Next-generation Sequencing Minimal Residual Disease Research Articles

Outcomes and Toxicities after Allogeneic Hematopoietic Cell Transplant for Remission Consolidation or Relapse Therapy after CD19 Chimeric Antigen Receptor T Cell Therapy in Children and Young Adults with Acute Lymphoblastic Leukemia

Introduction: CD19-directed chimeric antigen receptor-modified T cells (CART19) induce remarkably high remission rates in relapsed/refractory B-lymphoblastic leukemia (B-ALL), but 50% of children and young adults experience subsequent relapse. Allogeneic hematopoietic cell transplant (HCT) is frequently used after CART19, either for relapse prevention or to treat post-CART19 relapse. However, there is a paucity of safety and outcome data after HCT in this population. This gap is especially relevant because patients often undergo HCT post-CART19 later in their relapsed disease courses (i.e., CR3 or later) than historical patients undergoing HCT for B-ALL. Methods: We conducted a retrospective review of patients aged <30 years with B-ALL who underwent a first HCT for remission consolidation (preemptive cohort) or relapse therapy (relapse cohort) after CART19 at the Children's Hospital of Philadelphia between 2014-2022. The primary outcome was disease-free survival (DFS) from HCT, stratified by cohort. Secondary outcomes included overall survival (OS) and frequency of transplant-related mortality (TRM), graft-versus-host disease (GVHD), sinusoidal obstruction syndrome (SOS), and IgG replacement dependence. Results: The analysis included 41 patients: 19 in the preemptive cohort (pre-planned consolidative HCT, n=5; early loss of B cell aplasia, n=13; positive next-generation sequencing minimal residual disease [MRD], n=1) and 22 in the relapse cohort (morphologic relapse, n=18; detectable flow-based MRD, n=4) (Table 1). With one exception of a patient treated with a CD19/CD28-based product, all patients received investigational or commercial CD19/4-1BB CAR T cells. Median time from CART19 infusion to HCT was 5 months (range, 2-10) for the preemptive and 14 months (range, 5-40) for the relapse cohort. Between CART19 and HCT, 6/19 (32%) preemptive patients received bridging therapy (chemotherapy, n=3; blinatumomab, n=1; inotuzumab, n=2). In the relapse cohort, salvage therapies that successfully induced HCT-acceptable remissions (MRD <0.1%) included inotuzumab (n=11, 50%), cytotoxic chemotherapy (n=5, 23%), blinatumomab (n=2, 9%), CART22 (n=2, 9%), CART19 reinfusion (n=1, 5%), or pembrolizumab (n=1, 5%). At HCT, the majority (79%) of the preemptive cohort was in CR1 or CR2; whereas, the majority (77%) of the relapse cohort was in ≥CR3. All patients received myeloablative conditioning, either total body irradiation-based (n=39) or clofarabine-based (n=2), and 59% received a T cell depleted graft. In the preemptive cohort, with a median follow-up of 36 months, DFS at 1, 2, and 3 years after HCT was 100%, 93% (95% CI, 61-99), and 85% (95% CI, 51-96), respectively (Fig. 1). OS was 100% through 3 years. In the relapse cohort, with a median follow-up of 34 months, DFS was 58% (95% CI, 35-76) and OS was 68% (95% CI, 44-83) by 1 year, and remained the same through 3 years (Fig. 1). When stratified by CD19 immunophenotype at post-CART19 relapse, DFS by 1 year was 67% (95% CI, 34-86) for CD19-positive and 44% (95% CI, 14-72) for CD19-negative disease (p=0.235). Frequently observed toxicities included GVHD and SOS. In the preemptive cohort, 10 patients (53%) developed acute GVHD (3 [16%] were grade ≥3) and 7 (37%) developed chronic GVHD. In the relapse cohort, 11 patients (50%) developed acute GVHD (4 [18%] were grade ≥3) and 6 (27%) developed chronic GVHD. SOS was observed in 1 (5%) preemptive patient and 7 (32%) relapse patients. Four patients (18%), all in the relapse cohort, died of TRM between days +47-65 after HCT. Three had multisystem organ failure in the setting of multiple complications, including severe SOS. One had respiratory failure due to presumed adenoviral pneumonitis. At 1 year post-HCT, 9/18 (50%) preemptive and 8/12 (67%) relapse patients who remained event-free still required IgG replacement. Conclusion: In children receiving their first HCT for post-CART19 remission consolidation, DFS and OS were exceptionally good, without any observed TRM and with comparable rates of GVHD and other major complications to general pediatric HCT populations. For children who were able to undergo HCT following a post-CART19 relapse, of which 77% were in CR3 or later, DFS and OS were also favorable. The majority became long term survivors and all events occurred within 1 year after HCT. However, higher rates of SOS (likely associated with pre-HCT inotuzumab) and TRM were also observed in this cohort.

A multi-institutional prospective cohort study of minimal residual disease in peripheral T-cell lymphoma: Impact of autologous stem cell transplant.

