title: γH2AX assay in ex vivo irradiated tumour specimens: A novel method to determine tumour radiation sensitivity in patient-derived material.
abstract: OBJECTIVE
  To establish a clinically applicable protocol for quantification of residual γH2AX foci in ex vivo irradiated tumour samples and to apply this method in a proof-of-concept feasibility study to patient-derived tumour specimens.
  
  
  METHODS
  Evaluation of γH2AX foci formation and disappearance in excised FaDu tumour specimens after (a) different incubation times in culture medium, 4Gy irradiation and fixation after 24h (cell recovery), (b) 10h medium incubation, 4Gy irradiation and fixation after various time points (double strand break repair kinetics), and (c) 10h medium incubation, irradiation with graded single radiation doses and fixation after 24h (dose-response). The optimised protocol was applied to patient-derived samples of seminoma, prostate cancer and glioblastoma multiforme.
  
  
  RESULTS
  Post excision or biopsy, tumour tissues showed stable radiation-induced γH2AX foci values in oxic cells after >6h of recovery in medium. Kinetics of foci disappearance indicated a plateau of residual foci after >12h following ex vivo irradiation. Fitting the dose-response of residual γH2AX foci yielded slopes comparable with in situ irradiation of FaDu tumours. Significant differences in the slopes of ex vivo irradiated patient-derived tumour samples were found.
  
  
  CONCLUSIONS
  A novel clinically applicable method to quantify residual γH2AX foci in ex vivo irradiated tumour samples was established. The first clinical results suggest that this method allows to distinguish between radiosensitive and radioresistant tumour types. These findings support further translational evaluation of this assay to individualise radiation therapy.
authors: Department of Radiation Oncology, Medical Faculty and University Hospital, Eberhard Karls University Tübingen, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany and German Cancer Consortium (DKTK), Partner Site Tübingen, Germany.
authors: Apostolos
authors: A
authors: Menegakis
authors: German Cancer Research Center (DKFZ), Heidelberg, Germany and German Cancer Consortium (DKTK), Partner Site Dresden, Germany; Department of Radiation Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany; OncoRay - National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany.
authors: Cläre
authors: C
authors: von Neubeck
authors: Department of Radiation Oncology (Maastro), GROW-School for Oncology and Developmental Biology, Maastricht University Medical Centre, The Netherlands.
authors: Ala
authors: A
authors: Yaromina
authors: Department of Biostatistics, The University of Texas MD Anderson Cancer Center, Houston, USA.
authors: Howard
authors: H
authors: Thames
authors: Institute for Legal Medicine, Faculty of Medicine, University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany.
authors: Sandra
authors: S
authors: Hering
authors: Department of Urology, Medical Faculty and University Hospital, Eberhard Karls University Tübingen, Germany.
authors: Joerg
authors: J
authors: Hennenlotter
authors: Department of Pathology, Medical Faculty and University Hospital, Eberhard Karls University Tübingen, Germany.
authors: Marcus
authors: M
authors: Scharpf
authors: Department of Neurosurgery, Medical Faculty and University Hospital, Eberhard Karls University Tübingen, Germany.
authors: Susan
authors: S
authors: Noell
authors: German Cancer Research Center (DKFZ), Heidelberg, Germany and German Cancer Consortium (DKTK), Partner Site Dresden, Germany; Department of Radiation Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany; OncoRay - National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany; Institute of Radiooncology, Helmholtz-Zentrum Dresden - Rossendorf, Germany.
authors: Mechthild
authors: M
authors: Krause
authors: Department of Radiation Oncology, Medical Faculty and University Hospital, Eberhard Karls University Tübingen, Germany; German Cancer Research Center (DKFZ), Heidelberg, Germany and German Cancer Consortium (DKTK), Partner Site Tübingen, Germany.
authors: Daniel
authors: D
authors: Zips
authors: German Cancer Research Center (DKFZ), Heidelberg, Germany and German Cancer Consortium (DKTK), Partner Site Dresden, Germany; Department of Radiation Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany; OncoRay - National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Germany; Institute of Radiooncology, Helmholtz-Zentrum Dresden - Rossendorf, Germany.
authors: Michael
authors: M
authors: Baumann
mesh_terms: D000818:Animals; D001681:Biological Assay; D014408:Biomarkers, Tumor / Q000378:metabolism*; D004260:DNA Repair / Q000528:radiation effects; D005240:Feasibility Studies; D064593:Heterografts / Q000378:metabolism; D006657:Histones / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D009369:Neoplasms / Q000235:genetics* / Q000532:radiotherapy; D011836:Radiation Tolerance / Q000235:genetics*; D014183:Transplantation, Heterologous
chemical_list: D014408:Biomarkers, Tumor; C468783:H2AX protein, human; D006657:Histones
keywords: Biomarker; DNA repair; Radiotherapy; Tumour biopsy; γH2AX foci