Minimal Residual Disease Results Research Articles

Preliminary efficacy and safety of total neoadjuvant therapy combined with PD-L1 blockade in pMMR/MSS locally advanced rectal cancer (ESTIMATE): A prospective, single-arm, phase II trial.

e15602 Background: Patients with locally advanced rectal cancer (LARC) experience benefits from total neoadjuvant therapy (TNT). This study aims to explore the efficacy and safety of combining TNT with immunotherapy for pMMR/MSS LARC patients. Methods: ESTIMATE is a prospective, single-center, phase II study (ChiCTR2400079718) enrolling pMMR/MSS LARC patients with tumors located 10cm away from the anal margin. Treatment involved induction chemotherapy with immunotherapy ([mFOLFOX6, days 1-2 + Envafolimab 200mg, ih, day 1]; q2w; 2 cycles), long-course concurrent chemoradiotherapy (LCRT) with immunotherapy ([IMRT, 50Gy/25f, 2Gy + Cape 825mg/m2, bid]; 5w + Envafolimab 200mg, ih, day 1; q2w; 3 cycles), consolidation chemotherapy with immunotherapy ([mFOLFOX6 + Envafolimab; 2 cycles), and then total mesorectal excision (TME) 3 weeks later or watch and wait (W&W). The primary endpoint is the pathological complete response (pCR) rate. Results: Until Nov. 16th, 2023, 35 patients were enrolled, with 28 completing the TNT regimen. The efficacy analysis revealed that out of 28 patients, 12 (42.9%) achieved clinical complete response (cCR), with 7 individuals choosing the W&W strategy. 21 patients underwent TME, yielding 8 with a tumor regression grade (TRG) of 0, 7 with TRG of 1, and 6 with TRG of 2. The rates of pCR, major pathological response (MPR), and complete response (CR) were 38.1% (8/21), 71.4% (15/21), and 53.6% (15/28), respectively. 25 (89.3%) patients experienced treatment-related adverse events (AEs) of any grade. Non-hematological AEs included decreased appetite, fatigue, nausea, and vomiting, while hematological AEs mainly comprised thrombocytopenia, anemia, and leukopenia or neutrophilia. Grade III AEs were primarily leukopenia or neutrophilia (2/28, 7.1%). Additionally, circulating tumor DNA (ctDNA) levels were measured in 7 patients who chose the W&W strategy after achieving cCR. Of these, 6 tested negative for minimal residual disease (MRD), while 1 had a positive MRD result (ctDNA: 0.56 MTM/ml). As of Jan. 26th, 2024, with a median follow-up of 5 months (range: 3-6), no recurrence was observed in any of the patients. Conclusions: The TNT approach, integrating LCRT with mFOLFOX6 and PD-L1 blockade, demonstrated a favorable CR rate and acceptable tolerability, offering a potential avenue for organ preservation in low rectal cancer patients with pMMR/MSS. Long-term benefits need validation through further follow-up. Clinical trial information: ChiCTR2400079718. [Table: see text]

PD-1 and LAG-3 as early prognostic markers in the treatment of patients with chronic lymphocytic leukemia

Background. Recently, studies have been conducted all over the world to study the role of immune checkpoints in the pathogenesis of chronic lymphocytic leukemia (CLL) and the possibility of their use as prognostic markers. Of greatest interest are PD-1 (programmed cell death-1) and LAG-3 protein (lymphocyte-activation gene 3).Aim. To study the features of PD-1 (CD279) and LAG-3 (CD223) expression on blood B-cells of CLL patients and the possibility of their use as early markers for predicting the hematological response to therapy.Materials and methods. The blood of 30 patients with CLL in stage C according to Binet and 20 healthy individuals was studied by 10-color flow cytometry.Results. In patients with CLL, there were significant differences in the initial lymphocytes level, PD-1 and LAG-3 expression on B-lymphocytes, both with persons in the control group and among themselves with different hematological responses to therapy with rituximab according to the results of minimal residual disease monitoring.Conclusion. PD-1 and LAG-3 can be used as early markers for predicting the response of CLL patients to therapy. The combined use of initial lymphocytes level and PD-1 and LAG-3 expression on CD19+ blood cells has a greater prognostic value. New data obtained from the study of immune checkpoints PD-1 and LAG-3 may be useful in the development of targeted therapeutic agents.

Free from Drug Therapy in Multiple Myeloma (FREEDMM): MRD-Based Cessation of Maintenance

Background. Treatment of newly diagnosed multiple myeloma (NDMM) consists of induction therapy followed by autologous stem cell transplant (ASCT) and indefinite maintenance. Continuous lenalidomide carries both significant clinical and financial toxicity. Minimal residual disease (MRD) has proven to be a powerful surrogate for PFS and OS but is not yet used to guide treatment. We hypothesize that MRD could be used to predict a subset of patients who can safely stop maintenance therapy and enter into a disease surveillance phase. Study Design and Methods. This prospective phase 2 study (NCT 05192122) is designed to stop post-ASCT maintenance therapy once sustained MRD negativity is achieved. MM patients are eligible if they have completed ≥2 years of maintenance therapy post-ASCT, and meet IMWG criteria for ≥ PR. Bone marrow (BM) MRD testing is performed twice, one year apart, using NGS. Sustained MRD-negativity is defined at a threshold of 10 -6, with negative MRD sustained between the Year 1 and Year 2 interval. Patients with sustained MRD negativity and absence of new bony lesions on PET then undergo maintenance therapy cessation. MRD-positive patients are removed from study. In patients who discontinue maintenance, BM testing to re-assess for MRD is repeated yearly for 3 years. At the time of MRD testing, peripheral blood (PB) samples are collected for MALDI-TOF mass spectrometry testing and circulating multiple myeloma cell (CMMC) enumeration. These are done in parallel with MRD assessment. Health-related quality of life (HRQoL) using the EORTC QLQ-MY20 questionnaire is measured before and after maintenance cessation. This study plans to enroll 50 patients to meet the primary objective of assessing sustained MRD-negativity 1 year after maintenance cessation. For all patients, 5-mL PB and BM samples are collected, processed, and kept frozen until analysis. Cellular indexing of transcriptomes and epitopes (CITE-seq) to simultaneously quantify cell surface protein and transcriptome data is used to characterize differences in the immune microenvironment between MRD-negative, MRD-positive, as well as between patients with biochemical and/or clinical progression and patients with MRD-resurgence but biochemically and clinically stable. Preliminary Results. FREEDMM enrollment began December 2021, and 31 patients have been enrolled, 29 of whom have undergone Year 1 BM for MRD assessment. Median follow up of our cohort is 414 (0-577) days. The median age is 69.5 (40-78) years, and the majority are either Black (n=9; 29%) or Hispanic (n=11; 35%). 18 (58%) patients had R-ISS 1 or 2, while 2 patients had high risk disease with presence of 17p deletion. The median number of years of maintenance therapy completed prior to Year 1 BM is 3.4 (2-7.9) years. 28 (96.6%) were in CR at time of Year 1 BM. Year 1 MRD results are pending in 13 patients. At Year 1, 11 (68.8 %) of 16 patients were found to be MRD-negative, while 5 (31.3%) patients were found to be MRD-positive with a median of 206 (1-9,748) cells per 10 6 nucleated cells detected. 24 (85%) of 28 patients had a negative MALDI-TOF result at the time of the Year 1 BM. MALDI-TOF results were false negative in 3 MRD-positive patients. PB CMMC samples were concordant with MRD results in all 5 (100%) patients who were tested. There was concordance between MALDI-TOF and CMMC results in 9 (75%) of 12 patients. At year 2, 4 patients continued to be MRD negative, while 1 patient who was previously MRD-negative became MRD positive. At most recent follow-up, 3 (42.9%) of 7 MRD positive patients at Year 1 experienced clinical progression and were removed from the study. 2 (50%) of 4 patients with sustained MRD-negativity have undergone maintenance cessation, with the remaining 2 awaiting confirmation of MRD-negative results. 59 paired PB (n=29) and BM (n=30) samples have been collected for CITE-seq analysis. Pilot CITE-seq experiments captured 10,000-15,000 cells with 33,000-56,000 reads/ cell on one MRD-positive BM and paired PB sample. CITE-seq results for remaining patients will be presented at the conference. Conclusion: This phase 2 trial applies a novel clinical trial design with an adaptive strategy to explore use of MRD testing as a guide to maintenance therapy cessation in MM patients and represents one of the first studies introducing transcriptome analysis of the tumor and immune microenvironment in the maintenance setting. Ongoing results will be reported at the conference.

