Value Of Minimal Residual Disease Research Articles

The value of minimal residual disease and IKZF1 deletion for predicting prognosis in adult patients with B-cell acute lymphoblastic leukemia

Objective: To reassess the prognostic value of minimal residual disease (MRD) and IKZF1 gene deletions in adults with B-cell acute lymphoblastic leukemia (B-ALL) who received pediatric-specific chemotherapy regimens during the Nanfang Hospital PDT-ALL-2016 trial. Methods: We retrospectively analyzed the prognosis of 149 adult patients with B-ALL who were admitted to Nanfang Hospital from January 2016 to September 2020. Prognostic factors were identified using Cox regression models. Results: The complete remission rate was 93.2% in 149 patients, with a 5-year overall survival (OS) rate of (54.3±5.0) % and a cumulative incidence of relapse (CIR) of (47.5±5.2) %. The Cox regression analysis revealed that MRD positivity at day 45 (MRD(3)) after induction therapy was independently associated with relapse risk (HR=2.535, 95%CI 1.122-5.728, P=0.025). Deletion of IKZF1 gene was independently associated with mortality risk (HR=1.869, 95%CI 1.034-3.379, P=0.039). Based on MRD(3) and IKZF1 gene status, we categorized adult patients with B-ALL into the low-risk (MRD(3)-negative and IKZF1 gene deletion-negative) and high-risk (MRD(3)-positive and/or IKZF1 gene wild type) groups. The 5-year OS and CIR rates were (45.5±6.0) % vs (69.4±8.6) % (P<0.001) and (61.6±8.3) % vs (25.5±6.5) % (P<0.001), respectively, in the high-risk and low-risk groups, respectively. The multivariate analysis showed that the high-risk group was an independent risk factor for OS (HR=3.937, 95%CI 1.975-7.850, P<0.001) and CIR (HR=4.037, 95%CI 2.095-7.778, P<0.001) . Conclusion: The combined use of MRD and IKZF1 gene in prognostic stratification can improve clinical outcome prediction in adult patients with B-ALL, helping to guide their treatment.

Flow cytometric and cytomorphological definition of remission achievement in children with B-lineage acute lymphoblastic leukemia

The achievement of remission at the end of induction (EOI) chemotherapy in patients with acute lymphoblastic leukemia (ALL) is the key parameter of treatment effectiveness evaluation. The aim of the study – defining complete remission by multicolor flow cytometry (MFC) and bone marrow (BM) cytomorphology (CM) at the EOI chemotherapy in children with B-lineage ALL. The study was approved by the Independent Ethics Committee and the Scientific Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The study included patients of “ALL-MB 2008” and “ALL-MB 2015” trials for whom minimal residual disease (MRD) was evaluated by MFC at the EOI simultaneously with CM BM investigation. Less than 5% blasts in BM and MRD &lt; 1% were established as the remission achievement criteria for CM and MFC respectively. The study group included 1498 children aged from 1 to 18 years (median age was 4 years and 11 months) with B-cell precursor ALL. The overall concordance of MFC and CM was found to be 96.1% (1440 of 1498 patients). In 36 (2.4%) children with MRD ≥ 1%, M1 BM status was observed. In contrast, in 22 (1.5%) patients with M2/M3 BM status by CM, MRD value was below 1%. Treatment outcome was analyzed in 522 patients of “ALL-MB 2008” trial. Children with M2/M3 BM, as well as with MRD ≥ 1% demonstrated dramatically inferior outcome, in comparison to those who achieved remission. The presence of at least one of the mentioned criteria (M2/M3 status by CM or MRD ≥ 1% by MFC) defined a group of 23 (4.4%) patients with very low event-free survival (34.9%, standard error 11.0%) and very high cumulative incidence of relapse (56.4%, standard error 12.0%). For the evaluation of remission achievement, MFC and CM should be applied simultaneously at the EOI. High leukemic burden found by any of these methods is the clear definition of induction failure. MRD detection at the EOI should be implemented in any modern treatment protocol as an obligatory stage of treatment response monitoring and final risk group stratification. Considering the crucial importance of the MRD detection results, this study must be performed only in the reference laboratories of the study groups.

Prognostic Utility of Minimal Residual Disease (MRD) after Curative Intent Induction Therapy for DLBCL: A Prospective Real-World Ctdna Study

