angiogenesis in vivo and the degree of antiserum binding detected in vitro by both assays. Preparations containing TAF, whatever the source (i.e. human or animal tumours or tissue culture) shared common antigenic determinants. It is suggested that the quantitative assays should prove valuable in determining the clinical relevance of TAF as a tumour marker.
    • Quantitation of proliferative and cytotoxic precursor cells directed against human tumours: limiting dilution analysis in peripheral blood and at the tumour site.

      Vose, Brent M; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester, M20 9BX, UK (1982-08-15)
      Blood and tumour-infiltrating lymphocytes (TIL) from 16 cancer patients have been examined under limiting dilution conditions to determine the frequency of cells responding in mixed tumour-lymphocyte cultures (MLTC) to autologous tumour and Interleukin-2 (IL-2). Tumour-derived lymphocytes showed a high spontaneous response to IL-2 alone 1/1,900 in TIL; 1/6,000 in PBL suggesting the presence of "activated" T cells in situ. Proliferative frequencies were increased in MLTC in both blood (1/3,779) and TIL (1/1,084). Phenotypic analyses showed that total T-cell contents of the responder populations were comparable but TIL were enriched for the OKT8+ subset with a corresponding reduction in OKT4+. TIL showed increased numbers of OKMI+ and Tac+ lymphocytes. The major cytotoxic precursor expanding under these conditions was reactive against autologous tumour. K562 (NK) were present at a lesser frequency--particularly in TIL. The data show a concentration and activation of reactive lymphocytes at the tumour site and establish conditions for the clonal expansion of specifically cytotoxic T cells.
    • Quantitation of the radiotherapeutic importance of naturally-hypoxic normal tissues from collated experiments with rodents using single doses.

      Hendry, Jolyon H; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester (1979-07)
    • Quantitative analysis of biomarkers by LC-MS/MS.

      Cummings, Jeffrey; Unwin, Richard D; Veenstra, Timothy D; University of Manchester, Manchester, UK. jcummings@picr.man.ac.uk (2009-05-01)
    • Quantitative and qualitative alterations of heparan sulfate in fibrogenic liver diseases and hepatocellular cancer.

      Tátrai, Péter; Egedi, Krisztina; Somorácz, Aron; Van Kuppevelt, Toin H; Ten Dam, Gerdy; Lyon, Malcolm; Deakin, Jon A; Kiss, András; Schaff, Zsuzsa; Kovalszky, Ilona; et al. (2010-05)
      Heparan sulfate (HS), due to its ability to interact with a multitude of HS-binding factors, is involved in a variety of physiological and pathological processes. Remarkably diverse fine structure of HS, shaped by non-exhaustive enzymatic modifications, influences the interaction of HS with its partners. Here we characterized the HS profile of normal human and rat liver, as well as alterations of HS related to liver fibrogenesis and carcinogenesis, by using sulfation-specific antibodies. The HS immunopattern was compared with the immunolocalization of selected HS proteoglycans. HS samples from normal liver and hepatocellular carcinoma (HCC) were subjected to disaccharide analysis. Expression changes of nine HS-modifying enzymes in human fibrogenic diseases and HCC were measured by quantitative RT-PCR. Increased abundance and altered immunolocalization of HS was paralleled by elevated mRNA levels of HS-modifying enzymes in the diseased liver. The strong immunoreactivity of the normal liver for 3-O-sulfated epitope further increased with disease, along with upregulation of 3-OST-1. Modest 6-O-undersulfation of HCC HS is probably explained by Sulf overexpression. Our results may prompt further investigation of the role of highly 3-O-sulfated and partially 6-O-desulfated HS in pathological processes such as hepatitis virus entry and aberrant growth factor signaling in fibrogenic liver diseases and HCC.
    • A quantitative comparison of cytogenetic effects of anti-tumor agents.

