ClonoSEQ Assay Research Articles

Fixed-duration pirtobrutinib plus venetoclax with or without rituximab in relapsed/refractory CLL: the phase 1b BRUIN trial

AbstractPirtobrutinib is a highly selective, noncovalent (reversible) Bruton tyrosine kinase inhibitor (BTKi). Patients with relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) were treated with fixed-duration pirtobrutinib plus venetoclax (PV) or pirtobrutinib plus venetoclax and rituximab (PVR) in this phase 1b trial. Prior covalent BTKi therapy was allowed, but not prior treatment with venetoclax. Patients were assigned to receive PV (n = 15) or PVR (n = 10) for 25 cycles. Most patients (68%) had received prior covalent BTKi therapy. At the data cutoff date, the median time on study was 27.0 months for PV and 23.3 months for PVR. Overall response rates were 93.3% (95% confidence interval [CI], 68.1-99.8) for PV and 100% (95% CI, 69.2-100.0) for PVR, with 10 complete responses (PV: 7; PVR: 3). After 12 cycles of treatment, 85.7% (95% CI, 57.2-98.2) of PV and 90.0% (95% CI, 55.5-99.7) of PVR patients achieved undetectable minimal residual disease (<10-4) in peripheral blood. Progression-free survival at 18 months was 92.9% (95% CI, 59.1-99.0) for PV patients and 80.0% (95% CI, 40.9-94.6) for PVR patients. No dose-limiting toxicities were observed during the 5-week assessment period. The most common grade ≥3 adverse events (AEs) for all patients included neutropenia (52%) and anemia (16%). AEs led to dose reduction in 3 patients and discontinuation in 2. In conclusion, fixed-duration PV or PVR was well tolerated and had promising efficacy in patients with R/R CLL, including patients previously treated with a covalent BTKi. This trial was registered at www.clinicaltrials.gov as #NCT03740529.

Discontinuation of maintenance therapy in multiple myeloma guided by multimodal measurable residual disease negativity (MRD2STOP).

106 Background: Measurable residual disease (MRD) negativity is a potential marker for the absence of disease in multiple myeloma (MM), which could be used to guide treatment cessation. MRD2STOP (NCT04108624) is a prospective study investigating outcomes among patients with sustained multimodal MRD negativity who discontinue maintenance therapy. Methods: Discontinuation of maintenance was permitted if MRD negative by PET/CT, flow cytometry (limit of detection [LoD] 10-5), and NGS by clonoSEQ (threshold 10-6). Patients underwent serial blood testing along with clonoSEQ, flow, and PET/CT annually for 3 years. Concurrent BM aspirate samples also underwent CD138+ immunomagnetic enrichment analyzed using clonoSEQ to achieve MRD 10-7 sensitivity. Primary endpoints were MRD resurgence rate at the 10-6threshold, along with progression-free survival (PFS) and overall survival among those MRD negative by the standard non-enriched clonoSEQ. Results: 83 patients were screened, and 47 patients met eligibility to discontinue maintenance as of 1/21/24. Median age was 66 years (range 39-84). 17 (36%) had high-risk disease at diagnosis (8 with 1q copy abnormalities, 5 with ISS stage 3, 2 with t(4;14), 1 with t(14;16), and 3 with del17p). Most (45/47, 96%) had one line of therapy; 26 (55%) received a triplet and 19 (40%) a quadruplet. Prior autologous transplant was received by 30 (64%) and multi-drug consolidation in 36 (77%) prior to single-agent maintenance. 96% received lenalidomide as maintenance. Median duration of consolidation/maintenance therapy prior to discontinuation was 36 months (range 12-95), including 14 (30%) with &lt;27 months. Median follow-up was 30 months. Of 47 enrolled patients, 5 (11%) experienced disease progression and an additional 6 (13%) had MRD resurgence at 10-6. Of 11 MRD resurgent events, 4 (36%) were MRD 10-7 positive at baseline, and 3 (27%) were MRD 10-7 positive 1 year prior to MRD 10-6 resurgence. 2 second hematologic cancers occurred during follow-up: 1 Hodgkin lymphoma and 1 B-ALL, the latter which resulted in the only patient death on study. The estimated 3-year PFS was 85%, including 93% for patients MRD 10-7 negative (n=40) at baseline and 31% for those MRD 10-7 positive (n=7) at baseline (logrank p&lt;0.001). Among the MRD evaluable patients (n=45), the 3-year MRD-free survival (MRD-FS) was 68%; 78% for patients MRD 10-7 negative (n=38) and 33% for patients MRD 10-7 positive (n=7) (logrank p&lt;0.001). There were no differences in MRD-FS or PFS when stratified by high-risk disease, receipt of transplant, consolidation, or duration of maintenance. Conclusions: Discontinuation of maintenance therapy among patients with MM and multimodal MRD-negativity results in a high rate of sustained MRD-negativity and lack of disease progression. CD138+-enriched MRD samples using the clonoSEQ assay may help to even better identify patients who can discontinue therapy. Clinical trial information: NCT04108624 .

Abstract CT245: Circulating tumor DNA and nivolumab maintenance: A pilot study in diffuse large B cell lymphoma

Abstract Introduction: Molecular minimal residual disease (MRD) status correlates with disease relapse in patients (pts) with diffuse large B cell lymphoma (DLBCL). However, the clinical impact of early treatment intervention a time of detectable MRD (dMRD) prior to clinical relapse is unknown. Checkpoint inhibitor (CPI) therapy has activity in some pts with DLBCL. We hypothesized that early CPI therapy in patients with dMRD without clinical relapse may lower relapse rate. We conducted a pilot study in high risk pts in remission after most recent line of therapy (LOT) in whom maintenance nivolumab (MN) was offered at time of dMRD to assess effect on MRD clearance and relapse rate. Methods: Pts &amp;gt;18 years (yrs) with high-risk DLBCL (ABC-subtype, high grade B cell lymphoma (HGBCL), MYC translocation, relapsed/refractory disease, or Ki67 &amp;gt;90%) who had achieved CR on most recent LOT were eligible. Patients were monitored for detectable MRD (dMRD) based on the clonoSEQ assay for circulating tumor DNA (ctDNA). Pts had monthly MRD assessment and every 4 month imaging for 2 yrs. If conversion from uMRD to dMRD without clinical relapse, pts received MN (240 mg IV q2 weeks) for up 2 yrs. Pts with overt relapse at time of dMRD or dMRD at initial MRD assessment were ineligible for MN. The primary endpoint was rate of conversion from dMRD to undetectable MRD (uMRD). Results: 20 pts were screened and 15 pts enrolled between June 2018 and March 2022. 4 had transformed DLBCL (2 from FL, 1 from MZL, and 1 Richter’s transformation from CLL), 2 pts had HGBCL (one of which was also transformed). All pts with dMRD had radiographic relapse within 4 months of molecular relapse. No pts with uMRD by ctDNA had clinical relapse on study during 2 yrs follow up. Of the 8 pts who enrolled after first LOT, none had molecular or clinical relapse. Of the 7 pts with 2+ prior LOT, 3 had dMRD: two had clinical relapse at same time as dMRD and were taken off study (one had dMRD at baseline MRD assessment and the other 135 days post enrollment after initial period of uMRD). In the single patient eligible for MN, dMRD preceded radiographic relapse by 105 days, 410 days after enrollment. The pt received 2 cycles of MN before clinical progression and was taken off study. This pt failed to achieve conversion of dMRD to uMRD. Conclusion: Due to small sample size and lower than expected relapse, we were unable to demonstrate conversion of dMRD to uMRD using MN. However, this pilot study did confirm high correlation of uMRD and dMRD status with disease free survival and clinical relapse within 4 months, respectively. If strong negative predictive value is confirmed in a larger study, ctDNA MRD testing may be used to spare pts from imaging or to guide additional work up in pts with inconclusive findings on scans. Citation Format: Shazia Nakhoda, Tamarrah Sklarz, Cheyenne Pagan, Matthew Matasar, Zachary A. Frosch, Marcus Messmer, Asya Varshavsky Yanovsky, Rashmi Khanal, Carlyn Tan, Eli Mikkelsen, Henry Fung, Eric Ross, Mark Roschewski, Allison Jacob, Nadia Khan. Circulating tumor DNA and nivolumab maintenance: A pilot study in diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT245.