Background: The prognostic value of MRD using ultra-sensitive ctDNA assays after 1L DLBCL immunochemotherapy was recently reported (Roschewski, ASH 2022). However, real-world evidence (RWE) for the utility of ctDNA MRD assays to predict survival outcomes after standard-of-care (SOC) 1L treatment for DLBCL remains less clear. Here, we report longitudinally measured ctDNA levels, without use of tumor biopsies, in a real-world population of DLBCL patients treated with curative intent using current anthracycline-based induction regimens. Methods: We prospectively enrolled patients with DLBCLs from Samsung Medical Center undergoing 1L therapy for ctDNA profiling at 3 pre-defined milestones (Baseline, Interim after 3 cycles (C4D1), and end of treatment [EOT]). A total of 99 patients had baseline pretreatment plasma available with sufficient tumor burden for inclusion in this study. 364 samples were profiled in a blinded manner by Phased Variant Enrichment &amp; Detection Sequencing (PhasED-Seq, Foresight Diagnostics). Tumor-derived Phased Variants (PVs) were identified directly from baseline plasma (median 4 mL) without the use of tumor tissue, with matched constitutional DNA from paired PBMCs used to censor germline variants and CHIP. PVs were used to longitudinally assess ctDNA-MRD after therapy, including at the C4D1 and EOT timepoints. MRD levels were compared to standardized responses by PET/CT (Lugano 2014), PFS, and OS. Patients/samples were reported as MRD positive when ctDNA levels exceeded an analytical detection threshold (~1:10 6 cfDNA molecules) corresponding to 98% specificity. Results: Among the 99 evaluable patients, the median age was 58 years and 38% were women. 67% had stage III-IV disease, 53% had IPI scores ≥3, and 6% had MYC and BCL2 translocations. All patients were treated with anthracycline based therapy, with 94% receiving RCHOP and 6% receiving DA-EPOCH-R. The median follow-up was 44 months. We tested ctDNA performance at each landmark for predicting outcomes. ctDNA-MRD levels by PhasED-Seq at each landmark were prognostic for both PFS and OS. Specifically, when dividing pretreatment ctDNA levels at the median, ctDNA significantly predicted outcomes (PFS: P=0.004, HR=2.7; OS: P=0.0004, HR=5.1). At the C4D1 and EOT landmarks, we divided patients into those with detectable versus undetectable ctDNA-MRD. Both timepoints significantly stratified patients for outcomes including at C4D1 (PFS: P=0.0002, HR=5.0; OS: P=0.0046, HR=4.1) and at EOT (PFS: P&amp;lt;0.0001, HR=7.8; OS: P&amp;lt;0.0001, HR=10.8). As the current standard for assessing response in DLBCL is imaging, we also examined the prognostic value of PET/CT scans at the same time-point as ctDNA. While interim PET/CT scans in our cohort were prognostic for PFS, they were not significantly prognostic for OS (PFS: P=0.02, HR=2.5; OS: P=0.06, HR=2.4). At EOT, PET/CT scans were prognostic for both PFS and OS (PFS: P=0.011, HR=3.4; OS: P=0.011, HR=4.0). At both milestones, PET/CT appeared less discriminatory than ctDNA-MRD. Exemplifying this, the overall survival of patients at 24 months was better separated at EOT by ctDNA-MRD than it was by PET/CT scan (OS24 ctDNA-MRD+: 37%, ctDNA-MRD-: 94%, compared with PET/CT PR/SD/PD: 50%, PET/CT CR: 86%). Finally, to further compare the prognostic value of ctDNA-MRD in the context of the current standard PET/CT scan, we performed a Cox proportional hazards model to predict PFS and OS, using both PET/CT and ctDNA-MRD as univariate predictors, as well as a multivariate model considering both PET/CT and ctDNA. As expected, interim and EOT ctDNA-MRD and PET/CT scans were predictive of outcomes when considered in univariate analysis. However, when considered in combination, while the prognostic value of ctDNA-MRD for OS remained at both interim and EOT timepoints, PET/CT scan was no longer significantly prognostic of outcomes (Fig 1). Conclusions: These data both demonstrate the feasibility and the prognostic utility of ctDNA-MRD during and after SOC induction therapy for DLBCL in a real-world population using an ultrasensitive ctDNA-MRD assay. The higher predictive value and accuracy of detectable ctDNA-MRD as compared with PET/CT suggest opportunities for integration of such assays in lymphoma response criteria, to potentially inform future clinical decision making.

Predictive Value of Minimal Residual Disease for Efficacy of Rituximab Maintenance in Mantle Cell Lymphoma: Results From the European Mantle Cell Lymphoma Elderly Trial.

The outcome of older patients with mantle cell lymphoma (MCL) has improved by the introduction of immunochemotherapy, followed by rituximab (R)-maintenance. Assessment of minimal residual disease (MRD) represents a promising tool for individualized treatment decisions and was a prospectively planned part of the European MCL Elderly trial. We investigated how MRD status influenced the efficacy of R-maintenance and how MRD can enable tailored consolidation strategies. Previously untreated patients with MCL age 60 years or older have been randomly assigned to R versus interferon-alpha maintenance after response to rituximab, fludarabine, cyclophosphamide (R-FC) versus rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (R-CHOP). MRD monitoring was performed by real-time quantitative polymerase chain reaction (qPCR) following EuroMRD guidelines. A qPCR assay with a median sensitivity of 1 × 10-5 could be generated in 80% of 288 patients in an international, multicenter, multilaboratory setting. More extensive tumor dissemination facilitated the identification of a molecular marker. The efficacy of R-maintenance in clinical remission was confirmed for MRD-negative patients at the end of induction in terms of progression-free survival (PFS; hazard ratio [HR], 0.38 [95% CI, 0.21 to 0.63]) and overall survival (OS; HR, 0.37 [95% CI, 0.20 to 0.68]), particularly in R-CHOP-treated patients (PFS-HR, 0.23 [95% CI, 0.10 to 0.52]; OS-HR, 0.19 [95% CI, 0.07 to 0.52]). R-maintenance appeared less effective in MRD-positive patients (PFS-HR, 0.51 [95% CI, 0.26 to 1.02]) overall and after R-CHOP induction (PFS-HR, 0.59 [95% CI, 0.28 to 1.26]). R-FC achieved more frequent and faster MRD clearance compared with R-CHOP. MRD positivity in clinical remission after induction was associated with a short median time to clinical progression of approximately 1-1.7 years. The results confirm the strong efficacy of R-maintenance in patients who are MRD-negative after induction. Treatment de-escalation for MRD-negative patients is discouraged by our results. More effective consolidation strategies should be explored in MRD-positive patients to improve their long-term prognosis.

Performance of baseline FDG-PET/CT radiomics for prediction of bone marrow minimal residual disease status in the LyMa-101 trial

The prognostic value of 18F-Fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT) at baseline or the predictive value of minimal residual disease (MRD) detection appear as potential tools to improve mantle cell lymphoma (MCL) patients’ management. The LyMa-101, a phase 2 trial of the LYSA group (ClinicalTrials.gov:NCT02896582) reported induction therapy with obinutuzumab, a CD20 monoclonal antibody. Herein, we investigated the added prognostic value of radiomic features (RF) derived from FDG-PET/CT at diagnosis for MRD value prediction. FDG-PET/CT of 59 MCL patients included in the LyMa-101 trial have been independently, blindly and centrally reviewed. RF were extracted from the disease area with the highest uptake and from the total metabolic tumor volume (TMTV). Two models of machine learning were used to compare several combinations for prediction of MRD before autologous stem cell transplant consolidation (ASCT). Each algorithm was generated with or without constrained feature selections for clinical and laboratory parameters. Both algorithms showed better discrimination performances for negative vs positive MRD in the lesion with the highest uptake than in the TMTV. The constrained use of clinical and biological features showed a clear loss in sensitivity for the prediction of MRD status before ASCT, regardless of the machine learning model. These data plead for the importance of FDG-PET/CT RF compared to clinical and laboratory parameters and also reinforced the previously made hypothesis that the prognosis of the disease in MCL patients is linked to the most aggressive contingent, within the lesion with the highest uptake.