Daratumumab Bortezomib and Dexamethasone in Transplant Ineligible Newly Diagnosed Elderly Myeloma Patients - a Trial By Asian Myeloma Network

Background: Transplant ineligible (TI) newly diagnosed Multiple Myeloma (NDMM) patients have inferior outcomes compared to that of a transplant population. This led to investigation of therapies with novel mechanisms of action to improve outcomes in this group. MAIA and ALCYONE studies have showed impressive outcomes in this group of patients. However, significant toxicities are a concern when delivering multiple anti-myeloma drugs concurrently in a continuous schedule in elderly patients. In this context, the efficacy and safety of Daratumumab, Bortezomib (finite) and dexamethasone in NDMM remains unknown. Aims: In this study, we investigated the combination of Daratumumab Bortezomib (in a finite schedule), and dexamethasone (DVd) followed by Daratumumab as maintenance in transplant ineligible TI NDMM patients from Asia (NCT03695744). Methods: This is a prospective, open-label, single-arm, phase 2 study of TI NDMM patients conducted by Asian Myeloma Network across three hospitals in Singapore. The primary end point was overall response rate. Secondary end points included progression free survival, overall survival, toxicity & safety, duration of response, minimal residual disease (MRD) and sustained MRD negativity. The study was approved by SingHealth Centralised IRB (CIRB No 2018/2730). Patients with confirmed diagnosis of MM, fulfilling inclusion / exclusion criteria were enrolled after signing informed consent. Patients received IV daratumumab 16mg/kg weekly in weeks 1-9, Q3 weekly from weeks 10 to 24 and Q4 weekly from week 25 onwards. Subcutaneous Bortezomib 1.3mg/m 2 and Dexamethasone 40mg (20mg for patients >75 years old) once weekly were administered for 9 months, followed by daratumumab maintenance until progression or toxicity. Response assessments were as per IMWG criteria and serial QOL data was also collected. Regular safety reviews were performed from recruitment of first patient by an independent committee. Here we report the protocol specified interim analysis of safety and efficacy end points. Results: From Oct 2019 till Sep 2021, we enrolled 27 patients. Median age was 74.3 years (range 70.5 to 79.2 years; 13/27 patients were >75years). 13 male and 14 were female patients. Ethnic distribution was 20 Chinese, 3 Malay, 3 Indian and 1 Others. ECOG performance status was 0 in 5(19%), 1 in 16(59%) and 2 in 6(22%) patients respectively. 11(41%) and 6(22%) patients belonged to Stage II and III ISS respectively. 14(52%) patients had CrCl <60ml/Min. 26(96%) patients experienced any grade adverse event with 17 experiencing a grade 3/4 event. Most common adverse events were infections, gastrointestinal disorders, peripheral neuropathy, and infusion related reactions. All grade and grade 3/4 AEs were relatively low, particularly haematological, given the absence of drugs known to cause significant myelotoxicity. Detailed AEs and SAEs are tabulated in Table 1. Three patients have died, of which one was due to a treatment related infection, while two others were unrelated (1 - suicide secondary to known mental illness, 1 - accidental fall & cervical spine injury). At data cut off (15 Oct 2022), 26 out of 27 patients had a response with 4(14.8%) sCR, 3 (11.1%) CR, 14 (51.9%) VGPR and 5 (18.5%) PR (1 missing assessment) giving an ORR of 96.3%. Median duration on Daratumumab was 16.3 mos and Bortezomib was 8 mos. 6 patients (22.2%) have experienced disease progression. With a median follow-up of 17.6 months, the 18-month progression free survival and overall survival were 65.4% & 89% respectively (median PFS and OS not estimable). Follow-up is on-going and we aim to report long-term survival, FISH/ cytogenetic & MRD results, toxicity and QOL data in future. Conclusion: DVd with Bortezomib in a finite schedule and Daratumumab maintenance achieved highly efficacious responses in elderly TI NDMM. Though this is a small study, toxicity rates here compare favorably to that noted in larger Phase 3 studies with quadruplets (Alcyone) and continuous triplet regimen as in the MAIA study. Whilst using novel agents like CD38 monoclonals upfront clearly improves responses and survival, minimizing toxicities without compromising efficacy by careful limitation of long-term concurrent exposure to multiple anti-myeloma drugs in an elderly population could help improve safety and quality of life. DVd could be an effective alternative in some elderly TI NDMM patients.

Prognostic Impact of Minimal Disseminated Disease (MDD) and Minimal Residual Disease (MRD) in Chinese Pediatric Patients with ALK-Positive Anaplastic Large Cell Lymphoma (ALCL)

# Keyan Yang and Jing Yang contribute equally to this article. * Corresponding to both Qinlong Zheng (zhengql@gobroadhealthcare.com) and Yonghong Zhang (zhangyongh@gobroadhealthcare.com) Backgrounds: Anaplastic lymphoma kinase positive anaplastic large cell lymphoma (ALK+ALCL) is characterized by the occurrence of the ALK fusion and subsequently ALK fusion kinase protein expression. It has been reported that minimal disseminated disease (MDD) and minimal residual disease (MRD) detected by RQ-PCR are independent prognostic factors. Purpose & Methods: To explored the prognostic roles of MDD and MRD in Chinese pediatric patients with ALCL, we conduct a retrospective study in a cohort of 204 Chinese children and adolescent ALK+ALCL patients treated at multiple centers of the China Network of Childhood Lymphoma (CNCL). The median follow-up time was 39 months (95%CI: 34-44.7). MDD level as ALK fusion copy number was measured by quantitative RT-PCR at the initial diagnosis. Based on the Overall Survival (OS) data, cut-off values of 1.63 in peripheral blood (PB) and 1.43 in bone marrow (BM) were determined by using the maximally selected rank statistics, with the c-index values of 0.904 in PB and 0.895 in BM. Results: The prevalence of MDD in PB and/or BM of Chinese pediatric patients with ALK+ALCL was 24.8%, while the MDD level were significantly correlated between PB and BM (R=0.8, p<2.2e-16). In addition, the 3-year OS (PB, 87.8% vs. 100%; BM, 87.9% vs. 100%) and EFS (PB, 73.2% vs. 94.8%; BM, 80.0% vs. 93.2%) were revealed with significant difference, when analyzed by MDD level. The combined analyses of both MDD level (with the same cut-off value) and MRD results (positive vs. negative), either after 1 st course of treatment (MRD1) or 2 nd course of treatment (MRD2), indicated the further poor prognosis, both in 3-year OS (PB, MRD1, 64.8% vs. 100%, MRD2, 72.9% vs. 100%; BM, MRD1, 64.8% vs. 100%, MRD2, 68.8% vs. 100%) and EFS (PB, MRD1, 40.0% vs. 95.3%, MRD2, 45.0% vs. 95.1%; BM, MRD1, 40.0% vs. 94.7%, MRD2, 35.7% vs. 94.4%). Conclusion: Summary, in this work, MDD level detected by quantitative RT-PCR was revealed as a strong factor in prognostic evaluation of Chinese children and adolescent patients with ALK+ALCL, which showed its important role in clinical detection at initial diagnostic. In future, the further validation work could be carried out in different cohort of ALK+ALCL patients, even with larger enrolled population size and longer follow-up time.

Pediatric Patients with High-Risk B-Cell ALL in First Complete Remission May Benefit from Less Toxic Immunotherapy with Blinatumomab - Results from Randomized Controlled Phase 3 Trial AIEOP-BFM ALL 2017

Background: One of the key questions in trial AIEOP-BFM ALL 2017 focused on the reduction of treatment-related complications by replacing parts of the highly intensive consolidation phase by two courses of Blinatumomab (Blina) in a prospective randomized trial. Herein, we report the toxicity profile of B-cell acute lymphoblastic leukemia (B-ALL) patients (pts) with high-risk (HR) characteristics pending final outcome data. Patients and Methods: 728 pts with HR B-ALL were enrolled from July 15, 2018 to October 31, 2022 in this multicenter trial run in 8 different countries. Patients were included in the HR group in view of the presence of at least one of the following criteria: induction failure; high levels of minimal residual disease (MRD) at day 15 of induction, or at the end of induction (EOI), or at the end of consolidation (EOC); presence of KMT2A::AFF1, of IKZF1+ and positive MRD at EOI (Stanulla et al, JCO 2018), TCF3::HLF, or hypodiploidy. B-ALL pts with one or several of these characteristics represented 20.8% of all pts aged 0-18 years of age with B-ALL. After a 4-drug induction phase, and two weeks of consolidation treatment, patients were randomized to receive Bortezomib in addition to standard consolidation (not part of this report). Before treatment continued with intensive consolidation block HR-1', MRD was analyzed in order to identify patients eligible for subsequent hematopoietic stem cell transplantation (HSCT) based on MRD results. After block HR-1', all B-ALL HR pts were randomized to receive either two courses of consolidation (blocks HR-2', and HR-3'), or two 28-day courses of Blina at 15µg/m 2/d administered by continuous IV infusion (with a 2-week treatment-free interval). Two intrathecal injections of methotrexate (MTX) for CNS prophylaxis were given on days 1 and 29 of each Blina course, respectively. Out of 728 HR B-ALL pts, 619 pts were eligible for randomization [reasons for non-eligibility were: Event (death or relapse) before randomization was due (26), Down syndrome (25; scheduled for a non-randomized intervention with Blina), presence of TCF3::HLF (3; could receive any alternative therapy including Blina), discontinuation/substantial change of preceding therapy (23) or other protocol exclusion criteria (32)]. 572 pts were randomized (92.4% of those eligible). One pt assigned to the experimental arm (EA) and 4 pts assigned to the control arm (CA) received the other arms, respectively. Results: Toxicity was evaluated in randomized pts according to treatment administered during the randomized phase, respectively, until next element started: 268 pts were treated in the control and 281 in the EA (see Table). 16 of them switched in/after the first Blina cycle to the HR blocks due to toxicity or poor response to Blina (due to increasing MRD, >=1x10 -4). Medically relevant adverse reactions of special interest (ARSI) according to MedDRA (specified in the protocol) were evaluated in the randomized treatment phases (CA: HR-2‘/HR-3‘ block, EA: 2 Blina cycles). ARSI were recorded in 61 of 268 pts in the CA (22.8%, 71 ARSI) and in 29 of 281 pts in the EA (10.3% [p<0.001], 33 ARSI); 3 of the 33 ARSI in the EA were related to HR blocks and were observed in 3 of the 16 pts that switched to HR blocks. Life-threatening SAR were seen in 14 pts of the control group (5.2%) and in no patient in the EA (p<0.001). Conclusions: Taken together, these results show for the first time in newly diagnosed pts with HR B ALL, the favorable toxicity profile previously reported with Blinatumomab in pediatric relapsed ALL (Locatelli F et al, JAMA 2021; Brown P et al, JAMA 2021). We have demonstrated that the toxicity profile of Blinatumomab is much more favorable as compared to the intensive chemotherapy approach using HR-blocks. If upcoming analyses of outcome data will show no inferiority of the EA, blinatumomab replacement of some of the intensive chemotherapy blocks will become the new standard of care for treatment if newly diagnosed patients with HR B-ALL.

