• Vaccination of colorectal cancer patients with modified vaccinia Ankara delivering the tumor antigen 5T4 (TroVax) induces immune responses which correlate with disease control: a phase I/II trial.

      Harrop, Richard; Connolly, Noel B; Redchenko, Irina; Valle, Juan W; Saunders, Mark P; Ryan, Matthew G; Myers, Kevin A; Drury, Noel L; Kingsman, Susan M; Hawkins, Robert E; et al. (2006-06-01)
      PURPOSE: The highly attenuated strain of vaccinia virus, modified vaccinia Ankara (MVA), encoding the tumor antigen 5T4 (termed TroVax), has been evaluated in an open-label phase I/II study in colorectal cancer patients. The primary objectives were to assess the safety and immunogenicity of ascending doses of TroVax and to determine the biodistribution of the vector. EXPERIMENTAL DESIGN: TroVax was given to 22 patients with metastatic colorectal cancer. Seventeen patients received doses of TroVax ranging from 5 x 10(7) up to 5 x 10(8) plaque-forming units at 0, 4, and 8 weeks and were considered to be evaluable for assessment of immunologic responses. Both antibody and cellular responses specific for the tumor antigen 5T4 and the viral vector were monitored throughout the study. RESULTS: TroVax was well tolerated in all patients with no serious adverse events attributed to vaccination. Of 17 evaluable patients, 16 showed 5T4-specific cellular responses whereas 14 had detectable antibody levels following vaccination. TroVax was able to boost 5T4-specific immune responses in the presence of MVA neutralizing antibodies. Periods of disease stabilization ranging from 3 to 18 months were observed in five patients, all of whom mounted 5T4-specific immune responses. Furthermore, statistical analysis showed a positive association between the development of a 5T4 (but not MVA) antibody response and patient survival or time to disease progression. CONCLUSION: These data indicate that vaccination with TroVax is safe and well tolerated and that immune responses to 5T4 can be induced without any evidence of autoimmune toxicity. Furthermore, 5T4-specific antibody responses correlate with evidence of disease control.
    • Vaccination of healthy volunteers with human papillomavirus type 16 L2E7E6 fusion protein induces serum antibody that neutralizes across papillomavirus species.

      Gambhira, Ratish; Gravitt, Patti E; Bossis, Ioannis; Stern, Peter L; Viscidi, Raphael P; Roden, Richard B S; Department of Pathology, The Johns Hopkins University, Baltimore, Maryland 21231, USA. (2006-12-01)
      Oncogenic human papillomavirus (HPV) infection is a necessary cause of cervical cancer. Therefore, vaccination to prevent or eliminate HPV infection could reduce the incidence of cervical cancer. A fusion protein comprising HPV16 L2, E6, and E7 is a candidate combination preventive and therapeutic HPV vaccine. The L1- and L2-specific and neutralizing serum antibody titers and peripheral blood mononucleocyte antigen-specific proliferative responses generated by vaccination thrice at monthly intervals with HPV16 L2E7E6 were compared in two studies: a phase I randomized double-blind placebo controlled dose escalation trial in 40 healthy volunteers and a phase II trial of HPV16 L2E7E6 at the maximum dose in 29 women with high-grade anogenital intraepithelial neoplasia (AGIN). Vaccination of healthy volunteers induced L2-specific serum antibodies that were detected 1 month after the final vaccination (P(binomial) < 0.001). There was a significant trend to seroconversion for HPV16 and HPV18 neutralizing antibodies with increasing vaccine dose (P = 0.006 and P = 0.03, respectively). Seroconversion for HPV18 neutralizing antibodies showed a significant positive trend with increasing dose (P = 0.03) and was associated with seroconversion for HPV16 neutralizing antibodies (P(exact) = 0.04). The antigen-specific proliferative response of vaccinated healthy volunteers also showed a significant trend with increasing vaccine dose (P = 0.04). However, AGIN patients responded less effectively to vaccination than healthy patients for induction of HPV16 L2-specific antibody (P < 0.001) and proliferative responses (P < 0.001). Vaccination of healthy volunteers thrice with 533-mug HPV16 L2E7E6 at monthly intervals induced L2-specific serum antibodies that neutralized across papillomavirus species. Responses in AGIN patients were infrequent.
    • Vaccination of Metastatic Renal Cancer Patients with MVA-5T4: A Randomized, Double-Blind, Placebo-Controlled Phase III Study.