Minimal Residual Disease Detected by the 7NB-mRNAs ddPCR Assay Is Associated with Disease Progression in High-Risk Neuroblastoma Patients: A Prospective Multicenter Observational Study in Japan.

High-risk neuroblastoma (HR-NB) patients remain far from obtaining optimal outcomes, with more than 50% relapse/regrowth rate despite current intensive multimodal therapy. This originated from the activation/proliferation of chemoresistant minimal residual disease (MRD). MRD with a significant prognostic was reported by several quantitative PCR (qPCR) or droplet digital PCR (ddPCR) assays quantitating different sets of NB-associated mRNAs (NB-mRNAs). The 7NB-mRNAs ddPCR assay quantitating CRMP1, DBH, DDC, GAP43, ISL1, PHOX2B, and TH mRNAs was reported to outperform other qPCR assays by a retrospective in-house observational study. In the present study, the Japan Children's Cancer Group (JCCG) Neuroblastoma Committee conducted a prospective multicenter observational study aimed at evaluating a prognostic value of MRD in bone marrow (BM-MRD) and peripheral blood (PB-MRD) detected by 7NB-mRNAs ddPCR assay. Between August 2018 and August 2022, 7 HR-NB patients who registered for JCCG clinical trials (JN-H-11 and JN-H-15) were enrolled. A total of 19 BM and 19 PB samples were collected, and 4/15 BM and 4/15 PB samples were classified as progressive disease (PD)/non-PD samples. BM-MRD and PB-MRD estimated area under curve (AUC) of 0.767 and 0.800 with a significant accuracy (AUC > 0.7). The present study validated a prognostic value of BM-MRD obtained by a previous study (AUC 0.723) and revealed the significant accuracy of PB-MRD as well as BM-MRD.

P-075 Real-world evidence on prognostic value of minimal residual disease in multiple myeloma: Czech experience

P-075 Real-world evidence on prognostic value of minimal residual disease in multiple myeloma: Czech experience

Clinical applications of measurable residual disease monitoring by multi-parametric flow cytometry and next-generation sequencing in B-cell precursor acute lymphoblastic leukemia.

121 Background: Minimal residual disease (MRD) is accepted as the strongest independent prognostic factor in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The most frequently used methods for MRD monitoring include multi-parametric flow cytometry (MFC) and next-generation sequencing (NGS)-based IGH/IGK gene rearrangement. Both methods have been described to reach a low sensitivity threshold and be broadly applicable to most patients with BCP-ALL, although the clinical MRD cut-off of the two methods has not been fully defined. In this study, we compared MFC-MRD and NGS-MRD and established their clinical cut-off in BCP-ALL. Methods: Bone marrow aspirates with less than 5% blasts were obtained from 117 treated BCP-ALL patients. MFC-MRD assessment was performed using DuraClone RE ALB tubes (Beckman Coulter, Marseille, France) modified by adding 5 additional antibodies. In total, up to 1×105 cells were acquired using DxFLEX flow cytometer (Beckman Coulter), and all acquired data were analyzed using the Kaluza software v2.1. NGS-MRD was performed using the LymphoTrack IGH and IGK assay panels (InVivoScribe, San Diego, CA). We amplified 240 ng of high-quality DNA with a single multiplex master mix containing primers, and the library DNA was denatured and loaded onto a Miseq reagent kit v2. The FASTQ files were analyzed using the LymphoTrack MRD software v2.0.2. MRD evaluated using both of MFC and NGS in each sample, and the virtual sensitivity of both methods was 10-5. For 84 patients available, we analyzed the relapse-free survival (RFS) and overall survival (OS) rate for each MRD status and established a clinical cutoff. Results: We found a high correlation between methods (r=0.640) and a concordance rate of 81.2% for detecting MRD at 0.01% level. An MRD value of 0.01% proved to be a significant threshold value for 1-year RFS and OS. There was a difference in prognosis in both RFS and OS, but statistical significance was found only in RFS (MFC-MRD: 93.4±3.2% for MRD &lt;0.01% vs. 37.5±27.3% for MRD ≥0.01%, P=0.007; NGS-MRD: 93.7±3.6% for MRD &lt;0.01% vs. 68.8±14.2% for MRD ≥0.01%, P=0.016). In a Kaplan-Meier estimate of RFS at a threshold of 0.01%, patients who were negative for MRD by MFC and positive by NGS (n=12) had an intermediate prognosis between patients who were negative by both MFC and NGS (n=55) and patients who were positive by both MFC and NGS (n=11) (98.14±3.2%, 83.34±10.8%, 40.94±29.5%, respectively, P=0.017). Conclusions: An MRD threshold of 0.01% for both MFC and NGS is reasonable for clinical identification of poor-risk patients. NGS-MRD detection improves risk stratification for BCP-ALL.

MINIMAL DISSEMINATED AND MINIMAL RESIDUAL DISEASE IN CHILDHOOD AND ADOLESCENT NON‐HODGKIN LYMPHOMA

Introduction: Minimal disease determination might serve for risk stratification, response evaluation and follow-up disease monitoring in childhood Non-Hodgkin lymphomas. Besides helping to judge minimal residual disease (MRD) during therapy, detection of minimal disseminated disease (MDD) at diagnosis may be of prognostic value itself. However, different subtypes, high cure rates, low patient numbers, limited initial tumour material and early progression pose challenges. Methods: Pubmed literature search on MDD and MRD in pediatric lymphomas. Results: Current clinical applications of minimal disease determination differ between the subtypes. For lymphoblastic lymphomas, both flow-cytometry detecting aberrant immunophenotypes and PCR-based assays for TCR- or Ig-rearrangements have been used for minimal disease measurement. A prognostic value of MDD could not be clearly established yet. MRD has not been analysed in larger patient cohorts so far. In Burkitt-lymphoma and -leukemia, MYC-Ig-fusion sequences or Ig-rearrangements enable minimal disease detection. While conflicting data on the role of MDD as risk factor for Burkitt lymphoma have been published, the prognostic value of early MRD in Burkitt leukaemia treated by risk-adapted chemotherapy has been described by two study groups. However, its role in the rituximab-era needs to be confirmed. In ALK-positive anaplastic large cell lymphoma (ALCL), MDD and MRD determined by qualitative or quantitative PCR for ALK-fusion transcripts are validated independent prognostic parameters. They are standard of care parameters assessed in routine clinically practice and used for patient stratification in clinical studies. Early MRD might even serve as endpoint for clinical trials and for guiding individual therapy. Conclusions and future outlook: Validation of MDD and MRD as prognostic parameters is required for all subtypes but ALCL. Next-generation sequencing based methods and the use of circulating cell-free tumour DNA as medium may provide new options and applications for minimal disease evaluation in childhood lymphomas. Keywords: diagnostic and prognostic biomarkers, minimal residual disease, non-Hodgkin (pediatric, adolescent, and young adult) No conflicts of interests pertinent to the abstract.

