Role of clonoSEQ®, a Next-Generation Sequencing (NGS) Assay and PET/CT As a Measure of Minimal Residual Disease Negativity Among Patients with Multiple Myeloma
Authors: Nooka Ajay , Bradley Kyle , Joseph Nisha , Hofmeister Craig C , Dhodapkar Madhav V. , Boise Larry H. , Lonial Sagar , Li Shiyong , Kaufman Jonathan L. 
Published: February 2021

Background: The clonoSEQ® Assay (Adaptive Biotechnologies; Seattle, WA) is an in vitro diagnostic that uses next-generation sequencing (NGS) to identify and quantify rearranged IgH (VDJ), IgH (DJ), IgK, and IgL receptor gene sequences, as well as translocated BCL1/IgH (J) and BCL2/IgH (J) sequences in extracted DNA. clonoSEQ assay includes primers that amplify specific genomic regions present as diploid copies in normal genomic DNA (gDNA) to allow determination of total nucleated cell content. In the assessment of clonoSEQ Clonality (ID), the immune repertoire of the sample is checked for the presence of DNA sequences specific to "dominant" clone(s). For clonoSEQ Tracking MRD assessment, the complete immunoglobulin receptor repertoire is again assessed, and the previously identified dominant clonotype sequence(s) are detected and quantified to determine the sample MRD level. The MRD is expressed as a frequency that quantifies the level of residual disease based on the number of remaining copies of the initially dominant sequence(s) relative to the total number of nucleated cells in the sample. We have evaluated the rates of MRD negativity among myeloma patients achieving hematological response and its impact on the progression free survival.
Methods: We have identified 285 patients with MRD testing results available with a diagnosis of plasma cell dyscrasia (240 newly diagnosed (NDMM) and 42 relapsed/refractory myeloma (RRMM) patients, 1 light chain deposition disease, 1 solitary plasmacytoma and 1 patient with POEMS) treated at Emory University from June 2016 until March 2020. Data-cutoff was 4/13/2020. All patient, clinical data were obtained from the myeloma database approved by the Emory University Institutional Review Board. Of the results available for clonality on 392 samples, 58 (14.8%) failed quality control and were polyclonal in 31 (7.1%) leaving a positive clonal identification in 303 (77.3%) of the samples. Results for MRD testing were available for 343 samples altogether. MRD results were available for 285 patients at one time point, for 51 patients at two time points.
Results: The median age of the patients was 62 years (range: 36-77) for NDMM patients and 60 years (range: 45-77) for RRMM patients. Median age from diagnosis to MRD testing is 29 months (range: 1-154) and 56 months (range: 12-161) for NDMM and RRMM patients respectively. Hematological staging shows that 77.9% of NDMM patients achieved ≥CR (SCR: 70% and CR: 7.9%) and 70.7% of RRMM patients achieved ≥CR (SCR: 56.1% and CR: 14.6%). Among NDMM patients, MRD negativity was obtained in 119 patients (49.6%) at threshold of 10-6, 155 patients (64.6%) at 10-5, and 184 patients (76.7%) at 10-4 respectively. Similar numbers for the RRMM patients at thresholds of 10-6, 10-5, 10-4 were 16 (38.1%), 26 (61.9%) and 31 (73.8%), respectively. Correlation of hematologic response with MRD status and PET status was summarized in table 1. On univariate analysis attaining MRD negativity is prognostic at every threshold 10-6 (p-value: &lt;0.001)10-5 (p-value: &lt;0.001) 10-4 (p-value: &lt;0.001) as well as attaining PET negativity (p-value: 0.015) in NDMM patients. There were no events of progression observed in patients achieving a threshold 10-6 or 10-5 hence the additional impact of PET/CT could not be evaluated. However, addition of PET/CT improved the sensitivity at the threshold level of 10-4 (Figure 1). On multivariate analysis in NDMM, the hazards ratio for progression for Revised International Staging System (R-ISS) stage 3 disease and inability to achieve MRD negativity at threshold of 10-4 were 11.62 (95% CI 1.38-97.6, p=0.024) and 17.42 (95% CI 3.14-976.7, p=0.001) respectively. Among the 42 NDMM patients, that had MRD testing available 12 months apart to evaluate for sustained MRD negativity, 22 (88%) and 29 (97%) were MRD negative at 10-6 and10-5 thresholds, respectively.
Conclusions: Attaining MRD negativity is prognostic at every threshold (10-6, 10-5, 10-4)as well as attaining PET negativity in NDMM patients. clonoSEQ® MRD negativity rates at threshold level of 10-5 were attained in 75.9% of NDMM patients achieving ≥CR. Sustained MRD negativity with NGS MRD assessment 12 months apart at threshold level of 10-5 was possible in 97% of patients, while receiving maintenance. On multivariate analysis in NDMM, inability to attain MRD negativity of at least 10-4 holds the similar or worse hazards ratio for progression as R-ISS stage 3 disease.

Disclosures
Nooka: Oncopeptides: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria, Research Funding; GlaxoSmithKline: Consultancy, Honoraria, Other: Personal Fees: Travel/accomodations/expenses, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Karyopharm Therapeutics, Adaptive technologies: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Adaptive Technologies: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Spectrum Pharmaceuticals: Consultancy. Hofmeister:Karyopharm: Honoraria, Research Funding; Nektar: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Oncopeptides: Honoraria; Oncolytics Biotech: Research Funding; Imbrium: Honoraria; Janssen: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding. Dhodapkar:Kite: Membership on an entity's Board of Directors or advisory committees, Other; Lava Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees, Other; Celgene/BMS: Membership on an entity's Board of Directors or advisory committees, Other; Amgen: Membership on an entity's Board of Directors or advisory committees, Other; Janssen: Membership on an entity's Board of Directors or advisory committees, Other. Boise:AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genetech: Membership on an entity's Board of Directors or advisory committees. Lonial:Genentech: Consultancy; Onyx: Honoraria; Millennium: Consultancy, Honoraria; TG Therapeutics: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Consultancy; JUNO Therapeutics: Consultancy; Merck: Consultancy, Honoraria, Other: Personal fees; Abbvie: Consultancy; GSK: Consultancy, Honoraria, Other: Personal fees; Sanofi: Consultancy; Amgen: Consultancy, Honoraria, Other: Personal fees; Takeda: Consultancy, Other: Personal fees, Research Funding; Novartis: Consultancy, Honoraria, Other: Personal fees; BMS: Consultancy, Honoraria, Other: Personal fees, Research Funding; Janssen: Consultancy, Honoraria, Other: Personal fees, Research Funding. Kaufman:Tecnopharma: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; AbbVie: Consultancy; Sanofi/Genyzme: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria; Karyopharm: Membership on an entity's Board of Directors or advisory committees.