      Amato, R J; Hawkins, Robert E; Kaufman, H L; Thompson, J A; Tomczak, P; Szczylik, C; McDonald, M; Eastty, S; Shingler, W H; De Belin, J; et al. (2010-11-15)
      PURPOSE: The TroVax Renal Immunotherapy Survival Trial was a randomized, placebo-controlled phase III study that investigated whether modified vaccinia Ankara encoding the tumor antigen 5T4 (MVA-5T4) prolonged survival of patients receiving first-line standard-of-care (SOC) treatment for metastatic renal cell cancer. EXPERIMENTAL DESIGN: Patients with metastatic clear cell renal cancer, prior nephrectomy, and good or intermediate prognosis were randomized 1:1 to receive up to 13 immunizations of MVA-5T4/placebo in combination with either sunitinib, interleukin-2 or interferon-α. The primary end point was overall survival. Secondary end points included progression-free survival, overall response rate, and safety. RESULTS: Seven hundred thirty-three patients were recruited (365 MVA-5T4 and 368 placebo). Treatment arms were well balanced for SOC and prognosis. No significant difference in the incidence of adverse events or serious adverse events was observed. No significant difference in overall survival was evident in the two treatment arms (median 20.1 months MVA-5T4 versus 19.2 months placebo; P = 0.55). The magnitude of the 5T4-specific antibody response induced by vaccination with MVA-5T4 was associated with enhanced patient survival. Furthermore, exploratory analyses suggested a number of pretreatment hematologic factors that could identify patients who derive significant benefit from this vaccine. CONCLUSION: MVA-5T4 in combination with SOC was well tolerated, but no difference in survival was observed in the overall study population. Exploratory analyses indicate that there may be subsets of patients who could gain significant benefit from MVA-5T4, but such results would need to be confirmed in future randomized clinical studies. Clin Cancer Res; 16(22); 5539-47. ©2010 AACR.
    • Vaccination of patients with metastatic renal cancer with modified vaccinia Ankara encoding the tumor antigen 5T4 (TroVax) given alongside interferon-alpha.

      Hawkins, Robert E; Macdermott, Catriona; Shablak, Alaaeldin; Hamer, Caroline; Thistlethwaite, Fiona C; Drury, Noel L; Chikoti, Priscilla; Shingler, William; Naylor, Stuart; Harrop, Richard; et al. (2009-05)
      Approximately 90% of renal cell tumors overexpress the tumor antigen 5T4. The attenuated strain of vaccinia virus, modified vaccinia Ankara, has been engineered to express 5T4 (TroVax). We conducted an open-label phase 1/2 trial in which TroVax was administered alongside interferon-alpha (IFNalpha) to 11 patients with metastatic renal cell carcinoma. Antigen-specific cellular and humoral responses were monitored throughout the study, and clinical responses were assessed by measuring the changes in tumor burden by computed tomography scan (Response Evaluation Criteria In Solid Tumors). The primary objective was to assess the safety, immunogenicity, and efficacy of TroVax when given alongside IFNalpha. Treatment with TroVax plus IFNalpha was well tolerated with no serious adverse events attributed to TroVax. All 11 patients mounted 5T4-specific antibody responses and 5 (45%) mounted cellular responses. No objective tumor responses were seen, but the overall median time to progression (TTP) of 9 months (range: 2.1 to 26+ mo) was longer than expected for IFNalpha alone. For the 10 clear cell patients the TTP ranged from 3.9 to 26+ months, with a median TTP of 10.4 months. The high frequency of 5T4-specific immune responses and prolonged median TTP for clear cell patients compared with that expected for IFNalpha alone is encouraging and warrants further investigation.
    • Vaccination with HPV16 L2E6E7 fusion protein in GPI-0100 adjuvant elicits protective humoral and cell-mediated immunity.