Longitudinal Assessment of Minimal Residual Disease (MRD) in the ATLAS Randomized Phase 3 Trial of Post-Transplant Treatment with Carfilzomib, Lenalidomide, and Dexamethasone (KRd) Versus Lenalidomide (R) Alone in Patients with Newly Diagnosed Multiple Myeloma (NDMM)

Introduction The interim results of the ATLAS trial (Dytfeld, Lancet Oncology 2023) suggest a progression-free survival (PFS) benefit for NDMM patients after autologous hematopoietic stem cell transplantation (ASCT) treated with MRD-directed and risk-adapted maintenance using KRd compared to single-agent R. In order to gain a better understanding of the differences in response dynamics and depth achieved by the two regimens, we conducted a detailed analysis of MRD results assessed at multiple timepoints. Methods ATLAS is an ongoing phase 3 trial that randomized 180 patients with NDMM after ASCT to receive maintenance therapy with KRd or R. In the KRd arm, patients with standard-risk cytogenetics who achieved MRD-negativity after cycle 6 de-escalated therapy to receive R alone, starting from cycle 9. MRD assessments were performed using multicolor flow cytometry (MFC) with a limit of detection (LoD) of 10 -5 and next-generation sequencing (NGS) with LoD 10 -6 at screening, cycle 6 (C6), C12, C18, C24, and C36. Patients with MRD>10 -5 were considered MRD positive. When both MFC and NGS results were available, at least one positive result qualified a patient as MRD-positive. In line with the MRD reporting harmonization guidelines (Costa, Leukemia 2021), patients who have not reached a landmark timepoint for analysis were excluded from the denominator. MRD rates between the treatment arms were compared using the chi-square test. PFS and MRD-free survival (death, progression or MRD resurgence counted as an event) were analyzed using the Kaplan-Meier method to calculate survival rates, and the log-rank test was performed to compare the groups. Results We previously reported MRD rates at the end of 6 cycles (Dytfeld, Lancet Oncology2023). In this analysis, MRD results were evaluable for the following number of subjects at the respective timepoints: 180 at screening, 180 at C6, 180 at C12, 179 at C18, 175 at C24, and 160 at C36. All the reported data, including MRD results, response rates, and time to event, were obtained at the cut-off date 31-DEC-2021, as in the Lancet Oncology manuscript. In the landmark analysis, MRD-negativity after C6 was associated with improved PFS for the entire study patient population [HR=0.49 (95% CI: 0.27-0.90), p=0.01]. Although the proportion of MRD-negative patients at screening was higher in the R arm (46/87, 53%) compared to the KRd arm (36/93, 39%, p=0.06), MRD-negative rates were higher in the KRd arm in the subsequent landmark evaluations. The MRD-negativity rates for KRd and R were: 61/93 (66%) vs 41/87 (47%, p=0.01) at C6; 49/93 (53%) vs 41/87 (47%, p=0.46) at C12; 48/93 (52%) vs 35/86 (41%, p=0.14) at C18; 43/90 (48%) vs 26/85 (31%, p=0.02) at C24; 34/82 (41%) vs 16/78 (21%, p= 0.004) at C36. These findings were consistent with a higher number of patients in the KRd arm who converted from MRD-positivity at screening to MRD-negativity at any timepoint (Figure 1). Of the 50 patients with detectable MRD at screening in the KRd arm, 33 (66%) achieved MRD-negativity as their best response. Among them, 24 achieved MRD-negativity at the end of C6, with 2 patients converting as late as C24. The numbers were significantly lower for the R arm, with 14 out of 32 (44%) MRD-positive patients at screening achieving MRD-negativity (p=0.047). Sustained MRD-negativity for at least 12 months was reached in 51 out of 93 patients (55%) in the KRd arm and in 36 out of 87 (41%) in the R arm (p=0.06). Those who achieved sustained MRD-negativity have had significantly longer PFS (HR=0.35 (0.20-0.59), p<0.0001). Among patients with sustained MRD-negativity, patients in the KRd arm had superior PFS [HR=0.30 (0.12-0.79), p=0.01], and MRD-free survival [HR=0.42 (0.20-0.89), p=0.02, Figure 2]. In the KRd arm, sustained MRD-negativity was confirmed in 32 out of 41 (78%) patients who had de-escalated therapy from KRd to R. In this group there were 8 events (25%) of progressive disease or MRD resurgence, which compared favorably to 17/36 (47%, p=0.057) events observed in the R arm. Conclusions The improved PFS in patients with NDMM receiving post-ASCT KRd maintenance may be attributed to higher rates of MRD conversion and longer duration of MRD-negativity. Importantly, this benefit, compared to R alone, appears to be sustained even in the subset of patients who received only 8 cycles of MRD-directed, risk-adapted KRd and subsequently de-escalated to R alone.

Traverse: A Phase 2, Open-Label, Randomized Study of Acalabrutinib in Combination with Venetoclax and Rituximab in Patients with Treatment-Naive Mantle Cell Lymphoma

Background and Significance: Despite advances in treatment for mantle cell lymphoma (MCL), no curative option exists. Currently, the majority of patients with treatment-naive (TN) MCL receive chemoimmunotherapy, which can result in prolonged remissions, but is associated with significant toxicity. Almost all patients inevitably relapse, and therefore, frontline chemotherapy-free options are being increasingly explored. Acalabrutinib is a highly selective, second-generation Bruton tyrosine kinase inhibitor (BTKi) approved for the treatment of adult patients with relapsed/refractory (R/R) MCL. In a phase 1b study in patients with TN MCL, acalabrutinib in combination with venetoclax and rituximab (AVR) demonstrated high rates of clinical and molecular response with an acceptable toxicity profile, supporting its continued development as a chemotherapy-free treatment option (Wang et al. Blood. 2021; Wang et al. Blood. 2022). Furthermore, the occurrence of complete responses (CRs) with clearance of minimal residual disease (MRD) in MCL clinical trials suggests that treatment may be stopped in these patients, as observed in patients with chronic lymphocytic leukemia who received combination therapy with venetoclax and rituximab. Study Design and Methods: TrAVeRse (NCT#05951959) is a phase 2, open-label, randomized, multicenter, international study of AVR in patients with TN MCL. Patients aged ≥18 years with an Eastern Cooperative Oncology Group performance status of 0-2 (3 if due to lymphoma), and Ann Arbor stage II, III, or IV disease are eligible. Key exclusion criteria are active central nervous system involvement by lymphoma or leptomeningeal disease, severe/life-threatening illness or medical condition, clinically significant cardiovascular disease, stroke, or intracranial hemorrhage within 6 months of enrollment, and any active uncontrolled infection. Approximately 125 patients will be screened to achieve a study population of 100 patients. During the induction phase, patients will receive oral acalabrutinib 100 mg twice daily (BID) in 28-day cycles for 13 cycles, starting at cycle 1; oral venetoclax once daily, starting at cycle 2 at a dose of 20 mg daily, with ramp-up to a maximum of 400 mg daily over 5 weeks for a total of 12 cycles; and intravenous rituximab 375 mg/m 2 on day 1 of every cycle for 12 cycles, starting at cycle 1. Patients who complete 13 cycles of AVR induction will be centrally tested for MRD status and continue to receive acalabrutinib 100 mg BID for 1 additional cycle (cycle 14) while awaiting disease response assessment and MRD results. Patients who achieve an MRD-negative CR (in peripheral blood at a threshold of 10 -5) at the end of induction will be randomly assigned in a 1:1 ratio to either continue to receive acalabrutinib 100 mg BID or to observation from cycle 15. Randomization will be stratified according to the simplified MCL International Prognostic Index [sMIPI] score (low [score, 0-3] and intermediate risk [4-5] vs high risk [6-11]), histologic features (blastoid or pleomorphic vs classical histology), and TP53 status (mutated vs unmutated). Those with an MRD-positive CR, partial response (PR), or stable disease (SD) at end of induction will continue to receive acalabrutinib 100 mg BID until disease progression, death, or unacceptable toxicity. Patients who progress during observation may be retreated with acalabrutinib 100 mg BID at relapse. The primary endpoint of this study is the MRD-negative CR rate at the end of AVR induction (ie, end of cycle 13). Secondary endpoints include MRD-negative CR rate at any point during the study, objective response rate and CR rate, duration of response, time to next treatment, progression-free survival, event-free survival, overall survival, safety, and tolerability. The study will also assess the feasibility of response-adapted treatment cessation for those who achieve MRD-negative CR after AVR induction and the efficacy of acalabrutinib retreatment in the subgroup of patients randomized to observation who subsequently relapse. Patients will be followed for 36 months post last patient randomized. Enrollment is planned to begin Q3 of 2023 including sites in North America, Australia, and Europe. Summary: This study will evaluate the efficacy of AVR, a chemotherapy-free triplet combination therapy, in patients with TN MCL.