      Parkes, D J; Scott, David; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester, M20 9BX, UK (1982)
      The relative potency of different anti-tumor agents in inducing structural and numerical chromosome abnormalities and SCEs was assessed by making comparisons at equitoxic doses, measured in terms of colony forming ability, in cultured diploid human fibroblasts. At approximately 20% survival the relative potency of X-rays, daunorubicin, nitrogen mustard, adriamycin, and actinomycin D in inducing structural aberrations was 1.0, 0.85, 0.26, 0.22, and zero, respectively. SCE induction was quantitatively unrelated to the induction of chromosome aberrations. No numerical changes were observed. Accurate assessment of the yields of chromosome aberrations requires the use of multiple sampling times in asynchronous populations.
    • A quantitative description of the action of high-dose pulses of radiation on aerated acid solutions containing ferrous and chloride ions

      Bjergbakke, E; Navaratnam, S; Parsons, B; Swallow, A John; Riso National Laboratory, Post Box 49, DK-4000, Roskilde, Denmark (1987)
    • Quantitative development of adherent cell colonies in bone marrow cell culture in vitro.

      Mori, K; Fujitake, H; Okubo, H; Dexter, T Michael; Ito, Y (1979-04)
      Quantification of the formation of adherent cell colonies in bone marrow cell culture was attempted. By secondary transfer of the bone marrow cells as a single cell suspension after 4 days' culture of fine marrow fragments, a linear relationship was obtained between the number of adherent cell colonies developing and the number of cells secondarily inoculated into the culture bottle. This suggests that 4 days' culture of the bone marrow cells with close intercellular interactions is sufficient for the 'conditioning' of the cells to develop adherent cell colonies. Activity of such colonies to support haemopoietic stem cell proliferation was also shown.
    • A quantitative histometric murine in vivo model of radiation-induced oral mucositis.

      Wardley, Andrew M; Booth, Dawn; Roberts, Stephen A; Scarffe, J Howard; Potten, Christopher S; CRC Epithelial Biology Group, UK. (1998-07)
      Gastrointestinal toxicity is a limiting factor in the effectiveness of cancer therapy. This toxicity is most visible in the mouth. There is considerable interest in developing strategies involving growth-factor manipulation of the epithelial stem cells to afford protection to these cells during treatment and/or to speed up the regenerative process following treatment. In order for this to be achieved, studies have to be undertaken in animal systems to demonstrate the proof of principle and determine optimal protocols. Here, a murine model for oral mucositis based on measurements of tissue cellularity at various times after exposure to radiation was used to investigate cytotoxicity. Several sites in the mouth were analysed and the pronounced circadian rhythm in these various epithelial sites determined. The circadian rhythm is important in that it would determine the timing of administration of growth factors. A microscope with an interactive computer was used to define areas of epithelium and lengths of basal layer, within which, and along which, the total number of cell nuclei was determined over a range of times following exposure to 10, 20 and 30 Gy of X-rays. For various practical reasons, the ventral surface of the tongue was identified as the most appropriate tissue to analyse. Here, measurements of cellularity reached minimum values between 6 and 8 days following 20 Gy. Labelling of S-phase cells demonstrated foci of regeneration and a burst of proliferative regeneration that commenced at about 5 days and reached peak values at 8 days after irradiation. This burst of regenerative proliferation was coincident with the minimum in tissue cellularity on about day 8. The lower dose of radiation (10 Gy) had minimal effects on cellularity: after the higher dose (30 Gy), there was clearly a more severe level of cellular depletion. This quantitative model of oral mucositis could be used to study the effects of other cytotoxics, including combinations of agents, and the potential role of growth factors to reduce the severity of the cellular depletion and to speed up the kinetics of regeneration.
    • Quantitative imaging biomarkers in the clinical development of targeted therapeutics: current and future perspectives.