      Karanam, Balasubramanyam; Gambhira, Ratish; Peng, Shiwen; Jagu, Subhashini; Kim, Dae-Jin; Ketner, Gary W; Stern, Peter L; Adams, Robert J; Roden, Richard B S; Department of Pathology, The Johns Hopkins University, Baltimore, MD 21231, USA. (2009-02-11)
      A vaccine comprising human papillomavirus type 16 (HPV16) L2, E6 and E7 in a single tandem fusion protein (termed TA-CIN) has the potential advantages of both broad cross-protection against HPV transmission through induction of L2 antibodies able to cross neutralize different HPV types and of therapy by stimulating T cell responses targeting HPV16 early proteins. However, patients vaccinated with TA-CIN alone develop weak HPV neutralizing antibody and E6/E7-specific T cell responses. Here we test TA-CIN formulated along with the adjuvant GPI-0100, a semi-synthetic quillaja saponin analog that was developed to promote both humoral and cellular immune responses. Subcutaneous administration to mice of TA-CIN (20 microg) with 50microg GPI-0100, three times at biweekly intervals, elicited high titer HPV16 neutralizing serum antibody, robust neutralizing titers for other HPV16-related types, including HPV31 and HPV58, and neutralized to a lesser extent other genital mucosatropic papillomaviruses like HPV18, HPV45, HPV6 and HPV11. Notably, vaccination with TA-CIN in GPI-0100 protected mice from cutaneous HPV16 challenge as effectively as HPV16 L1 VLP without adjuvant. Formulation of TA-CIN with GPI-0100 enhanced the production of E7-specific, interferon gamma producing CD8(+) T cell precursors by 20-fold. Vaccination with TA-CIN in GPI-0100 also completely prevented tumor growth after challenge with 5x10(4) HPV16-transformed TC-1 tumor cells, whereas vaccination with TA-CIN alone delayed tumor growth. Furthermore, three monthly vaccinations with 125 microg of TA-CIN and 1000 microg GPI-0100 were well tolerated by pigtail macaques and induced both HPV16 E6/E7-specific T cell responses and serum antibodies that neutralized all HPV types tested.
    • Vaccine and antibody-directed T cell tumour immunotherapy.

      Dermime, Said; Gilham, David E; Shaw, David M; Davidson, Emma J; Meziane, El-Kahina; Armstrong, Anne C; Hawkins, Robert E; Stern, Peter L; Immunology, Cancer Research UK Groups, Paterson Institute for Cancer Research and University of Manchester, Christie Hospital NHS Trust, Manchester M20 4BX, UK. (2004-07-06)
      Clearer evidence for immune surveillance in malignancy and the identification of many new tumour-associated antigens (TAAs) have driven novel vaccine and antibody-targeted responses for therapy in cancer. The exploitation of active immunisation may be particularly favourable for TAA where tolerance is incomplete but passive immunisation may offer an additional strategy where the immune repertoire is affected by either tolerance or immune suppression. This review will consider how to utilise both active and passive types of therapy delivered by T cells in the context of the failure of tumour-specific immunity by presenting cancer patients. This article will outline the progress, problems and prospects of several different vaccine and antibody-targeted approaches for immunotherapy of cancer where proof of principle pre-clinical studies have been or will soon be translated into the clinic. Two examples of vaccination-based therapies where both T cell- and antibody-mediated anti-tumour responses are likely to be relevant and two examples of oncofoetal antigen-specific antibody-directed T cell therapies are described in the following sections: (1) therapeutic vaccination against human papillomavirus (HPV) antigens in cervical neoplasia; (2) B cell lymphoma vaccines including against immunoglobulin idiotype; (3) oncofoetal antigens as tumour targets for redirecting T cells with antibody strategies.
    • Vaccine development: From concept to early clinical testing.

      Cunningham, A; Garçon, N; Leo, O; Friedland, L; Strugnell, R; Laupèze, B; Doherty, M; Stern, Peter L; Westmead Institute, The Centre for Virus Research, 176 Hawkesbury Road, NSW 2145, Australia. (2016-10-18)
      In the 21st century, an array of microbiological and molecular allow antigens for new vaccines to be specifically identified, designed, produced and delivered with the aim of optimising the induction of a protective immune response against a well-defined immunogen. New knowledge about the functioning of the immune system and host pathogen interactions has stimulated the rational design of vaccines. The design toolbox includes vaccines made from whole pathogens, protein subunits, polysaccharides, pathogen-like particles, use of viral/bacterial vectors, plus adjuvants and conjugation technology to increase and broaden the immune response. Processes such as recombinant DNA technology can simplify the complexity of manufacturing and facilitate consistent production of large quantities of antigen. Any new vaccine development is greatly enhanced by, and requires integration of information concerning: 1. Pathogen life-cycle & epidemiology. Knowledge of pathogen structure, route of entry, interaction with cellular receptors, subsequent replication sites and disease-causing mechanisms are all important to identify antigens suitable for disease prevention. The demographics of infection, specific risk groups and age-specific infection rates determine which population to immunise, and at what age. 2. Immune control & escape. Interactions between the host and pathogen are explored, with determination of the relative importance of antibodies, T-cells of different types and innate immunity, immune escape strategies during infection, and possible immune correlates of protection. This information guides identification and selection of antigen and the specific immune response required for protection. 3. Antigen selection & vaccine formulation. The selected antigen is formulated to remain suitably immunogenic and stable over time, induce an immune response that is likely to be protective, plus be amenable to eventual scale-up to commercial production. 4. Vaccine preclinical & clinical testing. The candidate vaccine must be tested for immunogenicity, safety and efficacy in preclinical and appropriately designed clinical trials. This review considers these processes using examples of differing pathogenic challenges, including human papillomavirus, malaria, and ebola.
    • Vaccines in oncology: background and clinical potential.