7563 Background: Roughly 80% of pts with peripheral T-cell lymphomas (PTCL) respond to CHOP-based therapy but 5-yr survival ranges between 20-40%. The high relapse rate after complete response (CR) suggests more sensitive response assessment, such as minimal residual disease (MRD) testing, could have prognostic or predictive value. Next generation sequencing (NGS) of the T-cell receptor (TCR) can detect a known TCR clonotype at 10-5. In a prospective study, we evaluated the utility of MRD testing via TCR NGS in PTCL (NCT03297697). Here we report results for pts pre- and post-autologous stem cell transplant (ASCT). Methods: This investigator-initiated, multi-institutional, prospective cohort study assessed MRD testing by TCR NGS in PTCL. Pts with previously untreated PTCL not otherwise specified (NOS), angioimmunoblastic T-cell lymphoma (AITL), anaplastic large cell lymphoma (ALCL), PTCL with T-follicular helper (TFH) phenotype, or monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) treated with anthracycline-based curative-intent therapy were eligible. Baseline TCR (TRG or TRB) clonotypes were established from diagnostic tissue or peripheral blood if tissue was not available. TCR clonality was determined positive via the LymphoTrack TRG/TRB Assays – MiSeq (Invivoscribe, San Diego, CA) when top % reads were ≥2.5% and ≥2x above background. Samples were obtained at diagnosis (pre-treatment), following induction therapy (EOT/pre-ASCT), and serially post-ASCT. Results: Of 43 enrolled pts, 19 underwent consolidative ASCT, of which 10 had post-ASCT samples available for MRD testing (Table). Median age at ASCT was 62 years (range: 38-77). Baseline TCR clonotypes were identified in 10/10 pts. At pre-ASCT evaluation, 7/10 pts had detectable TCR MRD (‘pos’) and 3/10 pts had TCR MRD below the level of detection (‘neg‘). Of the 3 MRD neg pts pre-ASCT, 2 had CR and 1 had PR by PET-CT. All 7 TCR MRD pos pts pre-ASCT had CR by PET-CT. Of 7 MRD pos pts pre-ASCT, 6 remained MRD pos and 1 became MRD neg post-ASCT. Of the 4 MRD neg pts post-ASCT, none have relapsed at 52, 31, 34, and 31 mo post-ASCT. Of 7 MRD pos pts pre-ASCT, 3 have relapsed following ASCT (at 3, 9, and 29 mo post-ASCT). Conclusions: In this cohort of patients with PTCL receiving consolidative ASCT in CR1, all 4 pts with negative TCR MRD post-ASCT remain in remission at median follow up of 32.5 mo post-ASCT. The negative predictive value of TCR NGS MRD post-ASCT should be further evaluated. Clinical trial information: NCT03297697 . [Table: see text]

Comparison of Next-Generation Flow Cytometry (NGF) and Next-Generation Sequencing (NGS) in the Assessment of Minimal Residual Disease in Multiple Myeloma

Background:Next generation flow cytometry (NGF) and next generation sequencing (NGS) are standard methods for detection of minimal residual disease (MRD) in multiple myeloma (MM) according to domestic and international guidelines. However, due to the inability to standardize laboratory technology among hospitals, the results are unreliable. In order to meet the challenges of data evaluation and comparability between studies in multiple myeloma (MM) minimal residual disease (MRD) assessment，and to further confirm the role of MRD in evaluating deep remission，we provide a approach to evaluate and compare the efficacy of each patient after treatment by NGF and NGS concurrently. Methods: In a VRD clinical study in our center, a cohort analysis was performed on 82 patients with newly diagnosed multiple myeloma in the First Affiliated Hospital of Soochow University from September 01, 2019 to January 31, 2022.Bone marrow (BM) sample from 82 MM patients were analyzed by NGS and NGF MM MRD methods during treating procedure. Limit of detection (LOD) and tumor load (TL) were calculated, and the concept of MRD negative was defined. At the same time, step-by-step statistics and analysis of MRD results were carried out to confirm its role in evaluating the depth of remission, and to analyze and compare whether the two methods were consistent and the difference in detection rate, evaluating the prediction of patient survival by MRD detection. Results:Among the 82 patients, 100.00% of NGF specimens and 96.34% of NGS specimens met the LOD criteria of 1.0e-5. Non-detection of tumor cells at the 1.0e-5 level was defined as NGF-MRD-negative or NGS-MRD-negative. After induction therapy, the positive detection rate of NGS was higher than that of NGF (81.01% vs 51.22%). The patients who did not achieve VGPR serological response were all MRD positive. The positive rate of NGF-MRD was 42.25%, and the positive rate of NGS-MRD was 78.26% in patients with serological response reaching VGPR or above. With the deepening of serological efficacy: from PR to VGPR to CR and sCR, the positive rate of MRD gradually reduced（NGF：100.00%-70.37%-25.00%；NGS：100.00%-96.15%-67.44%). The median tumor load was progressively decreased (NGF: 10-3-10-4-10-6 level; NGS: 10-1-10-2-10-4 level). Compared with NGF, NGS not only has a higher positive detection rate, but also can detect more tumor load. Pairing the NGF-MRD and NGS-MRD results, the agreement between the two methods (NGF+NGS+ and NGF-NGS-) was 63.77%, and the Pearson correlation coefficient indicated a certain correlation in TL quantification (n=54 , r=0.4371, P=0.001). Conclusion: An MRD assessment (at least a 10-5 level) should be performed after obtaining a serologic response to VGPR and above. When analyzing MM-MRD, the LOD value should be marked and the definition of MRD negative should be stated. Compared with NGF, NGS not only has a higher positive detection rate of MRD (67.44% vs 25.00%), but also can detect more tumor load and has higher sensitivity in patients with CR or above after induction therapy. NGF MRD positivity predicts shorter PFS and earlier relapse after induction therapy. NGF and NGS MRD negativity predicts 100.00% in 2-year PFS. Keywords: multiple myeloma (MM); minimal residual disease (MRD); next-generation flow cytometry (NGF); next-generation gene sequencing (NGS); line of detection (LOD); tumor load (TL) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Pro-Active 4SCAR2.0 Therapy with NGS-MRD Disease Monitoring in High-Risk B Cell Leukemia Management

BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HCT) is a standard treatment for high-risk relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, about 30-40% of patients still relapse after HCT. Chimeric antigen receptor-modified T cell (CAR-T) therapy holds great promise as a salvage approach. Although CD19-targeted CAR-Ts have demonstrated a rapid and significant anti-tumor effect in patients with B-cell malignancies, many still suffer disease relapse due to the decrease or loss of CD19 antigen. Therefore, novel curative approaches are needed. PURPOSE: The aim of this study is to determine the best timing for applying CAR-T therapy in high-risk B-ALL patients based on high-sensitivity analysis of tumor cells by next-generation sequencing (NGS) detecting minimal residual disease (MRD). An innovative CAR-T therapy concept, CAR2.0, has been proposed, which uses multi-antigen-targeted CAR-Ts to prevent antigen escape and prolong remission. Based on NGS-MRD detection, patients with potential tumor relapse can be identified early and managed accordingly. On the other hand, if extended negative NGS-MRD is found, the patient is deemed at low risk and no further treatment is needed. The study proposes to treat high-risk B-ALL patients in the first remission period with CAR2.0 followed by continued disease monitoring by NGS-MRD to avoid HCT. METHODS: B-ALL patients who have high tumor burden and high-risk genetic indication is enrolled with the understanding that CAR-T therapy will be applied in the first remission and disease will be continuously monitored by NGS-MRD. The tumor cells from initial diagnosis will be analyzed for target antigens by immunostaining and to detect clonally rearranged immunoglobulin gene sequences by NGS-MRD with sensitivity ≤10-6. Patients will receive 4SCAR19 (CD19scFv-CD28/CD27/CD3ζ-iCasp9) therapy in the first remission and will be followed up for CAR-Ts, B cells and IgG/IgA/IgM in the blood, and bi-monthly NGS-MRD for residual tumor cells in the bone marrow (BM). A booster CAR-T treatment targeting additional tumor antigen such as CD22 will be applied 2 months later. A consolidation CAR-T treatment based on known target antigens will be applied 6 months after the first CAR-T infusion. The cyclophosphamide/fludarabine chemo-conditioning will be included prior to the first CAR-T treatment only. The CAR-T infusion dose is 1-5x106 CAR-Ts/kg. The quality of apheresis cells, efficiency of gene transfer, T cell proliferation, CAR-T infusion dose and blood CAR copies are quantitatively documented. The end point for this study is the establishment of NGS-MRD negativity for 1 year, with continued follow-up for 5 years. RESULTS: A 20 years old female was diagnosed with BCR/ABL1 P190 positive B-ALL, with 82.52% tumor blasts in the BM. NGS identified two high-frequency IgH clones at 66.29% and 30.41%. The patient received induction chemotherapy (VTCP, CAM, VP+MTX) combined with TKI therapy (Nilotinib) and achieved complete remission (CR) after the first round of treatment based on both flow cytometry and NGS MRD analyses. During the fourth round of chemotherapy (Methotrexate-Cytarabine-Dexamethasone), the patient suffered severe oral and intestinal mucositis, along with kidney and liver toxicity and required week-long dialysis. A decision was made to stop chemotherapy and start the first 4SCAR19 therapy of 3.53x106 CAR-Ts/kg, while maintaining the TKI therapy. The CAR-T cells in blood, the B cell count and Ig levels were monitored, and a booster CD22/CD19 dual CAR-Ts (4SCAR2219) was given at a dose of 3.35 x106 CAR-Ts/kg on day 96 (see figure). Consistent with 4SCART safety profile, the patient experienced no greater than grade 1 CRS. NGS-MRD detection of tumor cell clones remained negative on day 96 after the first CAR-T infusion and at 6 months into the remission. In summary, the 4SCAR2.0 therapy regimen appeared safe and effective in the innovative NGS-MRD/4SCAR2.0 B-ALL study regimen with in vivo persistency for more than three months. The simultaneous expansion of CD19 mono-specific and CD22/CD19 bi-specific CAR-Ts supports a stable maintenance treatment with good life quality as compared with conventional chemotherapy. CONCLUSIONS: The combination of 4SCAR2.0 with NGS-MRD detection of tumor clones in the management of high-risk B-ALL appears to be safe and may be a logical option for patients not elected for HCT therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

MM-361 The Prognostic Role and Utility of Highly Sensitive Minimal Residual Disease Assay in Multiple Myeloma: Is There Really a Difference?

The role of higher-sensitivity minimal residual disease (MRD) assays in predicting survival outcomes and guiding treatment decisions remains to be validated in patients with multiple myeloma (MM). To determine the predictive and prognostic role of high-sensitivity next-generation sequencing (NGS) MRD assays in patients with MM undergoing autologous stem cell transplant (ASCT). We performed a retrospective analysis on a cohort of patients diagnosed with MM between 2015 and 2020. All patients underwent front-line ASCT and were evaluated with an NGS MRD assay with a sensitivity of 10-6 at day 100 post-ASCT. Given the retrospective nature of the study, we used the time to next treatment (TTNT) as a surrogate marker for progression-free survival. Overall survival (OS) was included as a secondary analysis. A total of 186 patients were included in the analysis. With a mean follow-up time after ASCT of 30.2 months, patients who achieved MRD negativity at 10-6 had longer TTNT than patients achieving negativity at 10-5 or lower thresholds (HR: 0.289, 95% CI: 0.110-0.758, p=0.0116). On multivariable analysis, adjusting for the R-ISS stage, IMWG response category, and cytogenetic risk status, MRD negativity with a sensitivity of 10-6 was the strongest prognostic factor for longer TTNT. MRD alone did not predict OS, which may be associated with the relatively limited follow-up time. Additionally, patients who achieved sustained MRD negativity, defined as negative MRD confirmed after 1 year, had significantly longer TTNT in both the univariate (HR: 0.07, 95% CI: 0.01-0.63, p=0.0175) and multivariable (HR: 0.0109, CI: 0.0003-0.4199, p=0.0153) models. At cutoff, of the 23 patients with sustained MRD negativity, 13 (57%) had been off therapy for a mean duration of 14 months after the second measurement with no documented progressions or deaths. Overall, MRD negativity at a sensitivity of 10-6 was associated with a longer TTNT, regardless of induction therapy regimen or baseline characteristics. Higher-sensitivity MRD assays can help identify a subset of patients with very low disease burden who have a decreased risk of progression and could help guide therapy-related decisions, including treatment discontinuation.

