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Patients with chronic lymphocytic leukemia (CLL) who achieve blood or bone marrow (BM) undetectable minimal residual disease (U-MRD) status after first-line fludarabine, cyclophosphamide, and rituximab (FCR) have prolonged progression-free survival (PFS), when assessed by an assay with sensitivity 10-4 (MRD4). Despite reaching U-MRD4, many patients, especially those with unmutated IGHV, subsequently relapse, suggesting residual disease

Background: Measuring residual disease during the continuum of care is fundamental to oncology practice. In particular, minimal residual disease (MRD) assessments and trends over time can help inform clinical management, including change in treatment regimen or treatment discontinuation. In patients (pts) with plasma cell and lymphoid malignancies, next-generation sequencing (NGS)-MRD is a valuable tool for assessing MRD and depth of response to treatment. MRD status is strongly prognostic of time to relapse and overall survival in multiple myeloma (MM), acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL), and chronic lymphocytic leukemia (CLL). In this report, we summarize our 2-year experience with clinical implementation of NGS-MRD (clonoSEQ®) testing across a spectrum of plasma cell and lymphoid disease. Methods: This retrospective analysis summarizes our experience using the NGS-MRD Assay (Adaptive Biotechnologies, Seattle, WA) in plasma cell and lymphoid malignancies. The assay uses multiplex polymerase chain reaction (PCR) and NGS to identify, characterize, and monitor unique disease-associated sequence rearrangements or clonotypes of immunoglobulin (Ig) IgH (V-J), IgH (D-J), IgK, and IgL receptor gene sequences, and translocated BCL1/IgH (J) and BCL2/IgH (J) sequences in DNA extracted from high disease burden diagnostic (ID) and post-treatment (MRD) samples. PCR amplification bias control ensures a quantitative read-out of the full B-cell receptor repertoire present in the ID sample and provides direct measure of tumor burden. Our study included pts with plasma cell and lymphoid malignancies, including MM, ALL, CLL, and MCL treated at the Moffitt Cancer Center between March 2017 and March 2019 who had provided at least an ID sample for NGS-MRD testing. Results: A total of 423 ID tests using DNA from bone marrow (BM; n=407) or peripheral blood (PB; n=16) and 384 MRD tracking tests (BM, n=321; PB, n=63) were performed in 297 pts (Table). The median turnaround time from shipment arrival to assay initiation was 2.1 hours and from activation to report date was 7.1 days. For MM, ALL, MCL, and CLL, the numbers of tests ordered, calibration rates (defined as proportion of ID samples with trackable sequence[s]), and mean number of trackable sequences are shown in the Table. More ID tests were ordered than number of pts (range: 108-178%) due to multiple tests performed for each patient. Sequences analyzed for MRD tests included IgH, IgK/IgL, and T-cell receptors β and γ. The proportion of pts with detectable MRD is shown by indication in the Table. In MM, autologous stem cell transplant (autoSCT)-eligible pts or those who achieved excellent initial responses but were transplant-ineligible, were primarily considered for NGS-MRD testing as part of standard of care. NGS-MRD testing was performed prior to autoSCT and post-SCT before initiation of maintenance therapy for prognostication. More than 90% of MM cases with successful NGS-MRD results had trackable clones. Negative NGS-MRD assured excellent disease control and supported the decision to discontinue therapy in some pts with significant toxicities. In pts with ALL, treatment response after induction and/or consolidation guided decision-making for allogeneic (allo) SCT at first remission. MRD burden prior to alloSCT could potentially guide the decisions and timing on performing SCT or conditioning regimen intensity. In pts with MCL, treatment response evaluated by NGS-MRD following 6 cycles of therapy was a decision point in a randomized trial of auto-transplant + rituximab vs rituximab alone (ClinicalTrials.gov: NCT03267433). MRD is also being used to guide the duration of rituximab maintenance therapy. Updated data analysis for all indications, including CLL, is underway and will be presented at the meeting. Conclusions: The NGS-MRD Assay is a highly sensitive diagnostic tool for the observation of deeper disease response to therapy in multiple specimen types and in various lymphoid and plasma cell malignancies. NGS-MRD may assist in therapeutic decision-making or prognostication. NGS-MRD is a sensitive and powerful prognostic tool available for the majority of pts, which will help our understanding of the role of MRD in clinical management of plasma cell and lymphoid malignancies. Table Disclosures Srivastava: Adaptive Biotechnologies: Employment, Equity Ownership. Lee:Adaptive Biotechnologies: Employment, Equity Ownership. Nishihori:Novartis: Research Funding; Karyopharm: Research Funding. Shah:AstraZeneca: Honoraria; Pharmacyclics: Honoraria; Adaptive Biotechnologies: Honoraria; Spectrum/Astrotech: Honoraria; Novartis: Honoraria; Celgene/Juno: Honoraria; Kite/Gilead: Honoraria; Incyte: Research Funding; Jazz Pharmaceuticals: Research Funding. Alsina:Janssen: Speakers Bureau; Amgen: Speakers Bureau; Bristol-Myers Squibb: Research Funding. Baz:Merck: Research Funding; Sanofi: Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Research Funding. Pinilla Ibarz:Abbvie: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; Sanofi: Speakers Bureau; Bayer: Speakers Bureau; TG Therapeutics: Consultancy; Teva: Consultancy; Janssen: Consultancy, Speakers Bureau. Shain:Adaptive Biotechnologies: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Sanofi Genzyme: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees.

Introduction Patients (pts) with CLL who achieve blood or bone marrow (BM) undetectable minimal residual disease (U-MRD), assessed with a sensitivity of at least 10-4 (MRD4), have long PFS after 1st-line treatment with FCR. A proportion of pts with mutated immunoglobulin heavy chain variable gene (M-IGHV) may be cured of CLL, given the reported PFS beyond 10 yrs. In contrast, most pts with unmutated (UM)-IGHV relapse, despite achieving BM U-MRD4. The likelihood of relapse and relapse kinetics relate to CLL biology, including depth of remission and proliferation rate. MRD testing using next generation sequencing (NGS) can achieve sensitivity up to 10-6 (MRD6) and can also be used to test for circulating tumor DNA in plasma, which may reflect residual tumor in the tissue (eg. lymph nodes), not detectable in the cellular fraction of blood or BM. To better assess the depth of remission following first-line FCR treatment for CLL, we performed NGS MRD6 testing in BM, peripheral blood mononuclear cells (PBMC) and plasma of pts enrolled in a single center, prospective, phase II study. All pts had BM U-MRD4 by 4-color flow cytometry (FLC) at the end of treatment (EOT). Methods The following EOT samples from 62 pts were analyzed for MRD6: 57 BM, 29 PBMC and 32 plasma. Adaptive Biotechnologies' NGS MRD assay uses multiplex PCR and NGS to amplify rearrangements within the B cell receptor CDR3 and identify disease associated clonotypes. Specifically, the assay utilizes multiplexed primers complementing V-J rearrangements (IGH, IGK/L) and D-J (IGH) rearrangements. Pre-treatment samples were sequenced to determine the malignant clonotype, which was then quantitated in the EOT samples, within the polyclonal background. Results Regardless of assay sensitivity, U-MRD rates by HTS according to sample type were: 14/57 (25%) in BM; 16/29 (55%) in PBMC; 24/32 (75.0%) in plasma. All pts with MRD+ in plasma simultaneously had detectable disease in either BM or PBMC. Of 16 pts who had U-MRD in PBMC, 7/12 with simultaneous BM samples were MRD+ in BM. In all patients, there was a trend toward increased likelihood of U-MRD in pts with M-IGHV [12/30 (40%) vs 4/24 (17%), OR 3.3 (0.9-12.2), p = 0.06]. Assay sensitivity varies according to amount of DNA in the sample and with malignant