Abstract 5071: Minimal residual disease (MRD) status by peripheral blood mononuclear cells (PBMCs) and circulating tumor DNA (ctDNA) demonstrates rapid, deep, and sustained response in patients (Pts) with relapsed/refractory follicular lymphoma (R/R FL) treated with subcutaneous (SC) epcoritamab monotherapy in the pivotal phase 1/2 EPCORE™ NHL-1 trial

Abstract Introduction: Outcomes in high-risk R/R FL are poor. Epcoritamab SC, a CD3xCD20 bispecific antibody, demonstrated promising efficacy with a manageable safety profile in pts with R/R FL after ≥2 prior lines of therapy (phase 1/2 EPCORE NHL-1; NCT03625037). Here we evaluated MRD with PBMCs and ctDNA in pts with R/R FL and correlated the results with clinical findings. Methods: Adults with R/R FL 1-3A enrolled in EPCORE NHL-1 received epcoritamab SC in 28-day cycles. MRD analysis was performed on longitudinal plasma and PBMC samples collected at prespecified timepoints (clonoSEQ assay, Adaptive Biotechnologies). Screening tumor biopsies were used to identify trackable tumor clones; samples were quantified as tumor clones detected per mL plasma for ctDNA and per one million nucleated cells for PBMC. Overall response and progression-free survival (PFS) were assessed using Lugano criteria by independent review committee. Results: Of 128 pts in the R/R FL 1-3A cohort, 91 with PBMC data and 100 with ctDNA data were MRD evaluable. Overall MRD negativity was achieved in 67.0% [95% CI: 56.4-76.5] of pts per PBMC and 68.0% [95% CI: 57.9-77.0] of pts per ctDNA. Concordance of subject-level MRD status was high (79 of 89 with PBMC and ctDNA data; 88.8%). In most pts, MRD negativity was reached prior to radiographic complete response (PBMC, 76.4%; ctDNA 58.5%). MRD negativity by PBMC preceded ctDNA (median days to MRD negativity, 28 [PBMC] vs 57 [ctDNA]), suggesting a difference in methodology in detecting tumor DNA. Pts who became MRD negative had prolonged PFS (median not reached). Baseline MRD levels were not associated with clinical outcomes (PBMC; Wilcoxon P=0.59, ctDNA P=0.39). PFS was similar in pts achieving MRD negative status (median not reached, PBMC and ctDNA), independent of high-risk/difficult-to-treat disease (≥4 prior lines, Ann Arbor stage III-IV, bulky disease, double refractory, FLIPI 3-5, and POD24). The overall MRD negativity rate was also similar in high-risk pts, except those with ≥4 prior lines of therapy. Conclusions: Based on the EPCORE-NHL-1 study, one of the first pivotal studies to report MRD in pts with R/R FL, epcoritamab drives rapid, deep, and sustained responses as supported by MRD assessment by both PBMC and ctDNA. MRD results correlate with clinical outcome, and achieving MRD negativity is associated with longer PFS. PFS was similar among all pts who achieved MRD negativity, including those with high-risk disease. Citation Format: David Soong, Işıl Altıntaş, John Karavitis, Kim M. Linton, Wojciech Jurczak, Catherine Thieblemont, Kevin Zhao, Edith Szafer-Glusman, Daniela Hoehn, Elena Favaro, Mariana Sacchi, Maria Jure-Kunkel. Minimal residual disease (MRD) status by peripheral blood mononuclear cells (PBMCs) and circulating tumor DNA (ctDNA) demonstrates rapid, deep, and sustained response in patients (Pts) with relapsed/refractory follicular lymphoma (R/R FL) treated with subcutaneous (SC) epcoritamab monotherapy in the pivotal phase 1/2 EPCORE™ NHL-1 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5071.

Immunoglobulin High Throughput Sequencing (Ig-HTS) Minimal Residual Disease (MRD) Analysis is an Effective Surveillance Tool in Patients With Mantle Cell Lymphoma

IntroductionMantle cell lymphoma (MCL) accounts for 4% to 6% of B-cell non-Hodgkin lymphoma with historically poor outcomes. With the advent of intensive first-line, targeted, and cellular therapies, outcomes have improved, and initial remission can be 8 to 10 years or longer. As patients experience longer remissions, this raises the question of the optimal surveillance modality. Peripheral blood minimal residual disease (MRD) analysis offers a potential alternative to surveillance imaging that is sensitive, less costly, and eliminates the risk of radiation exposure. Materials and MethodsThe clonoSEQ assay (Adaptive Biotechnologies) is an FDA-cleared commercially available Ig-HTS MRD assay with a sensitivity of 1 cell in 1,000,000. We performed a retrospective analysis of 34 patients from 2015 to 2021, who underwent MRD testing after achieving remission with first-line therapy. ResultsWith a median follow-up of 6.5 years, 10-year progression free survival (PFS) was 60% and 10-year overall survival was 92% of the entire cohort. Among 12 patients who sustained a radiographic relapse, peripheral blood became MRD+ either at or prior to the time of relapse in 11 patients (92%). The first MRD+ test had a lead time of 0 to 24 months (median 34 days) prior to radiographic relapse. Only 1 patient had a MRD− result while being found to have progressive disease on imaging. Among 22 patients who sustained continuous clinical remission, 21 have remained MRD−. Several patients were able to enjoy 2 to 4-year intervals without surveillance imaging. ConclusionsOur data suggest that the clonoSEQ MRD assay is an effective surveillance tool for MCL patients following first-line therapy and is predictive of relapse prior to imaging.

Molecular Monitoring with Tumor DNA and Nivolumab Maintenance: A Pilot Study in Diffuse Large B-Cell Lymphoma

Introduction Molecular minimal residual disease (MRD) has been shown to correlate with disease relapse in patients (pts) with Diffuse Large B cell Lymphoma (DLBCL)[1-4]. However, the clinical impact of detectable MRD (dMRD) prior to clinical relapse is unknown. Checkpoint inhibitor (CPI) therapy has some activity in certain pts with DLBCL, particularly after CAR-T therapy attributed to effects on T cell activity [5-8]. We hypothesized that early intervention with CPI may lower relapse rate in pts with molecular recurrence only. Patients were monitored for detectable MRD (dMRD) by circulating tumor DNA (ctDNA) using the clonoSEQ assay monthly. We conducted a pilot study in high risk pts in remission after most recent line of therapy (LOT) in which maintenance nivolumab (MN) was offered at time of dMRD to assess effect on MRD clearance and relapse rate. Methods Pts &amp;gt;18 years with high-risk DLBCL (ABC-subtype, high grade B cell lymphoma (HGBCL), MYC translocation, relapsed/refractory disease, or Ki67 &amp;gt;90%) who had achieved CR on most recent LOT. Pts had monthly MRD and every 4 month CT or PET/CT and exam for 2 years. If conversion to dMRD without clinical relapse, pts received MN (240 mg IV q2 weeks) until clinical relapse or toxicity for up to 2 years (26 cycles). Pts with overt relapse at time of dMRD were ineligible for MN and taken off trial. The primary endpoint was rate of conversion from +MRD to undetectable MRD (uMRD) in pts receiving . Results 20 pts were screened and 15 pts were enrolled between June 2018 and March 2022. 5 pts had non-GCB subtype, 2 had GCB subtype, and two had unknown cell of origin. 4 had transformed DLBCL (2 from FL, 1 from MZL, and 1 Richter's), 2 pts had HGBCL (one of which was also transformed). Of the 8 pts who enrolled after 1 prior LOT, none had molecular or clinical relapse (Figure). In 7 pts who enrolled after 2+ prior LOT, 3 had dMRD. 2/3 had clinical relapse at same time as dMRD and were taken off study– one at initial enrollment with dMRD at first ctDNA screening and one converting from uMRD to dMRD after 135 days post enrollment. In the single patient who was eligible for MN, dMRD preceded radiographic relapse by 105 days, 410 days after enrollment. The pt went on to receive 2 cycles of MN before clinical progression and was taken off study. This pt failed to achieve conversion of dMRD to uMRD. Conclusions Due to small sample size and lower than expected relapse rates, we were unable to demonstrate conversion of dMRD to uMRD using MN. Of the 3 pts with dMRD, 2 were not eligible for MN due to clinical relapse at same time as dMRD and both died due to lymphoma progression. In the single pt who received MN, this intervention did not achieve uMRD or prevent relapse. However, this pt went on to achieve CR to subsequent CAR-T and remains in prolonged remission despite multiple relapsed/refractory disease. It is unknown if this patient's positive outcome was impacted by the effect of nivolumab on T cells and/or related to slower disease kinetics as this pt had a 3 month period between dMRD and radiographic detection and long duration of response to prior lines. A larger study is needed to adequately assess impact of early intervention MN using MRD kinetics, particularly in patients receiving CAR-T. All pts with dMRD had radiographic relapse within 4 months of molecular relapse. No pts with uMRD by ctDNA had clinical relapse on study during 2 year follow up, confirming uMRD correlates with disease free survival. If a strong negative predictive value is confirmed in a larger study, ctDNA MRD testing may be used to spare pts from imaging or to guide additional work up in pts with inconclusive findings on scans.