Impact of autologous hematopoietic cell transplantation on disease burden quantified by next-generation sequencing in multiple myeloma treated with quadruplet therapy.

The incremental impact of autologous hematopoietic cell transplantation (AHCT) on disease burden with quadruplet induction in newly diagnosed multiple myeloma (NDDM) can be reappraised with the serial assessment of minimal residual disease (MRD). We describe the impact of AHCT on MM burden assessed by next-generation sequencing (NGS) for patients enrolled in a clinical trial utilizing quadruplet induction, AHCT, followed by MRD-adapted consolidation. We describe quantitative changes in MRD burden with AHCT and explore patient and disease features influencing the magnitude of MRD reduction with AHCT. Among 123 included patients, 109 underwent AHCT and had MRD assessment pre and post AHCT. Forty percent achieved MRD < 10-5 post-induction, increasing to 70% after AHCT. Of the 65 patients (60%) who remained MRD positive post-induction, 54 (83%) had a reduction in MRD burden with AHCT. The median reduction in MRD with AHCT was 1.10 log10 (range, -1.26 to 3.41). Patients with high-risk cytogenetic abnormalities (HRCA) had greater reduction in MRD burden (p=.02) after AHCT. Median relative reduction was 0.91 log10 (range, -0.75 to 2.14), 1.26 log10 (range, -0.21 to 3.26) and 1.34 log10 (range, -1.28 to 3.41) for patients with 0, 1 and 2+ HRCA, respectively. The presence of HRCA was the only factor associated with greater than 1 log10 reduction in MRD burden with AHCT. Serial NGS MRD demonstrates the incremental effect of AHCT in MM marrow burden in the context of quadruplet induction, particularly in high-risk MM.

Minimal residual disease comparison between Ig/TCR PCR versus NGS assays in children with Philadelphia chromosome-positive acute lymphoblastic leukemia: A report from the COG AALL1631 study.

10023 Background: Minimal residual disease (MRD) assessment by immunoglobulin/T-cell receptor (Ig/TCR) polymerase chain reaction (PCR) is currently being used in the international pediatric Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) trial EsPhALL2017/AALL1631 for risk stratification. MRD concordance has previously been demonstrated between Ig/TCR PCR and flow cytometry in Ph+ALL. We sought to assess concordance of MRD assessment between conventional Ig/TCR PCR and next-generation sequencing (NGS) assays. Methods: MRD was assessed in all pts on AALL1631 by Ig/TCR PCR at end-induction IB; those with MRD &lt;5x10-4 were classified as standard-risk (SR) and randomized to treatment with imatinib and one of two chemotherapy regimens without hematopoietic stem cell transplant (HSCT), whereas pts with end-induction 1B MRD ≥ 5x10-4 were considered high-risk (HR) and assigned to HSCT after consolidation chemotherapy. Residual diagnostic and end-induction IB samples from consenting pts were assessed for NGS MRD by the clonoSEQ assay (Adaptive Biotechnologies) in blinded fashion and subsequently compared to Ig/TCR MRD to determine concordance as related to MRD-based HSCT recommendations ( ie, MRD ≥ 5x10-4 consistent with HR group assignment). MRD values were calculated using the kappa statistic for agreement above chance. Results: Sixty-seven pts had matched samples available for MRD assessment at end-induction 1B by both Ig/TCR PCR and NGS (Table). NGS MRD was evaluable for all 67 pts and stratified as 62 SR (&lt;5x10-4) and 5 HR (≥5x10-4). In contrast, Ig/TCR PCR results were inevaluable for 3 pts (unsatisfactory sample quality) and indeterminate (positive, but not quantifiable) in 4 pts. Of the remaining 60 pts, 55 met SR and 5 HR criteria using Ig/TCR PCR. There was only 1 discordant case between the two methods for MRD-based HSCT recommendation among these 60 pts with a kappa statistic for agreement above chance of 0.88. Conclusions: NGS and Ig/TCR PCR assays were highly concordant in MRD assessment for risk stratification at a threshold of 5x10-4 in pediatric pts with Ph+ALL enrolled on AALL1631. Of note, the NGS assay yielded MRD results amenable for risk stratification in 100% pts compared to 89.6% for the Ig/TCR PCR methodology. These data support the use of NGS MRD testing for risk stratification in pediatric Ph+ALL.[Table: see text]

Capture Rate of V(D)J Sequencing for Minimal Residual Disease Detection in Multiple Myeloma

Minimal residual disease (MRD) negativity is a strong predictor for outcome in multiple myeloma. To assess V(D)J clonotype capture using the updated Adaptive next-generation sequencing (NGS) MRD assay in a clinical setting, we analyzed baseline and follow-up samples from patients with multiple myeloma who achieved deep clinical responses. A total of 159 baseline and 31 follow-up samples from patients with multiple myeloma were sequenced using the NGS MRD assay. Baseline samples were also sequenced using a targeted multiple myeloma panel (myTYPE). We estimated ORs with 95% confidence intervals (CI) for clonotypes detection using logistic regression. The V(D)J clonotype capture rate was 93% in baseline samples with detectable genomic aberrations, indicating presence of tumor DNA, assessed through myTYPE. myTYPE-positive samples had significantly higher V(D)J clonotype detection rates in univariate (OR, 7.3; 95% CI, 2.8-22.6) and multivariate analysis (OR, 4.4; 95% CI, 1.4-16.9; P = 0.016). Higher disease burden was associated with higher probability of V(D)J clonotype capture, meanwhile no such association was found for age, gender, or type of heavy or light immunoglobulin chain. All V(D)J clonotypes detected at baseline were detected in MRD-positive samples indicating that the V(D)J clonotypes remained stable and did not undergo further rearrangements during follow-up. Of the 31 posttreatment samples, 12 were MRD-negative using the NGS MRD assay. NGS for V(D)J rearrangements in multiple myeloma offers a reliable and sensitive method for MRD tracking with high detection rates in the clinical setting.

Performance of Next Generation Sequencing for Minimal Residual Disease Detection for Pediatric Patients with Acute Lymphoblastic Leukemia: Results from the Prospective Clinical Trial DFCI 16-001

Performance of Next Generation Sequencing for Minimal Residual Disease Detection for Pediatric Patients with Acute Lymphoblastic Leukemia: Results from the Prospective Clinical Trial DFCI 16-001

Igh-V(D)J NGS-MRD Measurement in Pediatric B-Acute Lymphoblastic Leukemia

IntroductionB-acute lymphoblastic leukemia (B-ALL) is the most common cancer of childhood. Early response to induction chemotherapy is one of the important prognostic factors in B-ALL. However, the analytic sensitivity for flow cytometry (FC) is only 10 -4. The feasibility of using next-generation sequencing (NGS) of immunoglobulin for the determination of minimal residual disease (MRD) in B-ALL has been demonstrated. This study aimed to investigate the performance of NGS techniques measuring immunoglobulin heavy chain (IgH)-variable, diversity, and joining (V[D]J) clonal rearrangements compared with FC in detecting MRD for children with B-ALL and to predict the clinical outcome of B-ALL patients.MethodsNewly diagnosed younger than 18 years old B-ALL patients who received the treatment strategy of South China children's leukemia Group (SCCLG)-ALL 2016 were recruited. DNA extracted from bone marrow cells at all available time points for each patient was submitted to Simcere diagnostics for sequencing using Illumina NovaSeq platform. We performed IgH V(D)J NGS and FCM on the bone marrow serially obtained at diagnosis (D0), 15 days at induction therapy (D15), 33 days at induction therapy (D33) and then at the end of induction therapy (EOI). We defined MRD positive (MRD +) by IgH V(D)J NGS and FCM as more than 1 blast cell among 10 4 and 10 6 bone marrow cells, respectively. The sensitivity of MRD detection by IgH V(D)J NGS and FCM, and the association of MRD status with clinicopathological characteristics were investigated. Statistical analysis was performed through SPSS Statistics 22. Enumeration data and correlation between MRD data and clinicopathological characteristics were compared by Chi-square test or Fisher's exact test. This trial was registered at www.clinicaltrials.gov as # NCT04977895.ResultsAs of July 27, 2021, 22 patients (median age, 4.5 years; range, 3.0-7.3) were enrolled in the study. Three patients (13.6%) had a t (9;22) translocation consistent with Philadelphia chromosome positive disease. According to risk stratifications, 8 (36.4%), 8 (36.4%), and 2 (9.1%) patients were classified as low risk (LR), intermediate risk (IR), and high risk (HR) groups, respectively. The remaining 4 patients are still under treatment and have not been classified. We identified leukemic IgH clones in 100% of the diagnostic samples and 68.2% (15/22) of the patients were polyclonal. In 11 patients whose samples of all the four timepoints (D0, D15, D33, EOI) have been tested in parallel by FCM and IgH V(D)J NGS, the frequencies of patients with MRD + were 30.4% vs. 90.9% at D15 (P＜0.05) by FCM and IgH V(D)J NGS. IgH V(D)J NGS MRD monitoring could identify MRD + patients with frequency of 45.5% and 18.2% among patients achieved MRD negativity by FCM at D33 (P＜0.05) and EOI (P = 0.46). With an MRD detection limit of 10 -6, 90.9% (10/11), 36.4% (4/11) and 18.2% (2/11) patients were MRD negative by FCM but positive by the NGS test at D15, D33 and EOI, respectively. This suggested that the sensitivity of IgH V(D)J NGS was significantly higher than that of FCM. Correlation of the measured MRD between the two methods in the entire cohort (r = 0.7934, P < 0.0001) as well as in the concordant cases (r = 0.5558, P = 0.0032) was very high. There was a high discordant rate with NGS identifying more patients MRD + at this threshold. Furthermore, NGS MRD was positive but the FCM MRD was negative in 13 samples (P < 0.0001). In addition, positive MRD status of D33 by NGS was significantly associated with the age of B-ALL patients, patients under 6 years more frequently harbored detectable MRD compared with those ≥ 6 years old (87.5% vs. 11.1%, P < 0.01). There was no patient relapsed after a medium follow-up of 10.5 months.ConclusionsWe demonstrated the higher sensitivity of IgH-V(D)J NGS in MRD detection of B-ALL, which implies that NGS MRD monitoring could be helpful for more accurate risk stratifications and more precise treatment according to risk stratifications. Further study with a larger sample size and a longer follow-up period is need. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Retrospective Analysis of Minimal Residual Disease Testing By High Throughput Immunosequencing Versus High Sensitivity Flow Cytometry in Multiple Myeloma

Introduction:Detection of minimal residual disease (MRD) is one of the strongest predictors of outcome in multiple myeloma (MM). Until recently, the most commonly available method to detect MRD in clinical practice has been high sensitivity flow cytometry (FC) which can detect MRD with at 10 -5 sensitivity. In recent years, next-generation sequencing (NGS) has become a viable method to assess the MRD in MM patients with a 10 -6 sensitivity. NGS appears to have some advantages over HC-FC by circumventing subjectivity of analysis. However, real-world comparison between these two methodologies in the literature is limited and is important to inform daily hematopathology and oncology ordering practices.Methods:We retrospectively identified all cases of MM with NGS MRD data from bone marrow specimens at the Moffitt Cancer Center and collated corresponding flow MRD data and clinical data (OS, patient demographics) electronically and via chart review.10-color flow cytometry was performed on a Gallios System and analyzed on Kaluza (Beckman Coulter, IN). Two million events were collected on all cells. Validated lower limit of detection was at least 0.01%. Antibodies included CD28, CD81, CD56, CD138, CD319, CD20, CD19, CD117, CD38, CD45, CD27, CD200 (BD, Biolegend, Beckman Coulter).clonoSEQ ® (Adaptive Biotechnologies, Seattle, WA) testing was performed which uses multiplex polymerase chain reaction (PCR) and NGS to identify, characterize, and monitor 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) sequencesStatistical analysis was performed by Spearman correlation coefficient and Kaplan-Meier analysis.Results:192 samples from 122 unique patients were identified that had both NGS and FC data performed on the same sample. FC+ values ranged from 1x10 -7 to 0.39. NGS+ values ranged from 2.3 x 10 -7 to 0.15. Spearman correlation coefficient showed moderate concordance between NGS and FC at r=0.67 (p<0.001). Six samples were positive by FC (mean tumor burden (MTB)= 0.0007) but missed by NGS; whereas 59 samples were positive by NGS (MTB= 0.002) but missed by flow cytometry. Two cases by FC were equivocal and these were both definitively designated as MRD+ by NGS. Overall survival was worse for MRD+ (by NGS or FC) vs MRD(-) (Figure 1).Conclusion:Our study confirms the importance of MRD detection in MM and shows the robust utility of NGS for MRD detection in routine hematopathology practice. While both FC and NGS are complementary given that each can potentially detect MRD missed by another method, the data supports the increased sensitivity of NGS over FC. [Display omitted] DisclosuresNishihori: Novartis: Research Funding; Karyopharm: Research Funding. Hussaini: Stemeline Therapeutics: Honoraria.

New Alternatives for the Evaluation of Minimal Residual Disease (MRD) Detection By Next Generation Sequencing in Multiple Myeloma

New Alternatives for the Evaluation of Minimal Residual Disease (MRD) Detection By Next Generation Sequencing in Multiple Myeloma

Routine Evaluation of Minimal Residual Disease in Myeloma Using Next-Generation Sequencing Clonality Testing: Feasibility, Challenges, and Direct Comparison with High-Sensitivity Flow Cytometry

The 2016 International Myeloma Working Group consensus recommendations emphasize high-sensitivity methods for minimal residual disease (MRD) detection, treatment response assessment, and prognostication. Next-generation sequencing (NGS) of IGH gene rearrangements is highly specific and sensitive, but its description in routine clinical practice and performance comparison with high-sensitivity flow cytometry (hsFC) remain limited. In this large, single-institution study including 438 samples from 251 patients, the use of NGS targeting the IGH and IGK genes for clonal characterization and monitoring, with comparison to hsFC, is described. The index clone characterization success rate was 93.6% (235/251), which depended on plasma cell (PC) cellularity, reaching 98% when PC ≥10% and below 80% when PC <5%. A total of 85% of cases were successfully characterized using leader and FR1 primer sets, and most clones showed high somatic hypermutation rates (median, 8.1%). Among monitoring samples from 124 patients, 78.6% (147/187) had detectable disease by NGS. Concordance with hsFC was 92.9% (170/183). Discordant cases encompassed 8 of 124 hsFC MRD+/NGS MRD− patients (6.5%) and 4 of 124 hsFC MRD−/NGS MRD+ patients (3.2%), all with low-level disease near detection limits for both assays. Among concordant hsFC MRD−/NGS MRD− cases, only 5 of 24 patients (20.8%) showed subsequent overt relapse at 3-year follow-up. HsFC and NGS showed similar operational sensitivity, and the choice of test may depend on practical, rather than test performance, considerations.

Ofatumumab, Etoposide, and Cytarabine Intensive Mobilization Regimen in Patients with High-risk Relapsed/Refractory Diffuse Large B-Cell Lymphoma Undergoing Autologous Stem Cell Transplantation

BackgroundMore than one-half of high-risk patients with relapsed/refractory (rr) diffuse large B-cell lymphoma (DLBCL) relapse after autologous hematopoietic cell transplantation (auto-HCT). In this phase II study, we investigate the long-term outcomes of high-risk patients with rrDLBCL receiving intensive consolidation therapy (ICT) with OVA (ofatumumab, etoposide, and high-dose cytarabine) prior to auto-HCT. Patients and MethodsThe primary endpoints were the ability of OVA to mobilize peripheral stem cells and the 2-year progression-free survival (PFS) rate following OVA. Secondary endpoints included safety, 2-year overall survival (OS), impact of cell of origin (COO), and the prognostic utility of next-generation sequencing minimal residual disease (MRD) testing. We simultaneously retrospectively assessed the outcomes of DLBCL patients who underwent ICT with a similar regimen at our institution. ResultsTwenty-seven patients received salvage chemotherapy, with a response rate of 25% in patients with germinal center B-cell (GCB)-DLBCL versus 92% in patients with non-GCB-DLBCL (P = .003). Nineteen responding patients underwent ICT with OVA (100% successful stem cell mobilization). The 2-year PFS and OS rate was 47% and 59%, respectively, with no difference based on COO. Similar findings were observed when the study and retrospective cohorts were combined. Neutropenia was the most common toxicity (47%). MRD-negative patients at the completion of salvage had a median OS of not reached versus 3.5 months in MRD-positive patients (P = .02). ConclusionsOVA followed by auto-HCT is effective and safe for high-risk rrDLBCL. Patients with GCB-DLBCL had a lower response to salvage chemotherapy, but no difference in outcomes based on COO was seen after auto-HCT. MRD testing in the relapsed setting was predictive of long-term survival.

Interim Analysis from a Prospective Multicenter Study of Next-Generation Sequencing Minimal Residual Disease Assessment and CT Monitoring for Surveillance after Frontline Treatment in Diffuse Large B-Cell Lymphoma

Interim Analysis from a Prospective Multicenter Study of Next-Generation Sequencing Minimal Residual Disease Assessment and CT Monitoring for Surveillance after Frontline Treatment in Diffuse Large B-Cell Lymphoma

Next-Generation Sequencing Minimal Residual Disease of Mantle Cell Lymphoma in Autologous Stem Cell Grafts and Its Implication on Tumor Recurrence

Background &amp; Purpose The clinical course of mantle cell lymphoma (MCL) is often inflicted with tumor recurrence even though front-line autologous stem cell transplantation (ASCT) is the current standard of care. To elucidate the mechanism of post-transplant recurrence, this study aimed to interrogate the minimal residual disease (MRD) of MCL in the autologous grafts. Materials &amp; Methods Paired samples of 17 MCL patients' lymphoma diagnostic FFPE specimens were analyzed in parallel with their harvested autologous stem cell grafts. Extracted genomic DNA was subjected to LymphoTrackⓇ Dx IGH/IGK assay coupled with Illumina MiSeq sequencer to characterize the post-recombination immunoglobulin VDJ sequences. Positivity of MRD was defined for any identical sequences identified both in the diagnostic FFPE and in the autologous graft DNA. As the control for recombined VDJ protein motif analysis, diagnostic FFPE samples from 23 patients with diffuse large B cell lymphoma (DLBCL) were analyzed with the same platform. Results Of the 17 patients undertaking autologous stem cell harvest, 11 patients achieved complete response and 6 were in partial response before stem cell harvest. Sixteen of these actually underwent transplantation while one died of disease before transplantation. MRD was detected via next-generation sequencing (NGS) in 5 patients' autologous grafts with variable MRD loads and recombined VDJ stereotypes (Table 1). VH3-21 was the most prominent stereotype (41%), followed by VH4-59 (14%). The median somatic hypermutation rate was 0.88% (range 0 - 5.17%). Interestingly, a 46-amino-acid domain in recombined VDJ sequences, which was hydroxyl and amine group-rich, differed between MCL and DLBCL: hydrophilic amino acids were enriched in 6 positions of this domain in MCL (p&amp;lt;0.05). Among the five patients who had MRD in autologous grafts, the median MRD read number was 12,764 reads (range 89 - 33,554) with the median read depth of 195,865 reads (range 94,820 - 396,894). The MRD loads in the autologous grafts were inversely correlated with post-harvest PFS (Figure 1a and 1b). As for post-ASCT outcomes, patients with MRD had a trend toward worse PFS and OS than those without MRD (medians, 10 months vs 27 months, and 25 months vs 66.8 months, respectively). In subgroup analysis, a higher load of NGS-MRD (&amp;gt;1%) correlated with shorter post-ASCT PFS and OS than those without MRD (medians, 1.9 months vs 27 months (p=0.024) in Figure 1c, and 11.9 months vs 66.8 months (p=0.001) in Figure 1d, respectively). Conclusions Identification of MRD in autologous grafts by deep-sequencing VDJ recombination helped stratify MCL patients' post-ASCT outcomes. Higher MRD loads correlated with inferior post-ASCT PFS and OS. The implication of recombined VDJ stereotypes and their impact on ASCT outcomes warrants further investigation. Disclosures Ko: Roche: Honoraria.

Minimal Residual Disease in Multiple Myeloma: Targeted Mass Spectrometry in Blood Vs Next Generation Sequencing in Bone Marrow

Background Multiple myeloma (MM) is a plasma cell malignancy arising in the bone marrow that is characterized by secretion of a monoclonal immunoglobulin (M-protein). M-protein detection is an integral part of MM diagnostics, performed using electrophoretic methods. This approach is reliable, fast and inexpensive, but not sensitive enough for detecting deeper responses, characterized by low M-protein concentration. Novel therapies have led to an increasing number of patients obtaining complete remission, however no curative therapy is available for MM. Even in remission, minimal residual disease (MRD) is present and can develop towards a relapse. MRD analysis plays an important role as primary endpoint in clinical trials, powerful prognostic marker, and MRD-status is increasingly used to guide therapy-decisions. Methods currently used for MRD detection are based on bone marrow aspirates (e.g. flow cytometry or next generation sequencing (NGS)). Not only are repetitive bone marrow aspirations uncomfortable for the patient, they also introduce a risk of non-representative sampling due to the possible patchy occurrence of the cancer cells in MM. Aim The aim is to measure MRD in a cohort of 41 multiple myeloma patients during follow-up using a targeted mass spectrometry assay. Unique peptides from the M-protein are selected based on RNA sequence information. Patient-specific peptides from M-proteins will be measured using mass spectrometry and MRD status will be compared to available NGS data. Methods Multiple myeloma patients were selected from the IFM 2009 Study (ClinicalTrials.gov number: NCT01191060) with available RNA sequence information and MRD data by NGS. We have selected 41 patients with three serum samples per patient; a baseline serum sample and two follow-up time points for MRD assessment. For each patient we have selected at least two peptides unique for the M-protein that could be observed in the baseline sample using mass spectrometry; one peptide from the light chain, and one from the heavy chain.(Zajec et al, J Proteome Res, 2018) In the case of light chain MM (3 patients in this study), only light chain peptides were selected. Serum samples were diluted 500 times, digested with trypsin and directly measured using targeted Orbitrap mass spectrometry technology. Results For all 41 patients we were successful in selecting and targeting unique peptides for M-protein quantification using mass spectrometry. MRD was assessed in follow-up serum samples (n=82, two time points for each patient) using NGS and mass spectrometry. In these follow-up patients, seven patients were negative by both NGS and mass spectrometry. Additionally, eight patients were negative by NGS but positive by mass spectrometry; and ten patients were negative by mass spectrometry and positive by NGS. The remainder of the follow-up samples (n=57) were positive by both mass spectrometry and NGS. Conclusion Unique peptides of the M-protein were found in all 41 multiple myeloma patients. Using these peptides, it was feasible to measure the M-protein in blood samples from multiple myeloma patients during follow-up. NGS and mass spectrometry MRD assessment have a concordance of 78% (64/82) in the follow-up samples. These are preliminary results without any immunoglobulin enrichment. We expect that further optimization can improve the sensitivity of mass spectrometry measurements. Future perspective Described experiments were performed on non-enriched serum without optimization of mass spectrometry parameters. Future experiments should be adapted accordingly and will increase the sensitivity of the mass spectrometry measurements. Internal standards for the peptides will be synthesized for absolute quantification (g/L value) of the M-protein during MRD. Disclosures No relevant conflicts of interest to declare.

Impact of Minimal Residual Disease (MRD) By Multiparameter Flow Cytometry (MFC) and Next-Generation Sequencing (NGS) on Outcome: Results of Newly Diagnosed Transplant-Eligible Multiple Myeloma (MM) Patients Enrolled in the Forte Trial

Impact of Minimal Residual Disease (MRD) By Multiparameter Flow Cytometry (MFC) and Next-Generation Sequencing (NGS) on Outcome: Results of Newly Diagnosed Transplant-Eligible Multiple Myeloma (MM) Patients Enrolled in the Forte Trial

Comparison of NGS and MFC Methods: Key Metrics in Multiple Myeloma MRD Assessment.

In order to meet the challenges in data evaluation and comparability between studies in multiple myeloma (MM) minimal residual disease (MRD) assessment, the goal of the current study was to provide a step-by-step evaluation of next-generation sequencing (NGS) and multicolor flow cytometry (MFC) data. Bone marrow (BM) sample pairs from 125 MM patients were analyzed by NGS and MFC MM MRD methods. Tumor load (TL) and limit of detection (LOD) and quantification (LOQ) were calculated. The best-fit MRD cut-off was chosen as 1 × 10−5, resulting in an overall 9.6% (n overall = 12 (NGS n = 2, MFC n = 10)) nonassessable cases. The overall concordance rate between NGS and MFC was 68.0% (n = 85); discordant results were found in 22.4% (11.2% (n = 14) of cases in each direction. Overall, 55.1% (n = 60/109) and 49.5% (n = 54/109) of patients with a serological response ≥ very good partial response (VGPR) showed BM MRD negativity by NGS and MFC, respectively. A good correlation in the TL assessed by both techniques was found (correlation coefficient = 0.8, n = 40, p < 0.001). Overall, our study shows good concordance between MM BM MRD status and TL when comparing NGS and MFC at a threshold of 10–5. However, a sufficient number of analyzed events and calculation of MRD key metrics are essential for the comparison of methods and evaluability of data at a specific MRD cut-off.

The impact of total body irradiation-based regimens on outcomes in children and young adults with acute lymphoblastic leukemia undergoing allogeneic hematopoietic stem cell transplantation.

Total body irradiation (TBI)-based conditioning is the standard of care in the treatment of acute lymphoblastic leukemia (ALL) that requires allogeneic hematopoietic stem cell transplantation (HSCT). However, TBI is known to be associated with an increased risk of late effects, and therefore, non-TBI regimens have also been utilized successfully. A recent study showed that patients that were next-generation sequencing-minimal residual disease (NGS-MRD) negative prior to allogeneic HSCT had a very low risk of relapse, and perhaps could avoid exposure to TBI without compromising disease control. We examined outcomes at our institution in patients that received a TBI or non-TBI regimen, as well as explored the impact of NGS-MRD status in predicting risk of relapse post transplant. This retrospective analysis included 57 children and young adults with ALL that received their first myeloablative allogeneic HSCT from 2012 to 2017 at the University of California San Francisco. Our primary endpoint was the cumulative incidence of relapse at 3 years post transplant. We demonstrated similar cumulative incidence of relapse for patients treated with either a TBI or non-TBI conditioning regimen, while NGS-MRD positivity prior to transplant was highly predictive of relapse. The presence of acute graft-versus-host disease was associated with decreased relapse rates, particularly among patients that received a TBI conditioning regimen and patients that were NGS-MRD positive prior to HSCT. Our data suggest that the decision to use either a TBI or non-TBI regimens in ALL should depend on NGS-MRD status, with conditioning regimens based on TBI reserved for patients that cannot achieve NGS-MRD negativity prior to allogeneic HSCT.