      Armstrong, Anne C; Hawkins, Robert E; CRC Department of Medical Oncology, University of Manchester & Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK. (2001-11)
      Cancer is one of the leading causes of death in Western society. Despite improvements in screening, diagnosis and treatment of cancer, many patients ultimately succumb to their disease. Advances in molecular biology and our increased understanding of how the immune system functions have led to an intense interest in the development of cancer vaccines.
    • Vaccinia expression of Mycobacterium tuberculosis-secreted proteins: tissue plasminogen activator signal sequence enhances expression and immunogenicity of M. tuberculosis Ag85.

      Malin, Adam S; Huygen, Kris; Content, Jean; Mackett, Mike; Brandt, Lisa; Andersen, Peter; Smith, Steven M; Dockrell, Hazel M; Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, WC1E 7HT, London, UK. adam.malin@bigfoot.com (2000-11)
      There is increasing evidence to implicate a role for CD8(+) T cells in protective immunity against tuberculosis. Recombinant vaccinia (rVV) expressing Mycobacterium tuberculosis (MTB) proteins can be used both as tools to dissect CD8(+) T-cell responses and, in attenuated form, as candidate vaccines capable of inducing a balanced CD4(+)/CD8(+) T-cell response. A panel of rVV was constructed to express four immunodominant secreted proteins of MTB: 85A, 85B and 85C and ESAT-6. A parallel group of rVV was constructed to include the heterologous eukaryotic tissue plasminogen activator (tPA) signal sequence to assess if this would enhance expression and immunogenicity. Clear expression was obtained for 85A, 85B and ESAT-6 and the addition of tPA resulted in N-glycosylation and a 4-10-fold increase in expression. Female C57BL/6 mice were immunised using the rVV-Ag85 constructs, and interleukin-2 and gamma-interferon were assayed using a co-culture of immune splenocytes and recall antigen. There was a marked increase in cytokine production in mice immunised with the tPA-containing constructs. We report the first data demonstrating enhanced immunogenicity of rVV using a tPA signal sequence, which has significant implications for future vaccine design.
    • Vaccinia virus expression vectors.

      Mackett, Mike; Smith, G; Molecular Biology Department, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Withington, Manchester M20 9BX, UK. (1986-10)
    • A validated gene expression profile for detecting clinical outcome in breast cancer using artificial neural networks.

      Lancashire, Lee J; Powe, D G; Reis-Filho, J S; Rakha, E; Lemetre, Christophe; Weigelt, B; Abdel-Fatah, T M; Green, Anthony R; Mukta, R; Blamey, R; et al. (2009-04-04)
      Gene expression microarrays allow for the high throughput analysis of huge numbers of gene transcripts and this technology has been widely applied to the molecular and biological classification of cancer patients and in predicting clinical outcome. A potential handicap of such data intensive molecular technologies is the translation to clinical application in routine practice. In using an artificial neural network bioinformatic approach, we have reduced a 70 gene signature to just 9 genes capable of accurately predicting distant metastases in the original dataset. Upon validation in a follow-up cohort, this signature was an independent predictor of metastases free and overall survival in the presence of the 70 gene signature and other factors. Interestingly, the ANN signature and CA9 expression also split the groups defined by the 70 gene signature into prognostically distinct groups. Subsequently, the presence of protein for the principal prognosticator gene was categorically assessed in breast cancer tissue of an experimental and independent validation patient cohort, using immunohistochemistry. Importantly our principal prognosticator, CA9, showed that it is capable of selecting an aggressive subgroup of patients who are known to have poor prognosis.
    • Validated imaging biomarkers as decision-making tools in clinical trials and routine practice: current status and recommendations from the EIBALL* subcommittee of the European Society of Radiology (ESR)

