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Original Article
Discovery of methylated circulating DNA biomarkers for comprehensive non-invasive monitoring of treatment response in metastatic colorectal cancer
  1. Ludovic Barault1,2,
  2. Alessio Amatu3,
  3. Giulia Siravegna1,2,4,
  4. Agostino Ponzetti5,
  5. Sebastian Moran6,
  6. Andrea Cassingena3,
  7. Benedetta Mussolin2,
  8. Chiara Falcomatà1,2,
  9. Alexandra M Binder7,
  10. Carmen Cristiano5,
  11. Daniele Oddo1,2,
  12. Simonetta Guarrera8,
  13. Carlotta Cancelliere2,
  14. Sara Bustreo5,
  15. Katia Bencardino3,
  16. Sean Maden9,
  17. Alice Vanzati4,
  18. Patrizia Zavattari10,
  19. Giuseppe Matullo8,
  20. Mauro Truini3,
  21. William M Grady9,11,
  22. Patrizia Racca5,
  23. Karin B Michels7,
  24. Salvatore Siena3,12,
  25. Manel Esteller6,13,14,
  26. Alberto Bardelli1,2,
  27. Andrea Sartore-Bianchi3,
  28. Federica Di Nicolantonio1,2
  1. 1Department of Oncology, University of Torino, Torino, Italy
  2. 2Candiolo Cancer Institute-FPO, IRCCS, Candiolo, Italy
  3. 3Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy
  4. 4FIRC Institute of Molecular Oncology (IFOM), Milano, Italy
  5. 5Colorectal Cancer Unit, Medical Oncology Division 1, AOU Città della Salute e della Scienza, San Giovanni Battista Hospital, Turin, Italy
  6. 6Cancer Epigenetics and Biology Program, Bellvitge Biomedical Research Institute, L’Hospitalet, Barcelona, Spain
  7. 7Department of Epidemiology, Fielding School of Public Health, University of California, Los Angeles, California, USA
  8. 8Department of Medical Sciences, Italian Institute for Genomic Medicine – IIGM/HuGeF, University of Torino, Torino, Italy
  9. 9Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA
  10. 10Department of Biomedical Sciences, Unit of Biology and Genetics, University of Cagliari, Cagliari, Italy
  11. 11Department of Medicine, University of Washington School of Medicine, Seattle, Washington, USA
  12. 12Department of Oncology and Hemato-Oncology, Università degli Studi di Milano, Milan, Italy
  13. 13Department of Physiological Sciences II, School of Medicine, University of Barcelona, Barcelona, Catalonia, Spain
  14. 14Institucio Catalana de Recerca i Estudis Avançats, Barcelona, Catalonia, Spain
  1. Correspondence to Dr Ludovic Barault; ludovic.barault{at} and Federica Di Nicolantonio; federica.dinicolantonio{at}


Objective Mutations in cell-free circulating DNA (cfDNA) have been studied for tracking disease relapse in colorectal cancer (CRC). This approach requires personalised assay design due to the lack of universally mutated genes. In contrast, early methylation alterations are restricted to defined genomic loci allowing comprehensive assay design for population studies. Our objective was to identify cancer-specific methylated biomarkers which could be measured longitudinally in cfDNA (liquid biopsy) to monitor therapeutic outcome in patients with metastatic CRC (mCRC).

Design Genome-wide methylation microarrays of CRC cell lines (n=149) identified five cancer-specific methylated loci (EYA4, GRIA4, ITGA4, MAP3K14-AS1, MSC). Digital PCR assays were employed to measure methylation of these genes in tumour tissue DNA (n=82) and cfDNA from patients with mCRC (n=182). Plasma longitudinal assessment was performed in a patient subset treated with chemotherapy or targeted therapy.