clonotype. Sensitivity of at least 10-6 was achieved in 74% and 62% of samples in BM and PBMC, respectively. Among patients with U-MRD by NGS: 4/14 BM (1.06-3.2 x 10-6, median 1.96 x 10-6) and 8/16 PBMC (1.001 x 10-6 to 3.56 x 10-6, median 1.54 x 10-6) samples did not have sensitivity of 10-6. Notably, 3 pts also had detectable disease at a level Median follow-up was 82 months (mo) (range: 28-112.4). Fifty-six of 62 (90.3%) pts are alive; 28 are progression-free. Median PFS for the whole cohort was 89 mo. When PFS was analyzed according to whether MRD was detected or not (regardless of sensitivity), pts with U-MRD at the EOT had superior PFS vs pts with MRD+, whether the sample type used was BM (p=0.02, median NR vs 67 mo) or PBMC (p=0.02, median NR vs 74 mo), Figure A, B. When analyzed according to whether MRD was /=10-6 (MRD6), 40/53 (75%) in BM and 13/21 (62%) in PBMC were MRD6+. For this analysis, pts were excluded if they had U-MRD but assay sensitivity did not reach at least 10-6. Despite smaller n, there were trends toward shorter PFS for pts with MRD6+ vs U-MRD6 (Figure C, D). Discussion The majority of pts with BM U-MRD4 after first-line FCR were MRD6+ and these patients had shorter PFS; MRD analysis with a more sensitive assay may therefore more accurately assign prognosis. Not accounting for sensitivity, a higher proportion of BM than PBMC samples were MRD+. Plasma analysis was uninformative as all pts MRD+ in plasma were MRD+ in simultaneous BM or PBMC samples. Defining prognostically-relevant thresholds for MRD using more sensitive methodology is important, particularly if U-MRD is used a surrogate for PFS in clinical trials or as an endpoint for treatment cessation. MRD6 may become increasingly relevant if venetoclax-based regimens and the addition of novel agents to chemoimmunotherapy achieve U-MRD6 in more patients than FCR. In this retrospective study, a number of samples did not achieve 10-6 sensitivity; optimization of sample collection is important to achieve 10-6 sensitivity in most pts. Finally, additional studies will be required to determine the risk for and kinetics of relapse in patients with low-level MRD6+. Disclosures Thompson: AbbVie: Honoraria, Research Funding; Gilead Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees. Srivastava:Adaptive Biotechnologies: Employment. Hether:Adaptive Biotechnologies: Employment. O'Brien:Acerta: Research Funding; Sunesis: Consultancy, Research Funding; Regeneron: Research Funding; GlaxoSmithKline: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Pharmacyclics: Consultancy, Research Funding; Vaniam Group LLC: Consultancy; Abbvie: Consultancy; Aptose Biosciences Inc.: Consultancy; Pfizer: Consultancy, Research Funding; TG Therapeutics: Consultancy, Research Funding; Janssen: Consultancy; Astellas: Consultancy; Alexion: Consultancy; Kite Pharma: Research Funding; Gilead: Consultancy, Research Funding. Jain:ADC Therapeutics: Research Funding; Celgene: Research Funding; Abbvie: Research Funding; Seattle Genetics: Research Funding; Pharmacyclics: Research Funding; Incyte: Research Funding; Genentech: Research Funding; Infinity: Research Funding; Astra Zeneca: Research Funding; BMS: Research Funding; Pfizer: Research Funding; Servier: Research Funding; ADC Therapeutics: Research Funding; Verastem: Research Funding; Seattle Genetics: Research Funding; Cellectis: Research Funding; Incyte: Research Funding; Celgene: Research Funding; Adaptive Biotechnologioes: Research Funding; Astra Zeneca: Research Funding; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Research Funding; Verastem: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Cellectis: Research Funding; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologioes: Research Funding; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Verastem: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; ADC Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novimmune: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Wierda:Genentech: Research Funding; AbbVie, Inc: Research Funding.