Disease In Acute Lymphoblastic Leukemia Research Articles

Association of Interleukin-12 and Interleukin-18 Polymorphisms with Acute Lymphoblastic Leukemia Disease in Iraqi Patients

Acute lymphoblastic leukemia (ALL) is a blood cancer where high numbers of abnormal, immature lymphocytes called blasts start over-multiplying in the bone marrow. The lymphocyte cells and interleukins are crucial for controlling the immune response in tumor microenvironment. Many of the single nucleotide polymorphisms (SNPs) of some interleukin genes may change protein synthesis or function and regulate the immune response. A lot of these changes have been connected to a high risk of developing cancer. Aim of this study was to investigate the potential association between IL-12 (+1188 A/C) (rs3212227) and IL-18 (–607 A/C) (rs1946518) polymorphism with serum levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) and their genotypes association with the susceptibility to ALL disease in Iraqi patients. The study included 59 ALL patients and 30 healthy controls. The detection of IL-12 and IL-18 SNP genes was determined by ARMS-PCR methods and serum levels of IFN-γ and TNF-α were determined by utilizing enzyme linked immuno-sorbent assay (ELISA) kits. Results observed no significant association of IL-12 and IL-18 polymorphism with serum IFN-γ and TNF-α level. Genotyping frequencies of IL-12 (+1188A/C) CA, AA and CA+AA genotypes showed a strong association with the development of ALL disease, IL-18 genotypes showed no significant association with the disease progression.

Phase 2 study of inotuzumab ozogamicin for measurable residual disease in acute lymphoblastic leukemia in remission

AbstractThe detection of measurable residual disease (MRD) is the strongest predictor of relapse in acute lymphoblastic leukemia (ALL). Using inotuzumab ozogamicin in the setting of MRD may improve outcomes. Patients with ALL in first complete remission (CR1) or beyond (CR2+) with MRD ≥ 1 × 10−4 were enrolled in this phase 2 trial. Inotuzumab was administered at 0.6 mg/m2 on day 1 and 0.3 mg/m2 on day 8 of cycle 1, then at 0.3 mg/m2 on days 1 and 8 of cycles 2-6. Twenty-six consecutive patients with a median age of 46 years (range, 19-70 years) were treated. Nineteen (73%) were in CR1 and seven (27%) in CR2+; 16 (62%) had Philadelphia chromosome–positive ALL. Fifteen (58%) had baseline MRD ≥ 1 × 10−3. A median of 3 cycles (range, 1-6) were administered. Eighteen (69%) patients responded and achieved MRD negativity. After a median follow-up of 24 months (range, 9-43), the 2-year relapse-free survival rate was 54% and the 2-year overall survival rate was 60% in the entire cohort. Most adverse events were low grade; sinusoidal obstruction syndrome was noted in 2 patients (8%). In summary, inotuzumab ozogamicin resulted in favorable survival, MRD negativity rates, and safety profiles for patients with ALL and MRD-positive status. This study was registered at www.ClinicalTrials.gov as #NCT03441061.

Vidjil add-on for MRD quantification of samples processed using the EuroClonality-NGS protocol.

Assessment of minimal residual disease in acute lymphoblastic leukemia by immune repertoire NGS requires spiking CDR3 sequences at known quantities into the patient's sample. Recently, the EuroClonality-NGS group released one of the most comprehensive protocols for this purpose. ARResT/Interrogate is a closed-source software for processing these NGS libraries, developed by this same group. Vidjil, an open-source alternative, currently cannot handle libraries prepared using this protocol. Here, we present a Vidjil add-on to solve this issue. EuroClonality-NGS prepared samples analyzed with Vidjil and ARResT/Interrogate were highly concordant (r=0.998) and presented low error (root-mean-square error, RMSE=0.112).

Measurable residual disease in acute lymphoblastic leukemia: methods and clinical context in adult patients.