MINIMAL RESIDUAL DISEASE STATUS IMPROVED THE RESPONSE EVALUATION IN PATIENTS WITH WALDENSTRöM’S MACROGLOBULINEMIA

Minimal residual disease (MRD) has been recognized as an important prognostic factor for survival in hematological malignancies. However, the prognostic value of MRD in Waldenström’s macroglobulinemia (WM) remains largely unexplored. We analyzed 108 newly diagnosed WM patients receiving systematic therapy and assessed MRD by multiparameter flow cytometry (MFC) in the bone marrow. At best response, 34 patients (31.5%) achieved undetectable MRD (uMRD). Hemoglobin > 115 g/L (p = 0.03), serum albumin >35 g/L (p = 0.01), β2-MG ≤ 3 mg/L (p = 0.03), and low-risk International Prognostic Scoring System for WM (IPSSWM) stage (p < 0.01) were associated with a higher rate of uMRD. Improvements in IgM (p < 0.01) and hemoglobin (p = 0.03) were more evident in uMRD patients compared with MRD-positive patients. The 3-year progression-free survival (PFS) was better for uMRD patients compared with those MRD-positive patients (96.2% vs. 52.8%; p = 0.0012). Landmark analysis also showed that uMRD patients had a better PFS than MRD-positive patients after 6 and 12 months. Patients who achieved partial response (PR) and uMRD had a 3-year PFS of 100%, which was significantly higher than that patients with MRD-positive PR (62.6%, p = 0.029). Multivariate analysis showed that MRD positivity was an independent factor for PFS (HR 2.55, p = 0.03). Moreover, the combination of the 6th International Workshop on WM assessment (IWWM-6 Criteria) and MRD had a higher 3-year AUC compared with the IWWM-6 criteria only (0.71 vs. 0.67). MRD based on MFC was an independent prognostic factor for survival in WM, which could improve the precision of response evaluation, especially for patients who had obtained PR. Keywords: diagnostic and prognostic biomarkers, indolent non-Hodgkin lymphoma No conflicts of interests pertinent to the abstract.

Utilize protein markers and genomic features for minimal residual disease detection in hepatocellular carcinoma.

e15054 Background: Several studies have shown the clinical value of using circulating tumor DNA (ctDNA) for minimal residual disease (MRD) detection, which is more sensitive than traditional imaging methods. However, most MRD detection technologies focus on identifying mutations and require cancer tissue samples. These methods often have limitations, such as high cost, inaccessible cancer tissue, or inability to overcome tumor heterogeneity. SeekInCure is a cost-effective multi-dimensional MRD test that does not require cancer tissue analysis. The purpose of this prospective study was to evaluate the potential broad clinical utility of SeekInCure for detecting MRD in patients with hepatocellular carcinoma (HCC). Methods: 34 HCC patients undergoing radical surgery were prospectively enrolled in this study from Peking University Shenzhen Hospital. Peripheral blood samples of 8 mL were collected before and after surgery and analyzed using the SeekInCure assay, which integrates the protein tumor marker (AFP) and cancer genomic hallmarks: copy number aberrations and fragment size from ctDNA. Patients with positive results for AFP and/or ctDNA in the preoperative/postoperative samples were defined as MRD-positive (MRD+), while negative results were defined as MRD-negative (MRD-). Results: Of the 34 patients, 73.5% (25/34) were in early stages (stages I/II: 61.8%/11.8%), and 61.8% (21/34) received adjuvant therapies. The median follow-up was 279.5 days, ranging from 90 to 1,253 days. 82% (28/34) of the patients had MRD+ results in the preoperative samples, which showed remarkable sensitivity in a cohort with more than 70% of early-stage patients. Our previous findings were confirmed, with preoperative MRD+ patients having worse survival than MRD- patients1. After radical surgery, 26.5% (9/34) were MRD+. MRD+ patients had worse survival than MRD- patients, with a median overall survival (OS) time of 298 days for MRD+ patients, while the OS time for MRD- patients was not reached (P &lt; 0.01). 6 patients with MRD- in both pre- and post-operative samples had a very favorable outcome (100% OS). Conversely, 9 patients with both positive results had the worst outcome. We also found that MRD- patients receiving adjuvant therapies did not have a survival benefit compared to those without adjuvant therapies, but MRD+ patients receiving adjuvant therapies had better OS than those without adjuvant therapies (P = 0.026). Conclusions: This study confirms the excellent performance of our SeekInCure assay, which does not require cancer tissue analysis, in detecting MRD and the prognostic value of MRD in HCC patients. Adjuvant has no influence on survival, especially in MRD- patients, which indicates that amost one-third of HCC patients do not need additional adjuvant therapy.

The Prognostic Effect of IKZF1 Deletions in ETV6::RUNX1 and High Hyperdiploid Childhood Acute Lymphoblastic Leukemia.