Final Analysis of a Phase 2 Trial of Daratumumab, Carfilzomib, Lenalidomide, and Dexamethasone in Newly Diagnosed Multiple Myeloma (NDMM) without Autologous Stem Cell Transplantation (ASCT)

Introduction: A previous trial showed that Dara-KRd for 8 cycles without ASCT led to a minimal residual disease (MRD) negativity rate of 71% at 10 -5 in NDMM (Landgren et al. JAMA Oncology 2021). MRD-adapted Dara-KRd with ASCT led to an MRD(-) rate of 71% at 10 -6 with a 3-year progression-free survival (PFS) of 88%, 79%, and 50% in pts with 0, 1 or 2+ high-risk cytogenetic abnormalities (HRCA), respectively (Costa et al. EHA 2022). In this phase 2 study, we evaluated the safety and efficacy of 24 cycles of Dara-KRd without ASCT in pts with NDMM. Methods: Pts with NDMM with up to 1 cycle of prior therapy were enrolled regardless of ASCT eligibility. Pts received Dara-KRd for planned 24 cycles: daratumumab 16 mg/kg IV weekly for cycles (C) 1 & 2, q2weeks for C3-8, then q4weeks for C9-24; carfilzomib 20/36 mg/m 2 on days 1, 2, 8, 9, 15, 16 for C1-8, then 36 mg/m 2 on days 1, 2, 15, 16 for C9-24; lenalidomide 25 mg PO days 1-21 of a 28 day cycle for 24 cycles; dexamethasone 40 mg PO weekly (20 mg if age > 75) for C1-9, and 20 mg PO weekly for C9-24. Pts had the option to harvest stem cells (SC) to permit ASCT in the future. MRD testing was performed by NGS (clonoSEQ, Adaptive Biotechnologies) with sensitivity <10 -6 at the end of cycles 8 (C8), C12, and C24. Mass spectrometry (MS) of peripheral blood samples were also performed using MALDI and liquid-chromatography (LCMS) (The Binding Site Group). The primary endpoint was the rate of stringent CR and/or MRD(-) <10 -5 at the end of C8; the hypothesis was that >50% would meet the primary endpoint with Dara-KRd compared to historical 30% with KRd (85% power, one-sided alpha=0.10). Results: 42 pts enrolled from March 2019-January 2022. Median age was 58 years (range 39-79). 13 (31%) were Black and 5 (12%) were Hispanic. HRCA (t(4;14), t(14;16), t(14;20), +1q, del17p) were present in 24/42 (57%); 10 (24%) had 2+ HRCA. 34 pts (81%) underwent SC collection, all with plerixafor (median yield 8.91x10 6 CD34+/kg). 40 pts (95%) were evaluable for the primary endpoint at C8. Two pts withdrew before C8 for reasons unrelated to treatment. There were 32 (80%) MRD-evaluable pts at C8. The sCR and/or MRD(-) rate (<10 -5) was 30/40 (75%, 95% CI 61%-89%). The C8 10 -5 MRD(-) rate was 20/32 (63%) and at 10 -6 it was 12/32 (38%). Responses deepened over time; 1/12 (8%) and 6/20 (30%) pts converted from MRD(+) at C8 to MRD(-) at later timepoints at 10 -5 and 10 -6 thresholds, respectively, for a best MRD(-) rate of 21/32 (66%) at 10 -5 and 18/32 (56%) at 10 -6 . Sustained 10 -5 MRD(-) >12 months was achieved in 11 (26%) pts; 6 pts were <1 year from first MRD(-) result. Overall response rate at C8 was 38/40 (95%) with 27 (68%) sCR, 38 (95%) VGPR+, and 2 PD. Of the 39 pts with trackable mass spectra in the peripheral blood, the MALDI(-) rate was 22/39 (56%) after C8 and 24/39 (62%) as best response. The LCMS(-) rate was 7/39 (18%) after C8 and 12/39 (31%) as best response. Median follow-up was 27 months. 21 pts (50%) completed all 24 cycles, and 11 (26%) remain on protocol. 7 pts (17%) had disease progression (6 on protocol), including 4 pts who reached the primary endpoint at C8. 4 pts (10%) discontinued protocol therapy early (additional 2 pts due to nonadherence). 1 pt received ASCT after discontinuing while in response. 2 deaths occurred, both early in treatment and due to primary refractory disease. The 3-year PFS was 85%: 100% for standard-risk disease, 92% for 1 HRCA, and 60% for 2+ HRCA. Of the 7 pts with progression, 6 had at least one of: extramedullary disease (4), 2+ HRCA (4), or circulating plasma cells (1). Neither NGS nor mass spectrometry status at C8 were associated with PFS differences using the landmark method. The 3-year OS was 95%. All-grade/grade 3+ (G3) neutropenia occurred in 26%/21%, anemia in 59%/2%, and thrombocytopenia in 64%/26%. Notable nonhematologic adverse events were hyperglycemia (all 76%/ G3+ 7%), diarrhea (71%/5%), hypertension (57%/17%), and neuropathy (40%/0%). COVID infections occurred in 38% (2% G3) and upper respiratory infections in 45% (0% G3+). There was one case of thrombotic microangiopathy leading to discontinuation of carfilzomib. G3 atrial fibrillation and heart failure each occurred in 1 pt. Conclusions: Extended frontline Dara-KRd for NDMM without ASCT induced high rates of sCR and/or MRD(-) within 8 cycles, meeting the primary endpoint. The rate and depth of MRD(-) improved beyond C8. This ASCT-free approach led to lower PFS for pts with 2+ HRCA, as also seen with ASCT, but excellent PFS in those with standard-risk disease and 1 HRCA.

Results of the Phase III Randomized Iskia Trial: Isatuximab-Carfilzomib-Lenalidomide-Dexamethasone Vs Carfilzomib-Lenalidomide-Dexamethasone As Pre-Transplant Induction and Post-Transplant Consolidation in Newly Diagnosed Multiple Myeloma Patients