      O'Connor, James P B; Jackson, Alan; Asselin, Marie-Claude; Buckley, David L; Parker, Geoff J M; Jayson, Gordon C; Imaging Science and Biomedical Engineering, University of Manchester, Manchester, UK. james.o'connor@manchester.ac.uk (2008-08)
      Targeted therapeutics have challenged how imaging techniques assess tumour response to treatment because many new agents are thought to cause cytostasis rather than cytotoxicity. Advanced tracer development, image acquisition, and image analysis have been used to produce quantitative biomarkers of pathophysiology, with particular focus on measurement of tumour vascular characteristics. Here, we critically appraise strategies available to generate imaging biomarkers for use in development of targeted therapeutics. We consider important practical and technical features of data acquisition and analysis because these factors determine the precise physiological meaning of every biomarker. We discuss the merits of volume-based and other size-based metrics for assessment of targeted therapeutics, and we examine the strengths and weaknesses of CT, MRI, and PET biomarkers derived from conventional clinical data. We review imaging biomarkers of tumour microvasculature and discuss imaging strategies that probe other physiological processes including cell proliferation, apoptosis, and tumour invasion. We conclude on the need to develop comprehensive compound-specific imaging biomarkers that are appropriate for every class of targeted therapeutics, and to investigate the complementary information given in multimodality imaging studies of targeted therapeutics.
    • 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.
    • Quantitative phosphoproteome analysis of embryonic stem cell differentiation toward blood.

      Piazzi, M; Williamson, Andrew J K; Lee, Chia-Fang; Pearson, Stella; Lacaud, Georges; Kouskoff, Valerie; McCubrey, James A; Cocco, L; Whetton, Anthony D; Cell Signaling Laboratory, Department of Biomedical Science (DIBINEM), University of Bologna, Italy (2015-03-26)
      Murine embryonic stem (ES) cells can differentiate in vitro into three germ layers (endodermic, mesodermic, ectodermic). Studies on the differentiation of these cells to specific early differentiation stages has been aided by an ES cell line carrying the Green Fluorescent Protein (GFP) targeted to the Brachyury (Bry) locus which marks mesoderm commitment. Furthermore, expression of the Vascular Endothelial Growth Factor receptor 2 (Flk1) along with Bry defines hemangioblast commitment. Isobaric-tag for relative and absolute quantification (iTRAQTM) and phosphopeptide enrichment coupled to liquid chromatography separation and mass spectrometry allow the study of phosphorylation changes occurring at different stages of ES cell development using Bry and Flk1 expression respectively. We identified and relatively quantified 37 phosphoentities which are modulated during mesoderm-induced ES cells differentiation, comparing epiblast-like, early mesoderm and hemangioblast-enriched cells. Among the proteins differentially phosphorylated toward mesoderm differentiation were: the epigenetic regulator Dnmt3b, the protein kinase GSK3b, the chromatin remodeling factor Smarcc1, the transcription factor Utf1; as well as protein specifically related to stem cell differentiation, as Eomes, Hmga2, Ints1 and Rif1. As most key factors regulating early hematopoietic development have also been implicated in various types of leukemia, understanding the post-translational modifications driving their regulation during normal development could result in a better comprehension of their roles during abnormal hematopoiesis in leukemia.
    • Quantitative proteomic analysis reveals maturation as a mechanism underlying glucocorticoid resistance in B lineage ALL and re-sensitization by JNK inhibition.

      Nicholson, L; Evans, Caroline A; Matheson, E; Minto, L; Keilty, C; Sanichar, M; Case, M; Schwab, C; Williamson, D; Rainer, J; et al. (2015-08-27)
      Glucocorticoid (GC) resistance is a continuing clinical problem in childhood acute lymphoblastic leukaemia (ALL) but the underlying mechanisms remain unclear. A proteomic approach was used to compare profiles of the B-lineage ALL GC-sensitive cell line, PreB 697, and its GC-resistant sub-line, R3F9, pre- and post-dexamethasone exposure. PAX5, a transcription factor critical to B-cell development was differentially regulated in the PreB 697 compared to the R3F9 cell line in response to GC. PAX5 basal protein expression was less in R3F9 compared to its GC-sensitive parent and confirmed to be lower in other GC-resistant sub-lines of Pre B 697 and was associated with a decreased expression of the PAX5 transcriptional target, CD19. Gene set enrichment analysis showed that increasing GC-resistance was associated with differentiation from preB-II to an immature B-lymphocyte stage. GC-resistant sub-lines were shown to have higher levels of phosphorylated JNK compared to the parent line and JNK inhibition caused re-sensitization to GC. Exploiting this maturation may be key to overcoming GC resistance and targeting signalling pathways linked to the maturation state, such as JNK, may be a novel approach.
    • Quantitative proteomic analysis using isobaric protein tags enables rapid comparison of changes in transcript and protein levels in transformed cells.