IKZF1 deletions are an established prognostic factor in childhood acute lymphoblastic leukemia (ALL). However, their relevance in patients with good risk genetics, namely ETV6::RUNX1 and high hyperdiploid (HeH), ALL remains unclear. We assessed the prognostic impact of IKZF1 deletions in 939 ETV6::RUNX1 and 968 HeH ALL patients by evaluating data from 16 trials from 9 study groups. Only 3% of ETV6::RUNX1 cases (n = 26) were IKZF1-deleted; this adversely affected survival combining all trials (5-year event-free survival [EFS], 79% versus 92%; P = 0.02). No relapses occurred among the 14 patients with an IKZF1 deletion treated on a minimal residual disease (MRD)-guided protocols. Nine percent of HeH cases (n = 85) had an IKZF1 deletion; this adversely affected survival in all trials (5-year EFS, 76% versus 89%; P = 0.006) and in MRD-guided protocols (73% versus 88%; P = 0.004). HeH cases with an IKZF1 deletion had significantly higher end of induction MRD values (P = 0.03). Multivariate Cox regression showed that IKZF1 deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in HeH ALL (hazard ratio of relapse rate [95% confidence interval]: 2.48 [1.32-4.66]). There was no evidence to suggest that IKZF1 deletions affected outcome in the small number of ETV6::RUNX1 cases in MRD-guided protocols but that they are related to higher MRD values, higher relapse, and lower survival rates in HeH ALL. Future trials are needed to study whether stratifying by MRD is adequate for HeH patients or additional risk stratification is necessary.

Combined analysis of PHOX2B at two time points and its value for further risk stratification in high-risk neuroblastoma.

Risk stratification of high-risk neuroblastoma (NB) is crucial for exploring treatments. This study aimed to explore the value of minimal residual disease (MRD) based on PHOX2B levels for further risk stratification in high-risk NB. The expression of PHOX2B was monitored at two time points (after two and six cycles of induction chemotherapy, TP1 and TP2, respectively) by real-time polymerase chain reaction (RT-PCR). The clinical characteristics between groups and survival rates were analyzed. The study included 151 high-risk patients. Positive expression of PHOX2B at diagnosis was seen in 129 cases. PHOX2B was mainly expressed in patients with high lactate dehydrogenase (LDH) and neuron-specific enolase (NSE) levels (p<.001), bone marrow metastasis (p<.001), more than three metastatic organs (p<.001), 11q23 loss of heterozygosity (LOH) (p=.007), and when more events occurred (p=.012). The 4-year EFS rate was significantly lower in patients with positive PHOX2B expression compared to thenegative group at diagnosis (32.9%±6.2% vs. 74.5%±10.1%, p=.005). We stratified the 151 patients into three MRD risk groups: low high-risk (low-HR), with TP1 less than 10-4 and TP2 less than 10-4 ; ultra-HR, with TP1 greater than or equal to 10-2 or TP2 greater than or equal to 10-4 , and others classified as intermediate-HR. Patients in ultra-HR had the worst survival rate compared with other two groups (p=.02). In a multivariate model, MRD risk stratification based on PHOX2B levels at TP1 and TP2 was an independent prognostic factor for high-risk patients (p=.001). Patients in ultra-HR were associated with 11q23 LOH (p<.001), more than three organs of metastasis (p=.005), bone marrow metastasis (p<.001), and occurrenceofmore events (p=.009). MRD risk stratification based on PHOX2B levels at two time points (after two and six cycles of induction chemotherapy) provideda stratification system for high-risk NB, which successfully predicted treatment outcomes. Our results present an effective method for further stratification of high-risk NB.

Digital Droplet PCR for Minimal Residual Disease Assessment in Philadelphia-Positive Acute Lymphoblastic Leukemia Using IG/TR Genes and BCR/ABL1 as Markers. Preliminary Results of a Comparative Analysis

Beldinanzi M, Della Starza I, contributed equally Background. The Philadelphia (Ph) chromosome is the most frequent genetic abnormality in adult acute lymphoblastic leukemia (ALL). BCR/ABL1 fusion transcript levels and clonal immunoglobulin/T-cell receptor (IG/TR) gene rearrangements are used as biomarkers for the study of minimal residual disease (MRD) in Ph+ ALL, with IG/TR used mainly in pediatric cohorts. There is evidence suggesting a poor concordance between these two molecular markers and there is a growing interest in evaluating MRD using a "double-hit strategy", especially in the adult setting where data are lacking. The real-time quantitative polymerase chain reaction (RQ-PCR) represents the current gold standard for MRD monitoring. It carries, however, some intrinsic limitations that digital droplet PCR (ddPCR), an innovative evolution of standard method, could overcome. Aim of this study. To carry out in adult Ph+ ALL patients a parallel MRD monitoring at early time points by both molecular targets - BCR/ABL1 gene fusion and clonal IG/TR gene rearrangements - by ddPCR. For BCR/ABL1 this was carried out by both RQ-PCR and ddPCR, in order to determine the concordance between the MRD values. Materials and Methods. The study comprised samples derived from 17 adults with newly diagnosed Ph+ ALL enrolled in the ongoing phase III GIMEMA LAL2820 clinical trial. Bone marrow (BM) samples were collected at the end of the induction phase (day +70) with ponatinib in the experimental arm or with a chemotherapy backbone plus imatinib in the control arm. At diagnosis, all patients underwent also a IG/TR gene rearrangements screening and MRD monitoring was performed by RQ-PCR and ddPCR for the quantification of BCR/ABL1 transcript levels and by ddPCR for IG/TR rearrangements, as described. MRD results were considered discordant in case of a difference greater than one log10 or in case of positivity/negativity. Results. The comparison between MRD values obtained in all samples by RQ-PCR and ddPCR using BCR/ABL1 mRNA level as biomarker showed an excellent correlation degree (R2=0.99). At variance, by IG/TR screening, it was possible to identify a clonal rearrangement only in 11/17 cases (65%), while 6/17 (35%) had no marker (NM) (3 p190, 3 p210; 2 IKZF1plus, 2 IKZF1 loss only and 2 IKZF1 WT). Moreover, of the 11 patients with a clonal rearrangement, in 3 (27%) (3 p190; 1 IKZF1plus, 2 IKZF1 WT) it was not possible to design a sensitive allele-specific oligonucleotide (ASO) for MRD monitoring. Overall, 8 samples were evaluated using both molecular markers by ddPCR, showing a poor correlation (R2=-0.21) and a low concordance rate. Indeed, 3/8 (37.5%) samples were concordant by both targets, either having highly positive (2/3) or negative (1/3) MRD values, while 5/8 (62.5%) were discordant; among the discordant cases, 1/5 was MRD-positive by both targets but outside the concordance range, while in 4/5 IG/TR MRD monitoring was negative, while BCR/ABL1 MRD monitoring was positive (range 10-3-10-1). Finally, in the 9 patients who failed IG/TR MRD monitoring, the BCR/ABL1 RQ-PCR analysis revealed a quantifiable MRD positivity in 6 cases (ranged 10-1-10-3), 2 proved "positive non-quantifiable" (PNQ) and 1 was MRD-negative (Figure 1). Conclusions. In adult Ph+ ALL, ddPCR with BCR/ABL1 as marker proved to be a sensitive and reliable tool for MRD monitoring at least comparable to RQ-PCR, showing a very high concordance rate. At variance, at least at early time points, the degree of concordance between BCR/ABL1 fusion transcript levels and IG/TR clonal rearrangements appeared suboptimal. Although IG/TR gene rearrangements are the most widely used biomarker for MRD monitoring in pediatric patients, the rate of failure in IG/TR marker identification in the adult Ph+ ALL setting is higher than in other subtypes. These findings suggest that the BCR/ABL1 fusion transcript remains the main sensitive target in adult Ph+ ALL and lead to hypothesize that in in this setting IG/TR clonal rearrangements represent a surrogate marker. Cohort expansion is warranted and clinical correlations are ongoing. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Predictive Value of Minimal Residual Disease on Efficacy of Rituximab Maintenance in Mantle Cell Lymphoma: Results from the European MCL Elderly Trial