Impact of Sequence Uniqueness on MRD Monitoring in NGS Immunoglobulin Sequencing: An Analysis of Ig Loci Among >1200 Diffuse Large B-Cell Lymphoma Patients Tested By Clonoseq

Introduction Diffuse Large B-Cell Lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma that most commonly affects older adults. While ~80% of patients achieve complete response to frontline therapy, roughly half are cured while the other half relapse and receive salvage treatment such as transplant or CAR-T therapy, either of which may be curative. Significant efficacy in later lines has been demonstrated with the recently approved bispecific therapies. Due to the aggressiveness of DLBCL and currently available therapies that offer a potential cure, there is growing interest in a monitoring tool that provides additional information to standard disease monitoring where clinical imaging may fall short. clonoSEQ Ò is a next-generation sequencing CLIA LDT which monitors measurable residual disease (MRD) levels from ctDNA of DLBCL patients Methods The clonoSEQ assay sequences the V(D)J regions (specifically, the CDR3) of Ig loci. A baseline (calibration) sample containing high tumor burden is sequenced to determine the clonotypic/dominant sequence(s). The clonoSEQ algorithm assigns a uniqueness score to each dominant sequence based on analyses of Ig locus V, D, and J segments and non-templated nucleotides. This score reflects the probability a that sequence could be independently recreated in a non-malignant cell, including in another person, and is incorporated into the limit of detection (LOD). Consequently, sequences with a lower uniqueness must be observed at a higher rate to be above the LOD and reflect confidence they are tumor related in subsequent MRD samples. Previous reports have explored the uniqueness distribution of Ig loci, but did not consider the impact of LOD on report interpretation. Using data from our database, we assessed ID sample calibration rates and Ig loci uniqueness among patients with orders from the clinical offering and from clinical trials. Our database contains 3657 dominant sequences from 1268 patients. 969 of these patients are from 27 clinical studies, and 302 are from use of the commercially available CLIA LDT assay. Results: Overall, 1535 ID samples have been received and included several different sample tissues and preparation methods. 1285 (83.7%) of these calibrated. Calibration was most successful in FFPE and gDNA preparations from lymphatic tissue or extranodal masses with 806/883 (91.3%). Least successful sample types included whole blood (48/106 [45.3%] calibrated) and skin (9/28 [32.1%] calibrated) which is likely a reflection of disease burden not being present in these tissues among a large proportion of the patients tested. For samples run on the clinical offering where the sample requirements are more stringent, the most common and most successfully calibrated sample type is FFPE lymph node tissue with a calibration rate of 95.2%. Across the population of 1268 DLBCL patients and 3657 dominant sequences, there was a range of 1-9 dominant sequences (avg 2.89; med 3). We assessed the distribution of uniqueness across the Ig loci in these patients. Fig 1a reflects a higher median uniqueness of IgH (VJ), IgH (DJ), and IgL clonotypes relative to IgK. Fig 1b indicates the uniqueness score for each patient with only one dominant sequence, with just 31 (2.4%) of all subjects with expected ‘backgrounds’ of &amp;gt; 1/100,000 of all Ig sequences. Conclusions Our analysis of &amp;gt;1200 DLBCL patients demonstrates that identification of dominant Ig sequences is more successful using lymph tissue and that the majority of these patients rely on multiple dominant sequences for disease tracking. In a small subset of patients (2.4%), disease is only trackable using one lower uniqueness sequence. In these cases, the LOD is particularily informative in providing guidance to contextualize the probability the sequence may not be associated with the disease being monitoried.

Using Next Generation Sequencing to Identify Trackable Clonotypic Sequences for Minimal Residual Disease Testing in AL Amyloidosis

Introduction: Recent studies have shown that deeper free light chain responses are associated with improved outcomes in patients with systemic light chain (AL) amyloidosis, yet the optimal long-term goal remains unclear.(N Engl J Med 2018; 378:518-528) Unlike multiple myeloma, where the role of minimal residual disease (MRD) testing is well established, in AL amyloidosis the methods to best identify MRD, and whether achieving MRD would benefit disease outcomes, warrant further investigation. (Blood Cancer J. 2021 Jun 21;11(6):117; PLoS ONE 15(7): e0235713. https://doi.org/10.1371/journal.pone. 0235713) In AL amyloidosis, the toxic misfolded oligomers and amyloid deposits are both due to clonal immunoglobulin light chains and cause organ damage and death. Hence, the tracking of clonotypic light chain sequences could present a promising approach for detecting the maintenance of profound therapeutic responses. Our study aimed to assess the frequency with which next generation sequencing (NGS) detects light-chain clones consistent with the patient's clonal light-chain isotype and to determine whether the light-chain sequences identified were encoded by light-chain variable region genes considered AL-related.(Journal of Clinical Oncology 2019 37:15_suppl, 8010; Blood (2017) 129 (3): 299-306; PLoS One 2022 Feb 25;17(2):e0264407) Methods: Ninety patients diagnosed with AL amyloidosis between September 2016 and July 2023 at Tufts Medical Center and University of California San Francisco were included. Bone marrow samples from the 90 patients were sent to Adaptive Biotechnologies Inc. (Seattle, WA) for initial clone identification using the clonoSEQ Assay. IMGT/BlastSearch database (Version 1.2.1) was used to identify the clonotypic immunoglobulin light chain variable domains detected by the clonoSEQ assay. Samples were then divided in to two groups based on the consistency of paraprotein isotype with the trackable sequences, and Mann-Whitney U test was used to compare the difference of the numbers of clonotypic sequences. Results: Of the 90 patients with bone marrow samples sent for clone identification, 85 (94.4%) had at least one trackable clonotypic sequence, with a median number of 3 and a maximum of 11. Of the 85 patients with trackable sequences, 67 (78.8%) had an AL paraprotein isotype consistent with the detected clonotypic sequence isotype, 48 with light chain and 19 with heavy chain isotypes. Compared to the inconsistent ones, patients who had consistency in paraprotein and trackable sequences exhibited higher numbers of clonotypic sequences ( P = 0.001). With respect to marrow plasma cell infiltrations, both consistent and inconsistent groups had a median of 15% marrow plasma cells with no statistically significant difference between them by two-tailed Mann-Whitney. Of note, consistent with the restricted pattern of germline gene use in AL, the 48 patients also used AL-related variable region Ig light chain genes; the variable region light chain genes identified were AL-related and included IGKV1-33 (8 identified sequences), IGKV4-1 (4), IGKV1-16 (3), IGKV1-5 (1), GKV1-13 (1), IGLV6-57 (8), IGLV3-21 (6), IGLV1-44 (5), IGLV3-1 (4), IGLV1-40 (3), IGLV2-14 (3), IGLV1-51 (2). Conclusion: In patients with AL amyloidosis, the ClonoSEQ assay can identify a sequence consistent with the patient's light or heavy chain isotype almost 80% of the time. Of those cases in which a clonal light chain sequence was consistent with the patient's isotype, the genes identified were from the well described AL-related light chain variable region gene repertoire. These findings indicate that the size of the AL clone does not affect this outcome and suggest that other factors related to the adaptive immune dysregulation of AL or to the multiplex PCR process may be involved. ClonoSEQ can identify trackable clonotypic light chain sequences that are AL-related 56% of the time in patients with AL, a finding that may be relevant to patients with myeloma.