Background. The current standard treatment for transplant-eligible (TE) patients (pts) with newly diagnosed multiple myeloma (NDMM) consists of quadruplet induction with proteasome inhibitors, immunomodulatory agents, dexamethasone and anti-CD38 monoclonal antibody followed by high-dose melphalan and autologous stem-cell transplant (MEL200-ASCT), with subsequent consolidation. The phase III IsKia trial assessed efficacy and safety of isatuximab-carfilzomib-lenalidomide-dexamethasone (IsaKRd) as pre-ASCT induction and post-ASCT consolidation vs KRd. Methods. TE NDMM pts aged <70 years were enrolled and randomized. IsaKRd pts received 4 28-day cycles of Isa: 10 mg/kg IV days 1, 8, 15, 22 cycle 1, followed by 10 mg/kg days 1, 15 cycles 2-4; K: 20 mg/m 2 IV day 1 cycle 1, followed by 56 mg/m 2 IV days 8, 15 cycle 1 and days 1, 8, 15 cycles 2-4; R: 25 mg PO daily days 1-21; d: 40 mg PO days 1, 8, 15, 22; MEL200-ASCT and 4 consolidation cycles with IsaKRd at the same schedule. KRd pts received 4 KRd induction cycles; MEL200-ASCT and 4 KRd consolidation cycles (K, R and d at the same schedule as in the IsaKRd arm). The primary endpoint was the rate of minimal residual disease (MRD) negativity by next-generation sequencing (NGS; 10 -5) after consolidation in the intention-to-treat (ITT) population. MRD was tested in all pts who achieved at least a very good partial response (≥VGPR). Key secondary endpoints were the rate of NGS MRD negativity (10 -5) after induction and PFS. MRD rates were evaluated in an ITT analysis (pts with missing MRD data or who achieved ≤PR were considered as MRD positive). The data cut-off for the analysis was May 22, 2023. Results. 302 pts were enrolled and randomized (151 in both the IsaKRd and KRd arms). Pt characteristics were well balanced between the two arms: median age was 61 vs 60 years, respectively; 18% vs 19% of pts had high-risk (HiR) cytogenetic abnormalities (CA) [del(17p) and/or t(4;14) and/or t(14;16)]; 9% vs 8% had ≥2 HiR CA [double hit; including del(17p), t(4;14), t(14;16) and gain/amp(1q)]. In ITT analysis, the rates of MRD negativity at the 10 -5 cut-off after consolidation (primary endpoint) were 77% vs 67% (OR 1.67; p=0.049) with IsaKRd vs KRd; the respective rates of MRD negativity at the 10 -6 cut-off were 67% vs 48% (OR 2.29; p<0.001); consistent MRD results were detected by next-generation flow. ≥VGPR after consolidation was 94% in both arms; ≥CR 74% vs 72% and sCR 64% vs 67% in the IsaKRd vs KRd arms. The MRD negativity advantage, both at 10 -5 and 10 -6, was retained in all subgroups analyzed ( Figure), with similar benefit in pts with standard-risk (SR) and HiR features. In particular, the 10 -5 MRD negativity rates with IsaKRd were 76% in HiR and 77% in double-hit pts, comparable to the one in SR pts (79%). In the KRd arm, the 10 -5 MRD negativity rates were 58% in HiR and 53% in double-hit pts, inferior to the one in SR pts (70%). The 10 -6 MRD negativity rates with IsaKRd were 72% in HiR, 77% in double-hit pts and 67% in SR pts. The MRD negativity rate after induction (first key secondary endpoint) was also significantly higher with IsaKRd vs KRd (10 -5: 45% vs 26%, OR 2.34, p<0.001; 10 -6: 27% vs 14%, OR 2.36, p=0.004), with a consistent benefit in all subgroups. After induction, the MRD negativity rates in HiR and double-hit pts treated with IsaKRd were: 10 -5, HiR 60%, double-hit 54%; 10 -6, HiR 40%, double-hit 31%. The MRD negativity rates after ASCT were also significantly better with IsaKRd vs KRd (10 -5: 64% vs 49%, OR 1.93, p=0.006; 10 -6: 52% vs 27%, OR 3.01, p<0.001), with a consistent advantage in all subgroups. At the current follow-up (median, 20 months, IQR 18-23), there was no difference in PFS (95% at 1 year in both arms). 55% of pts had ≥1 hematologic adverse events (AEs) with IsaKRd vs 43% with KRd; main grade 3-4 hematologic AEs in IsaKRd vs KRd were neutropenia (37% vs 22%) and thrombocytopenia (15% vs 17%). 41% of pts had ≥1 non-hematologic AEs with IsaKRd vs 37% with KRd, including infections (16% vs 12%), gastrointestinal (7% vs 5%), vascular (2% vs 7%) and cardiac events (1% vs 4%). Discontinuation for toxicity was 6% in IsaKRd vs 5% in KRd arms; treatment-related deaths were 4 with IsaKRd (2 COVID, 1 pneumonia, 1 pulmonary embolism) and 1 with KRd (septic shock). Conclusion. In TE NDMM pts, the addition of isatuximab to KRd induction and consolidation significantly increased MRD negativity rates in every treatment phase as compared to KRd, with no new safety concerns. This benefit was retained in HiR pts.

Minimal residual disease in patients with diffuse large B-cell lymphoma undergoing autologous stem cell transplantation.

Improved biomarkers are required to guide the optimal use of autologous stem cell transplantation (ASCT) in patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL). We hypothesized that minimal residual disease (MRD) identified using immunoglobulin high-throughput sequencing in apheresis stem cell (ASC) samples, post-ASCT peripheral blood mononuclear cell (PBMC), and plasma samples could predict relapse. We studied 159 patients with R/R DLBCL who underwent ASCT, of whom 98 had an ASC sample and 60 had post-ASCT surveillance samples. After a median post-ASCT follow-up of 60 months, the 5-year progression-free survival (PFS) was 48%. MRD was detected in of 23/98 (23%) ASC samples and was associated with very poor PFS (5-year PFS 13% vs 53%, P<.001) and inferior overall survival (52% vs 68%, P= .05). The sensitivity and specificity of ASC MRD positivity for progression and death were 36% and 93%, respectively. Positive ASC MRD remained a significant predictor of PFS in multivariable analysis (hazard ratio [HR], 3.7; P< .001). Post-ASCT surveillance MRD testing of plasma, but not PBMC samples, reliably identified patients with an impending relapse. A positive plasma MRD result was associated with inferior PFS (HR, 3.0; P= .016) in a multivariable analysis. The median lead time from MRD detection to relapse was 62 days (range, 0-518 days). In conclusion, the detection of MRD in ASC samples is associated with a very high risk of relapse, justifying alternative treatment strategies or trials of novel consolidation options in these patients. Furthermore, post-ASCT MRD monitoring may facilitate the evaluation of the early initiation of treatment at molecular relapse. This trial has been registered at www.clinicaltrials.gov as #NCT02362997.

Clinical validation of next-generation sequencing-based clonality assays for minimal residual disease tracking in multiple myeloma.

e20037 Background: The prognosis of multiple myeloma (MM) patients is dependent on minimal residual disease (MRD) status after treatment. Multicolor flow cytometry (MFC) as a clinical MRD evaluation method has certain limitations regarding sample requirement, sensitivity and specificity. Next-generation sequencing (NGS)-based B cell receptor (BCR) clonality assessment has an edge in lymphoid clone MRD detection, with easier sample preparation and higher sensitivity. NGS-based BCR clonality assays were performed to evaluate curative effect and track MRD of MM patients, and its performance in testing samples with limited DNA contents was explored. Methods: In this retrospective study, newly diagnosed or relapsed MM patients with paired sample collected before and after initial treatment were included, and OriMIRACLE L NGS assay were used for BCR clonality detection in genomic DNA (gDNA) extracted from clinical residual marrow smear samples. The quantitative accuracy of NGS was compared to MFC. The concordance of MRD between MFC, NGS and clinical response assessment were analyzed. Results: From December 2016 to April 2022, 29 patients were included with a median age of 65 years old. The gDNA input of the sample collected before treatment was 100.0 ng, and the median DNA content of the sample collected after treatment was 1673.4 ±594.8 ng. The quantitative results of NGS and MFC were consistent in samples with a frequency of cancer cells of above 1x10-4 among all nucleated cells. BCR clonality detection in 23 newly diagnosed and 6 relapsed cases showed a 100% sensitivity at diagnosis. For post-treatment monitoring, patients with positive MRD by both MFC and NGS (n = 18), the MRD results were consistent with clinical response assessment. One of the patients achieved complete response (CR), while the remaining patients were unable to achieved CR. The CR patient was relapsed after 134 days. The clinical responses of 11 cases with negative MRD by MFC and positive MRD by NGS were reviewed. There were 7 cases with CR, 3 cases with very good partial response or partial response, and one case with stable disease. Among those CR patients, except for one patient who was lost to follow-up, 4 patients who underwent continuous treatment were still CR and 2 patients who had treatment discontinued recurred at 67 and 134 days. Conclusions: OriMIRACLE L NGS assay showed the higher sensitivity and specificity of MRD evaluation than MFC. For patients with negative MRD by MFS and positive MRD by NGS, recurrence might occur in case of treatment discontinuation. It is recommended to consider carefully that whether maintenance treatment is required in such cases.

Long-term follow-up of rituximab plus bendamustine and cytarabine in older patients with newly diagnosed MCL

AbstractThe combination of rituximab, bendamustine, and low-dose cytarabine (R-BAC) has been studied in a phase 2 prospective multicenter study from Fondazione Italiana Linfomi (RBAC500). In 57 previously untreated elderly patients with mantle cell lymphoma (MCL), R-BAC was associated with a complete remission rate of 91% and 2-year progression-free survival (PFS) of 81% (95% confidence interval [CI], 68-89). Here, we report the long-term survival outcomes, late toxicities, and results of minimal residual disease (MRD) evaluation. After a median follow-up of 86 months (range, 57-107 months), the median overall survival (OS) and PFS were not reached. The 7-year PFS and OS rates were 55% (95% CI, 41-67), and 63% (95% CI, 49-74), respectively. Patients who responded (n = 53) had a 7-year PFS of 59% (95% CI, 44-71), with no relapse or progression registered after the sixth year. In the multivariate analysis, blastoid/pleomorphic morphology was the strongest adverse predictive factor for PFS (P = .04). Patients with an end of treatment negative MRD had better, but not significant, outcomes for both PFS and OS than patients with MRD-positive (P = 0.148 and P = 0.162, respectively). There was no signal of late toxicity or an increase in secondary malignancies during the prolonged follow-up. In conclusion, R-BAC, which was not followed by maintenance therapy, showed sustained efficacy over time in older patients with MCL. Survival outcomes compare favorably with those of other immunochemotherapy regimens (with or without maintenance), including combinations of BTK inhibitors upfront. This study was registered with EudraCT as 2011-005739-23 and at www.clinicaltrials.gov as #NCT01662050.