Measurable residual disease (MRD) is the most powerful independent predictor of risk of relapse and long-term survival in adults and children with acute lymphoblastic leukemia (ALL). For almost all patients with ALL there is a reliable method to evaluate MRD, which can be done using multi-color flow cytometry, quantitative polymerase chain reaction to detect specific fusion transcripts or immunoglobulin/T-cell receptor gene rearrangements, and high-throughput next-generation sequencing. While next-generation sequencing-based MRD detection has been increasingly utilized in clinical practice due to its high sensitivity, the clinical significance of very low MRD levels (<10-4) is not fully characterized. Several new immunotherapy approaches including blinatumomab, inotuzumab ozogamicin, and chimeric antigen receptor T-cell therapies have demonstrated efficacy in eradicating MRD in patients with B-ALL. However, new approaches to target MRD in patients with T-ALL remain an unmet need. As our MRD detection assays become more sensitive and expanding novel therapeutics enter clinical development, the future of ALL therapy will increasingly utilize MRD as a criterion to either intensify or modify therapy to prevent relapse or de-escalate therapy to reduce treatment-related morbidity and mortality.

SOHO State of the Art Updates and Next Questions: Measurable Residual Disease in Acute Lymphoblastic Leukemia - Optimization and Innovation in 2022 and Beyond.

Measurable residual disease (MRD) is an established component of acute lymphoblastic leukemia (ALL) management in both children and adults. Society guidelines and expert consensus documents include assessment of MRD as the standard of care following induction therapy, consolidation therapy, and at additional time points, depending on the treatment regimen administered. Further, the approval of blinatumomab for MRD+ B-ALL has advanced the concept of MRD response as a clinical endpoint in ALL. Although the utility of MRD in ALL has been well defined over the last decades, several questions remain. In this review we focus on areas of ongoing controversy and exploration in ALL MRD, including the following: (1) Does increasing the depth of MRD assessment add prognostic value? (2) Is there a role for ongoing MRD monitoring once patients achieve MRD response? (3) Can MRD assessment of the peripheral blood be substituted for bone marrow? (4) Should MRD assays be applied to the analysis of the central nervous system (CNS)? Ongoing studies should answer the majority of these questions in the coming years.

Hyperactive CREB subpopulations increase during therapy in pediatric B-lineage acute lymphoblastic leukemia.

Persistence of residual disease in acute lymphoblastic leukemia (ALL) during the initial stages of chemotherapy is associated with inferior survival. To better understand clonal evolution and mechanisms of chemoresistance, we used multiparameter mass cytometry, at single-cell resolution, to functionally characterize pediatric B-ALL cells at disease presentation and those persisting during induction therapy. Analysis of ALL cells from presentation samples (n=42) showed that the most abundant phosphosignals were pCREB, pH2AX and pHH3 and we identified JAK-STAT and RAS pathway activation in five of six patients with JAK or RAS genetic aberrations. The clonal composition of ALL was heterogeneous and dynamic during treatment but all viable cell clusters showed pCREB activation. Levels of pCREB in ALL cells were increased or maintained during therapy and high dimensional analysis revealed a subpopulation of ALL cells at presentation that was positive for pCREB/pHH3/pS6 which increased during treatment in some patients, implicating this signaling node in conferring a survival advantage to multi-agent induction therapy. The small molecule CREB inhibitor, 666-15, was shown to reduce CREB transcriptional activity and induce apoptosis in ALL patient-derived xenograft cells of varying cytogenetic subtypes in vitro, both in the presence and absence of stromal support. Together, these data suggest that the cAMP signaling pathway may provide an opportunity for minimal residual disease-directed therapy for many patients at high risk of relapse.

LymphoTrack Is Equally Sensitive as PCR GeneScan and Sanger Sequencing for Detection of Clonal Rearrangements in ALL Patients.

Monoclonal rearrangements of immunoglobulin (Ig) genes and T-cell receptor (TCR) genes are used for minimal measurable disease in acute lymphoblastic leukemia (ALL). The golden standard for screening of gene rearrangements in ALL has been PCR GeneScan and Sanger sequencing, which are laborsome and time-consuming methods. More rapid next-generation sequencing methods, such as LymphoTrack could possibly replace PCR GeneScan and Sanger sequencing for clonality assessment. Our aim was to evaluate to what extent LymphoTrack can replace PCR GeneScan and Sanger sequencing concerning sensitivity and quantifiability in clonality assessment in 78 ALL samples. With LymphoTrack, clonality assessment was based on the %Total reads, where ≥10% was used as cut off for clonal rearrangements. The patients displayed 0 to 4 clonal rearrangements per assay. The detection rate (rearrangements detected with PCR GeneScan and/or Sanger sequencing, also detected with LymphoTrack) was 85/85 (100%) for IGH, 64/67 (96%) for IGK, 91/93 (98%) for TCRG and 34/35 (97%) for TCRB. Our findings demonstrate that LymphoTrack was equally sensitive in detecting clonal rearrangements as PCR GeneScan and Sanger Sequencing. The LymphoTrack assay is reliable and therefore applicable for clonal assessment in ALL patients in clinical laboratories.

Clinical Value of Measurable Residual Disease in Acute Lymphoblastic Leukemia

Measurable (minimal) residual disease (MRD) status in acute lymphoblastic leukemia (ALL) has largely superseded the importance of traditional risk factors for ALL, such as baseline white blood cell count, cytogenetics, and immunophenotype, and has emerged as the most powerful independent prognostic predictor. The development of sensitive MRD techniques, such as multicolor flow cytometry (MFC), quantitative polymerase chain reaction (PCR), and next-generation sequencing (NGS), may further improve risk stratification and expand its impact in therapy. Additionally, the availability of highly effective agents for MRD eradication, such as blinatumomab, inotuzumab ozogamicin, and chimeric antigen receptor (CAR) T-cell therapies, enabled the development of frontline regimens capable of eradicating MRD early in the treatment course. While long-term follow-up of this approach is lacking, it has the potential to significantly reduce the need for intensive post-remission treatments, including allogeneic bone marrow transplantation, in a significant proportion of patients with ALL.

Measurable Residual Disease in Acute Lymphoblastic Leukemia: Optimization and Innovation in 2021 and Beyond

Introduction Measurable residual disease (MRD) is an established component of acute lymphoblastic leukemia (ALL) management in both children and adults. Society guidelines and expert consensus documents include assessment of MRD as the standard of care following induction therapy, consolidation therapy, and at additional time points, depending on the treatment regimen administered.1–3 Further, the approval of blinatumomab for MRD+ B-ALL has advanced the concept of MRD response as a clinical endpoint in ALL. Although the utility of MRD in ALL has been well defined over the last decades, several questions remain. This extended abstract and the associated presentation focus on areas of ongoing controversy and exploration in ALL MRD, including the following.1) Does increasing the depth of MRD assessment add prognostic value? 2) Is there a role for ongoing MRD monitoring once patients achieve MRD– response? 3) Can MRD assessment of the peripheral blood be substituted for bone marrow? 4) Should MRD assays be applied to the analysis of the central nervous system (CNS)? Measurable residual disease (MRD) is an established component of acute lymphoblastic leukemia (ALL) management in both children and adults. Society guidelines and expert consensus documents include assessment of MRD as the standard of care following induction therapy, consolidation therapy, and at additional time points, depending on the treatment regimen administered.1–3 Further, the approval of blinatumomab for MRD+ B-ALL has advanced the concept of MRD response as a clinical endpoint in ALL. Although the utility of MRD in ALL has been well defined over the last decades, several questions remain. This extended abstract and the associated presentation focus on areas of ongoing controversy and exploration in ALL MRD, including the following.1) Does increasing the depth of MRD assessment add prognostic value? 2) Is there a role for ongoing MRD monitoring once patients achieve MRD– response? 3) Can MRD assessment of the peripheral blood be substituted for bone marrow? 4) Should MRD assays be applied to the analysis of the central nervous system (CNS)?

Analytical Quality Controls for ddPCR Detection of Minimal Residual Disease in Acute Lymphoblastic Leukemia

Abstract Background Droplet digital PCR (ddPCR) is a promising technique for absolute quantification of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), but there is no comprehensive quality assurance program to enable its application in clinical laboratories. Current guidelines for real-time quantitative PCR (qPCR) assays targeting immunoglobulin/T-cell receptor (Ig/TCR) gene rearrangements needed adaptation for ddPCR to cover droplet generation, intraassay variation, and interassay variation in the absence of standard curves. Methods Six qPCR MRD assays for Ig/TCR gene rearrangements and a standard albumin control gene assay were migrated to a ddPCR platform and used to test 82 remission samples from 6 patients with ALL. Three analytical quality controls (QC) were developed and evaluated for ddPCR MRD detection. Results Analytical QC for droplet number generation (DN-QC), for albumin ddPCR assay performance (Alb-QC) and for patient-specific marker assay performance (PS-QC) were established with pass/fail limits and corresponding QC rules. Compared to established qPCRs, the ddPCR assays had comparable sensitivity and quantitative range. Overall, there was close agreement (91%) of MRD results between qPCR and ddPCR (κ = 0.86, P &amp;lt; 0.0001) and stronger concordance in 32 quantifiable samples (R2 = 0.97, P &amp;lt; 0.0001). Conclusions The use of this newly developed quality control system for ddPCR MRD testing avoids the need to repeat standard curves and provides reliable results comparable to standardized qPCR methods for MRD detection in ALL.

Molecular and cellular markers for measurable residual disease in acute lymphoblastic leukemia.

Acute leukemia is the leading cause of death in children worldwide, particularly in developing countries where the growing number of cases with unfavorable prognosis and high risk of early relapse have positioned pediatric cancer as a priority. The late and imprecise diagnosis, malnutrition and unfavorable environmental conditions, and toxicity-associated therapy are some of the factors that compromise the success of the treatment and affect survival rates in vulnerable regions. An early and exhaustive classification of malignant neoplasms at the clinical debut and the proper follow-up of treatment's response constitute one of the most powerful prognostic factors. Remarkably, the ultrasensitive detection of residual and relapse clones that determine the minimal/measurable residual disease (MRD) has been a milestone in the comprehensive management of hematologic malignancies that favorably improve the complete remission cases. In this review, we discuss the scientific and technological advances applied to laboratory diagnosis in MRD determination: from the multiparametric immunophenotyping to next-generation sequencing and cytomics. As a result of multidisciplinary research in the main concentration oncology centers and laboratories, residual leukemia detection strategies that combine molecular analysis and cellular markers are recommended as the most valuable tools, making them the paradigm for stratification campaigns in vulnerable regions.

Minimal Residual Disease in Acute Lymphoblastic Leukemia: Current Practice and Future Directions.

Simple SummaryAcute lymphoblastic leukemia minimal residual disease (MRD) refers to the presence of residual leukemia cells following the achievement of complete remission, but below the limit of detection using conventional morphologic assessment. Up to two thirds of children may have MRD detectable after induction therapy depending on the biological subtype and method of detection. Patients with detectable MRD have an increased likelihood of relapse. A rapid reduction of MRD reveals leukemia sensitivity to therapy and under this premise, MRD has emerged as the strongest independent predictor of individual patient outcome and is crucial for risk stratification. However, it is a poor surrogate for treatment effect on long term outcome at the trial level, with impending need of randomized trials to prove efficacy of MRD-adapted interventions.Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer and advances in its clinical and laboratory biology have grown exponentially over the last few decades. Treatment outcome has improved steadily with over 90% of patients surviving 5 years from initial diagnosis. This success can be attributed in part to the development of a risk stratification approach to identify those subsets of patients with an outstanding outcome that might qualify for a reduction in therapy associated with fewer short and long term side effects. Likewise, recognition of patients with an inferior prognosis allows for augmentation of therapy, which has been shown to improve outcome. Among the clinical and biological variables known to impact prognosis, the kinetics of the reduction in tumor burden during initial therapy has emerged as the most important prognostic variable. Specifically, various methods have been used to detect minimal residual disease (MRD) with flow cytometric and molecular detection of antigen receptor gene rearrangements being the most common. However, many questions remain as to the optimal timing of these assays, their sensitivity, integration with other variables and role in treatment allocation of various ALL subgroups. Importantly, the emergence of next generation sequencing assays is likely to broaden the use of these assays to track disease evolution. This review will discuss the biological basis for utilizing MRD in risk assessment, the technical approaches and limitations of MRD detection and its emerging applications.

Minimal Residual Disease in Acute Lymphoblastic Leukemia

Minimal Residual Disease in Acute Lymphoblastic Leukemia

Validation of a PCR-Based Next-Generation Sequencing Approach for the Detection and Quantification of Minimal Residual Disease in Acute Lymphoblastic Leukemia and Multiple Myeloma Using gBlocks as Calibrators

Detection of minimal residual disease (MRD) to guide therapy has been a standard practice in treatment of childhood acute lymphoblastic leukemia (ALL) for decades. In multiple myeloma (MM), a clear correlation is found between absence of MRD and longer survival. Quantitative allele-specific oligonucleotide (qASO)-PCR is the standard molecular method for MRD detection in these hematologic malignant tumors. However, this technique has some drawbacks that can be overcome by next-generation sequencing (NGS). In this study, NGS is validated as an alternative method for qASO-PCR for MRD detection in both ALL and MM. MRD results obtained by NGS and qASO-PCR were compared in 59 and 39 bone marrow samples of 33 and 14 patients with ALL and MM, respectively. Our results indicate that the use of gBlocks as calibrators makes the NGS approach a powerful tool to quantify MRD. With an input of 400 ng of DNA (corresponding to approximately 7×104 cells), a limit of detection of 0.01% can be achieved. The specificity of the NGS-MRD technique was 100%, and a correlation with qASO-PCR for quantifiable MRD results of 0.93 and 0.91 was found in ALL and MM, respectively. Especially for MM, the higher applicability (100%) of the NGS-MRD protocol, compared with qASO-PCR (57%), was clearly demonstrated. These results demonstrate that NGS is an even better alternative to qASO-PCR.

How I investigate minimal residual disease in acute lymphoblastic leukemia.

Minimal Residual Disease (MRD) is the most important independent prognostic factor in acute lymphoblastic leukemia (ALL) and refers to the deep level of measurable disease in cases with complete remission by conventional pathologic analysis, especially by cytomorphology. MRD can be detected by multiparametric flow cytometry, molecular approaches such as quantitative polymerase chain reaction for immunoglobulin and T-cell receptor (IG/TR) gene rearrangements or fusion genes transcript, and high-throughput sequencing for IG/TR. Despite the proven clinical usefulness in detecting MRD, these methods have differences in sensitivity, specificity, applicability, turnaround time and cost. Knowing and understanding these differences, as well as the principles and limitations of each technology, is essential to laboratory standardization and correct interpretation of MRD results in line with treatment time points, therapeutic settings, and clinical trials. Here, we review the methodological approaches to measure MRD in ALL and discuss the advantages and limitations of the most commonly used techniques.

Central Nervous System (CNS) Involvement in Adult Acute Lymphoblastic Leukemia (ALL) Assessed Solely By Flow Cytometry Has an Adverse Impact on Survival

Introduction: Several studies published in recent years dealing with the detection of Central Nervous System (CNS) disease in Acute Lymphoblastic Leukemia (ALL) have demonstrated superiority in terms of sensitivity of Flow Cytometry (FCM) over standard cytology (Tawfik et al, 2018). However, whether CNS activity detected by FCM has an impact on the clinical outcome of ALL adult patients, is still a matter of debate. (Del Principe et al, 2019) Methodology: We analyzed a retrospective cohort of newly diagnosed ALL adult patients in an oncologic center in Mexico, who had a CNS analysis by FCM and conventional cytology, diagnosed between 2017 and 2019. The primary endpoint was to assess the impact on the survival of patients with CNS disease detected by FCM. We defined positive FCM (FCM+) when the patient had &amp;gt;10 phenotypically pathologic events and positive cytology (Cyt+) when the patent had confirmed CNS-2 or CNS-3 activity as defined by NCCN and ESMO; double-negative (DN) patients were considered when neither FCM nor cytology demonstrated disease as described above. Baseline characteristics were grouped in tables and summarized as medians and ranges for quantitative variables or frequencies in qualitative variables. Basal characteristics and different outcomes were compared by the chi-square test for binary variables, and the Mann-Whitney U test for quantitative variables. Overall Survival (OS) and Disease-Free Survival (DFS) analysis were made by Kaplan-Meier curves. A comparison between groups was performed using the log-rank test, with a significant P value less than 0.05 and a 95% confidence interval (CI). We performed a Cox regression model to obtain an HR with a 95% confidence interval for survival to identify risk associations. Results: Of the 92 evaluable patients, the median age was 28 years. 88% of all ALL were ALL-B. The median count of leucocytes was 11.65 x109/L (range 4.71-38.24 x109/L); median LDH was 758 UI/L (range 314-1242 UI/L). 63% of patients had an unfavorable karyotype (hypodiploidy, complex karyotype, or KMT2A). At diagnosis, 27 (28%) were positive by FCM and negative by cytology. The median of the analyzed events was 14,733 (5,025-50,000) with a viability of 38.5% (22-61.75). Only 6 patients were positive by both techniques. 45% of patients with CNS involvement had neurological symptoms (mainly headache). The most used treatment was BFM in 56.7% of patients, followed by HyperCVAD in 37.8%. Patients with documented CNS infiltration by FCM or cytology were treated with triple intrathecal chemotherapy two times per week. Three patients had no response after two weeks of treatment, one received radiotherapy without any response and the other two were dead for chemotherapy toxicity. We found gender as the only statistically significant variable associated with a higher FCM+ (77% males vs 23% females, p&amp;lt;0.01). For the entire cohort, the complete remission (CR) after induction was 84% and 63% had a negative minimal residual disease (MRD) by FCM in the bone marrow. 15% of the patients were refractory. There were no differences between CR (73% versus 88%, p= 0.16), MRD negativity (46% versus 69%, p=0.11), refractory disease (26% versus 10%, p=0.13) or relapsed disease (50% versus 43%, p=0.65) between FCM(+) and ND groups. The OS for the entire cohort was 11.3 months. Patients with FCM+ had a lower survival of 5.6 months [CI95% (0.72-10.67)] compared with 11.8 months [CI95% (0.47-11.95)] in the DN group. The median DFS for the FCM+ patients was 4.6 months [CI95% (3.42-5.92)] versus 16.4 months [CI95% (6.21-26.75)] for DN group. In the Cox regression model, the HR for survival in patients with FCM+ was 1.85 (CI95% 1.004-3.421; p=0.04). Conclusions: Despite the retrospective nature of this study, the detection of CNS involvement was increased from 6% to 28% employing FCM compared with cytology. As mentioned previously, in other studies, FCM has greater sensitivity than cytology (Ranta et al. 2014). According to our results, the presence of CNS disease, detected by FCM has an adverse impact on OS in adult patients with newly diagnosed ALL. These results dictate consideration of FCM as a routine study in the diagnosis of ALL. Prospective studies are needed to evaluate more intensive treatment in these patients. Figure Disclosures No relevant conflicts of interest to declare.

Minimal residual disease in Acute Lymphoblastic Leukemia in the context of hematopoetic stem cell transplantation

Minimal or measurable residual disease (MRD) is considered the most important independent prognostic factor in acute lymphoblastic leukemia (ALL). MRD status after clinical remission has been used to establish the risk of relapse and therapeutic stratification, identifying patients who can benefit from therapeutic intensification, including allogeneic stem cell transplantation (alloSCT). The pre alloSCT MRD also identifies patients eligible for transplant and those with low or high risk of relapse after transplantation, according to the level of MRD detected. However, MRD status post-alloSCT has been shown to be a more powerful predictor of relapse than pre-transplant MRD. In addition, it is important to take into account that there are some factors to be considered to better interpret MRD information, these include: the method used for MRD assessment and its sensitivity and specificity, which may vary according to each specific time point of evaluation; the treatment regimen used; and the identification of genetic lesions that combined with MRD information can further improve the management of patients with ALL.

Analytical evaluation of the clonoSEQ Assay for establishing measurable (minimal) residual disease in acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma

BackgroundThe clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines.MethodsSensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay’s ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels.ResultsLoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low.ConclusionsThese studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.

Evaluation and management of measurable residual disease in acute lymphoblastic leukemia.

With standard chemotherapy regimens for adults with acute lymphoblastic leukemia, approximately 90% of patients achieve complete remission. However, up to half of patients have persistent minimal/measurable residual disease (MRD) not recognized by routine microscopy, which constitutes the leading determinant of relapse. Many studies in pediatric and adult populations have demonstrated that achievement of MRD negativity after induction chemotherapy or during consolidation is associated with significantly better long-term outcomes, and MRD status constitutes an independently prognostic marker, often superseding other conventional risk factors. Persistence of MRD after intensive chemotherapy is indicative of treatment refractoriness and warrants alternative therapeutic approaches including allogeneic stem cell transplantation, blinatumomab, or investigational therapies such as inotuzumab ozogamicin or chimeric antigen receptor T cells. Furthermore, the incorporation of novel monoclonal antibodies or potent BCR-ABL1 tyrosine kinase inhibitors, such as ponatinib into frontline treatment may have the advantage of achieving higher rates of MRD negativity while minimizing chemotherapy-related toxicities. Many studies are therefore ongoing to determine whether this strategy can improve cure rates without the need for allogeneic stem cell transplantation.

Effect of Dose and Schedule of L-Asparaginase Administration on Early Minimal Residual Disease in Acute Lymphoblastic Leukemia

Abstract Background and Objectives: L-asparaginase has become the backbone of acute lymphoblastic leukemia induction. In Berlin–Frankfurt–Munster (BFM) 95/2000 protocols, L-asparaginase was given twice weekly for initial 4 weeks. While sufficient L-asparaginase levels are important, there is no apparent correlation between high L-asparaginase levels and minimal residual disease (MRD). In view of toxicities of L-asparaginase, we planned to study the effect of dose and schedule of Escherichia coli-derived L-asparaginase on early MRD by phasing the same total dose, once a week over 8 weeks. Methods: This prospective, observational study enrolled 45 children and young adults ≤40 years. Modified BFM 95 protocol was followed. Weekly 5000 IU/m2 L-asparaginase was given intravenously, and MRD was analyzed at the end of 4 weeks (MRD1) and at 8 weeks (MRD2), using multicolor flow cytometry. MRD positive was defined as residual blasts ≥0.01%. Results: Thirty-one patients were eligible for final analysis. Nine could receive scheduled eight doses of L-asparaginase and 22 patients received less than eight doses. We analyzed age, gender, diagnosis, prednisone response, cytogenetics, central nervous system status, BFM risk group, MRD2, and relapse. L-asparaginase dose association was not statistically significant with respect to MRD2 (P = 0.237). There were no cases of pancreatitis, hypersensitivity, bleeding, or thrombosis. Reasons for patients receiving less than the scheduled eight doses were low serum fibrinogen levels and liver dysfunction. This study revealed 8 MRD1-negative and 13 MRD2-negative patients. Conclusion: L-asparaginase dose intensity does not affect early MRD. Phasing L-asparaginase over 8 weeks could lead to the achievement of more MRD2-negative status and thereby improve long-term outcome. This strategy may also reduce the incidence of adverse drug events.