title: Epigenetic profiling to classify cancer of unknown primary: a multicentre, retrospective analysis.
abstract: BACKGROUND
  Cancer of unknown primary ranks in the top ten cancer presentations and has an extremely poor prognosis. Identification of the primary tumour and development of a tailored site-specific therapy could improve the survival of these patients. We examined the feasability of using DNA methylation profiles to determine the occult original cancer in cases of cancer of unknown primary.
  
  
  METHODS
  We established a classifier of cancer type based on the microarray DNA methylation signatures (EPICUP) in a training set of 2790 tumour samples of known origin representing 38 tumour types and including 85 metastases. To validate the classifier, we used an independent set of 7691 known tumour samples from the same tumour types that included 534 metastases. We applied the developed diagnostic test to predict the tumour type of 216 well-characterised cases of cancer of unknown primary. We validated the accuracy of the predictions from the EPICUP assay using autopsy examination, follow-up for subsequent clinical detection of the primary sites months after the initial presentation, light microscopy, and comprehensive immunohistochemistry profiling.
  
  
  RESULTS
  The tumour type classifier based on the DNA methylation profiles showed a 99·6% specificity (95% CI 99·5-99·7), 97·7% sensitivity (96·1-99·2), 88·6% positive predictive value (85·8-91·3), and 99·9% negative predictive value (99·9-100·0) in the validation set of 7691 tumours. DNA methylation profiling predicted a primary cancer of origin in 188 (87%) of 216 patients with cancer with unknown primary. Patients with EPICUP diagnoses who received a tumour type-specific therapy showed improved overall survival compared with that in patients who received empiric therapy (hazard ratio [HR] 3·24, p=0·0051 [95% CI 1·42-7·38]; log-rank p=0·0029).
  
  
  CONCLUSIONS
  We show that the development of a DNA methylation based assay can significantly improve diagnoses of cancer of unknown primary and guide more precise therapies associated with better outcomes. Epigenetic profiling could be a useful approach to unmask the original primary tumour site of cancer of unknown primary cases and a step towards the improvement of the clinical management of these patients.
  