Minimal Residual Disease (MRD) Status Predicts Outcomes in Patients with Follicular Lymphoma (FL) Treated with Chemo-Immunotherapy on SWOG S0016

Background. FL is associated with a heterogeneous clinical course, and currently available prognostic indicators are unable to reliably separate patients who exhibit early therapy resistance (POD24, defined as progression within 24 months from diagnosis) from those who achieve “functional cure” following initial therapy. NGS methods developed in recent years enabled quantification of MRD. The ClonoSeq assay (Adaptive Biotechnology, Inc.) has been FDA-cleared to track MRD in several hematologic malignancies. Here we demonstrate that MRD analysis by ClonoSeq assay predicts outcomes in a prospective cohort of patients with advanced stage FL enrolled on SWOG S0016 clinical trial. Methods. SWOG S0016 was a phase III randomized study that compared the safety and efficacy of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) with CHOP-RIT (CHOP + I 133-tositumomab) in patients with FL. Between 2001 and 2008, 531 patients with previously untreated advanced-stage FL (bulky stage II, III-IV) of any pathologic grade were randomly assigned to receive 6 cycles of R-CHOP or CHOP-RIT. We used high-throughput sequencing (HTS) of the IGH and IGK/L loci (ClonoSeq assay) from tumor biopsy specimenstaken prior to treatment to define CDR3 rearrangements that tag the tumor. Subsequently, 1-year bone marrow specimens were screened for residual disease. The baseline dominant clone was defined as ≥10 estimated number of genomes (ENG) and comprised ≥5% of the library. MRD-positive state (MRDp) was defined as a minimum Hamming distance (HD) ≤6 in a patient's post-library (in any clone also detected in a pre-library) and conserved pre-existing SHM (if HD&amp;gt;0). Undetectable MRD (MRDu, at &amp;lt;10 -4) was defined as CRD3 HD&amp;gt;6 in a post-library with a depth &amp;gt;10,000 ENG. Five-year progression-free survival was the primary endpoint (PFS; defined as the time from registration to the first observation of progression or death due to any cause). Landmark analysis was defined at 1 year after registration and used for imputing the 5-year PFS and OS. Overall survival (OS) was defined as the time from registration to date of death due to any cause or last follow-up. PFS and OS estimates and 95% confidence interval were calculated using the Kaplan-Meier method and compared for MRDp and MRDu disease using two-sided log-rank test. HR and 95% CI were calculated using Cox regression model. Results. Tumor biopsy tissue was available for 189 patients. Of these, 24 patients were not evaluable (16 did not consent for banking, 6 were ineligible and 4 had an early event). In 36 patients, pre-libraries did not yield a trackable clone. In total, MRD status could not be ascertained in 47 patients due to absence of a clear trackable clone in either pre- or post-library, contributing to a 28.8% failure rate. Of 116 patients which yielded a trackable clone in both libraries, 83 were MRDu and 33 were MRDp. Baseline characteristics (age, sex, race, β2M, B symptoms, bulky disease, grade 3, stage, and FLIPI risk) were similar between MRDp and MRDu patients; however, MRDp patients were more likely to exhibit morphologic baseline bone marrow involvement by FL (82% vs. 55%, p=0.01). The estimated 5-year PFS was 72% in MRDu patients and 30% in MRD+ patients (Figure); 10-year PFS was 56% and 17%, correspondingly. Meanwhile, 5-year OS was not different (96% for MRDu, 81% for MRD+, p=0.91). Patients with MRDu had a significantly higher best overall response rate than MRDp patients (96% vs 79%, p=0.005), but complete response (CR) rates were similar (41% vs. 30.3%). Patients who received CHOP-RIT had better PFS regardless of MRD status (HR=0.48, p=0.01 for MRDu; HR=0.38, p=0.02 for MRDp), while OS was not statistically different. Five-year PFS was superior in patients who achieved MRDu (75% for CR and 70% for PR) compared with MRDp patients, regardless of response (34%). Patients who had MRDp status had a significantly increased risk of experiencing POD24 relative to MRDu patients (RR=6.5, 95% CI 3.0-14.0; p&amp;lt;0.0001). Conclusion. Here we for the first time demonstrate that MRDu status, as assessed by Clonoseq, predicts improved 5- and 10-year PFS in patients with FL initially treated with chemoimmunotherapy. MRD+ status in the bone marrow at a 1-year timepoint was associated with POD24. Despite a relatively high failure rate to detect trackable sequences using current technology (~29%), MRD assessment by NGS is a promising prognostic tool in FL. *co-senior authors

A Single Center, 10-Year Experience of CALGB-10403 Regimen in AYA Population with Philadelphia-Chromosome Negative Acute Lymphoblastic Leukemia

INTRODUCTION: The use of pediatric-inspired regimens for the treatment of acute lymphoblastic leukemia (ALL) has now become the standard of care in adolescents and adults (AYA). One such regimen, CALGB 10403, had higher rates of event free survival (EFS) and overall survival (OS) compared with historical controls using adult ALL regimens. It was shown to be feasible and efficacious in fit patients up to the age of 40 (Wendy Stock et al., Blood 2019). In 2012, based on emerging data on the improved efficacy of pediatric-inspired regimens in AYA patients, CALGB 10403 was adopted as the standard-of-care regimen in this population at Karmanos Cancer Institute (KCI), an adult cancer treatment center. Here we report our single center, real world experience of using this regimen in a Philadelphia chromosome negative (Ph-) AYA population, approximately 10 years after its adoption. METHODS: We conducted a retrospective chart review of consecutive AYA (ages 18-39) patients with Ph- ALL treated with CALGB 10403 regimen between January 2012 and June 2023 at KCI. Patients who received alternative regimens due to distance from KCI were excluded. Baseline demographic, clinical and molecular characteristics were noted. High-risk cytogenetics were defined as KMT2A rearrangements, hypodiploidy with a chromosome number ≤43, and complex karyotype. Testing for Ph-like ALL was not available in most patients. Beginning in 2019, clonoSEQ assay was used to test for minimal residual disease (MRD), with MRD negativity defined at a threshold of less than 10 -4. Incidence of adverse effects and treatment related mortality were reported to describe safety. Complete response (CR) rate, OS and PFS were used as efficacy outcomes. Kaplan-Meier curves were used to estimate OS and PFS. RESULTS: A total of 17 Ph- AYA patients were treated at KCI with a median age at diagnosis of 25 years (range 18-34). Males and females had a near equal representation (47% and 53%). The patients were predominantly White (64%). The majority of patients had a good ECOG performance status of 0 or 1 (88%). The diagnosis was B-ALL in 9 (53%) and T-ALL in 8 (47%) patients. High risk cytogenetics were present in 3 (80%) of the evaluable patients. None of the patients had CNS involvement at diagnosis. Baseline laboratory parameters expressed as medians were bone marrow blast percentage 80%, WBC count 9.2 x1000/mm 3, hemoglobin 8.4 g/dL, platelets 109 x1000/mm 3, and LDH 480. The median duration of follow-up was 54 months. 15 (88%) patients had CR with induction, and MRD was negative in 4 (57%) of the 7 evaluable patients. 2 (12%) patients had primary refractory disease. One of the patients with refractory disease underwent further therapy with blinatumomab, inotuzumab, CAR-T and allogeneic stem cell transplantation and is alive at 16 months from diagnosis. Another underwent salvage with hyper-CVAD and allogeneic stem cell transplant, and is currently alive at 88 months from diagnosis. Two additional patients were MRD positive after induction (course I) but subsequently attained MRD negativity. One of these eventually relapsed 7.5 months from diagnosis with a mixed phenotypic (T/myeloid) leukemia and died at 10 months, while the other is currently under treatment. An additional patient with CR1 and unknown MRD status had CNS relapse at 35 months, underwent successful hyper-CVAD, reintroduction of intrathecal chemotherapy and allo-HSCT salvage and is alive at 122 months. The 5-year PFS and OS were 74.5% and 94% respectively and the medians were not reached for both. Noteworthy adverse effects include neutropenic fever (65%), liver function tests (LFT) abnormalities (70%), and neuropathy from vincristine (70%). Peg-asparaginase administration was associated with pancreatitis in 12%, thromboembolism in 30% and hypersensitivity reactions in 24% requiring switching to alternative forms of asparaginase products (erwinia and Rylaze). These toxicities were manageable and there were no treatment-related deaths in our cohort. CONCLUSIONS: The pediatric-inspired regimen CALGB 10403 was safe and efficacious in our AYA Ph- ALL population with no deaths due to treatment and a 5-year overall survival of 94%. Toxicities, especially from peg-asparaginase, should be closely monitored. While most patients will attain durable remission/cure, the minority with refractory or relapsed disease can still be successfully salvaged to achieve long term survival.

