Original Article

Leukemia (2016) 30, 929–936; doi:10.1038/leu.2015.313; published online 29 January 2016

Minimal residual disease

A complementary role of multiparameter flow cytometry and high-throughput sequencing for minimal residual disease detection in chronic lymphocytic leukemia: an European Research Initiative on CLL study

A C Rawstron1, C Fazi2, A Agathangelidis2, N Villamor3, R Letestu4, J Nomdedeu5, C Palacio6, O Stehlikova7, K-A Kreuzer8, S Liptrot9, D O'Brien9, R M de Tute1, I Marinov10, M Hauwel11, M Spacek12, J Dobber13, A P Kater13, P Gambell14, A Soosapilla15, G Lozanski16, G Brachtl17,18, K Lin19, J Boysen20, C Hanson20, J L Jorgensen21, M Stetler-Stevenson22, C Yuan22, H E Broome23, L Rassenti23, F Craig24, J Delgado3, C Moreno5, F Bosch6, A Egle17, M Doubek7, S Pospisilova7, S Mulligan25, D Westerman14, C M Sanders26, R Emerson26, H S Robins26, I Kirsch26, T Shanafelt20, A Pettitt19, T J Kipps23, W G Wierda21, F Cymbalista4, M Hallek8, P Hillmen27, E Montserrat3 and P Ghia2,28 on behalf of ERIC (European Research Initiative on CLL)

  1. 1HMDS, St James's Institute of Oncology, Leeds, UK
  2. 2IRCCS San Raffaele Scientific Institute, Milano, Italy
  3. 3Hospital Clínic, IDIBAPS, Barcelona, Spain
  4. 4Hôpital Avicenne, Assistance Publique-Hôpitaux de Paris (AP-HP), Bobigny, France
  5. 5Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
  6. 6Hospital Universitari Vall d'Hebron, Barcelona, Spain
  7. 7Department of Internal Medicine—Hematology and Oncology, University Hospital Brno and CEITEC, Masaryk University, Brno, Czech Republic
  8. 8University Hospital, Cologne, Germany
  9. 9St James Hospital, Dublin, Ireland
  10. 10Institute of Hematology and Blood Transfusion, University Hospital Prague, Prague, Czech Republic
  11. 11University Hospital Geneva, Geneva, Switzerland
  12. 12General University Hospital, Prague, Czech Republic
  13. 13Department of Hematology, Academic Medical Centre, Amsterdam, Netherlands
  14. 14Peter MacCallum Cancer Centre, Melbourne, Australia
  15. 15Hematology and Flow Cytometry, Laverty Pathology, Sydney, Australia
  16. 16Ohio State University Medical Center, Columbus, OH, USA
  17. 173rd Medical Department, Paracelsus Medical University, Salzburg, Austria
  18. 18Experimental and Clinical Cell Therapy Institute, Spinal Cord and Tissue Regeneration Center, Paracelsus Medical University, Salzburg, Austria
  19. 19Royal Liverpool and Broadgreen University Hospitals NHS Trust, Liverpool, UK
  20. 20Divisions of Hematology and Hematopathology, Mayo Clinic, Rochester, MN, USA
  21. 21MD Anderson Cancer Center, Houston, TX, USA
  22. 22National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
  23. 23Moores Cancer Center, University of California, San Diego, La Jolla, CA, USA
  24. 24Un1iversity of Pittsburgh, Pittsburgh, PA, USA
  25. 25Department of Haematology, Royal North Shore Hospital, Sydney, Australia
  26. 26Adaptive Biotechnologies, Seattle, WA, USA
  27. 27Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, UK
  28. 28Università Vita-Salute San Raffaele, Milan, Italy

Correspondence: Dr AC Rawstron, HMDS, St James's Institute of Oncology, Leeds Teaching Hospitals NHS Trust, Bexley Wing, Beckett Street, Leeds LS9 7TF, UK. E-mail: andy.rawstron@hmds.org.uk

Received 8 May 2015; Revised 14 August 2015; Accepted 14 September 2015
Accepted article preview online 7 December 2015; Advance online publication 29 January 2016



In chronic lymphocytic leukemia (CLL) the level of minimal residual disease (MRD) after therapy is an independent predictor of outcome. Given the increasing number of new agents being explored for CLL therapy, using MRD as a surrogate could greatly reduce the time necessary to assess their efficacy. In this European Research Initiative on CLL (ERIC) project we have identified and validated a flow-cytometric approach to reliably quantitate CLL cells to the level of 0.0010% (10−5). The assay comprises a core panel of six markers (i.e. CD19, CD20, CD5, CD43, CD79b and CD81) with a component specification independent of instrument and reagents, which can be locally re-validated using normal peripheral blood. This method is directly comparable to previous ERIC-designed assays and also provides a backbone for investigation of new markers. A parallel analysis of high-throughput sequencing using the ClonoSEQ assay showed good concordance with flow cytometry results at the 0.010% (10−4) level, the MRD threshold defined in the 2008 International Workshop on CLL guidelines, but it also provides good linearity to a detection limit of 1 in a million (10−6). The combination of both technologies would permit a highly sensitive approach to MRD detection while providing a reproducible and broadly accessible method to quantify residual disease and optimize treatment in CLL.