      Unwin, Richard D; Pierce, Andrew; Watson, Rod B; Sternberg, David W; Whetton, Anthony D; Department of Faculty of Medical and Human Sciences, University of Manchester, Christie Hospital, Withington, Manchester, M20 9BX, United Kingdom. (2005-07)
      Isobaric tags for relative and absolute quantitation, an approach to concurrent, relative quantification of proteins present in four cell preparations, have recently been described. To validate this approach using complex mammalian cell samples that show subtle differences in protein levels, a model stem cell-like cell line (FDCP-mix) in the presence or absence of the leukemogenic oncogene TEL/PDGFRbeta has been studied. Cell lysates were proteolytically digested, and peptides within each sample were labeled with one of four isobaric, isotope-coded tags via their N-terminal and/or lysine side chains. The four labeled samples are mixed and peptides separated by two-dimensional liquid chromatography online to a mass spectrometer (LC-MS). Upon peptide fragmentation, each tag releases a distinct mass reporter ion; the ratio of the four reporters therefore gives relative abundances of the given peptide. Relative quantification of proteins is derived using summed data from a number of peptides. TEL/PDGFRbeta leukemic oncogene-mediated changes in protein levels were compared with those seen in microarray analysis of control and transfected FDCP-mix cells. Changes at the protein level in most cases reflected those seen at the transcriptome level. Nonetheless, novel differences in protein expression were found that indicate potential mechanisms for effects of this oncogene.
    • Quantitative proteomics analysis demonstrates post-transcriptional regulation of embryonic stem cell differentiation to hematopoiesis.

      Williamson, Andrew J K; Smith, Duncan L; Blinco, David; Unwin, Richard D; Pearson, Stella; Wilson, Claire L; Miller, Crispin J; Lancashire, Lee J; Lacaud, Georges; Kouskoff, Valerie; et al. (2008-03)
      Embryonic stem (ES) cells can differentiate in vitro to produce the endothelial and hematopoietic precursor, the hemangioblasts, which are derived from the mesoderm germ layer. Differentiation of Bry(GFP/+) ES cell to hemangioblasts can be followed by the expression of the Bry(GFP/+) and Flk1 genes. Proteomic and transcriptomic changes during this differentiation process were analyzed to identify mechanisms for phenotypic change during early differentiation. Three populations of differentiating Bry(GFP) ES cells were obtained by flow cytometric sorting, GFP-Flk1- (epiblast), GFP+Flk1- (mesoderm), and GFP+Flk1+ (hemangioblast). Microarray analyses and relative quantification two-dimensional LCLC-MS/MS on nuclear extracts were performed. We identified and quantified 2389 proteins, 1057 of which were associated to their microarray probe set. These included a variety of low abundance transcription factors, e.g. UTF1, Sox2, Oct4, and E2F4, demonstrating a high level of proteomic penetrance. When paired comparisons of changes in the mRNA and protein expression levels were performed low levels of correlation were found. A strong correlation between isobaric tag-derived relative quantification and Western blot analysis was found for a number of nuclear proteins. Pathway and ontology analysis identified proteins known to be involved in the regulation of stem cell differentiation, and proteins with no described function in early ES cell development were also shown to change markedly at the proteome level only. ES cell development is regulated at the mRNA and protein level.