Sequential T-Cell Engagement for Myeloma (“STEM”) Trial: A Phase 2 Study of Cevostamab Consolidation Following BCMA CAR T Cell Therapy

Background and significance: The BCMA-targeted CAR T products idecabtagene vicleucel (ide-cel) and ciltacabtagene autoleucel (cilta-cel) are currently approved for relapsed/refractory multiple myeloma (RRMM) patients with ≥4 prior lines of therapy, including an IMID, proteasome inhibitor, and CD38 antibody. However, despite unprecedented response rates, CAR T cells are not curative in these late-line patients, even for those in complete remission. Mechanisms of resistance may include lack of persistence or poor function of persisting CAR T cells, as well as BCMA-low or -negative residual tumor cells that serve as a reservoir for relapse. FcRH5 is another MM cell surface antigen, with expression independent of BCMA. Cevostamab is an FcRH5-targeted, T cell-engaging bispecific antibody (bsAb) with demonstrated activity in RRMM, including in patients with prior BCMA-directed therapies (Trudel et al, ASH 2021, #157). We hypothesize that consolidating BCMA CAR T cell therapy with a bsAb targeting a different antigen may re-invigorate persisting CAR+ T cells against residual BCMA+ tumor cells, while also activating endogenous T cells against FcRH5+, BCMA-low/negative tumor cells, ultimately improving rates of sustained minimal residual disease (MRD)-negativity and durability of response. Study design and methods: This is a single-institution, investigator-initiated study (NCT05801939) sponsored by the University of Pennsylvania, with funding support from Genentech. Targeted population are patients with RRMM who have received a commercially available CAR T cell product (ide-cel or cilta-cel) according to the FDA label, within the past 8 weeks, with stable disease or better. Major inclusion criteria include absolute neutrophil count ≥ 1, hemoglobin ≥ 7, platelets ≥ 50, and creatinine clearance ≥ 30 ml/min. Major exclusion criteria include prior cytokine release syndrome or ICANS ≥ grade 3, or any grade hemophagocytic lymphohistiocytosis (HLH) or Parkinsonism, or any active infection. The study schema is shown in Figure 1. Cevostamab is initiated 8-10 weeks after CAR T cell infusion. This timepoint was chosen to allow recovery from acute CAR T cell-related toxicities, but while CAR T cells may still be detectable in circulation. Cevostamab is given at a step-up dose of 3.6mg intravenously (IV) on Cycle 1, Day 1 (C1D1), followed by full dose of 160mg IV on C1D8. Subjects are hospitalized for 48 hours after each C1 dose to monitor for CRS and ICANS. They then continue cevostamab every 3 weeks for total of 8 cycles. If they are in an MRD-negative complete response (CR) after 8 cycles (Adaptive Clonoseq assay, at 10e-5 sensitivity), they stop therapy and are observed. If not, or if bone marrow results are indeterminate, they get another 8 cycles of cevostamab, then stop and are observed. The primary endpoint is frequency of MRD-negative CR at 12 months post-CAR T cell therapy. Assuming a roughly equal proportion of patients enrolling after ide-cel and cilta-cel, the null hypothesis is that the true MRD-negative CR rate at 12 months is 35%. Twenty-six evaluable subjects will be accrued in a single-stage design. The null hypothesis will be rejected if 14 or more subjects meet the primary endpoint. This design yields a one-sided type I error rate of 0.05 and power of 0.84 for an exact test when the true 12-month MRD-negative CR rate is 60%. Secondary endpoints include feasibility, safety/tolerability, and other efficacy measures (overall and CR rates, PFS, OS). Exploratory endpoints include 1) impact of cevostamab on pre- and post-therapy frequency and phenotype of both CAR+ and CAR-negative T cells in blood and marrow, assessed by multiparameter flow cytometry; 2) pre- and post-therapy expression of BCMA and FcRH5 on myeloma cells (when available), serum concentrations of soluble BCMA and FcRH5, and relationship of these factors to clinical outcome measures, and 3) pre- and post-therapy genotypic and phenotypic make-up of bone marrow microenvironment, assessed by single cell RNA sequencing (scRNAseq) and multiparameter flow cytometry, and relationship of these factors to clinical outcome measures. Conclusions: This phase 2 study is exploring the efficacy, safety, and feasibility of cevostamab consolidation following BCMA-directed CAR T cell therapy for RRMM, with the goal of sequential T cell engagement against 2 different antigens to eliminate residual disease. Accrual started in July 2023.

Blinatumomab Therapy for Low Level Minimal Residual Disease Detected By Next-Generation Sequencing in Adult B-Cell Acute Lymphoblastic Leukemia

Introduction: Blinatumomab, a bi-specific T-cell engager, has shown clinical benefit in patients with relapsed-refractory and hematologic complete remission with minimal residual disease (MRD)-positive Acute Lymphoblastic Leukemia (ALL). The BLAST trial (Gökbuget N et al., Blood. 2018) demonstrated improved overall survival (OS) and disease-free survival (DFS) in patients with MRD-positive disease (≥0.1%) who achieved MRD response with Blinatumomab. We sought to evaluate the utility of Blinatumomab in low levels of MRD (&amp;lt;0.1%) using clonoSEQ with a sensitivity of (0.0001%). Methods: Adult (18+) B- ALL patients that underwent treatment for B-cell ALL at Norris Comprehensive Cancer Center (NCCC) between 2016 and 2022 who had residual disease tracked using the Next Generation Sequencing (NGS)-based clonoSEQ assay (Adaptive Biotechnologies, Seattle, WA) were included. Patients who received Blinatumomab for MRD positivity were included only if they had a tracked clonoSEQ sequence within one month before starting treatment. Patients who didn't receive Blinatumomab were included if they had an MRD-positive clonoSEQ report. Those who were relapse/refractory when found to be MRD positive were excluded. Cumulative incidence of relapse (CIR) was evaluated using competing risk regression (Fine-Gray method), with death as a competing outcome. At the same time, OS and DFS were analyzed using Cox proportional hazards model. The start time was set to the date of the MRD report, and patients were censored until the last follow-up. Confounder-adjusted KM curves were calculated using direct standardization. Results: 47 patients were included with a median age at diagnosis of 45 years (range: 21-70). Most were Hispanic (N = 33, 70.2%), and the median follow-up time was 12 months. The 3-year OS, DFS, and CIR were 95.4% (95% CI 89.3-100%), 75.1% (95% CI 60.4-93.5%), and 20.3% (95% CI 7.2-38.0%). Twenty-six patients were treated with Blinatumomab (median two cycles, range 1-9), with most (N= 14, 53.8%) having low levels of MRD (&amp;lt;0.1%). Fourteen patients (53.8%) achieved MRD negativity with a median time to MRD negativity of 3.1 months. There were no relapses or death in patients who achieved MRD negativity in the treated group. However, non-responders with Blinatumomab had one death and two relapses. All patients continued Maintenance therapy after receiving Blinatumomab and did not proceed with the transplant. The patients who received Blinatumomab were more likely to be MRD positive on multiparameter flow cytometry (MFC) (60.0% vs. 28.6%, P = 0.042) but had a similar distribution of patient age, treatment type, and receipt of transplant. Additionally, there was a trend toward increased median percent residual cells on clonoSEQ (0.018% vs. 0.003% P =0.244) and flow (0.02% vs 0% P =0.066) in the Blinatumomab treatment group. On univariate analysis, significant predictors of CIR and DFS were flow percent (DFS: HR = 1.54; 95% CI 1.07-2.23; P = 0.019; CIR: HR = 1.83; 95% CI 1.38-2.41; P&amp;lt;0.001) and clonoSEQ residual cell count(DFS: HR = 1.58; 95% CI 1.04-2.41; P = 0.031, CIR: HR = 1.88; 95% CI 1.18-2.97 P=0.008). When controlling for the number of residual cells on close, patients who received Blinatumomab had significantly better CIR(HR = 0.11; 95% CI 0.02-0.69; P = 0.019) and a trend towards improved DFS(HR = 0.21; 95% CI 0.04-1.28; P = 0.092), but no difference in OS. On subgroup analysis of the 31 patients with low levels of MRD (&amp;lt;0.1%), there was no improvement in DFS for patients who were treated with Blinatumomab (HR = 0.41; 95% CI 0.04-3.91; P = 0.44). Of those with low levels of MRD, only two relapsed, and both did not receive Blinatumomab. Conclusions: In B cell ALL, Blinatumomab may offer a CIR benefit to patients with low levels of MRD as detected on clonoSEQ.

Next-generation sequencing-based MRD in adults with ALL undergoing hematopoietic cell transplantation.

Measurable residual disease (MRD) is an adverse prognostic factor in adult patients with acute lymphoblastic leukemia (ALL) undergoing hematopoietic cell transplant (HCT). Next-generation sequencing (NGS) can detect MRD with a sensitivity of 10-6, but the prognostic value of NGS-based MRD in adult patients with ALL undergoing HCT remains minimally studied. To evaluate the prognostic value of NGS-based MRD in adult patients with ALL undergoing HCT, patients aged ≥18 years with ALL who underwent allogeneic HCT at Stanford University or Oregon Health & Science University between January 2014 and April 2021 and were evaluated for MRD using the NGS-based clonoSEQ assay were included in this study. MRD was assessed before HCT (MRDpre) and up to 1 year after HCT (MRDpost). Patients were followed up for leukemia relapse and survival for up to 2 years after HCT. In total, 158 patients had a trackable clonotype for MRD monitoring. The cumulative incidence of relapse was increased at all levels of MRDpre, including in patients who had low MRDpre of <10-4 (hazard ratio [HR], 3.56; 95% confidence interval [95% CI], 1.39-9.15). In multivariable analysis, MRDpre level remained significantly prognostic; however, detectable MRDpost was the strongest predictor of relapse (HR, 4.60; 95%CI, 3.01-7.02). In exploratory analyses limited to patients with B-cell ALL, the detection of post-HCT immunoglobulin H (IgH) MRD clonotypes, rather than non-IgH MRD clonotypes, was associated with relapse. In this analysis across 2 large transplant centers, we found that the detection of MRD by NGS at a level of 10-6 offers significant prognostic value in adults with ALL undergoing HCT.

Comparison of next-generation sequencing and flow cytometry in detecting minimal residual disease in adult acute lymphoid leukemia: Evaluating clinical outcomes in a single-center study.

7033 Background: Detection of measurable residual disease (MRD) is an important biomarker to direct treatment for adult patients with acute lymphoblastic leukemia (ALL). Currently, multiparameter flow cytometry (MFC) is standard of care and can detect residual leukemia cells in 1 out of 100,000 cells (1 × 10−5). Routine use of next-generation sequencing (NGS) assays with higher levels of sensitivity may improve disease-free survival and overall survival in adult ALL patients. The clonoSEQ MRD assay (Adaptive Biotechnologies Corporation, USA) can detect MRD at a sensitivity of 1 × 10−6 . In this study, we compare the detection of MRD by clonoSEQ assay and MFC in adult ALL patients at a single institution. Methods: We performed a retrospective study evaluating 92 adult patients with B- or T-ALL admitted to Norris Comprehensive Cancer Center between 2014 and 2022. Patients were excluded if they did not achieve complete remission (N=9) or if they presented with prior relapse (N=27). Median time to first complete remission (CR1) was 2.38 months. Among our cohort (N=56 patients), all patients underwent pre-treatment bone marrow sampling which was used for Clonoseq assay. 52 patients had contemporaneous MFC which was used for comparative analysis. MRD on MFC was defined as residual leukemia cells at a sensitivity of 1 × 10−5. MRD on Clonoseq assay was evaluated at 1 × 10−6; residual cells under this limit were considered MRD−. Results: Demographics are summarized in the table. Median time to follow-up from MRD assessment was 18.0 months. 10 patients relapsed (17.9%), 3 patients died (5.4%), and 20 patients (35.7%) underwent allogeneic transplant during first remission. Among 52 patients analyzed by both MFC and NGS at CR, 26 (50%) were concordantly MRD− by NGS and NGF, 13 (25%) were discordantly MRD− by NGF but MRD+ by NGS, and 13 (25%) concordantly MRD+ by NGS and NGF. Residual cell levels between NGS and NGF were strongly correlated (R-Squared = 0.6744, p&lt;0.001), however, MRD status was significantly different between NGS and NGF (p&lt;0.001 using Fisher’s Exact Test). This difference was likely driven by disparities in the limits of detection between both methods. Conclusions: Assessment of MRD using the clonoSEQ assay over a standard MFC-based approach adds crucial prognostic information among adult ALL patients who achieved CR. [Table: see text]

Five-year follow-up update of defined-composition CD19 CAR T-cell therapy for relapsed/refractory CLL.

7511 Background: We have described high response rates in patients (pts) with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL) treated with CD19-targeted CAR T cells. We now report comprehensive analyses of factors associated with duration of response (DOR) with a 68-month median follow-up. Methods: We included 49 pts with R/R CLL and/or Richter’s transformation (n = 10) treated on a phase I/II clinical trial of defined-composition CD19 CAR T-cell therapy (1:1 ratio of CD8+:CD4+). Response rates were investigator-assessed by 2018 iwCLL criteria. Measurable residual disease (MRD) in bone marrow (BM) was evaluated by multiparameter flow cytometry (MFC) and IGH next-generation sequencing (NGS; clonoSEQ assay). CAR T-cell enumeration was assessed by MFC. Serum cytokine concentrations (20 analytes) were measured by Luminex assay. Univariate associations between duration of response (DOR) and patient, disease, CAR T-cell manufacturing, and treatment-related variables were estimated using Cox regression (50+ variables). Association between CAR T-cell in vivo persistence and DOR was modeled with a time-dependent Cox model including blood CAR transgene levels (copies/µg genomic DNA). Results: Median age was 61 years (IQR, 55-67). Forty-six pts (94%) had complex karyotype and/or 17p deletion; 46 (94%) received cyclophosphamide/fludarabine lymphodepletion. In the response-evaluable cohort (n = 47), median follow-up was 68.3 months (IQR, 54.3-81.6). Overall and complete response rates at day+28 were 70% and 17%, respectively, and the median DOR was 22.2 months (95% CI, 10.7-not reached [NR]). MRD-negativity at day+28 by MFC occurred in 33/47 (70%). In pts with MRD-negativity by MFC and available data, MRD negativity by NGS was seen in 18/29 (62%). In NGS MRD-negative responders, the median DOR was 56.8 months (95% CI, 39.5-NR). In all evaluable patients, the 5-year OS and PFS were 35.5% (95% CI, 24.0%-52.5%) and 21.5% (95% CI, 12.0%-38.5%), respectively. In univariate Cox regression, day+28 MRD-negativity by MFC (HR = 0.03, 95% CI, 0.01-0.18, p &lt; 0.001), day+28 MRD-negativity by NGS (HR = 0.21, 95% CI, 0.07-0.61, p = 0.004), peak CD8+ CAR T-cell expansion (HR = 0.49, 95% CI, 0.24-1.00, p = 0.05), CAR T-cell persistence (HR = 0.71, 95% CI, 0.54-0.91, p = 0.01), and lower peak MIP-1β (HR = 3.12, 95% CI, 1.13-8.65, p = 0.03) were associated with longer DOR. Peak MIP-1β remained associated with DOR (HR = 3.83, 95% CI, 1.35-10.9, p = 0.012) in a multivariable model including pre-LD CLL cells in BM, tumor cross-sectional area, peak CD8+ CAR T-cell expansion, and day+28 MRD by MFC. Conclusions: CD19 CAR T-cell therapy achieved durable remissions in R/R CLL pts. Day+28 MRD, CAR T-cell peak expansion, and CAR T-cell persistence were strongly associated with DOR. Lower peak serum levels of MIP-1β, a cytokine known to be produced by CLL cells upon BCR activation, was strongly and independently associated with DOR. Clinical trial information: NCT01865617 .