      de Souza, NM; Achten, E; Alberich-Bayarri, A; Bamberg, F; Boellaard, R; Clement, O; Fournier, L; Gallagher, F; Golay, X; Heussel, CP; et al. (q)
      Observer-driven pattern recognition is the standard for interpretation of medical images. To achieve global parity in interpretation, semi-quantitative scoring systems have been developed based on observer assessments; these are widely used in scoring coronary artery disease, the arthritides and neurological conditions and for indicating the likelihood of malignancy. However, in an era of machine learning and artificial intelligence, it is increasingly desirable that we extract quantitative biomarkers from medical images that inform on disease detection, characterisation, monitoring and assessment of response to treatment. Quantitation has the potential to provide objective decision-support tools in the management pathway of patients. Despite this, the quantitative potential of imaging remains under-exploited because of variability of the measurement, lack of harmonised systems for data acquisition and analysis, and crucially, a paucity of evidence on how such quantitation potentially affects clinical decision-making and patient outcome. This article reviews the current evidence for the use of semi-quantitative and quantitative biomarkers in clinical settings at various stages of the disease pathway including diagnosis, staging and prognosis, as well as predicting and detecting treatment response. It critically appraises current practice and sets out recommendations for using imaging objectively to drive patient management decisions.
    • Validation of an ELISA for the determination of rituximab pharmacokinetics in clinical trials subjects.

      Hampson, G; Ward, Timothy H; Cummings, Jeffrey; Bayne, M; Tutt, A L; Cragg, M S; Dive, Caroline; Illidge, Timothy M; Clinical and Experimental Pharmacology Laboratory, University of Manchester, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester, M20 4BX, UK. (2010-06-11)
      Rituximab is a chimeric anti-CD20 monoclonal antibody that has revolutionised the treatment of many B-cell malignancies, and is now increasingly being used in non-malignant conditions such as auto-immune disorders. Serum rituximab levels are highly variable in patients receiving similar 'standard' approved doses. Little is known regarding the factors that affect serum rituximab concentration and that in turn may influence clinical outcome. In order to provide a tool that may ultimately enable patient specific dosing of rituximab therapy, we have validated a reliable, robust ELISA for the quantitation of serum rituximab levels to provide accurate pharmacokinetic (PK) data that will guide the optimisation of rituximab dosing regimes. Extensive validation of the assay was performed in order to utilise the assay for clinical applications. The within and between day plate coating reproducibility was tested and proved a robust starting platform for the assay. The within day precision for the assay was determined using spiked serum samples and was shown to have a coefficient of variation (CV) of <10% with an accuracy between 91 and 125%. The between day precision (CV) was <25% with an accuracy between 95 and 109%. Dilution linearity and parallelism were demonstrated. Spike recovery for all concentrations and donors was shown to be within +/-15% on average, with a CV below 10%. This assay is highly accurate and reproducible in determining the levels of rituximab in spiked serum samples. It meets stringent acceptance criteria, is fit for purpose, and is currently being applied to several clinical trials incorporating rituximab in the treatment of lymphoma. This assay represents a useful tool for clinical application of this widely used therapeutic.
    • Validation of pharmacodynamic assays to evaluate the clinical efficacy of an antisense compound (AEG 35156) targeted to the X-linked inhibitor of apoptosis protein XIAP.