Minimal Residual Disease Detection Using Gene Scanning Analysis, Fluorescent Fragment Analysis, and Capillary Electrophoresis for IgH Rearrangement in Adult B-Lineage Acute Lymphoblastic Leukemia: A Cross-Sectional Study.

Minimal residual disease (MRD) is considered the greatest prognostic factor in acute lymphoblastic leukemia (ALL). MRD is a valuable tool for anticipating impending relapse and treatment response assessment. The objective of the present study was to investigate whether the detection of IgH gene rearrangement using polymerase chain reaction (PCR)-based GeneScan analysis could be a complementary method to monitor MRD along with the quantitative realtime PCR (qPCR). In this cross-sectional study, we valued the MRD levels, based on the GeneScanning analysis (GSA), and then compared the data with quantitative real-time polymerase chain reaction at different time points in peripheral blood (PB) samples of adult B-lineage ALL patients (n=35). The specific polymerase chain reaction (PCR) primers for IGH gene FR-1 and fluorescence-labeled J-primer were used and analyzed by capillary gel electrophoresis on a sequencer. The results of this study were compared with the previously reported MRD results obtained by the IGH rearrangements allele-specific oligonucleotide (ASO) -qPCR methods. The total concordance rate was 86.7%, with a P<0.001. MRD results obtained by GSA and ASO-qPCR methods were concordant in all diagnostic samples and samples on the 14th and 28th days of induction therapy. The results of these 2.5 years' follow-ups demonstrated a significant correlation between the two techniques (r=0.892, P<0.001). It seems that the PCR-based GeneScan analysis of IGH gene rearrangement detection may be a valuable molecular technique to distinguish monoclonality from polyclonality. And also, it may be a precise tool to detect the residual leukemic DNA in the PB follow-up samples of patients.

Free from Maintenance Drug Therapy in Multiple Myeloma (The FREEDMM Trial): A Pilot Study of Minimal Residual Disease (MRD)-Driven Discontinuation of Maintenance

Background Induction therapy followed by autologous stem cell transplant (ASCT) and maintenance is the current standard of care for the treatment of newly diagnosed multiple myeloma (NDMM). However, indefinite maintenance therapy after ASCT carries both significant clinical and financial toxicities. While minimal residual disease (MRD) testing has become part of routine standard of care, its use in guiding treatment decisions, in particular cessation of therapy, has not been established in the maintenance setting. We hypothesize that MRD monitoring could be used to guide clinical decision making, justifying cessation of maintenance therapy in a sub-group of patients with sustained MRD-negative remission. Study Design and Methods This is a phase 2 single-arm adaptive design study (NCT 05192122) for decision-making regarding maintenance therapy (Figure 1). Patients are eligible if they have a diagnosis of active MM, have completed at least 2 years of maintenance therapy post-ASCT, and meet International Myeloma Working Group (IMWG) criteria for very good partial response (VGPR) or complete response (CR). MRD testing is performed twice, at least one year apart, on bone marrow aspirate using next-generation sequencing (NGS) and MRD-negative status is defined at a threshold of 10-6. Patients with negative PET scan in VGPR or CR and sustained (i.e., twice, 1 year apart) MRD negative status discontinue maintenance therapy. MRD-positive patients continue continue maintenance and are taken off study. In patients who are MRD-negative and discontinue maintenance therapy, MRD testing will be re-assessed yearly for 3 years. At each time of MRD testing, MALDI-TOF spectrometry and measurement of circulating multiple myeloma cells (CMMCs) will be done in parallel to compare against MRD results. Health-related quality of life (HRQoL) will be assessed throughout and results on maintenance compared with those after discontinuation using the EORTC QLQ-MY20 questionnaire. This study will enroll 50 patients to address the primary objective of assessment for sustained MRD-negative status at 12 months after stopping maintenance therapy. For all patients, a 5-mL peripheral blood and bone marrow sample is collected and stored at the time of each standard of care bone marrow biopsy and will be used to characterize the immune microenvironment by Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) of paired peripheral blood and bone marrow in MRD-negative and MRD-positive patients as well as of patients progressing after stopping therapy. Preliminary Results Enrollment into FREEDMM trial began in December 2021, and to date 20 patients in CR have undergone a first bone marrow for MRD assessment, with results pending in 9 patients. The median age is 66.5 (range: 39-76) years, and the majority of patients are either Black (n=7; 35%) or Hispanic (n=7; 35%). One patient has high risk disease (17p deletion). The median number of years of maintenance therapy completed is 4.2 (range: 2.14-7.9) years. 17 (85%) patients had a negative MALDI-TOF test at the time of the first marrow. Seven (63.6%) of 11 patients were found to be MRD-negative. MALDI-TOF failed to accurately correlate with MRD results in 2 patients. 40 paired peripheral blood (n=20) and bone marrow aspirate (n=20) samples have been collected for CITE-seq analysis. Pilot CITE-seq experiments captured 6,700-11,000 cells with between 38,000-68,000 reads per cell on one MRD-negative and one MRD-positive bone marrow aspirate and paired peripheral blood sample. CITE-seq results for these 20 patients will be presented at the conference. Conclusion: This phase 2 trial applies a novel clinical trial design with an adaptive strategy to explore use of MRD testing as a guide to discontinuing maintenance therapy in myeloma and represents one of the first studies to explore in parallel various measures of disease monitoring (i.e., MRD, MALDI-TOF, CMMCs, CITE-seq). Ongoing results will be reported at the conference. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dissecting MRD Kinetics By Automated Computational Analysis to Improve Outcome Prediction in Mantle Cell Lymphoma: A Bioinformatic Substudy from the Fondazione Italiana Linfomi (FIL) MCL0208 Clinical Trial

Background and Aims. Minimal residual disease (MRD) detection is a validated outcome predictor in mantle cell lymphoma (MCL). Recently, the clinical relevance of repeated MRD monitoring has been described in a prospective clinical trial [Ferrero, Blood 2022]. Nonetheless, complex patterns of MRD kinetics over time and availability of results obtained by heterogeneous tissues (bone marrow, BM, vs peripheral blood, PB) might generate substantial interpretation issues and hamper an easy-to-use application of this predictive biomarker. To overcome the limitation of empirical analysis we tested CONNECTOR, a novel automated computational framework to facilitate the interpretation of MRD kinetics and stratify patients in risk classes based on a solid, algorithm-derived, classification. Patients and methods. ASO RQ-PCR MRD data already generated from BM and PB samples in the FIL MCL0208 trial [Ferrero, Blood 2022], offering first-line high dose chemoimmunotherapy and autologous transplantation (ASCT) to youngers MCL patients, were employed. CONNECTOR (https://qbioturin.github.io/connector/) is a data-driven framework based on Functional Data Analysis for analyzing longitudinal high-dimensional data in a straightforward and revealing way. CONNECTOR allows the aggregation of time-series data through an unsupervised approach in informative clusters. Moreover, we developed a classification approach to predict the risk class for new MRD data. The new MRD curves were allocated to existing risk clusters defined on a training set of MRD data. Results. Without knowledge of any clinical or outcome data, CONNECTOR was run on 117 patients characterized by at least 3 MRD timepoints in BM (median 6, 3-9) and separately on 95 patients with at least 4 MRD timepoints in PB (median 7, 4-9). Starting from BM MRD data, CONNECTOR identified four patient risk Clusters (CrC), based on MRD kinetics, Figure1A. CrC_A comprised 73 patients with an overall quick and stable MRD negativization; CrC_B accounted for 14 patients characterized by later MRD negativization, alternating MRD results, or low-level MRD persistence; the 14 patients in CrC_C were always characterized by early MRD negativization followed by late MRD reappearance; CrC_D was composed of 16 patients with stable MRD positivity or only transient MRD negativization followed by early MRD reappearance. The different trends of MRD kinetics dissected by CONNECTOR fairly predicted patients’ outcomes: median TTP was not reached for CrC_A and B, 36 months for CrC_C and 27 months for CrC_D, respectively (p<0.0001), Figure1B. Similar data were generated when CONNECTOR was run on MRD results from PB samples: CrC allocation of BM vs PB was concordant for 89% of patients and survival analysis on PB results confirmed patients stratification in two groups: favorable MRD kinetics (CrC_A and B, median TTP not reached) vs unfavorable MRD kinetics (CrC_C and D, median TTP 35 months, p<0.0001), as depicted in Figure1B. Finally, we tested the performance of pooling together BM and PB results, to increase the number of evaluable patients (irrespective of eventual missing BM samples). Only the BM point at 12 months after ASCT was considered, together with the PB MRD results of all the available time points. CONNECTOR was used to verify if this mixed MRD pattern was able to correctly reclassify patients in the same CrC. Actually, 89 out of 95 patients (94%) were correctly reclassified (p_mean=0.96, p_sd= 0.07). Discussion. CONNECTOR allowed unsupervised identification of patients clusters with distinct MRD kinetics and highly significant different clinical outcome. Such clusterization proved effective using BM, PB and mixed tissues, as well. Validation of CONNECTOR performance in large, independent prospective trials is currently ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inotuzumab Ozogamicin Induction Followed By Standard Chemotherapy Yields High Remission Rates and Promising Survival in Older (>55 Years) Patients with De Novo B-Lymphoblastic Leukemia (GMALL-Initial1 Trial)