Evaluation of ctDNA in a phase I/II trial in relapsed or refractory large B‐cell lymphoma of epcoritamab, a novel, subcutaneous CD3xCD20 bispecific T‐cell–engaging antibody

Introduction: The emergence of novel, more potent therapeutics in the treatment of patients with large B-cell lymphoma (LBCL) has resulted in higher rates of responses and highlighted a need for additional methods, such as minimal residual disease (MRD), to determine the depth and quality of response and potentially guide duration of treatment. Epcoritamab (DuoBody®-CD3xCD20) is a subcutaneously (SC) administered bispecific antibody (bsAb) that simultaneously binds to CD3 on T cells and CD20 on malignant B cells, inducing activation and cytotoxic activity of T cells for the killing of malignant CD20 B cells. In the pivotal epcoritamab trial, a correlation was demonstrated between MRD by clonoSEQ® (Adaptive Biotechnologies) and long-term outcomes. These findings led to additional analyses to further clarify the clinical use of ctDNA. As the evaluation of ctDNA in LBCL has not been standardized, we have expanded our investigation of the utility of ctDNA measurements from the initial analysis with the clonoSEQ assay to the AVENIO ctDNA assay (Roche). Here we present additional exploratory analyses of ctDNA using the AVENIO assay. Methods: Patients with relapsed/refractory (R/R) LBCL enrolled in EPCORE NHL-1 expansion phase (NCT03625037) received SC epcoritamab administered in 28-d cycles. ctDNA was measured using the clonoSEQ and AVENIO ctDNA assays at protocol-specified timepoints. Specimens were collected in EDTA and Streck cfDNA tubes, respectively, based on the recommended sample type at the time of study initiation. Plasma ctDNA levels were quantified per sample as count per mL (CPM) and mutant molecules per mL (MMPM) for clonoSEQ and AVENIO, respectively. Results: ctDNA quantification was concordant between the assays when within the dynamic range of the clonoSEQ assay (>80% in paired samples tested); however, a greater sensitivity of the AVENIO assay was observed, allowing further exploratory analyses of ctDNA using this platform. Consistent with prior observations, baseline ctDNA levels were associated with key clinical parameters, including LDH, TMTV, ECOG, and IPI. In patients who responded to epcoritamab, ctDNA levels decreased rapidly, with the majority of patients achieving CR having a deep ctDNA response by C3D1. Additional patients achieved MRD-negative status at later evaluations with continued therapy. MRD-negative status at C3D1, measured as absolute or relative change from baseline, was associated with longer progression free survival (PFS) and sustained MRD negativity throughout the duration of treatment. The research was funded by: Genmab A/S and AbbVie Keywords: Aggressive B-cell non-Hodgkin lymphoma, Liquid biopsy, Minimal residual disease Conflicts of interests pertinent to the abstract. D. Soong Employment or leadership position: Genmab C. Thieblemont Consultant or advisory role: AbbVie, Amgen, BMS/Celgene, Cellectis, Gilead Sciences, Kite, Novartis, Roche, Incyte, Janssen, Takeda Honoraria: AbbVie, Amgen, Bayer, Cellectis, Gilead Sciences, Incyte, Janssen, Kite, Novartis, Takeda Research funding: BMS/Celgene, Hospira, Roche Educational grants: AbbVie, Amgen, BMS/Celgene, Cellectis, Gilead Sciences, Kite, Novartis, Roche J. Karavitis Employment or leadership position: Genmab B. Hess Consultant or advisory role: ADC Therapeutics, BMS P. Caimi Consultant or advisory role: ADC Therapeutics, BMS, Beigene, Genentech, Lilly, Takeda, Kite pharma, Incyte, Novartis, MEI pharma Research funding: Genentech, Abbvie, ADC Therapeutics T. Feldman Consultant or advisory role: AbbVie, AstraZeneca, Epizyme, Genmab, Gilead/Kite, Karyopharm, Takeda, Seagen Other remuneration: Seagen: Speakers Bureau M. Hutchings Consultant or advisory role: AbbVie, Celgene, Genmab, Janssen, Roche, Takeda Research funding: Celgene, Genentech, Genmab, Incyte, Janssen, Novartis, Roche, Takeda (All Paid to Institution) A. Sureda Employment or leadership position: AbbVie Consultant or advisory role: Takeda, BMS/Celgene, Novartis, Janssen, MSD, Amgen, GSK, Sanofi, Kite, Mundipharma, Bluebird Honoraria: Takeda, BMS/Celgene, Novartis, Janssen, MSD, Amgen, GSK, Sanofi, Kite Research funding: Takeda Educational grants: Takeda, BMS/Celgene, Roche Other remuneration: Takeda, BMS/Celgene, Novartis, Janssen, MSD, Amgen, GSK, Sanofi, Kite: Speakers Bureau E. Szafer-Glusman Employment or leadership position: AbbVie M. Sacchi Employment or leadership position: Genmab M. Jure-Kunkel Employment or leadership position: Genmab N. Adya Employment or leadership position: Genmab C. Chiu Employment or leadership position: Genmab

HSR23-121: Assessment of Sustained MRD Negativity and Reversion in Real-World Multiple Myeloma Patients Using NGS (clonoSEQ Assay)

HSR23-121: Assessment of Sustained MRD Negativity and Reversion in Real-World Multiple Myeloma Patients Using NGS (clonoSEQ Assay)

Specificity & Precision of Minimal Residual Disease Monitoring in DLBCL Using Ig-HTS

Background: Dynamic minimal residual disease (MRD) monitoring in the diffuse large B-cell lymphoma (DLBCL) setting has been shown to predict treatment failure at early timepoints and relapse at later timepoints. Precise and reliable approaches are critically important for accurate detection of residual disease. We sought to evaluate the precision of heavy-chain (IGH) and light-chain (IGK/L) MRD measurement within a standard clinical workflow. Methods: We developed a database of clinical clonoSEQ (Adaptive Biotechnologies) and clonoSIGHT reports (Sequenta, Inc.) identifying the dominant malignant IGH or IGK/L clonotypes from the tumor biopsy samples of 285 patients collected as a part of routine clinical care for MRD monitoring. Included patients had been diagnosed with DLBCL at either Stanford University or the National Cancer Institute, 130 of whom had samples sequenced with the clonoSEQ assay and 155 with the clonoSIGHT assay. A control database was generated from all publicly available immunoSEQ (Adaptive Biotechnologies) B-cell repertoires with IGH or IGK/L sequencing, spanning both malignant and healthy patient contexts. The patients within the clinical DLBCL and control databases were distinct without any overlap. We assessed whether each malignant IGH or IGK/L clonotype from the clinical DLBCL database shared overlap with clonotypes present in the control IGH or IGK/L repertoires. A match was considered positive only if the sequence context of the malignant clonotype was an exact match with the sequence of a control clonotype; exact substrings were allowed given the differences in potential primer sites between assays. Results: The clinical database included 341 dominant IGK/L sequences from 187 patients and 271 dominant IGH sequences from 195 patients, where dominance was determined by the clinical assay. We generated a control IGK/L database of 618,775 sequences from 71 subjects and a control IGH database of 65,017,932 sequences from 1698 subjects. IGH demonstrated high precision (99.3%) with only two clinically dominant sequences demonstrating overlap with the control repertoire. Despite the smaller control database, IGK/L demonstrated significantly reduced precision of 92% (Fisher's exact p-value < 0.001) with 29 sequences demonstrating overlap with the control repertoire, all of which were limited to the subset of patients who had been sequenced by clonoSEQ. At the patient level, 19 of the 105 patients with clonoSEQ IGK/L sequencing had clinically trackable (Fig. 1A), putatively malignant sequences that were shared with the control database (precision 84.7%). The frequency of these clinically dominant sequences in the control repertoires ranged from 8.27x10-8 to 0.575 with a mean frequency of 0.000598. Most non-unique clonotypes were shared across multiple control subjects (range 1-37, mean 13, Fig. 1B). Conclusions: The high rate of overlap seen between putatively malignant, dominant IGK/L clonotypes and a publicly available control repertoire suggests substantially reduced validity of approaches relying on IGK/L tracking for minimal residual disease monitoring. The overlapping light chain clonotypes were not only present at relatively high frequencies in both the clinical samples and control repertoire, but were also seen across multiple control subjects, suggesting MRD assays that track only IGK/L may be unable to reliably differentiate malignant clonotypes from background light chain repertoires. Caution should be used when MRD is detected using IGK/IGL given reduced specificity and precision of these sequences. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clonotypic Mass Spectrometry with Easym Assay for Detection of Measurable Residual Disease in Multiple Myeloma

The presence of measurable residual disease (MRD) is an important prognostic factor for multiple myeloma. However, MRD tests in current clinical use require bone marrow (BM) samples that are uncomfortable, expensive, and time-consuming for patients. BM-based assays are also limited by sampling of a single location within the marrow, which may not accurately represent overall disease burden. Consequently, there is an urgent unmet need for peripheral blood-based MRD testing that would allow for convenient, repeated assessment of MRD. M-protein quantification using mass spectrometry (MS) is promising candidate methodology for peripheral blood MRD monitoring due to its exceptional analytical sensitivity. Specifically, "clonotypic" MS techniques can identify and track a specific M-protein "peptide fingerprint", allowing sensitive and specific measurement of MRD that may exceed BM-based techniques. However, clinical data regarding this approach is limited and no clonotypic MS assays are currently in clinical practice. Consequently, we performed a pilot study to assess the use of clonotypic MS-based MRD testing using the EasyM system (Rapid Novor, Kitchener, ON). We retrospectively identified a subset of patients in our institutional tissue bank who met the following criteria: 1) IgG multiple myeloma, 2) M-protein ≥0.5 g/dL by serum protein electrophoresis (SPEP) at diagnosis, and 3) adequate samples for analysis. Baseline and follow up serum samples were analyzed by the study sponsor in a blinded fashion. EasyM results were subsequently compared to SPEP and, when available, next generation sequencing MRD testing with the clonoSeq assay (Adaptive, Seattle, WA). We used receiver-operator curve analysis to identify cutoffs for percent reduction in EasyM to predict BM-based MRD using the R package "cutpointr". 19 patients were identified for analysis. Median age was 62 and 52% were male. All patients received proteasome inhibitor-based induction and 17 patients received lenalidomide. All underwent autologous stem cell transplant (ASCT) in first remission. A clonotypic signature was identified in all 19 patients from the baseline sample. 54 follow up samples were analyzed. Percent change in EasyM and SPEP were well correlated (Pearson R = 0.808; p < 0.01). 37 timepoints had MRD testing available. Of these, 14 and 9 timepoints were negative at 10-5 and 10-6, respectively; only 3 (8%) had undetectable M-protein by EasyM, all MRD negative at 10-6. Using receiver-operator curve analysis, we identified optimal cutoffs for percent reduction in EasyM by area under the curve for BM-based MRD at 10-5 (99.0%) and 10-6 (99.3%) thresholds. The PPV and NPV for EasyM at 10-5 were 91% and 80%. The PPV and NPV for EasyM at 10-6 were 92% and 58%. Given the half-life of IgG (2 - 3 weeks), we also performed an exploratory analysis for MRD at 10-5 separating day 100 samples (n = 18) from later samples (n = 18). This analysis yielded slightly different optimal cutoffs for percent reduction by EasyM for day 100 (98.2%) versus later timepoints (99.4%), with similar PPV (91% and 92%) and improved NPV (100% for both), suggesting time of sample collection may impact the predictive value of EasyM. At day 100 after ASCT, 7 patients were in CR, 9 in VGPR and 3 in PR. 7 patients progressed during the follow up period. One patient had undetectable EasyM at day 100 after ASCT and received only 4 months of post-ASCT lenalidomide, but remains in CR over 3 years after transplant. Two patients achieved undetectable EasyM at 9 and 55 months, respectively, post-ASCT and remain in CR at 52 and 64 months. Our study provides initial data regarding clonotypic MS with the EasyM assay for peripheral blood MRD testing. We demonstrated excellent correlation between percent reduction in M-protein by SPEP and EasyM. Interestingly, 79% of samples that were MRD negative at 10-5 by clonoSeq had detectable M-protein by clonotypic MS, suggesting EasyM may be a more sensitive test for residual disease. After optimization, EasyM showed excellent PPV and NPV for BM-based MRD negativity, suggesting clonotypic MS be able to identify patients who can forego a bone marrow biopsy for MRD monitoring. We are currently validating this assay in non-IgG isotypes and light chain disease. Overall, clonotypic MS represents a promising new tool for myeloma monitoring and should be performed in larger retrospective cohorts and integrated into prospective trials for further validation.

MRD Assessment Using Next-Generation Sequencing (clonoSEQ® Assay) Among Fee-for-Service Medicare Beneficiaries with Newly Diagnosed Multiple Myeloma Who Received Transplant

Introduction The assessment of minimal residual disease (MRD) for multiple myeloma (MM) has been increasingly incorporated into both clinical trials and patient management. MRD assessment, using an assay with a minimum sensitivity of 10-5 or better, is now recommended within NCCN guidelines following the completion of treatment phases. The clonoSEQ Assay (Adaptive Biotechnologies; Seattle, WA) is currently the only FDA cleared test available for bone-marrow MRD assessment in MM patients, and its use in routine clinical practice across the US facilitates an important new ability to evaluate MRD patterns and outcomes in the real-world setting. This study examined a cohort of Medicare MM patients who underwent transplant, and described characteristics of the treatments and MRD assessment these patients received. Claims-based analyses exploring MM treatments in the real-world have been published (Song 2016, Fonseca 2020), however to our knowledge this is the first study to match distinct claims datasets with corresponding MRD testing patterns and results. Methods The study population includes MM patients who had been assessed for MRD with clonoSEQ, who were successfully matched with 100% Medicare claims data. Newly diagnosed MM (NDMM) patients were identified as adult fee-for-service (FFS) beneficiaries with a diagnosis of MM between January 2017 to December 2021 (the first observed MM diagnosis is Index Diagnosis), with continuous coverage in Medicare FFS Parts A/B/D of at least 6 months during pre-index and 12 months during post-index period. Patients were further required to have initiated MM treatment within 60 days of Index Diagnosis, received a transplant, had at least one MRD assessment with clonoSEQ, and had no enrollment in clinical trials nor prior MM treatment pre-index. Phases of treatment within frontline therapy and line of therapy (LOT) were identified following established rules from the literature. Charlson Comorbidity Index (CCI) was calculated during the 6 months pre-index. We describe patient characteristics, treatment journey, and assessment of MRD test results at a sensitivity of 10-5 in accordance with IMWG guidance. Results The study identified 102 NDMM transplant patients meeting the above criteria, among which 99 received transplant as part of frontline therapy. The mean age of the cohort was 69 years, 53% were male, and 89% were white. The mean CCI was 0.97. One third of patients were diagnosed between 2017-2018. Both the mean and median follow-up time for this cohort are 32 months. Among the 102 patients, approximately 20% received consolidation and 50% received maintenance, as part of their frontline regimen; 44% received second or later LOT. The most common induction therapies used in this cohort were a PI+IMiD+Corticosteroid triplet (53%) or a PI+IMiD+CD38+Corticosteroid quadruplet (16%). On average, transplant patients started treatment 20 days from Index Diagnosis and received transplant 249 days from Index Diagnosis. A total of 171 MRD assessment were performed among this population with an average of 1.7 assessments and a maximum of 4 assessments per patient. Approximately 45% of transplant patients had >=2 MRD assessments and the median interval between adjacent MRD assessment is 196 days. Most of the patients (80%) were being assessed for MRD during frontline therapy, while 29% also received MRD assessment during later lines. Of the 99 patients who received transplant during frontline therapy, 66 (67%) patients had an MRD assessment within 6 months of transplant. Among them, 28 (42%) were MRD negative. Conclusions This is the first study to map treatment phases and line of therapy with Medicare claims data and examine the characteristics of MRD assessment in NDMM transplant patients. The study provides insight into real-world MRD testing patterns and outcomes within the Medicare population using next-generation sequencing. Further applications of this methodology to evaluate therapeutic response and understand MRD-supported decision making in the real-world setting are planned. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal