Minimal Residual Disease Measurement Research Articles

Minimal Residual Disease Measurement in Multiple Myeloma: Survey of Clinician Practices

Minimal residual disease (MRD) in multiple myeloma (MM) is evaluated by multiparametric flow cytometry (MFC), next generation sequencing (NGS), and positron emission tomography (PET-CT). MRD negativity is associated with improved survival, but there is no consensus on its value, modality, and timing. We surveyed MM clinicians regarding MRD measurement in MM.

Quality of Response in Acute Myeloid Leukemia: The Role of Minimal Residual Disease.

In the acute myeloid leukemia (AML) setting, research has extensively investigated the existence and relevance of molecular biomarkers, in order to better tailor therapy with newly developed agents and hence improve outcomes and/or save the patient from poorly effective therapies. In particular, in patients with AML, residual disease after therapy does reflect the sum of the contributions of all factors associated with diagnosis and post-diagnosis resistance. The evaluation of minimal/measurable residual disease (MRD) can be considered as a key tool to guide patient’s management and a promising endpoint for clinical trials. In this narrative review, we discuss MRD evaluation as biomarker for tailored therapy in AML patients; we briefly report current evidence on the use of MRD in clinical practice, and comment on the potential ability of MRD in the assessment of the efficacy of new molecules.

Clinical Significance of Minimal Residual Disease at the End of Remission Induction Therapy in Childhood Acute Lymphoblastic Leukemia.

BACKGROUND:Detection of minimal residual disease (MRD) in the early phase of therapy is the most powerful predictor of relapse risk in children with acute lymphoblastic leukaemia (ALL).AIM:We aimed to determine the significance of MRD at the end of remission induction therapy in the prediction of treatment outcome in children with ALL.METHODS:Sixty-four consecutive patients aged 1-14 years with newly diagnosed ALL were enrolled in this study from January 2010 to October 2017. All patients were treated according to the ALL IC BFM 2002 protocol. MRD was detected at the end of remission induction therapy (day 33) by multiparameter 6-colour flow cytometry performed on bone marrow specimens with a sensitivity of 0.01%.RESULTS:Overall, 42.2% of patients had detectable MRD on day 33 of therapy. MRD measurements were not significantly related to presenting characteristics but were associated with a poorer blast clearance on day 8 and 15 of remission induction therapy. Patients with negative MRD status on day 33 had a 5-year event-free survival of 94.6% compared with 76.1% for those with positive MRD status (P = 0.044).CONCLUSION:MRD levels at the end of remission induction therapy measured by multiparameter flow cytometry have clinical significance in childhood ALL. High levels of MRD are strongly related to poor treatment outcome.

Clinical Utilization of Next-Generation Sequencing-Based Clonality Assays for Immunoglobulin Heavy Chain in Evaluating Suspected B-Cell Proliferation

Abstract Objectives B-cell clonality assays of the immunoglobulin heavy chain (IGH) and light chain (IGK) genes have been used widely in the workup of B-cell neoplasms. PCR is the current gold standard test for clonality. Recently, NGS has been suggested to both improve sensitivity and identify the specific V-(D-)J sequence required to track clones. Here we report our experience with using NGS for complementing our current procedures. Methods Our validated NGS test is performed by amplifying patients’ DNA using IGH-FR1 assay panel. Pooled amplicons get sequenced. The sequence data then get analyzed using in-house bioinformatics software, which sorts sequences and generates frequency distributions. Each sequence is ranked by its frequency. A clonal population is designated if the top sequence read is at least 1% of the total reads. For the standard PCR gene rearrangement test, we use a BIOMED-2 PCR-based B-cell assay. Results We retrospectively searched our database for all cases in which IGH and IGK clonality was tested by either standard PCR or NGS from 8/2016 to 7/2017. A total of 253 cases were tested by PCR while 21 cases were tested by NGS, which showed higher success rates. Of the 253 cases tested by conventional PCR, 39% (n = 98) were clonal (either IGH, IGK, or both), 34% (n = 86) were polyclonal, and the rest 28% (n = 69) showed a weak inconclusive signal or failed. Of the 21 cases analyzed by NGS, 71% (n = 15) were clonal, 24% (n = 5) were polyclonal, and just 5% (n = 1) failed. Conclusion Our experience indicates that NGS for IGH clonality, even more expensive, is an effective and more sensitive tool that produces higher informational content and yields higher success rates when compared to conventional PCR assay. NGS captures multidimensional data and provides a more precise determination of the clonal relationship between different lymphoid proliferations, which also is essential for the detection and measurement of minimal residual disease.

Minimal Residual Disease Assessment Within the Bone Marrow of Multiple Myeloma: A Review of Caveats, Clinical Significance and Future Perspectives.

There is an increasing clinical interest in the measure and achievement of minimal residual disease (MRD) negativity in the bone marrow of Multiple Myeloma (MM) patients, as defined equally either by Multicolor Flow Cytometry (MFC) or by Next Generation Sequencing (NGS) technologies. At present, modern technologies allow to detect up to one on 104 or on 105 or even on 106 cells, depending on their throughput. MFC approaches, which have been progressively improved up to the so-called Next Generation Flow (NGF), and NGS, which proved clear advantages over ASO-PCR, can detect very low levels of residual disease in the BM. These methods are actually almost superimposable, in terms of MRD detection power, supporting the lack of unanimous preference for either technique on basis of local availability. However, some technical issues are still open: the optimal assay to use to detect either phenotype (e.g., next generation multidimensional flow cytometry, imaging) or genotype aberrations (e.g., ASO-RQ PCR, digital droplet PCR, NGS) and their standardization, the sample source (BM or peripheral blood, PB) and its pre-processing (red-cell lysis vs. Ficoll, fresh vs. frozen samples, requirement of CD138+ cells enrichment). Overall, MRD negativity is considered as the most powerful predictor of favorable long-term outcomes in MM and is likely to represent the major driver of treatment strategies in the near future. In this manuscript, we reviewed the main pitfalls and caveats of MRD detection within bone marrow in MM patients after front-line therapy, highlighting the improving of the currently employed technology and describing alternative methods for MRD testing in MM, such as liquid biopsy.

Quality control and quantification in IG/TR next-generation sequencing marker identification: protocols and bioinformatic functionalities by EuroClonality-NGS

Assessment of clonality, marker identification and measurement of minimal residual disease (MRD) of immunoglobulin (IG) and T cell receptor (TR) gene rearrangements in lymphoid neoplasms using next-generation sequencing (NGS) is currently under intensive development for use in clinical diagnostics. So far, however, there is a lack of suitable quality control (QC) options with regard to standardisation and quality metrics to ensure robust clinical application of such approaches. The EuroClonality-NGS Working Group has therefore established two types of QCs to accompany the NGS-based IG/TR assays. First, a central polytarget QC (cPT-QC) is used to monitor the primer performance of each of the EuroClonality multiplex NGS assays; second, a standardised human cell line-based DNA control is spiked into each patient DNA sample to work as a central in-tube QC and calibrator for MRD quantification (cIT-QC). Having integrated those two reference standards in the ARResT/Interrogate bioinformatic platform, EuroClonality-NGS provides a complete protocol for standardised IG/TR gene rearrangement analysis by NGS with high reproducibility, accuracy and precision for valid marker identification and quantification in diagnostics of lymphoid malignancies.

ClonoSEQ assay for the detection of lymphoid malignancies

ABSTRACTIntroduction: Immunoglobulin rearrangement studies by molecular methods are routinely used to detect clonality and to follow up patients with lymphoid malignancies. The design of a next-generation sequencing (NGS) panel using a comprehensive pool of primers, the establishment of a straightforward analytical protocol, a bioinformatic pipeline and the availability of the results of clinical studies have allowed the Clonoseq platform to be licensed as the first assay to measure minimal residual disease (MRD) both in acute lymphoblastic leukemia (ALL) and in multiple myeloma (MM).Areas covered: An extensive literature review (Pubmed search) on the applicability of the high-throughput sequencing (HTS) approach in lymphoid malignancies was conducted. This review discusses recent data in the field and compares this emerging molecular technique with standardized technologies.Expert Opinion: Real-time quantitative (RQ)-PCR and multiparametric flow cytometry (MPFC) are still the gold standard methods of minimal residual disease assessment. New HTS methods as Clonoseq show a high concordance with the above-mentioned techniques and at the same time it provides potential advantages to detect clonal changes. Clonoseq could be helpful to optimize risk-stratification and adjusting treatments in lymphoid malignancies. Moreover, HTS could also be applied to the detection of circulating tumor DNA (ctDNA) on the plasma in lymphomas.

S130 TRANSIENT SUBCLONES CARRYING NT5C2 MUTATIONS DEFINE A HIGH‐RISK PATIENT GROUP WITH POOR OUTCOME IN PEDIATRIC RELAPSED B‐CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

Background:Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Despite an overall favorable prognosis, 15–20% of patients suffer a relapse. In the treatment of relapsed ALL, resistance to chemotherapy is a major challenge. The purine nucleotide metabolizing cytosolic 5́‐nucleotidase II (NT5C2) harbors activating mutations in 20% of pediatric relapsed T‐ALL and in 3–10% of relapsed B‐cell precursor ALL cases, which are considered to drive relapse formation by causing resistance to purine analogue‐based maintenance treatment of ALL. However, the impact of NT5C2 mutations for second‐line treatment and long‐term outcome of relapsed patients is unknown.Aims:To elucidate the clinical significance of NT5C2 mutations in relapsed ALL, we studied the NT5C2 gene in a large cohort of 455 patients with relapsed B‐cell precursor ALL and assessed correlations with clinical presentation, genetics, response to treatment and outcome.Methods:We detected NT5C2 mutations by Sanger sequencing and amplicon‐based next generation sequencing. To detect subclonal NT5C2 mutations, we established allele‐specific quantitative real‐time PCR assays following minimal residual disease measurement guidelines. All relapsed patients were treated according to the German ALL‐REZ BFM 2002 protocol.Results:By sequencing, we found n = 36 NT5C2 mutations in n = 34 samples. About 30% of NT5C2 mutations appeared subclonal with an estimated allele frequency of less than 20%. The variants p.R39Q (8/36, 22%) and p.R367Q (14/36, 39%) were the most prevalent mutations in our cohort. Using allele‐specific quantitative real‐time PCR targeted to p.R39Q and p.R367Q, we identified additional 69 subclonal NT5C2 mutations. In total, NT5C2 mutations were present in 16% of patients with relapsed B‐cell precursor ALL (74/455), but the majority of cases (66%) showed subclonal mutations only. NT5C2 mutations most frequently occurred in relapses of so‐called B‐other‐ALL that lacks established cytogenetic changes (P < 0.001) and were correlated with CDKN2A/B loss. In addition, predominant, but not subclonal NT5C2 mutations were associated with NRAS mutations indicating that RAS signaling may be involved in NT5C2‐driven relapse formation. Both, subclonal and predominant NT5C2 mutations, were associated with inferior event‐free survival rates of relapsed patients compared to wild‐type NT5C2 (0.189±0.058 and 0.333 ± 0.096, respectively, versus 0.575 ± 0.026; P < 0.001). Response to relapse treatment was particularly poor in relapsed patients with subclonal NT5C2 mutations only. This group showed an increased rate of nonresponse of more than 30% (P < 0.001). Moreover, patients of this group who responded to relapse treatment, showed high minimal residual disease levels at the end of relapse induction treatment in 80% of cases. To investigate whether outgrowth of NT5C2 mutant subclones during relapse treatment contributes to the poor response and outcome, we analyzed follow‐up samples and found that subclonal NT5C2 mutations never grew out to predominant mutations at the time of nonresponse or second relapse. In fact, analysis of samples taken after relapse induction treatment showed that NT5C2 mutant clones were frequently eradicated early during relapse treatment (14/20, 70%), whereas NT5C2 wild‐type leukemic cells persisted at minimal residual disease levels higher than 10E‐03 (16/20, 80%).Summary/Conclusion:Subclonal NT5C2 mutations define a patient group with poor response to treatment and outcome in relapsed B‐cell precursor ALL, but due to their transient character they may not play a driving role in this clinical phenotype.

PB1766 ASSESSMENT OF MINIMAL RESIDUAL DISEASE IN ADULT EGYPTIAN ACUTE MYELOID LEUKEMIA PATIENTS

Background: The term minimal residual disease (MRD) is used to describe residual disease after suboptimal induction chemotherapy, but at the same time refers to the lowest levels of disease potentially compatible with cure or to molecularly defined relapse after long term remission. This study assesses the minimal residual disease in relation to the outcome in acute myeloid leukemia patients after 6 months of treatment of chemotherapy. Aims: The aim of this study is assessment of measurable minimal residual disease after treatment to identify acute myeloid leukemia patients who are at high risk of poor outcome. Methods: Minimal Residual Disease was measured using Flow cytometry in 30 newly diagnosed a 30 newly diagnosed acute myeloid leukemia (AML) patients. The patients were recruited from clinical hematology department at Ain shams university hospital over the period from May 2017 to November 2018. Results: A total number of 30 acute myeloid leukemia patients were recruited from Ain Shams University hospital, hematology and Oncology Unit outpatient clinic, with age ranging from 18-60 years old (median age 40 years), 14 of them were males representing 46.6% of the total number and 16 were females representing 53.3% of the total number. There was no statistical significance between the age of the studied group and the MRD with P-value 0.147, the sex of the patients doesn’t contribute to the risk stratification with a P-value of 0.200. The number of cases with negative MRD after induction was 13 representing 43.3% of the total number, and was 12 patients after 6 months representing 70.5% of the remitted patients, considering that the cut out value of MRD is 0.1. There was a significant relationship between TLC and MRD, the higher the TLC the more positive the MRD. Positive MRD is associated with higher mortality rates, exhibiting a statistical significance with P-value of 0.005. Summary/Conclusion: We concluded that assessment of minimal residual disease in AML is of great value in determination of prognosis of the disease and its outcome and it is of great value to determine the levels of minimal residual disease (MRD) during the course of therapy and to stratify patient to identify high risk patient and to plan the therapeutic program accordingly.

Guidelines Insights: Acute Lymphoblastic Leukemia, Version 1.2019.

Survival outcomes for older adults with acute lymphoblastic leukemia (ALL) are poor and optimal management is challenging due to higher-risk leukemia genetics, comorbidities, and lower tolerance to intensive therapy. A critical understanding of these factors guides the selection of frontline therapies and subsequent treatment strategies. In addition, there have been recent developments in minimal/measurable residual disease (MRD) testing and blinatumomab use in the context of MRD-positive disease after therapy. These NCCN Guidelines Insights discuss recent updates to the NCCN Guidelines for ALL regarding upfront therapy in older adults and MRD monitoring/testing in response to ALL treatment.

The assessment of minimal residual disease versus that of somatic mutations for predicting the outcome of acute myeloid leukemia patients

BackgroundIn addition to morphological and cytogenetic features, acute myeloid leukemias are characterized by mutations that can be used for target-therapy; also the minimal/measurable residual disease (MRD) could be an important prognostic factor. The purpose of this retrospective study was to investigate if somatic mutations could represent an additional prognostic value in respect of MRD alone.MethodAt baseline, 98 patients were tested for NPM1, FLT3, and for WT1 expression; 31 for ASXL1, TET2, IDH1, IDH2, N-RAS, WT1, c-KIT, RUNX1, and DNMT3A. The same genes have been also tested after induction and consolidation.ResultsOverall, 60.2% of our patients resulted mutated: 24.5% carried mutations of FLT3-ITD, 38.7% of NPM1, 48.4% of c-KIT, 25.8% of N-RAS and 19.3% of IDH2. The probability of achieving a complete response (CR) was higher for younger patients, with low ELN risk score, NPM1-mutated, with low WT1 levels, and without FLT3. The presence of additional mutations represented a poor predictive factor: only 19% of these cases achieved CR in comparison to 43% of subjects without any of it. Concerning survival, it was conditioned by a lower ELN risk score, younger age, reduction > 1 log of the NPM1 mutational burden, disappearance of FLT3 mutations and lower WT1 expression. Regarding the role of the additional mutations, they impaired the outcome of 20% of the already MRD-negative patients. Concerning the possibility of predicting relapse, we observed an increase of the NPM1 mutational burden at the time-point immediately preceding the relapse (about 2 months earlier) in 50% of subjects. Similarly concerning WT1, an increase of its expression anticipated disease recurrence in 64% of cases.ConclusionsWe demonstrated that additional somatic mutations are able to impair outcome of the already MRD-negative subjects. About MRD, we suggest a prognostic role also for the WT1 expression. Finally, we considered as relevant the assessment of NPM1 quantity clearance instead of the presence/absence of mutations alone. Still remains in doubt the utility in terms of long-term prognosis of a baseline more complex mutational screening; we could hypothesize that it would be useful for those patients where other markers are not available or who reached the MRD negativity.

Measurable residual disease monitoring using Wilms tumor gene 1 expression in childhood acute myeloid leukemia based on child-specific reference values.

Measurable/minimal residual disease (MRD) monitoring can predict imminent hematological relapse in acute myeloid leukemia (AML). The majority of childhood AML patients do not harbor fusion genes or mutations applicable as MRD markers and overexpression of Wilms tumor gene 1 (WT1) may constitute a useful monitoring target. However, age-specific reference values in healthy hematopoiesis and standardization of WT1 assessment are prerequisites for clinical utility. We investigated WT1 expression across age in hematologically healthy controls (n=109), during suspected infection (n=90) and bone marrow (BM) regeneration (n=13). WT1 expression in AML at diagnosis (n=91) and during follow-up (n=30) was compared with age-specific reference values. WT1 expression correlated with age and showed higher levels in both BM and peripheral blood (PB) in children compared with adults (P<0.001 and P=0.01). WT1 expression from healthy hematopoiesis was lower in PB compared with BM (WT1BM /WT1PB =8.6, 95% CI: 5.3-13.7) and not influenced by infection nor BM regeneration. At AML diagnosis, 66% had more than 20-fold WT1 overexpression in PB or BM (PB 74%; BM 45%). WT1 was quantified in 279PB samples during follow-up. All 11 patients with PB sampling within 4 months of disease recurrence displayed WT1 overexpression by a median of 1.9 months (range, 0.7-9.7) before hematological relapse. This study defines child-specific reference values for WT1 expression in healthy hematopoiesis and demonstrates that WT1 expression in PB is a useful post-treatment monitoring tool in childhood AML. Based on these observations, we propose definitions for childhood AML molecular relapse using WT1 overexpression.

Blinatumomab in the Treatment of Acute Lymphoblastic Leukemia: Russian Multicenter Clinical Trial

Background. Recent advances in the treatment of relapsed/refractory acute lymphoblastic leukemia (R/R ALL) are attributed to the implementation of immunotherapy methods which include blinatumomab, the bispecific engager of a patient's endogenous T-cells (Blincyto™, Amgen®) (BC). Aim. To assess BC efficacy and toxicity in the treatment of R/R ALL patients with persistence of minimal tumor clone before and after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Materials &amp; Methods. The trial included 66 B-ALL patients with CD19+ aged 18 to 72 years, 23 (35 %) of them with measurable minimal residual disease (MRD+) and 43 (65 %) with R/R ALL. In 18 (27 %) patients BC was administered after prior allo-HSCT. Results. In the overall group 2-year overall survival (OS) and disease-free survival (DFS) in patients with response to BC treatment were 53 % and 38 % respectively. In the R/R ALL group complete remission (CR) was achieved in 29 (67 %) patients including 24 (83 %) patients with negative MRD. CR rate was higher in standard cytogenetic risk group (73 %) in comparison with high-risk group (59 %). In patients with more or less than 50 % blast cells in bone marrow CR rate was 85 % and 61 %, respectively. When BC was administered after prior allo-HSCT and without it CR rate was 80 % and 60 %, respectively. In R/R ALL patients with response to BC 2-year OS and DFS were 40 % and 26 %, respectively, in the MRD+ group of ALL patients they were 66 % and 51 %, respectively. Relapse rate was lower in the group with allo-HSCT than in the group without it, i.e. 21 % vs. 55 %. Adverse events of grade 3-4 were observed in 25 (38 %) patients. In 11 (16 %) patients BC therapy had to be discontinued, in 5 (7 %) patients it was terminated prior to the scheduled date. Conclusion. BC efficacy is higher in the MRD+ group and in R/R ALL patients with smaller tumor mass. BC treatment after allo-HSCT yields remissions in most patients and can be combined with immune-adoptive therapy.

MRD Detection in B-Cell Non-Hodgkin Lymphomas Using Ig Gene Rearrangements and Chromosomal Translocations as Targets for Real-Time Quantitative PCR.

Minimal residual disease (MRD) diagnostics is of high clinical relevance in patients with indolent B-cell non-Hodgkin lymphomas (B-NHL) and serves as a surrogate parameter to evaluate treatment effectiveness and long-term prognosis. MRD diagnostics performed by real-time quantitative PCR (RQ-PCR) is still the gold standard and currently the most sensitive and the most broadly applied method in follicular lymphoma (FL) and mantle cell lymphoma (MCL). Alternatively, droplet digital PCR (ddPCR) can be used for MRD monitoring in multiple myeloma, mantle cell lymphoma, and follicular lymphoma with comparable sensitivity, accuracy, and reproducibility.The most broadly applicable MRD target in B-NHL is the junctional regions of the rearranged immunoglobulin heavy chain gene (IGHV). Chromosomal translocations like the t(14;18) translocation in FL and t(11;14) translocation in MCL can be used as MRD target in selected lymphoma subtypes. In patients with B-cell chronic lymphocytic leukemia, both flow-cytometry and RQ-PCR are equally suited for MRD assessment as long as a sensitivity of 10-4 shall be achieved.MRD diagnostics targeting the IGHV gene is complex and requires extensive knowledge and experience because the junctional regions of each lymphoma have to be identified before the patient-specific RQ-PCR assays can be designed for MRD monitoring. In addition, the presence and load of somatic hypermutation (SHM) within the rearranged IG heavy variable (IGHV) gene occurring as during B-cell development of germinal center and post-germinal center lymphomas may hamper appropriate primer binding leading to false-negative results. The translocations mentioned above have the advantage that consensus forward primers and probes, both placed in the breakpoint regions of chromosome 18 in FL and chromosome 11 in MCL, can be used in combination with a reverse primer placed in the IGH joining region of chromosome 14. RQ-PCR-based methods can reach a good sensitivity (≤10-4). This chapter provides all relevant background information and technical aspects for the complete laboratory process from detection of the clonal IGHV gene rearrangement and the chromosomal translocations at diagnosis to the actual MRD measurements in clinical follow-up samples of B-NHL. However, it should be noted that MRD diagnostics for clinical treatment protocols has to be accompanied by regular international quality control rounds to ensure the reproducibility and reliability of the MRD results.

Assessment of plasma cell myeloma minimal residual disease testing by flow cytometry in an international inter-laboratory study: Is it ready for primetime use?

Minimal/measurable residual disease (MRD) testing by flow cytometry (FC) has been proposed as a potential surrogate clinical endpoint in plasma cell myeloma (PCM) clinical trials. As a result, effort has gone into standardizing this approach on PCM patients. To assess inter-laboratory variation in FC MRD testing of PCM patients in an independent inter-laboratory study. A dilution series of five stabilized bone marrow samples manufactured to contain 0%, 0.1%, 0.01%, 0.001%, and 0.0001% neoplastic plasma cells (PCs) were tested blind, using standardized FC PCM MRD assays by 10 international laboratories. Laboratories' assays broadly adhered to the consensus guidelines; however, some deviations were identified in panel design, fluorochrome conjugates, and lysis reagents. Despite this, all laboratories that returned results detected neoplastic PCs down to 0.001% of leucocytes. 6/8 laboratories detected neoplastic PCs at a level of 0.0001%. Quantitative data returned by laboratories showed good consensus and linearity with increasing variation at lower levels of MRD. However, examples of analytical and post analytical error were identified. Broadly standardized PCM MRD FC assays can attain the lower limit of detection (LOD) required by current and future clinical trials, an important consideration in establishing PCM MRD testing as a surrogate clinical marker in PCM clinical trials. Laboratories' assays showed good linearity, encouraging the prediction of survival based on log reduction in neoplastic PC populations in future clinical trials. However, the deviations from consensus guidelines identified in this study would suggest that if PCM MRD assays are further standardized interlaboratory variation could be reduced. © 2018 International Clinical Cytometry Society.

Flow-cytometric minimal residual disease monitoring in blood predicts relapse risk in pediatric B-cell precursor acute lymphoblastic leukemia in trial AIEOP-BFM-ALL 2000.

Flow-cytometric monitoring of minimal residual disease (MRD) in bone marrow (BM) during induction of pediatric patients with acute lymphoblastic leukemia (ALL) is widely used for outcome prognostication and treatment stratification. Utilizing peripheral blood (PB) instead of BM might be favorable, but data on its usefulness are scarce. We investigated 1303 PB samples (days 0, 8, 15, 33, and 52) and 285 BMs (day 15) from 288 pediatric ALL patients treated in trial AIEOP-BFM ALL 2000. MRD was assessed by four-color flow cytometry and evaluated as relative, absolute, and kinetic result. In B-ALL only, PB measures from early time points correlated with relapse incidence (CIR). Best separation occurred at threshold <1blast/μL at day 8 (5-year CIR 0.02±0.02 vs 0.12±0.03; P=0.044). Patients with highest relapse risk were not distinguishable, but PB-MRD at days 33 and 52 correlated with prednisone response and postinduction BM-MRD by PCR (P<0.001). Kinetic assessment did not convey any advantage. In multivariate analysis including day 15 BM-MRD, PB-MRD measures lost statistical power. In summary, PB-MRD in pediatric B-ALL correlates with outcome and risk parameters, but its prognostic significance is not strong enough to substitute for BM assessment in AIEOP-BFM trials. It might, however, be valuable in treatment environments not using multifaceted risk stratification with other MRD measures.

Measurable Residual Disease Assessment By qPCR in Peripheral Blood Is an Informative Tool for Disease Surveillance in Childhood Acute Myeloid Leukemia