Introduction: Acute lymphoblastic leukemia (ALL) represents approx. 5% of all newly diagnosed leukemias in patients (pts) between 55 and 70 years of age. Incorporation of novel antibody-based therapies in the first line induction therapy bears the potential of a significantly increased response rates and survival together with a reduction of treatment toxicity. Methods: This open label phase II study (ClinicalTrials.gov, identifier: NCT03460522) of the German Multicenter Study Group on Adult Acute Lymphoblastic Leukemia was conducted at 13 centers. Pts aged >55 years with newly diagnosed Ph/BCR-ABL negative B-precursor ALL (at least CD20 positivity of leukemic blasts) were eligible. The 1st induction cycle consisted of InO 0.8mg/m2 on day1 and 0.5mg/m2 on d8 and d15 together with dexamethasone 10 mg/m2 (day7-8, day14-17) and one intrathecal injection (i.th.) of methotrexate (MTX), cytarabine (AraC) and dexamethasone (Dex). The 2nd and 3rd induction cycle consisted of InO 0.5mg/m2 on day 1, 8 and 15 plus i.th. MTX/AraC/Dex. Pts achieving a complete remission (CR) were offered to receive 5 conventional consolidation therapies (3 x ID-MTX/asparaginase; 2 x ID-AraC) and one reinduction therapy (idarubicine/ AraC/ cyclophosphamide / Dex.) in combination with rituximab (for CD20+ ALL), followed by a maintenance therapy with 6-MP/MTX. The primary endpoint was event free survival (EFS) at 12-months follow-up. An event was defined as any of the following: persisting bone marrow blasts after 2 cycles of InO, relapse or death. An event rate of ≤40% at 12 months follow-up was considered to qualify the experimental treatment as very promising for additional testing. Results: 43 out of the 45 pts included were analyzed regarding the primary end point. Two pts were excluded per protocol from further analysis, as they received only one cycle of induction or withdraw consent. At diagnosis, median age was 64 years (range 56-80 years, 20 pts ≥65 years and 12 pts ≥70 years), median bone marrow blast count was 80% and median CD22 expression on leukemic blasts was 69% (range 21-99). 38 pts were diagnosed with a common- and 5 with a pro-B ALL. 42/43 pts completed all 3 cycles of induction therapy. All 43 pts achieved a complete remission (CR/CRi), mainly after the 1st induction. Median time between start of 1st induction to start 2nd induction, from 2nd to 3rd induction and from 3rd induction to 1st consolidation therapy was 21 days, 28 days and 30 days, respectively. Results of minimal residual disease (MRD), measured by real-time quantitative PCR, are available for all 43 pts, with 23/43 (53%) and 31/42 (74%) pts being MRD negative after 2nd and 3rd induction therapy, respectively. With a median follow-up of 697 days, the probability of OS at 1 and 2 years is 91% (95% C.I. 82-99%) and 81% (95% C.I. 69-94%), respectively. No patient died within the first 6 months after start of induction therapy. 5 events occurred within the first year after study inclusion, 3 deaths in CR and 2 relapsed ALL, resulting in event rate of 12% and a probability of EFS at 1 year (primary endpoint of the study) of 88% (95% CI 79-98). EFS at 2 years was 73% (95% CI 59-88). So far, 7 pts with persisting/reappearance of MRD (2 pts) or relapse (5 pts) were treated with blinatumomab. 9 pts received an aSCT (4 pts in CR1 and 5 pts >CR1), 6 of these pts are alive in CR. Regarding adverse events (AEs) during induction cycles 1, 2 and 3, most common AEs ≥CTC grade 3 reported were leukocytopenia (in 74%, 19% and 2% of all cases, respectively), anemia (37%, 5%, 0%), thrombocytopenia (49%, 7%, 2%) and elevation of liver enzymes (14%, 5%, 0%). One patient was reported with suspected veno occlusive disease (after 2nd induction therapy). Classical factors which may be associated with inferior survival outcomes like age, diagnosis of pro-B ALL, disease burden at diagnosis, co-morbidity or no MRD negative CR after induction had no significant impact on OS or EFS. Conclusion: Three cycles of InO as induction therapy for the treatment of older pts with de novo acute B-lymphoblastic leukemia resulted in a very high remission rate. Given that our study met the primary end point (predefined event rate at 1 year ≤40%, observed event rate 12%), these promising study results could be a rationale for integrating InO induction therapy in future treatment recommendations. Ideally, this novel induction therapy should be evaluated in prospective randomized trials. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Clinical Impact of Next Generation Flow in Bone Marrow Vs Qip-Mass Spectrometry in Peripheral Blood to Assess Minimal Residual Disease in Newly Diagnosed Multiple Myeloma Patients Receiving Maintenance As Part of the GEM2014MAIN Trial

Introduction: GEM2014MAIN is a phase 3, multicenter, open-label, randomized trial exploring maintenance treatment with lenalidomide and dexamethasone (Rd) vs lenalidomide, dexamethasone and ixazomib (IRd) after autologous stem cell transplantation in newly diagnosed patients with multiple myeloma (MM). The study includes yearly analysis of minimal residual disease (MRD) in bone marrow (BM) by Next Generation Flow (NGF) and the results at year 2 guide further management. Mass spectrometry methods measuring low-level immunoglobulins have shown potential for peripheral-blood (PB) based MRD assessment in MM. Aim: In this study, we analyzed and compared the results and clinical impact of MRD evaluation in BM by NGF and in PB by quantitative immunoprecipitation mass-spectrometry (QIP-MS) during maintenance in patients enrolled in the GEM2014MAIN trial. Patients and Methods: Patients achieving at least stable disease at the end of the GEM2012MENOS65 trial were included in the GEM14MAIN, and randomized to receive Rd (lenalidomide 15mg po od days 1-21 and dexamethasone 20mg po od days 1-4 and 9-12) or IRd (Rd plus ixazomib 4mg po od days 1, 8 and 15) in cycles of 28 days. After two years of treatment, BM samples were analyzed by NGF (sensitivity of 3x10-6) as per protocol: if MRD was undetectable, maintenance was discontinued; if detectable, Rd with reduced dexamethasone at 20mg po od only days 1-4 was continued for 3 additional years. At the same time point, MRD was assessed in PB by QIP-MS with anti IgG/A/M, total k and total l beads using the EXENT system (The Binding Site, in development). The association of MRD detection by either method with progression-free survival (PFS) was assessed by the Kaplan-Meier method. Landmark analyses of PFS at 2 years post-maintenance were performed to minimize lead-time bias. Additional survival analyses were carried out to evaluate the clinical impact of MRD evolution during the study period. Results: 156 patients with paired BM/PB samples available after 2 years of maintenance were included in the analysis. NGF detected residual disease in 29% (45/156) of cases and EXENT in 24% (37/156). The results of both techniques were concordant in 85% of cases and discordances were due to 16 patients (10%) NGF+/EXENT- and 8 (5%) NGF-/EXENT+. Of note, 8/16 cases NGF+/EXENT- corresponded to light-chain MM (anti-free light chain beads were not used in this study). Therefore, overall, and considering NGF as a reference, the negative predictive value (NPV) of EXENT was 87% and the positive predictive value (PPV) of 78% (p<0.0001). With a median follow-up of 66 months from the start of the GEM14MAIN, MRD detection by either method associated with a shorter PFS at 5 years as compared to patients with undetectable MRD: NGF: 57% vs 89%, respectively; HR= 6.54 (2.92-14.67); p<0.0001; EXENT: 62%% vs 86%, respectively; HR= 4.79 (1.99-11.53); p=0.0005. We also analyzed the evolution of MRD between enrollment and after 2 years of maintenance, since we already had MRD results by both methods at the end of the GEM2012. Patients were grouped into 1) those with persistent MRD positivity at the two time points or converting from MRD- to + by either method and 2) those with sustained MRD negativity at the two time points or converting from MRD+ to - by either method. Both NGF and EXENT showed that persistent MRD+ or conversion from MRD- to MRD+ were associated with a shorter PFS at 66 months, compared to patients who remained MRD- or converted from MRD+ to MRD-: NGF: 54% vs 89%, respectively; HR = 8.9 (3.55-22.08); p <0.0001; EXENT: 61% vs 86%, respectively; HR: 4.95 (1.91-12.9); p=0.0010. Conclusions: Our study shows that, during maintenance, the identification of MRD by NGF and EXENT and its evolutionary pattern are both associated with a comparable clinical value in terms of PFS. Supported by a NPV of 87% as compared to NGF, mass spectrometry could be used to define the optimal time-point for BM-based MRD assessments as well as, alongside with standard methods, to introduce MRD adapted treatment approaches.