On behalf of the European MCL Network Introduction: Rituximab (R) maintenance after immunochemotherapy represents standard of care in older mantle cell lymphoma (MCL) patients. The European MCL Elderly trial has shown the efficacy of R maintenance after R-CHOP in terms of PFS and OS (Kluin-Nelemans et al., JCO 2020). Induction with R-FC showed similar response rates and failure-free survival but inferior OS as compared to R-CHOP. The assessment of minimal residual disease (MRD) represents a promising tool for individualized treatment decisions. Within the European MCL Elderly trial, we investigated whether MRD status predicts the efficacy of maintenance and aimed to identify MRD-based trigger time points for targeted intervention. Patients and methods: The MCL Elderly trial included patients with previously untreated, advanced stage MCL aged 60 years or older and not suitable for autologous stem cell transplantation. MRD assessment was done at predefined time points during and after treatment until clinical progression for patients recruited in France or Germany with baseline samples and at least one follow-up sample. Three central MRD labs performed marker screening (BCL1-JH t(11;14) translocation or IGH rearrangement) and real-time quantitative PCR (qPCR) following standardized ESG guidelines, as previously reported (Pott et al., Blood 2010). MRD status was pooled from bone marrow and peripheral blood results by classifying a patient as MRD negative if all available samples at the respective time point were negative. Predictive value of MRD status at end of induction for efficacy of maintenance was analysed with Cox regression for PFS including the interaction term of MRD status and treatment group. To assess the prognostic value of MRD status for clinical progression, landmarks were defined at midterm induction (MI), end of induction (EOI), and during four 6-months periods following EOI. Results: A molecular marker for MRD assessment was generated in 85% of 288 screened patients and in 80% a qPCR assay fulfilling standardized criteria could be established. Patients in whom a molecular marker could be identified had more frequently generalized disease compared with those without molecular marker (85% vs. 51% stage IV, 78% vs. 37% with bone marrow involvement, median LDH/ULN quotient: 0.93 vs. 0.81). Among patients without clinical progression during induction, MRD-negativity was achieved more frequently and earlier with R-FC (MI: 63% of 82 patients; EOI: 80% of 82) than with R-CHOP (MI: 26% of 84, p<0.0001; EOI: 53% of 89, p=0.0002). The efficacy of maintenance was clearly seen in patients MRD negative at EOI (PFS-HR 0.38, 95% CI 0.21-0.63, Figure 1A) and was slightly less pronounced in MRD positive patients (HR 0.51, 95% CI 0.26-1.02; interaction p=0.48, Figure 1B). Especially after R-CHOP, the efficacy of R-maintenance was higher among MRD-negative patients (HR 0.23, 95% CI 0.10-0.52) than among MRD-positive patients (HR 0.59, 95% 0.28-1.26, interaction p=0.097). Landmark analyses confirmed the prognostic value of MRD status during and after R-FC and during maintenance/follow-up after R-CHOP. Median time from MRD-positivity 6/12/18/24 months from EOI to clinical progression was consistently short with 2.2/1.2/1.6/1.4 years after R-CHOP and 1.0/1.0/0.7/0.7 years after R-FC. Conclusions: The results confirm the strong efficacy of R-maintenance in MRD-negative patients after induction. Omitting or stopping maintenance based on MRD-negativity is thus discouraged. Considering the short time to progression, more effective treatment strategies should be explored in MRD-positive patients to improve the long-term prognosis of patients with MCL. Figure 1: Progression-free survival (PFS) from end of induction (EOI) by maintenance randomization (R vs. no R) in patients MRD-negative (A) or MRD-positive (B) at EOIFigure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immunoparesis Recovery in Transplant-Ineligible Newly Diagnosis Multiple Myeloma Patients: An Independent Prognosis Factor That Could Complement Prognostic Value of Minimal Residual Disease