Background: Relapse remains a serious event and main obstacle to permanent cure in childhood acute myeloid leukemia (AML). Reduction of measurable/minimal residual disease (MRD) assessed by real-time quantitative PCR (qPCR) during therapy is predictive of outcome in adults but persistent MRD positivity may be observed despite long-term remission and hamper accurate risk assignment. Sequential MRD determinations, rather than analysis at single landmark time points, may better capture the dynamics of leukemia eradication and identify relapse at molecular levels when applied in the post-therapy setting. We investigated the post-induction qPCR MRD kinetics in peripheral blood (PB) and bone marrow (BM) in a large cohort of childhood AML patients and demonstrate the utility of serial assessments for disease surveillance after therapy completion.Methods: We collected the results from qPCR MRD analyses of 774 samples (BM 347, PB 427) from 75 children with AML harboring recurrent fusion transcripts (32 RUNX1-RUNX1T1, 24 CBFB-MYH11, 16 KMT2A-MLLT3 and 3 KMT2A-ELL). Patients were treated according to The Nordic Society for Paediatric Haematology and Oncology (NOPHO)-AML 2004 or NOPHO-DBH AML 2012 protocols (2004 - 2016), or AML-BFM 2012 protocol (2014 - 2016). Only patients who achieved complete remission (CR) during induction therapy and received standard-risk consolidation without allografting were eligible for the study.Risk of relapse as a function of time of MRD positivity in sequential samples from BM or PB during consolidation therapy was modeled using restricted cubic splines. Considering shifting from MRD negative to positive in PB and MRD increments in BM above 5x10-4 as time-dependent variables, the Mantel-Byar method was applied to evaluate the prognostic impact of MRD kinetics during follow-up.Results: Risk of relapse was independent of MRD persistence in BM during therapy, but showed a strong correlation with time of MRD positivity in PB where 8/8 patients with detectable MRD after first consolidation course relapsed (Figure 1A and 1B).At therapy completion, MRD level in BM did not correlate to outcome, HR=0.64/MRD log reduction, 95% confidence interval (CI):0.32 - 1.26 (P=0.19), and there was no difference in 3-year cumulative incidence of relapse (CIR) according to MRD status (P=0.51, Figure 2A). Four patients remained MRD positive throughout consolidation therapy and all subsequently relapsed, whereas 7/28 patients who were MRD negative in PB at the end of therapy suffered relapse, 3-year CIR=27%, CI:14% - 49% (P<0.001, Figure 2B).Shifting from negative to positive in PB during follow-up predicted subsequent relapse in 10/10 patients. All 253 PB samples collected during follow-up from 36 patients in continuous CR were MRD negative. In core binding factor (CBF) AML, persistent low-level MRD positivity in BM was frequent (detected in 38% of patients tested within six months of therapy completion) but an increment to above 5x10-4 heralded subsequent relapse in 12/12 patients. Pre-relapse MRD kinetics were delineated in 16 patients and revealed a median log increment/30 days of 0.8 (range:0.4 - 1.5) in CBF patients versus 2.2 (range:1.1 - 3.7) in patients with KMT2A-MLLT3 (P=0.008).Perspectives: This study demonstrates that qPCR MRD monitoring in PB, rather than BM, represents an accurate discriminator of prognosis and is a highly informative tool for disease surveillance in childhood AML. Early relapse detection during follow-up may facilitate preemptive therapy strategies of molecular relapse, possibly improving relapse treatment outcomes. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Favorable Outcome in Patients with Acute Myeloblastic Leukemia (AML) with NPM1 Mutation Who Present an Inadequate Clearance or Relapse of Minimal/Measurable Residual Disease (MRD): Results of a Preemptive Intervention Policy (CETLAM-2012 Protocol)

IntroductionPatients diagnosed with AML with NPM1mutation (NPM1mut AML) included in the European LeukemiaNet favorable genetic risk category (ELNfav, i.e., without FLT3-ITD or with a low allelic burden FLT3-ITD comutation [FLT3-ITD/FLT3wt <0.5; FLT3-ITDLOW]) do not benefit from an allogeneic stem cell transplantation (alloSCT) in first complete remission (CR1). However, a significant proportion of these patients fail to frontline chemotherapy and require salvage therapy. Persistence or detection of MRD after post-CR treatment is associated with a high relapse risk and worse prognosis. With this background, the cooperative group CETLAM proposed an early therapeutic intervention in CETLAM-2012 protocol for patients with ELNfavNPM1mut AML patients not achieving a sustained MRD clearance after consolidation therapy, defined as molecular failure (MF). Herein we analyzed the outcome and predictive risk factors of MF.MethodsAll patients diagnosed with ELNfav NPM1mut AML treated according to CETLAM-12 protocol who achieved CR after 1 or 2 courses of induction chemotherapy were included in the study. Intended post-CR therapy consisted of 3 courses of high-dose cytarabine chemotherapy (HDAC). MRD was assessed in bone marrow samples after each chemotherapy course and thereafter, at regular 3-month interval during 3 years, in reference labs from CETLAM group following standard NPM1-mutation specific RQ-PCR. MF was defined as failure to achieve a molecular response (molCR) after consolidation therapy (i.e., NPM1mut/ABL·100 ratio > 0.05) or MRD reappearance after molCR. All MFs were confirmed in a second sample collected at least 4 weeks apart from previous sample. For patients who present a MF, alloSCT was recommended, without a predefined indication of debulking previous salvage chemotherapy.ResultsOut of 145 patients with ELNfav NPM1mut AML (75 M/70 F; median age, 56, 18-74), 132 (91%) achieved CR after 1 (n=124) or 2 courses (n=8). After a median follow-up of 25 months, 2-year overall survival (OS), event-free survival (EFS), leukemia-free survival (LFS) of the entire cohort were 79.9% (±3.6), 71.8% (±4) and 79.4% (±3.8), respectively. Among patients with available complete MRD information (n=89), 33 patients developed an hematological and/or molecular failure (Mol/Hem failure), resulting into a Mol/Hem failure-free survival at 2 years of 62% (±5.6%); median time from CR to Mol/Hem failures was 8.2 months (2-43). Fourteen patients presented with an overt hematological relapse (hREL), preceded by a detectable MF in 8. Overall, 14 received salvage therapy in morphological CR, MRD(+) status (MF), and 14 For hREL. Among MF, 5 patients received HDAC-type salvage treatment (followed by alloSCT in 3) and 9 proceeded directly to alloSCT. In 5 additional MF patients with MRD persistence after consolidation, subsequent MRD monitoring showed decreasing MRD levels until complete clearance (n=4) or intermitent detection (n=1) and have not received further therapy. After salvage therapy, 12/14 (86%) patients with MF achieved molCR, and 10/14 (71%) treated for hREL achieved CR2 (MRD negative in 7, 50%), followed by alloSCT in 9. OS after Mol/Hem failure was 64.8% (± 8.4) at 2 years, 88.8 (±7.5%) in patients treated for MRD(+)-status (MF) and 32.1% (±13.6%) in patients treated with overt hREL (p<0.001; figure 1).Potential predictive factors of Mol/Hem failure were investigated. Interestingly, a ratio of NPM1mut/ABL*100 ≥1 after induction allowed distinction of two patient subpopulation with strikingly different Mol/Hem failure risk: Mol/Hem failure-free survival at 2 years of 84±6% vs. 33±10% in patients with a lower (<1) and higher (³1) tumor burden (p<0.001; figure 2). Remarkably, concurrence of FLT3-ITDLOWdid not correlate with outcome or Mol/Hem failure risk.ConclusionDespite a significant proportion of Mol/Hem failures, NPM1mut AML patients allocated in the ELN favorable risk group presented a favorable overall outcome. NPM1mut-based MRD surveillance is able to anticipate most hematological relapses, and a MRD-driven early intervention policy, at time of MF, allowed a successful rescue of a significant proportion of patients. Moreover, an early measure of residual tumor burden, after induction therapy, might identify those patients with a high risk of molecular or hematological subsequent failure, allowing the potential implementation of preemptive intensification strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Phase II Study of Blinatumomab in Patients with B-Cell Lineage Acute Lymphocytic Leukemia with Positive Minimal/Measurable Residual Disease

Phase II Study of Blinatumomab in Patients with B-Cell Lineage Acute Lymphocytic Leukemia with Positive Minimal/Measurable Residual Disease

The Impact of Minimal Residual Disease (MRD) Status at Day 28 in Predicting Relapse in Adults with Acute Lymphoblastic Leukemia in the Real World Setting

BackgroundAcute lymphoblastic leukemia (ALL) requires intensive systemic and intrathecal (IT) therapy for a prolonged period to prevent relapse [1, 2]. MRD negativity has been associated with long-term disease-free survival.[3] However, most published data are in patients treated in a clinical trial setting. Real-world data are lacking. We performed a retrospective cohort analysis of our database of ALL patients to determine the impact of MRD measurement on the pattern of relapse.[3-5]MethodsWe conducted a retrospective cohort analysis of patients with ALL treated at our institution between December 2001 and September 2015. We interrogated the database for patient and disease characteristics, treatment protocols, and outcomes. In all cases, MRD reported was obtained by way of multi-color flow cytometry on bone marrow biopsy samples. Data were stored in the RedCap database and simple, descriptive statistics were utilized for analysis. Chemotherapy consisted of standard regimens including E2993, Hyper-CVAD, the Vanderbilt ALL protocol, multiple pediatrics protocols, and multiple regimens not otherwise specified. [3-5]ResultsWe identified 147 patients over the time period specified, consisting of 91 males and 56 females. The median age of the cohort was 42 years. The most common chemotherapy regimens used included Hyper-CVAD (31%), E2993 (24%), the Vanderbilt ALL protocol (23%) and pediatric protocols (19%). Among the patients in morphologic CR, 55 (37%) had MRD measured at day 28 (+/- 7 days). Of those patients, 38 (83%) were MRD-negative and 17 (13%) were MRD-positive. In the MRD-negative cohort, 2 (5.3%) relapses occurred within the first year while 8 (21%) ocured later. In the MRD-positive cohort, 3 (38%) relapsed within the first while 2 (12%) occurred later.DiscussionOur results underscore the importance of early measurement of MRD in ALL patients in predicting early relapse. In those patients with no detectable MRD on flow cytometry of the bone marrow at day 28, only 5.3% relapsed within one year compared to 38% of those with detectable MRD. This difference is actually more pronounced than those observed in some clinical trials, validating early MRD measurement across treatment regimens in the real world setting. Notably, 2 patients with detectable MRD at day 28 were continued on the same protocol and did not relapse at 7-year follow-up. This suggests a potential role for serial MRD measurement in select, low-risk patients with MRD close to negative. Our study is limited as it is a retrospective analysis with a small sample size and at a single tertiary academic medical center which may diminish its applicability to other practice settings.