      Cummings, Jeffrey; Ward, Timothy H; Lacasse, Eric; Lefebvre, C; St-Jean, M; Durkin, J; Ranson, Malcolm R; Dive, Caroline; Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK. jcummings@picr.man.ac.uk (2005-02-14)
      The inhibitor of apoptosis protein, XIAP, is frequently overexpressed in chemoresistant human tumours. An antisense oligonucleotide (AEG 35156/GEM 640) that targets XIAP has recently entered phase I trials in the UK. Method validation data are presented on three pharmacodynamic assays that will be utilised during this trial. Quantitative RT-PCR was based on a Taqman assay and was confirmed to be specific for XIAP. Assay linearity extended over four orders of magnitude. MDA-MB-231/U6-E1 cells and clone X-G4 stably expressing an RNAi vector against XIAP were chosen as high and low XIAP expression quality controls (QCs). Within-day and between-day coefficients of variation (CVs) in precision for cycle threshold (CT) and delta CT values (employing GAPDH and beta 2 microglobulin as housekeepers) were always less than 10%. A Western blotting technique was validated using a GST-XIAP fusion protein as a standard and HeLa cells and SF268 (human glioblastoma) cells as high and low XIAP expression QCs. Specificity of the final choice of antibody for XIAP was evaluated by analysing a panel of cell lines including clone X-G4. The assay was linear over a 29-fold range of protein concentration and between-day precision was 29% for the low QC and 23% for the high QC when normalised to GAPDH. XIAP protein was also shown to be stable at -80 degrees C for at least 60 days. M30-Apoptosense plasma Elisa detects a caspase-cleaved fragment of cytokeratin 18 (CK18), believed to be a surrogate marker for tumour cell apoptosis. Generation of an independent QC was achieved through the treatment of X-G4 cells with staurosporine and collection of media. Measurements on assay precision and kit-to-kit QC were always less than 10%. The M30 antigen (CK18-Asp396) was stable for 3 months at -80 degrees C, while at 37 degrees C it had a half-life of 80-100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited.
    • Validation of the comet-X assay as a pharmacodynamic assay for measuring DNA cross-linking produced by the novel anticancer agent RH1 during a phase I clinical trial

      Danson, Sarah; Ranson, Malcolm R; Denneny, Olive; Cummings, Jeffrey; Ward, Timothy H; Clinical and Experimental Pharmacology, Paterson Institute for Cancer Research, University of Manchester, Paterson Laboratories, Wilmslow Road, Manchester, M20 4BX, UK. (2007-11)
      PURPOSE: RH1 is a novel anticancer agent with potent DNA-cross linking activity. RH1 has the potential to be activated within tumors over expressing NQO1, giving maximal antitumour activity with reduced toxicity in normal tissues. RH1 has recently completed a Cancer Research UK sponsored phase I clinical trial at two different centers in the United Kingdom. The comet-X assay was a secondary endpoint in this trial and assay validation was necessary. We describe here this validation process. Whilst it is impossible to cover all variations/conditions of a pharmacodynamic assay, we have strived to evaluate and demonstrate that this assay conforms to the three R's of validation, that is robustness, reliability and reproducibility. METHODS: K562 and peripheral blood mononuclear cells were treated with either radiation alone, or with a combination of radiation and drug. These samples were then embedded in low melting point agarose and subjected to a modified version of the alkaline single cell gel electrophoresis (Comet) assay, described here as the comet-X assay. Variations in the preparation, electrophoresis, storage and scoring of these samples was investigated. In addition radiation and drug dose response curves were constructed. Finally stability of QC standards was investigated over a 30-month period. RESULTS: We have demonstrated a linear radiation-dose response in cells up to 20 Gy and drug induced DNA cross-linking up to 50 nM. From the radiation dose response curves we were able to show that the relative inaccuracy measured against a global mean value was less than 25% and the relative (within day) imprecision was less than 30% over all doses. Between day runs produced an intra assay imprecision of 21.2%. Variables involved in the electrophoresis process showed the voltage across all slides in the tank ranged from 3.1 to -2.0 (mV) whilst the current ranged from 0.8-5.5 mA. QC standards were prepared from PBMCs of healthy donors and frozen at -80 degrees C. The stability of these frozen QC standards was measured over a 30-month period. No significant deterioration in any of the control, irradiated or drug treated samples was observed. CONCLUSIONS: The comet-X assay has been shown to be a robust, reliable and reproducible assay. It is ideally suited for the evaluation of the pharmacodynamic effects of DNA cross-linking agents undergoing early clinical trials. Furthermore, this assay may provide valuable data, in conjunction with pharmacokinetics, when measuring toxicity and efficacy as part of the RH1 phase I clinical trial.
    • The value of local therapy in treatment of solitary melanoma progression upon immune checkpoint inhibition

      Versluis, J. M.; Hendriks, A. M.; Weppler, A.; Brown, L.; de Joode, K.; Suijkerbuijk, K. P. M.; Zimmer, L.; Kapiteijn, E.; Allayous, C.; Johnson, D. B.; et al. (2020)
      Background: Patients (pts) with stage IV melanoma can achieve long-term disease control through treatment with immune checkpoint inhibition (ICI). Disease progression in a single tumor lesion (solitary progression, SP) after initial response to ICI is often treated with local therapy. This study aimed to evaluate the benefits of local therapy for the treatment of SP during (on) or after cessation of (off) ICI. Methods: Pts with stage IV melanoma with at least stable disease (SD) as best overall response (BOR) upon ICI and SP as first progressive event were retrospectively included from 17 centers in 9 countries. Results: We included 294 pts with SP on anti-PD-1 (67%), anti-CTLA-4 (13%), anti-PD- 1 + anti-CTLA-4 (15%) and other ICI combinations (5%). BOR prior to SP was SD (15%), partial response (55%) and complete response (30%). Local therapy was mainly surgery (56%), radiotherapy (35%) or both (5%). Median follow-up from start ICI was 43 months (m), median time to SP 13m and median time to second progression after treatment of SP (TTSP) 33m. Median overall survival (OS) was not reached, estimated 3-year OS was 79%. SP occurred in 143 pts on ICI (median 11m) and in 151 pts off ICI (median 17m from start ICI, 9m from stop ICI). SP was treated with local + systemic therapy (42%), local therapy (36%) or systemic therapy only (18%). Second progression was mostly progression at multiple sites (64% and 65% for SP on and off ICI). In pts with SP on ICI, median TTSP was 29m. Local therapy + ICI continuation (N¼94) resulted in similar 3-year TTSP as local therapy (N¼15) or ICI continuation only (N¼14, P¼0.971). OS at 3 years was superior for local therapy + ICI continuation (P¼0.020). In pts with SP off ICI, median TTSP was 35m. ICI restart + local therapy (N¼22, 85%) resulted in superior TTSP compared to local therapy (N¼90, 41%) or ICI restart (N¼18, 56%, P¼0.002), without OS differences so far. Conclusions: In pts with SP off ICI, the combination of local therapy + ICI restart was most successful in delaying further progression, but did not improve OS so far compared to single modality treatment. Local therapy + ICI continuation in pts with SP on ICI did not improve TTSP, but did improve OS. This indicates that local therapy can benefit pts.
    • Variability and regulation of O6-alkylguanine-DNA alkyltransferase.

      Margison, Geoffrey P; Povey, Andrew C; Kaina, Bernd; Santibanez-Koref, Mauro F; Cancer Research Uk Carcinogenesis Group, Paterson Institute for Cancer Research, Manchester M20 4BX, UK. gmargison@picr.man.ac.uk (2003-04)
      O(6)-Alkylguanine-DNA alkyltransferase (ATase) confers resistance to many of the biological effects of certain classes of alkylating agents by repairing the DNA lesions responsible. The role of ATase in the mutagenic and toxic effects of the carcinogenic and antitumour alkylating agents are of interest in relation to the prevention and treatment of cancer in man. In this commentary we specifically focus on the variation in ATase levels and our current understanding of the factors involved in the regulation of ATase expression.
    • Variance in resistance to natural and antibody-dependent cellular cytotoxicity and to complement-mediated lysis among K562 lines.

      Kimber, Ian; Moore, Michael; Roberts, Kevan; Department of Immunology,Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX (1983-08-15)
      Isolation of sub-populations of the NK-sensitive erythro-leukaemic cell line K562 by limiting dilution techniques has revealed marked clonal variation in susceptibility to natural cytotoxicity. Detailed examination of two such lines (E10/P2 and F9/P2) which differed significantly in their susceptibility to both native and activated NK-cell-mediated lysis revealed that their differences were stable and independent of culture conditions. The resistant (F9/P2) and sensitive (E10/P2) lines had comparable cold-inhibitory and effector-cell adsorption capacities, indicating that differential susceptibility was not attributable to variable expression of NK target structures. F9/P2 was also less susceptible to antibody-dependent cellular cytotoxicity (ADCC) and complement-mediated lysis, indicating the existence of variants with a generalized capacity to resist several immunolytic processes.
    • Variations in chromosome numbers and their possible relationship to the development of 8-azaguanine resistance in V79 Chinese hamster cells.

      Fox, Margaret; Radacic, M; Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester, M20 9BX, United Kingdom (1982-01)