Results Methylation in at least one marker was detected in all tumour tissue samples and in 156 mCRC patient cfDNA samples (85.7%). Plasma marker prevalence was 71.4% for EYA4, 68.5% for GRIA4, 69.7% for ITGA4, 69.1% for MAP3K14-AS1% and 65.1% for MSC. Dynamics of methylation markers was not affected by treatment type and correlated with objective tumour response and progression-free survival.

Conclusion This five-gene methylation panel can be used to circumvent the absence of patient-specific mutations for monitoring tumour burden dynamics in liquid biopsy under different therapeutic regimens. This method might be proposed for assessing pharmacodynamics in clinical trials or when conventional imaging has limitations.

  • colorectal cancer
  • methylation
  • tumour markers
  • screening
  • chemotherapy

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  • Contributors LB, AB, ASB and FDN participated in the design of the study. AA, AP, AC, ABi, CCr, SB, KB, PR, KBM, SS and ASB contributed to the collection and retrieval of clinical data of blood samples. AA, AC, AV, MT, SS and ASB contributed to the collection and retrieval of tissue samples. LB, AP, CF, CCr, AC, BM, GS and DO contributed to blood sample preparation and processing. LB, SMo, SMa, SG, GM, WG and ME contributed to the genome-wide methylation experiments. PZ contributed to establishing the pipeline for in silico and wet validation of GRIA4. LB performed the bioinformatics analyses of the genome-wide methylation experiments. LB, CF and DO performed methylation analyses in cfDNA samples. BM and GS performed genetic alteration analyses in cfDNA samples. LB, BM and GS interpreted the results in cfDNA. LB and FDN wrote the manuscript. All authors critically reviewed and commented the manuscript.

  • Funding Research in the authors’ laboratory was supported by grants AIRC IG n. 17707 (FDN); AIRC IG n. 16788 (AB); Fondo per la Ricerca Locale (ex 60%), Università di Torino, 2014 and 2017 (FDN); grant Fondazione Piemontese per la Ricerca sul Cancro—ONLUS Farmacogenomica 5 per mille 2009 MIUR (FDN), grant Fondazione Piemontese per la Ricerca sul Cancro-ONLUS 5 per mille 2011 Ministero della Salute (AB and FDN); European Community’s Seventh Framework Programme under grant agreement no. 602901 MErCuRIC (AB and FDN); H2020 grant agreement no. 635342-2 MoTriColor (AB); IMI contract n. 115749 CANCER-ID (AB); Fondazione Piemontese per la Ricerca sul Cancro-ONLUS 5 per mille 2014 Ministero della Salute (AB). Investigators at Niguarda Cancer Centers are supported by the following grants: Terapia Molecolare dei Tumori (AS-B and SS) and Dynamic of Tumor Evolution & Therapy (AS-B) from Fondazione Oncologia Niguarda Onlus; Associazione Italiana per la Ricerca sul Cancro (AIRC) 2010 Special Program Molecular Clinical Oncology 5x1000, project 9970 (SS and AB). PZ is partly funded by Fondazione Banco di Sardegna (2012) and Regione Autonoma della Sardegna (CRP-79303). Partly supported also by NIH grants 1R01CA194663, 1R01CA189184, U01CA152756 and P30CA15704 to WMG. Ludovic Barault was the recipient of a postdoctoral fellowship from Fondazione Umberto Veronesi in 2013 and 2015, and of ‘Assegno di Ricerca’ from the University of Torino in 2016.

  • Competing interests AB reports personal fees (scientific advisory board member) from Horizon Discovery, personal fees (scientific advisory board member) from Biocartis, personal fees (Consultant) from Novartis, personal fees (Consultant) from Roche, personal fees (Consultant) from Illumina. AB and FDN reports grants from Trovagene, outside the submitted work. In addition, FDN and PZ have a patent 102017000072650 pending. All the other authors have nothing to disclose.

  • Ethics approval Niguarda Cancer Center ethics commitee; San Giovanni Battista Hospital ethics commitee.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Author note AS-B and FDN contributed equally as co-senior authors.