Next Generation Sequencing (NGS) Based Minimal Residual Disease (MRD) Testing Is Highly Predictive of Overall and Progression Free Survival in the Total Therapy Trials and Shows Different Prognostic Implications in High Vs Standard Risk Multiple Myeloma
Authors: Schinke Carolina , Deshpande Shayu , Wang Hongwei , Carlton Victoria , Kong Katherine A. , Moorhead Martin , Willis Tom , Patel Purvi , Thanendrarajan Sharmilan , Mohan Meera , Mathur Pankaj , Susanibar Sandra , Hoque Shadiqul , Radhakrishnan Muthukumar , Tytarenko Ruslana , Stephens Owen W , Rasche Leo , van Rhee Frits , Zangari Maurizio , Petty Nathan , Alapat Daisy V. , Johnson Sarah K. , Epstein Joshua , Barlogie Bart , Davies Faith E , Hoering Antje , Weinhold Niels , Morgan Gareth J 
Published: October 2019

Abstract
Introduction
Achieving complete remission (CR) improves outcomes in multiple myeloma (MM). We have shown that 50% of patients enrolled in Total Therapy (TT) trials achieve CR within one year of enrollment irrespective of risk as defined by the GEP70 risk signature. Nevertheless, the majority of patients with high risk (HR) MM show an early relapse with grim prognosis, while most standard risk (SR) patients tend to relapse late, most often years after completion of maintenance therapy. Evaluation of minimal residual disease (MRD) has been shown to be of prognostic relevance in patients in CR with MRD negativity being associated with better outcomes. Here we report the impact of next generation sequencing (NGS)-based MRD assessment in TT patients who have achieved at least a very good partial response (VGPR) at two different time points during their treatment protocol: first after auto stem cell transplant (ASCT at 4-8 months of enrollment) and secondly during maintenance therapy (12-24 months).
Materials and methods
In brief, our TT protocols incorporate induction therapy consisting of Velcade and Thalidomide in conjunction with chemotherapy (Cisplatin, Doxorubicin, Cytoxan, Etoposide) followed by ASCT, consolidation and three years of maintenance with Velcade, Revlimid and Dexamethasone. HR and SR were assigned using the GEP70 risk signature. For MRD testing, we included 119 patients who were treated on our TT3b -TT6 protocols and who achieved at least VGPR and had bone marrow samples available at 4-8 months post ASCT and 12-24 months into maintenance. Thirty-eight patients had HR MM (32%), 75 patients had SR MM (63%) and no GEP70 data was available for 6 patients (5%).
For NGS-based MRD assessment (Adaptive Biotechnologies Corp), genomic DNA was amplified using locus-specific primer sets for immunoglobulin heavy-chain complete (IGH-VDJH) and incomplete (IGH-DJ) as well as for immunoglobulin κ locus (IGκ). The amplified products underwent sequencing and clonal gene rearrangements were analyzed. MRD levels were calculated at a sensitivity level of 1 x 10-5. PET-CT and/or MRI were obtained at same time point as MRD assessment to evaluate presence of focal lesions.
Results
Using MRD status post-ASCT and during maintenance as a predictor, there were significant differences for PFS and OS in HR and SR MM. In HR disease, MRD positivity post ASCT was associated with significantly worse clinical outcomes with 2 year PFS/OS at 33% and 52% for MRD positive patients compared to 82%and 83% in MRD negative patients. Further 5 year PFS and OS for the same patient group were 11% and 24% for MRD positivity compared to 45% and 75% for HR patients that had achieved MRD negativity.
In contrast to the HR group, there was no significant difference between MRD positivity and negativity in SR patients at the time point post ASCT we have investigated. In this latter patient group a significant difference only became obvious later during maintenance with 5 year PFS and OS at 55% and 63% for MRD positive patients compared to 83% and 93% for MRD negative patients.
Of the 54 SR patients that were MRD positive post ASCT, 25 (46%) became negative later during maintenance, while in HR disease only 4 of 21 MRD positive patients post ASCT became negative during maintenance (19%).
Importantly, a stratified analysis of our MRD data showed that even during maintenance the majority of cases in the favorable CD-2 subgroup were MRD positive, indicating that MRD results should further be interpreted in the context of patients' molecular subgroup.
Conclusions
Our data suggest that HR patients that do not achieve MRD negativity after their first ASCT have a very high likelihood of disease progression and death within 24 months. In contrast, SR patients that are still MRD positive post ASCT tend to have continuing response to treatment and MRD testing only becomes prognostic during maintenance. Of interest is that a high proportion of HR patients that achieve MRD negativity post ASCT and a smaller proportion of MRD negative SR patients still relapse within 5 years of enrollment suggesting that remaining residual MM cells are not detected by molecular MRD testing. Imaging studies with PET-CT and MRI could further stratify these patients as they detect any residual focal lesions that are not assessed by molecular MRD testing. We have combined PET-CT/MRI data for all of the 119 patients that were included in this study and analyzed data will be presented at ASH 2016.

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