Introduction: Immunoparesis (IP) or the suppression of polyclonal uninvolved immunoglobulins (Ig) is one of the immune system alterations present in patients with multiple myeloma (MM) at diagnosis. Several studies have shown that the lack of IP recovery after treatment is associated with worse prognosis. However, these are observational retrospective studies and patients were not homogeneously treated. On the other hand, the depth of response, specially reaching minimal residual disease (MRD) negative, is one of the most important prognostic factors in patients with MM. We decided to study, in a prospective way, the prognostic value of IP recovery in a group of elderly patients not suitable for transplantation and treated in a similar way and compare it with the depth of response to treatment, especially with MRD status. Methods: We analyze patients included and treated in the PETHEMA/GEM2010MAS65 study (registered at www.clinicaltrial.gov as #NCT01237249) who reached CR or VGPR. All patients included in this trial were older than 65 years old and transplant-ineligible and were planned to receive 9 cycles of bortezomib plus melphalan and prednisone (VMP) and 9 cycles of lenalidomide plus dexamethasone (LD). Polyclonal Ig were analyzed by nephelometry or turbidimetry at diagnosis, at the end of the treatment (after 18 cycles) and during subsequent follow up. Minimal residual disease (MRD) was analyzed at the end of the treatment by multidimensional flow cytometry (MFC) with a sensitivity level of 10-5. Results: Of the 233 patients included in the study, 161 reached CR or VGPR, but the protocol design did not included determination of uninvolved Igs at diagnosis and for this reason they were only available in 120 patients. Only 9 cases (7,5%) had normal Ig at diagnosis while 111 patients (92,5%) had IP. There were no differences in progression free survival (PFS) and overall survival (OS) between patients with or without IP. We had Ig determination of 117 cases at the end of the treatment and during follow up. Those patients who had IP at diagnosis and recover it after treatment had better PFS (p<0,001; HR 0,33) and better OS (p=0,001; HR 0,35) than those who do not recover it (Figure 1A). We found no differences between those patients who had recovery of IP at cycle 18 than those who recover IP during follow up. When we focused the analysis in CR patients and VGPR patients separately, in both groups recovery of IP was associated with a better PFS and OS. In fact, when we compared both groups, we found that those patients with VGPR and complete recovery of IP had better PFS (p=0,001; HR 0,12) and OS (p=0,015; HR 0,22) than those with CR and no recovery of IP. These data suggest that IP recovery could be a prognostic factor equal or more important than conventional response. MRD information by MFC was available in 112 cases. As previously reported, in this trial those patients with MRD negative (-ve) had longer PFS and OS than MRD positive (+ve). When we analyzed IP recovery in MRD -ve patients we found that patients with MRD -ve and IP recovery (33 cases) had better PFS (p=0,004; HR 0,30) and better OS (p=0,010; HR 0,21) than patients with MRD -ve without IP recovery (11 cases), although it should be recognized that the number of patients within this latter subgroup was rather small (Figure 1B). We performed a multivariate analysis including other prognostic factors, and we found that both MRD and IP recovery have independent prognostic value for PFS, but only MRD and no IP recovery, is an independent prognosis factor for OS. Conclusions: Recovery of IP after non-continuous treatment in elderly patients with CR or VGPR confers better prognosis (PFS and OS). Prognostic value of IP recovery could be equal or more important than conventional response to treatment. Prognostic value of MRD could be complemented when combined with IP recovery, but these data must be confirmed in other series. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recovery of Uninvolved Heavy/Light Chain Pair Immunoparesis during Maintenance Is an Independent Prognostic Factor for Multiple Myeloma and Could Complement Prognosis Value of Minimal Residual Disease

Recovery of Uninvolved Heavy/Light Chain Pair Immunoparesis during Maintenance Is an Independent Prognostic Factor for Multiple Myeloma and Could Complement Prognosis Value of Minimal Residual Disease

Prognostic Relevance of Minimal Residual Disease in Therapy Related and Secondary Acute Myeloid Leukemia Receiving CPX-351 or Fludarabine-Based Induction

Background: The high prognostic value of minimal residual disease (MRD) assessment in Acute Myeloid Leukemia patients (AML) undergoing intensive induction therapy has been confirmed by several studies. The most widely adopted MRD techniques include multicolor flow cytometry (MFC), RT-PCR for recurrent genetic lesion and, for patients lacking specific markers, RT-PCR for the pan-leukemic marker WT1. However, mostly younger patients receiving a conventional 3+7 induction are included in clinical studies evaluating MRD in AML. As a consequence, secondary AML, evolving from a previous myelodisplastic syndrome (s-AML) or therapy-related AML (t-AML), which usually affect elderly AML patients are usually under-represented in those trials. Recently, CPX-351 was approved for the treatment of s-AML and t-AML, following the positive results of the Phase III trial by Lancet et al. However, as MRD was not assessed in this trial, only a limited ammount of data are available on the kinetics and the prognostic value of MRD in elderly s- AML and t-AML patients receiving CPX-351 induction. Aims: In order to assess the prognostic value and to define the best time points for MRD assessment, MFC-based and WT1 based MRD was evaluated in a cohort of elderly s-AML or t-AML patients receiving CPX-351 induction. Timepoints for assessment were after induction (TP1), after first consolidation (TP2) and at the end of treatment (TP3). As a comparison, we also evaluated MRD in a cohort of elderly s-AML and t-AML patients who received an age-adjusted fludarabine-containing regimen (FLAI3). MRD analysis in this cohort was performed with the same methodics and at the same timepoints. Methods: A total of 151 elderly (>60 year, median age 68, range 60-77) patients were analyzed in this study. Patients were treated between January 2005 and January 2020 in our Center, either with CPX-351 (n=50) or fludarabine- high dose cytarabine-idarubicin (FLAI), with (n=72) or without (n =29) gemtuzumab-ozogamicin (GO). MRD was analyzed in all patients achieving hematological complete remission (CR) with both MFC and WT1 expression levels. All patients were affected by s-AML or t-AML, defined according to the WHO 2016 criteria. Results: After first induction cycle, 95 patients (59%) achieved CR. Patients treated with CPX-351 had a significantly higher CR rate, if compared to patients receiving FLAI (40/50, 80% and 55/101, 54.5%, respectively, p<0.05). Adding GO to FLAI did not increase CR rate. Overall, in the two cohorts, an MRD negative CR was achieved in 51 (53.7%) and 53 responding patients (55.8%) assessed with MFC or WT1-based methods, respectively. The probability of achieving MFC MRD negativity was significantly higher among patients treated with CPX-351, when compared to patients receiving FLAI with or without GO (MFC MRD negativity rate of 26/40, 65% and 25/55, 45%, respectively, p<0.05). In both cohort, the most informative timepoint in terms of survival prediciton was after first cycle (TP1). WT1-based MRD assessment led to superimposable results. In multivariate analysis, MRD was significantly correlated with an higher probability of longer Overall Survival (OS) in all treatment group (2-year OS of 34% and 77% in patients with or without residual MFC MRD after induction, respectively, p<0.05). Allogeneic stem cell transplantation consolidation (HSCT) was also significantly correlated with longer OS in both treatment groups (p<0.05). Notably, 12/40 (30%) CR patients treated with CPX 351 underwent HSCT, whereas only 8 CR patients proceeded to HSCT in the FLAI group (15%) Conclusions: MRD assessment has a strong prognostic value also in the context of elderly s-AML and t-AML patients. WT1-based and MFC-based MRD assessment lead to similar conclusions thus allowing to obtain MRD data from virtually all AML patients treated in the selected time period. Overall, CPX-351 resulted in higher CR rate with deeper MRD responses, if compared to FLAI3. This increased efficacy translated in a significantly higher number of elderly AML patients proceeding to HSCT in CR, which is the only curative approach in this particular AML setting.

