Flowcytometric Minimal Residual Disease Assessment in the EMN-02/HOVON-95 MM Trial: Used Methods and a Comparison of Their Sensitivity
Authors: Hofste op Bruinink Davine , Oliva Stefania , Rihova Lucie , van der Holt Bronno , Gilestro Milena , te Marvelde Jeroen G. , Vsianska Pavla , Schmitz Alexander , Høholt Helle , Johnsen Hans Erik , Boccadoro Mario , Hajek Roman , Sonneveld Pieter , Palumbo Antonio , Omedè Paola , van der Velden Vincent H.J. 
Published: October 2019

Abstract
Background
The introduction of novel treatment strategies against multiple myeloma (MM) has resulted in a major improvement in the overall outcome, which has led to an increased need for highly sensitive methods to detect minimal residual disease (MRD) in each patient. MRD assessment by multicolor flowcytometry (MFC) has been shown to be of prognostic value in many treatment protocols over the last decade, making it an attractive method to assess response in clinical trials. However, it is currently not known (1) what the best timing is to perform MFC MRD analysis in the context of a treatment protocol including induction, intensification, consolidation and maintenance treatment, (2) which patients should be selected for this analysis, and (3) what its feasibility is in a large international trial. The ongoing EMN-02 MRD Study aims to answer these questions within the framework of the EMN-02/HOVON-95 MM trial. Here, we describe our methods and the results of our first quality assessment round to compare the sensitivity of the used protocols.
Methods
The EMN-02/HOVON-95 MM trial is a randomized, multicenter, phase 3 trial in which newly diagnosed MM patients 18-65 years received 4 cycles of bortezomib, cyclophosphamide and dexamethasone (VCD) as induction treatment, followed by a first randomization between either 4 cycles of bortezomib, melphalan and prednisone (VMP), or high dose melphalan (HDM) and 1 or 2 ASCT as intensification treatment. Subsequently, patients were randomized between 2 cycles of bortezomib, lenalidomide and dexamethasone (VRD) or no consolidation treatment, followed by lenalidomide maintenance treatment for all until progression or toxicity occurred. Patients undergoing a bone marrow (BM) aspiration for complete response (CR) confirmation according to the International Myeloma Working Group (IMWG) criteria (Rajkumar et al. - Blood 2011) anytime during the trial were eligible for the EMN-02 MRD Study. BM samples from patients from 13 European countries were sent to 4 central MFC MRD laboratories in the Netherlands (A), Czech Republic (B), Denmark (C) and Italy (D), either using the strict Euroflow protocol (A) (Van Dongen et al. - Leukemia 2012) or Euroflow-based methods (B, C & D).
In order to check compatibility between protocols, 5 bone marrow samples from MM patients with a clinical response ranging from progressive disease (PD) to CR were each divided in equal volumes and sent to the respective laboratories on 3 different days. MFC MRD analysis was performed on a FACS Canto II (BD) (A-C) or Coulter Navios flowcytometer (D). Protocols A, B & C used the Euroflow Plasma Cell Disorder (PCD) tube 1 and 2 combination of antibodies, containing the backbone markers CD138-PO, CD38-FITC, CD45-PB and CD19-PE-Cy7, with CD56-PE, B2micro-PerCP-Cy5.5, cyIgK-APC and cyIgL-APC-C750 in tube 1, and CD28-PE, CD27-PerCP-Cy5.5, CD117-APC, CD81-APC-H7 in tube 2. Protocol D had the same backbone markers (CD138-PerCP-Cy5.5, CD38-PB, CD45-KO and CD19-PE-Cy7), together with CD27-PE, CD81-FITC and CD20-APC in tube 1 and cyIgK-FITC, cyIgL-PE, CD56-APC and CD117-APC-AF 750 in tube 2. Bulk lysis was performed in protocols A, B and D. Every laboratory acquired at least 2x10e6 leukocytes (or at least 1x10e4 plasma cells) and performed data-analysis in Infinicyt version 1.6 or higher (A, B & C) or Navios Kaluza (D), using a threshold ranging from 10-25 aberrant plasma cell events as cutoff for MFC MRD positivity.
Results
Acquisition of events occurred the day after BM aspiration for all samples. The total number of acquired events per sample was dependent on the level of MRD, ranging from 3x10e5 to 2x10e7 leukocytes. MFC MRD results were very comparable between labs with a 1:1 correlation between results at every level of residual disease, being 1x10e-2, 1x10e-4, 1x10-4, 1x10e-5 and 1 MRD negative sample at the level of <1x10e-5. Based on these findings, protocols have been further harmonized and a second quality assessment round will be organized in Fall 2016 to validate the suggested improvements.
Conclusions
This is the first time that a European framework has been set up between laboratories to test MFC MRD analysis in the context of an international trial. The sensitivity of the protocols has been compared in a quality assessment round, which showed a high correlation of results.

Disclosures
Oliva: Amgen: Honoraria; Takeda: Honoraria; Celgene: Honoraria. Boccadoro:CELGENE: Honoraria, Research Funding; Mundipharma: Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Abbivie: Honoraria; SANOFI: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; BMS: Honoraria, Research Funding. Hajek:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sonneveld:Celgene: Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria, Research Funding. Palumbo:Takeda: Employment, Honoraria; Janssen Cilag: Honoraria.