Comparison of MRD Detection By MFC, NGS and PET-CT in Patients at Different Treatment Stages for Multiple Myeloma
Authors: Susanibar Sandra , Carlton Victoria , Thanendrarajan Sharmilan , Mohan Meera , Mathur Pankaj , Hoque Shadiqul , Radhakrishnan Muthukumar , van Rhee Frits , Zangari Maurizio , Davies Faith E , Kong Katherine A. , Morgan Gareth J , Alapat Daisy V. , Schinke Carolina 
Published: November 2019

Abstract
Introduction
Theachievement of Minimal Residual Disease (MRD) negativity has become a new standard of care for patients with multiple myeloma (MM). However the methods used for MRD detection, including multi-color flow-cytometry (MFC) and next generation sequencing (NGS), although both highly sensitive have different levels of sensitivity and are prone to a different range of errors. In addition MM is not distributed evenly through the marrow and biopsies are taken only from a single site. In contrast imaging studies using PET-CT can review an extended area and will detect focal lesions and extra-medullary disease. Studies incorporating all of these modalities are rare despite the importance of understanding how they perform together.
We report on the comparison and clinical relevance of MRD testing by MFC, NGS and PET-CT in patients at different stages of their disease, including newly diagnosed MM (NDMM) as well as relapsed/refractory MM (RRMM) and in patients in long standing remission.
Methods
We investigated 100 patients, who all had at least achieved a VGPR and underwent MRD detection by 8 color MFC, NGS using the ClonoSEQ assay (Adaptive Biotechnologies Corp.) and imaging with PET-CT. Patients were at different disease stages and were categorized as either 1) undergoing upfront therapy for NDMM (n=53 including induction, post Stem Cell Transplant [SCT] and maintenance), 2) remission after completed therapy (n=32) or 3) RRMM (n=5). Detection thresholds were 1 MM cell in 105 using 8 parameter MFC and 1MM cell in 106 by NGS.
Results
Of the 100 patients analyzed, a myeloma clonal rearrangement was detected in 90 patients (90%). For patients with long-term MM history, clonal rearrangements could be identified in BM samples up to 13 years ago. Fifty-three of 90 patients (59%) were undergoing upfront therapy for newly diagnosed MM and the proportion of MRD negativity by MFC and NGS increased with treatment stage and was highest during maintenance. Of these 53 patients, six were undergoing induction therapy, none of them had achieved MRD negativity by MFC or by NGS. Eight patients were post-transplant and MFC was negative in 6 (75%) and 2 were negative by NGS (25%). Thirty-nine of the 53 patients were undergoing maintenance therapy. Of these 26 were negative by MFC (50%) and 17 (44%) by NGS.
32 of the 90 patients (35.5%) were off any therapy after achieving complete remission during treatment and completion of maintenance. MFC was negative in 19 of these 32 patients (60%), yet only 15 patients had an undetectable tumor burden by NGS (47%). For 14 patients with very long term remission (>5years) and who had remained off treatment for at least 2 years, MRD by MFC was negative in all of them and 11 showed no residual tumor burden by NGS (78.5%).
Five patients were status post treatment for relapsed and refractory disease (5.5%). Of interest is that MRD burden by molecular analysis was overall very low, with 4 patients having <0.01% detectable tumor load by MFC and NGS, yet PET-CT showed active and newly involving lesions in 4 of these 5 patients suggesting a typical pattern of macro-focal disease. Apart from these 4 relapsed patients, PET-CT was positive in 5 patients that were undergoing upfront therapy. In these cases however, activity and number of focal lesions was improving to prior imaging, indicating that response to treatment was ongoing.
Conclusions
The study shows:
1) Almost half of patients who have achieved remission and completed treatment for NDMM are still MRD positive by NGS. Further evaluation will be necessary to determine whether MRD positivity in this patient population inevitably leads to clinical relapse and 6 months follow up of this patient population will be presented at ASH 2016.
2) The proportion of patients that achieve MRD negativity increases with treatment and is highest during and after maintenance. Detection of MRD should hence be measured sequentially throughout treatment to identify the time point of best response, which could further have an impact of overall prognosis.
3) Relapsed/refractory patients can present with non-secretory focal lesions without any significant MM burden on random aspirate. In these patients imaging studies, such as PET-CT, are crucial for the detection and monitoring of their disease.

Disclosures
Carlton: Adaptive Biotechnologies: Employment, Equity Ownership. Davies:Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Kong:Adaptive Biotechnologies Corp: Employment, Equity Ownership. Morgan:Univ of AR for Medical Sciences: Employment; Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria; Bristol Meyers: Consultancy, Honoraria; Janssen: Research Funding.