The Prognostic Effect of IKZF1 Deletions in ETV6::RUNX1 and High Hyperdiploid Childhood Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most prevalent childhood malignancy and recent cure rates approach 90% on first line therapy. IKZF1 deletions have been reported as an unfavorable prognostic factor and are rare in ETV6::RUNX1 and high hyperdiploid (HeH) ALL, the two cytogenetic subgroups with the most favorable prognosis. Due to its rarity, the prognostic effect of IKZF1deletions within this subset of patients remains unclear. Therefore, we assessed the prognostic impact of IKZF1deletions in 939 ETV6::RUNX1 and 968 HeH ALL patients. We performed this retrospective analysis on data of children and adolescents of 1-18 years with B-cell precursor ALL diagnosed in 1991-2016 and treated on one of 16 trials from 9 study groups, of which 6 were minimal residual disease (MRD) guided. Cytogenetic, fluorescence in-situ hybridization, and RT-PCR analyses of pre-treatment bone marrow samples to determine ploidy and fusion gene status were performed locally. Trials using MRD for risk stratification applied either PCR or flow cytometry analyses. The IKZF1 status was determined by multiplex ligation-dependent probe amplification by each individual study group. 3% of ETV6::RUNX1 (n=26) cases had an IKZF1 deletion; this affected survival adversely overall (5-year event free survival [EFS]: 79% vs. 92%, p = 0.02) (Table 1). However, in minimal residual disease (MRD) guided protocols, IKZF1 did not affect outcome in ETV6::RUNX1 cases ( 5-year EFS: 100% vs. 93%, p = 0.34) (Table 1). 9% of HeH cases had an IKZF1 deletion (n=85); this adversely affected survival overall (5-year EFS: 76% vs. 89%, p = 0.006), and in MRD-guided protocols (73% vs. 88%, p = 0.004) (Table 1). Multivariate cox regression showed that IKZF1deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in HeH ALL (Hazard ratio of EFS [95% CI]: 2.00 [1.20-3.30]) (Table 1). As the prognostic effect of IKZF1 deletions has been shown to vary by MRD risk group, we examined the prognostic effect of IKZF1deletions in HeH cases with detectable MRD <5% at end of induction in more detail. Of these cases, 10% carried an IKZF1deletion (n=34) which negatively affected outcome (5-year EFS: 78% vs. 92%, p = 0.01) Multivariate cox regression showed that IKZF1deletions negatively affected survival independent of sex, age, and white blood cell count at diagnosis in this intermediate MRD HeH ALL (Hazard ratio of EFS [95% CI]: 2.90 [1.20-6.80]). However, cases with an IKZF1 deletion had significantly higher MRD values than their wild-type counterparts (p = 0.03, Figure 1). In conclusion, our analysis of a large composite cohort consisting of 16 trials shows that IKZF1deletions do not affect survival in ETV6::RUNX1 ALL when treated on MRD-guided protocols. In contrast, our data shows that in HeH ALL IKZF1deletions are associated with lower survival rates, higher relapse rates, and higher MRD values. Future results of current trials such as the ALLTogether will likely reveal whether risk stratification predominantly reliant on MRD is adequate for HeH patients or whether stratification by copy number alteration profile, including IKZF1 status, or by other methods would be more suitable. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Minimal residual disease detection by next-generation sequencing in multiple myeloma: Promise and challenges for response-adapted therapy.

Assessment of minimal residual disease (MRD) is becoming a standard diagnostic tool for curable hematological malignancies such as chronic and acute myeloid leukemia. Multiple myeloma (MM) remains an incurable disease, as a major portion of patients even in complete response eventually relapse, suggesting that residual disease remains. Over the past decade, the treatment landscape of MM has radically changed with the introduction of new effective drugs and the availability of immunotherapy, including targeted antibodies and adoptive cell therapy. Therefore, conventional serological and morphological techniques have become suboptimal for the evaluation of depth of response. Recently, the International Myeloma Working Group (IMWG) introduced the definition of MRD negativity as the absence of clonal Plasma cells (PC) with a minimum sensitivity of <10−5 either by next-generation sequencing (NGS) using the LymphoSIGHT platform (Sequenta/Adaptative) or by next-generation flow cytometry (NGF) using EuroFlow approaches as the reference methods. While the definition of the LymphoSIGHT platform (Sequenta/Adaptive) as the standard method derives from its large use and validation in clinical studies on the prognostic value of NGS-based MRD, other commercially available options exist. Recently, the LymphoTrack assay has been evaluated in MM, demonstrating a sensitivity level of 10−5, hence qualifying as an alternative effective tool for MRD monitoring in MM. Here, we will review state-of-the-art methods for MRD assessment by NGS. We will summarize how MRD testing supports clinical trials as a useful tool in dynamic risk-adapted therapy. Finally, we will also discuss future promise and challenges of NGS-based MRD determination for clinical decision-making. In addition, we will present our real-life single-center experience with the commercially available NGS strategy LymphoTrack-MiSeq. Even with the limitation of a limited number of patients, our results confirm the LymphoTrack-MiSeq platform as a cost-effective, readily available, and standardized workflow with a sensitivity of 10−5. Our real-life data also confirm that achieving MRD negativity is an important prognostic factor in MM.