  
  BACKGROUND
  European Research Council (ERC), Cellex Foundation, the Institute of Health Carlos III (ISCIII), Cancer Australia, Victorian Cancer Agency, Samuel Waxman Cancer Research Foundation, the Health and Science Departments of the Generalitat de Catalunya, and Ferrer.
authors: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet, Barcelona, Catalonia, Spain.
authors: Sebastian
authors: S
authors: Moran
authors: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet, Barcelona, Catalonia, Spain.
authors: Anna
authors: A
authors: Martínez-Cardús
authors: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet, Barcelona, Catalonia, Spain.
authors: Sergi
authors: S
authors: Sayols
authors: Department of Pathology, Hospital Universitari Germans Trias i Pujol, C/ Ctra de Canyet s/n, Badalona, Barcelona, Catalonia, Spain.
authors: Eva
authors: E
authors: Musulén
authors: Medical Oncology, Catalan Institute of Oncology (ICO), University Hospital Germans Trias i Pujol, Badalona, Barcelona, Catalonia, Spain.
authors: Carme
authors: C
authors: Balañá
authors: Medical Oncology, Catalan Institute of Oncology (ICO), University Hospital Germans Trias i Pujol, Badalona, Barcelona, Catalonia, Spain.
authors: Anna
authors: A
authors: Estival-Gonzalez
authors: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet, Barcelona, Catalonia, Spain.
authors: Cátia
authors: C
authors: Moutinho
authors: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet, Barcelona, Catalonia, Spain.
authors: Holger
authors: H
authors: Heyn
authors: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet, Barcelona, Catalonia, Spain.
authors: Angel
authors: A
authors: Diaz-Lagares
authors: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet, Barcelona, Catalonia, Spain.
authors: Manuel Castro
authors: MC
authors: de Moura
authors: Cardiothoracic and Vascular Department, Pneumology Unit, IRCCS Policlinico San Matteo Foundation, Pavia, Italy.
authors: Giulia M
authors: GM
authors: Stella
authors: Institute for Cancer Research at Candiolo, Candiolo, Italy.
authors: Paolo M
authors: PM
authors: Comoglio
authors: IRBLleida Biobank, Lleida, Catalonia, Spain.
authors: Maria
authors: M
authors: Ruiz-Miró
authors: Department of Pathology and Molecular Genetics/Oncologic Pathology Group, Hospital Universitari Arnau de Vilanova, Universitat de Lleida, IRBLleida, Lleida, Catalonia, Spain.
authors: Xavier
authors: X
authors: Matias-Guiu
authors: Medical Oncology Service, Hospital Miguel Servet, Zaragoza, Spain.
authors: Roberto
authors: R
authors: Pazo-Cid
authors: Medical Oncology Service, Hospital Miguel Servet, Zaragoza, Spain.
authors: Antonio
authors: A
authors: Antón
authors: Medical Oncology Service, Complejo Hospitalario Universitario de Santiago, Santiago de Compostela, Spain.
authors: Rafael
authors: R
authors: Lopez-Lopez
authors: Medical Oncology, Catalan Institute of Oncology (ICO), Hospital Duran i Reynals, L'Hospitalet de Llobregat, Barcelona, Catalonia, Spain.
authors: Gemma
authors: G
authors: Soler
authors: Medical Oncology Service, Hospital Universitario Ramon y Cajal, Madrid, Spain.
authors: Federico
authors: F
authors: Longo
authors: Biobanco Vasco, Hospital Universitario de Araba, Vitoria, Spain.
authors: Isabel
authors: I
authors: Guerra
authors: Biobanco Vasco, Hospital Universitario de Basurto, Bilbao, Spain.
authors: Sara
authors: S
authors: Fernandez
authors: Division of Epigenomics and Cancer Risk Factors at the German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors: Yassen
authors: Y
authors: Assenov
authors: Division of Epigenomics and Cancer Risk Factors at the German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors: Christoph
authors: C
authors: Plass
authors: Oncology Department, Vall d'Hebron University Hospital, Universitat Autònoma de Barcelona (UAB), Barcelona, Catalonia, Spain; Oncology Department, Vall d'Hebron Institute of Oncology (VHIO), Barcelona, Catalonia, Spain.
authors: Rafael
authors: R
authors: Morales
authors: Oncology Department, Vall d'Hebron University Hospital, Universitat Autònoma de Barcelona (UAB), Barcelona, Catalonia, Spain; Oncology Department, Vall d'Hebron Institute of Oncology (VHIO), Barcelona, Catalonia, Spain.
authors: Joan
authors: J
authors: Carles
authors: The Peter MacCallum Cancer Centre, Melbourne, VIC, Australia; The Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia; The Department of Pathology, University of Melbourne, Parkville, VIC, Australia.
authors: David
authors: D
authors: Bowtell
authors: The Peter MacCallum Cancer Centre, Melbourne, VIC, Australia; The Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Victoria, Australia; The Department of Pathology, University of Melbourne, Parkville, VIC, Australia.
authors: Linda
authors: L
authors: Mileshkin
authors: Liver Cancer Program, Division of Liver Diseases, Tisch Cancer Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
authors: Daniela
authors: D
authors: Sia
authors: The Peter MacCallum Cancer Centre, Melbourne, VIC, Australia; The Department of Pathology, University of Melbourne, Parkville, VIC, Australia.
authors: Richard
authors: R
authors: Tothill
authors: Oncology Department, Vall d'Hebron University Hospital, Universitat Autònoma de Barcelona (UAB), Barcelona, Catalonia, Spain; Oncology Department, Vall d'Hebron Institute of Oncology (VHIO), Barcelona, Catalonia, Spain.
authors: Josep
authors: J
authors: Tabernero
authors: Liver Cancer Translational Research Laboratory, Barcelona Clinic Liver Cancer (BCLC) Group, Liver Unit, IDIBAPS, Hospital Clínic, CIBERehd, Barcelona, Catalonia, Spain; School of Medicine, University of Barcelona, Barcelona, Catalonia, Spain; Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Catalonia, Spain; Liver Cancer Program, Division of Liver Diseases, Tisch Cancer Institute, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
authors: Josep M
authors: JM
authors: Llovet
authors: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L'Hospitalet, Barcelona, Catalonia, Spain; School of Medicine, University of Barcelona, Barcelona, Catalonia, Spain; Institucio Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Catalonia, Spain. Electronic address: mesteller@idibell.cat.
authors: Manel
authors: M
authors: Esteller
mesh_terms: D019175:DNA Methylation; D044127:Epigenesis, Genetic; D066246:ErbB Receptors / Q000235:genetics; D005260:Female; D006801:Humans; D008297:Male; D009382:Neoplasms, Unknown Primary / Q000145:classification / Q000235:genetics* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics; D012189:Retrospective Studies
chemical_list: C117307:KRAS protein, human; D066246:ErbB Receptors; D016283:Proto-Oncogene Proteins p21(ras)