Stem Cell Autograft Minimal Residual Disease Negativity Improves Outcomes after Autotransplant for Multiple Myeloma

Background: Autologous hematopoietic stem cell transplantation (AHCT) in the up-front setting is the standard-of-care for newly diagnosed multiple myeloma (NDMM). Contemporary induction regimens generate deeper responses, with higher rates of minimal residual disease (MRD) negativity in the bone marrow (BM) leading to improved progression-free survival (PFS) and overall survival (OS). However, it is unclear whether stem cell autograft (AG) contamination with myeloma cells and/or their precursors contributes to relapse after AHCT. To address this, we examined MM contamination of AG by next-generation flow cytometry (NGF) to determine the predictive value of AG MRD status on outcomes after AHCT. Methods: This study included 119 NDMM patients with prospectively collected BM and AG MRD results by NGF who underwent stem cell collection and up-front AHCT at our institution between Sept 2017 and Feb 2020. AG MRD status was assessed in peripheral blood at the time of stem cell collection (SCC). BM MRD status was assessed post-induction/pre-SCC and at 3-months post-AHCT. Additional BM MRD results were also available at 1- and 2-years post-AHCT for most patients. Patients were dichotomized based on AG MRD status (negative vs positive). A Kaplan-Meier model compared median PFS and OS between cohorts. A cox proportional hazards model determined prognostic impact, adjusting for high-risk cytogenetics by FISH and induction therapy. Results: Baseline characteristics were balanced between the MRD-negative and positive groups, with median age at AHCT of 61 (range, 55-67) years in the MRD-negative group and 60 (range, 54-67) years in the MRD-positive group. Patients received a median of 6 (range, 2-12) induction cycles with daratumumab, carfilzomib, lenalidomide, and dexamethasone (D-KRd, n=20), KRd (n=38), bortezomib, lenalidomide, and dexamethasone (VRD, n=51), or other therapy (n=10). Patients with MRD-positive AG were not enriched for high-risk cytogenetics. AG MRD-negativity rates after D-KRd, KRd, VRd, and other therapy were 95.0%, 78.9%, 58.8%, and 50%, respectively. All patients with MRD-positive AG (n=35) also had MRD-positive pre-AHCT BM, whereas 71% of patients with MRD-negative AG had MRD-positive pre-AHCT BM (p <0.001). Sustained or conversion to MRD-negativity in the BM at 3-months and 1-year post-AHCT was observed more for patients with MRD-negative AG than MRD-positive AG (40% vs 9%). At a median follow-up of 40 months (range 33- 46) after AHCT, patients with MRD-negative AG, compared with MRD-positive AG counterparts, had a significantly improved PFS (3-y PFS 78%, CI: 69 ‒ 87, vs. 54%, CI: 39 ‒ 73, p=0.021)(Fig1A) and trend toward better OS (3-y OS 96%, CI: 92 ‒ 100, vs. 82%, CI: 70 ‒ 96)(Fig1B) . The 3-year PFS of the MRD-negative AG group treated with D-KRd, KRd, VRd, and other therapy were 84% (CI: 69 ‒ 100), 79% (CI: 65 ‒ 96), 76% (CI: 71 ‒ 98), and 60% (CI:29 ‒ 100), respectively, while for the MRD-positive AG group treated with KRd, VRd, and other therapy (no D-KRd in this group), 3-year PFS rates were 73% (CI: 47 ‒ 100), 57% (CI: 39 ‒ 83), and 20% (CI:3.5 ‒ 100), respectively (p = 0.4). The 3-year PFS for patients with or without high-risk cytogenetics were 59% (CI: 44 ‒79) and 90% (CI: 81 ‒ 100), respectively, in the MRD-negative AG group, and 40% (CI: 22 ‒ 74) and 67% (CI: 49 ‒ 93), respectively, in the MRD-positive AG group (p = 0.002). The 3-year OS rates were 93% (CI: 85 ‒ 100) and 100% (CI: 100 ‒ 100) for the MRD-negative AG group and 76% (CI: 61 ‒ 100) and 84% (CI: 69 ‒ 100) for the MRD-positive group (p not calculated). On univariate analysis of PFS, the presence of high-risk cytogenetics (HR 3.2, CI: 1.55 ‒ 6.57), R-ISS stage (HR 3.01, CI 1.42 ‒6.41 for R-ISS=3), and AG MRD positive (HR 2.14, CI: 1.10 ‒ 4.16) were significant prognostic factors. Multivariate analysis identified high-risk cytogenetics (HR 3.16, CI: 1.51 ‒ 6.60) as the only significant factor for PFS. Conclusion: Patients with MRD-negative AG have improved PFS and OS following AHCT. These findings support the potential utility of AG MRD status as a prognostic parameter for clinical outcomes, with implications for guiding post-AHCT therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Dissecting Ph-like ALL: The Role of Genomic Lesion and Minimal Residual Disease in Refining Outcome

Cardinali D and Della Starza I contributed equally Foà R and Chiaretti S contributed equally Background. Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL) is a subgroup of B-lineage ALL characterized by a gene expression profile (GEP) that resembles that of Ph-positive ALL but lacks the BCR/ABL1 transcript. Ph-like ALL cases are characterized by CRLF2 overexpression, JAK-STAT pathway mutations, IKZF1 deletions and rearrangements involving cytokine receptors and tyrosine kinases. To identify Ph-like ALL cases, our group developed a predictive tool named "BCR/ABL1-like predictor", which is based on the quantification by quantitative PCR of 10 transcripts, specifically overexpressed by Ph-like ALL cases1. ALL is the first neoplasm where the assessment of early response to therapy by minimal residual disease (MRD) monitoring is pivotal for guiding therapeutic choices. Real-time-quantitative PCR (RQ-PCR) of IG/TR gene rearrangements is the most widely used molecular method for MRD assessment. The GIMEMA LAL2317 is a clinical trial designed for newly diagnosed adult B-lineage Ph-negative ALL that includes two cycles of blinatumomab in the consolidation phase. MRD is evaluated at established time points (TPs), the most important being TP2, i.e., after the first consolidation cycle with high-dose chemotherapy, and TP3, after the first cycle of blinatumomab. Aim of this study. To refine risk relapse categories of Ph-like ALL cases based on their genomic features at presentation in combination with the MRD status. Methods. We performed this sub-analysis on a cohort of Ph-like ALL cases enrolled in the GIMEMA LAL2317 protocol identified according to the BCR/ABL1-like predictor assay and that underwent a centralized comprehensive molecular screening at diagnosis by IG/TR gene rearrangements and targeted DNA/RNA sequencing. MRD status was evaluated at specific TPs, i.e., TP2 and TP3, by RQ-PCR and digital droplet PCR (ddPCR). Results. A Ph-like profile was documented in 31/109 evaluable patients (28.4%); 11 of them (35.5%) experienced a relapse. By targeted RNA sequencing, 9/11 presented a gene fusion at presentation: in 4/9 a CRLF2-P2RY8 gene fusion was identified, whereas the remaining cases had PAX5-ZCCHC7, ETV6-BCL21L4, IKZF1-DDC, JAK2-r and ABL-class fusion in 1 case each (Figure 1A). The MRD status was evaluated at TP2 and TP3 by RQ-PCR in 23/31 patients: 15/23 were negative at both TPs, while 8/23 were positive at TP2 and negative at TP3; in the remaining 8 cases, MRD was not evaluated because of refractoriness (n=3), early death (n=2) or loss to follow-up (n=3). DdPCR analysis was performed in 9 patients and compared to RQ-PCR: 6/9 were concordant by both methods, while 3/9 resulted discordant with RQ-PCR being negative at TP3 while being positive by ddPCR. On the basis of the Ph-like signature and MRD results, we defined 3 subgroups: i) Ph-like ALL cases (n=12, 39%) that did not relapse, being MRD-negative at both TP2 and TP3; 3 cases were positive for fusion genes; ii) relapsed Ph-like ALL cases (n=8, 26%) who were MRD-positive at TP2 but became MRD negative at TP3, with 7 harboring a fusion gene; iii) relapsed Ph-like ALL cases (n=3, 10%), always MRD-negative at both TPs, with 2/3 positive for fusion genes (Figure 1B). Finally, by flow cytometry analysis, available in 7 patients at relapse, 6 maintained CD19 positivity and only 1 proved CD19-negative. Conclusions. Ph-like ALL patients tend to relapse early even after blinatumomab treatment and despite becoming MRD-negative. Relapse is not related to a mechanism of CD19 escape. Based on the gene fusions at presentation and on the MRD status, we could identify 3 subgroups of Ph-like ALL. These findings suggest that Ph-like ALL cases should be followed with different markers in addition to IG/TR, particularly in cases with well-defined fusion genes, who are at a very high risk of relapse. In this sense, a refined and rapid genetic characterization at presentation of Ph-like ALL cases is warranted for a more personalized and targeted patient management. 1. Chiaretti S et al., BJH 2018 Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal