Flow Cytometric Evaluation Of Minimal Residual Disease In Adult Acute Lymphoblastic Leukemia Using a Simplified, Single-Tube Approach

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1378-1378
Author(s):  
Roger Belizaire ◽  
Olga Pozdnyakova ◽  
Daniel J. DeAngelo ◽  
Betty Li ◽  
Karry Charest ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Turnaround Time ◽  
Tube Flow ◽  
Flow Cytometry Assay ◽  
Single Tube ◽  
Negative Results ◽  
Minimal Residual

Abstract Flow cytometry for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) has been widely used in pediatric patients to quantify therapeutic response and to assess the risk of relapse. Flow cytometry for MRD provides roughly the same level of sensitivity (0.01%) as molecular methods but at lower cost and with faster turnaround time. MRD assessment in ALL currently requires an evaluation of 20 or more parameters divided among multiple tubes. In part due to the assessment complexity, the use of flow cytometry for MRD detection in adult ALL patients has been relatively limited. We developed a 6-color, single-tube, flow cytometry assay to detect MRD in bone marrow (BM) aspirate specimens from adult ALL patients. The 73 patients included 52 patients with B-ALL (71%), 19 patients with T-ALL (26%) and 2 patients with T/myeloid leukemia (3%) and were treated with one of several standard chemotherapeutic regimens or targeted therapies. Patients were tested for MRD by flow cytometry after induction or re-induction therapy and serially thereafter. The 6-marker MRD panel was customized for each patient based on the 18-20-marker diagnostic immunophenotype. Sixty-three percent of B-ALL patients (n=33) had lymphoblasts with an aberrant immunophenotype; expression of a myeloid marker (e.g., CD13, CD15 or CD33) was the most common aberrancy. The remaining 37% of B-ALL patients (n=19) had disease with a hematogone immunophenotype, which comprised surface expression of CD10, CD19, CD20, CD34, CD38 and CD45; in the majority of these cases, leukemic cells were distinguishable from normal hematogones based on the intensity of surface marker expression. Forty-seven percent of T-ALL patients (n=9) had an aberrant immunophenotype, most often characterized by CD33 expression. One-hundred forty-six consecutive specimens analyzed for MRD by flow cytometry were classified as positive (23%), negative (72%) or uncertain (5%). Of the 34 samples classified as positive, 14 (41%) showed morphologic (i.e., BM aspirate or biopsy) evidence of disease; nineteen (65%) samples did not show morphologic evidence of disease and 1 sample did not have a concurrent morphologic assessment. Of the 105 samples classified as negative by flow cytometry, 103 (98%) were also negative by morphology and 1 sample did not have a concurrent morphologic assessment. One sample that was negative by flow cytometry had morphologic evidence of disease in the biopsy (10-20% blasts) but not the aspirate, suggesting that aspirate sampling artifact was responsible for the discrepancy. None of the 7 samples classified as uncertain by flow cytometry had morphologic evidence of disease; five out of 7 uncertain classifications were in B-ALL patients with hematogone immunophenotypes. Overall, MRD flow cytometry showed 86% concordance with the results of morphologic assessment. We evaluated outcomes in all patients with negative morphologic results and any positive MRD flow cytometry result(s). Of the 73 patients in this study, 61 had morphology-negative results that were either MRD-negative (n=45) or MRD-positive (n=16). Patients in this group were at various points of treatment post-induction or re-induction. Four out of 45 patients (9%) with MRD-negative results relapsed during a median follow-up period of 22 months, and 8 out of 16 patients (50%) with an MRD-positive result relapsed during a median follow-up period of 15 months (odds ratio for relapse 10.3, 95% confidence interval 2.5-42.4, P=0.001). In addition, relapse-related and overall mortality (Figure 1) were higher in patients with MRD-positive results (P=0.0023 and P=0.0016, respectively, by the log-rank test). In summary, we present a simplified, single-tube, flow cytometry assay that can be used to detect MRD in adult ALL at relatively low cost with rapid turnaround time; our approach was applicable to cases with either hematogone or aberrant immunophenotype, yielding a definitive result in 95% of cases. Notably, the presence of MRD was associated with relapse and mortality, suggesting that our method of MRD assessment could be used to guide treatment of adult ALL. Further analysis of the correlations between MRD results, clinical management and patient outcomes is ongoing. Disclosures: No relevant conflicts of interest to declare.

A Single-Tube Seven-Colour Flow Cytometry Assay for Detection of Minimal Residual Disease In Myeloma.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1688-1688
Author(s):  
Soraya Wuilleme ◽  
Nelly Robillard ◽  
Steven Richebourg ◽  
Marion Eveillard ◽  
Laurence Lodé ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Cell ◽  
Plasma Cells ◽  
Residual Disease ◽  
Flow Cytometry Assay ◽  
Single Tube ◽  
Cell Identification ◽  
Colour Flow ◽  
Multiparameter Analysis ◽  
Minimal Residual

Abstract Abstract 1688 The eradication of minimal residual disease (MRD) in myeloma predicts for improved outcome. A number of different approaches to myeloma MRD detection are available; these vary widely in sensitivity and cost. Flow cytometric assessment of MRD may be preferable in practice because of lower cost and easier feasibility. Myeloma MRD flow cytometry requires at least three markers for plasma cell identification (CD38, CD138 and CD45) and combination of several additional markers to detect phenotypic abnormality including CD19, CD20, CD27, CD28, CD45, CD56 and CD117. Also, assessment of immunoglobulin light-chain restriction (cytoplasmic K and L) combined with myeloma-associated phenotypic plasma cell abnormalities, is very important. Four-tube four-colour flow cytometry combine markers CD38/CD138/CD45 with markers for plasma cell phenotypic abnormalities and clonality. Six –colour flow cytometry combines the same markers (markers for plasma cell identification) plus clonality markers; it potentially increases the sensitivity of the method through coincident multiparameter analysis. However, the single-tube six-colour flow cytometry, proposed by others studies, excludes the myeloma-associated phenotypic plasma cell abnormalities and consequently decreases specificity of the assay. We propose a new single-tube seven-colour flow cytometry, including plasma cell identification antigens, clonality markers and myeloma-associated phenotypic plasma cell abnormalities markers. In this new method, PCs are stained with antibodies: (i) CD38, CD138, CD45 used for identified plasma cells and percentage plasma cells to total leucocytes. (ii) CD19 and CD56+CD28 used to identify normal and abnormal plasma cells; and (iii) cy-IgK and cy-IgL, for confirm the plasma cells clonality. We analysed normal bone marrow provided from healthy individuals. Our results showed a presence myeloma-associated phenotypic plasma cell abnormalities at low levels in healthy individual. The monotypy studies confirm polyclonality of this normal plasma cells. Then we compared MRD assessement with single-six colour flow cytometry assay (plasma cells markers, clonality markers and exluding myeloma-associated phenotypic markers) and seven-colour flow cytometry assay (including myeloma-associated phenotypic markers). Six –colour flow cytometry has a better sensitivity and showed efficacy for quantification MRD in myeloma patients. However, the single-tube six-colour flow cytometry excluded the myeloma-associated phenotypic plasma cell abnormalities and in some cases the seven-colour flow cytometry will be more informative because it detected myeloma-asociated phenotypic marquers combined with clonality marquers. Finally, the single-tube seven colour flow cytometry assay provides reduction in antibody cost and increases sensitivity and specificity of the method through coincident multiparameter analysis. Disclosures: No relevant conflicts of interest to declare.

Minimal Residual Disease Monitoring in Childhood Precursor B Lymphoblastic Leukemia with t(12;21)(p13;q22); ETV6-RUNX1: A Comparison Between Quantitative RT-PCR and Flow Cytometry

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3774-3774
Author(s):  
Sofie J Alm ◽  
Charlotte Engvall ◽  
Julia Asp ◽  
Lars Palmqvist ◽  
Jonas Abrahamsson ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Fusion Transcript ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Time Points ◽  
Qrt Pcr ◽  
Minimal Residual

Abstract The translocation t(12;21)(p13;q22) resulting in the fusion gene ETV6-RUNX1, is the most frequent gene fusion in childhood precursor B lymphoblastic leukemia (pre-B ALL), affecting about one in four children with pre-B ALL. In the NOPHO ALL-2008 treatment protocol, treatment assignment in pre-B ALL is based on clinical parameters, genetic aberrations, and results from analysis of minimal residual disease (MRD) at day 29 and 79 during treatment (where MRD >0.1% leads to upgrading of treatment). For pre-B ALL, in this protocol MRD analysis is performed using flow cytometry as the method of choice. In this study, we also analyzed MRD in t(12;21)(p13;q22) cases with quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the fusion transcript ETV6-RUNX1 in parallel with routine MRD analysis with flow cytometry, to determine if qRT-PCR of the ETV6-RUNX1 fusion transcript would be a reliable alternative to FACS. Bone marrow samples were collected at diagnosis and at day 15, 29 and 79 during treatment from 31 children treated according to the NOPHO ALL-2000 (n = 3) and NOPHO ALL-2008 (n = 28) protocols in Gothenburg, Sweden, between 2006 and 2013. Samples were analyzed in parallel with qRT-PCR for ETV6-RUNX1 fusion transcript and with FACS. For qRT-PCR, mRNA was isolated, cDNA synthesized, and qRT-PCR performed with GUSB as reference gene. MRD-qRT-PCR was defined as the ETV6-RUNX1/GUSB ratio at the follow-up time point (day 15/29/79) divided with the ETV6-RUNX1/GUSB ratio at diagnosis (%). MRD analysis with FACS was performed, after lysis of erythrocytes, using antibodies against CD10, CD19, CD20, CD22, CD34, CD38, CD45, CD58, CD66c, CD123, and terminal deoxynucleotidyl transferase, and when applicable also CD13 and CD33. Results of MRD-FACS were expressed as % of all cells. In total, 83 samples were analyzed with both methods in parallel; 31 from day 15 in treatment, 28 from day 29, and 24 from day 79. Overall, MRD-qRT-PCR showed good correlation with MRD-FACS. In total, 31 samples were positive with qRT-PCR and 24 with FACS, with concordant results (positive with both methods or negative with both methods) in 89% of samples, when the limit of decision (positive/negative MRD) was set to 0.1%. The concordance was especially high at the treatment stratifying time points, i.e. day 29 and 79; 89% and 100%, respectively. No samples at these time points were positive with FACS but negative with qRT-PCR. During the follow-up period (6-81 months), one patient relapsed (with negative MRD with both methods at stratifying time points), and two succumbed from therapy-related causes. Our results show that there is a significant relationship between the results of MRD analysis using FACS and MRD analysis using qRT-PCR of ETV6-RUNX1 fusion transcript. The high concordance between the methods indicates that negative MRD using qRT-PCR is as reliable as negative MRD using FACS, and that qRT-PCR could therefore be an alternative to FACS in cases where FACS is not achievable. In comparison to quantitative PCR of TCR/Ig gene rearrangements, which is the current backup MRD method for cases with pre-B ALL in NOPHO ALL-2008, qRT-PCR of ETV6-RUNX1 is much less time and labor consuming, making it appealing in a clinical laboratory setting. Disclosures No relevant conflicts of interest to declare.

scholarly journals Re-Emergence of Minimal Residual Disease Detected by Flow Cytometry Predicts an Adverse Outcome in Pediatric Acute Lymphoblastic Leukemia

2021 ◽  
Vol 10 ◽  
Author(s):  
Yu Wang ◽  
Yu-Juan Xue ◽  
Yue-Ping Jia ◽  
Ying-Xi Zuo ◽  
Ai-Dong Lu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Lymphoblastic Leukemia ◽  
Minimal Residual Disease ◽  
Residual Disease ◽  
Adverse Outcome ◽  
Lymphoblastic Leukemia ◽  
Negative Results ◽  
Cell Counts ◽  
Pediatric All ◽  
Minimal Residual

PurposeWhile the role of minimal residual disease (MRD) assessment and the significance of achieving an MRD-negative status during treatment have been evaluated in previous studies, there is limited evidence on the significance of MRD re-emergence without morphological relapse in acute lymphoblastic leukemia (ALL). We sought to determine the clinical significance of MRD re-emergence in pediatric ALL patients.MethodsBetween 2005 and 2017, this study recruited 1126 consecutive patients newly diagnosed with ALL. Flow cytometry was performed to monitor MRD occurrence during treatment.ResultsOf 1030 patients with MRD-negative results, 150 (14.6%) showed MRD re-emergence while still on morphological complete remission (CR). Patients with white blood cell counts of ≥50 × 109/L (p = 0.033) and MRD levels of ≥0.1% on day 33 (p = 0.012) tended to experience MRD re-emergence. The median re-emergent MRD level was 0.12% (range, 0.01–10.00%), and the median time to MRD re-emergence was 11 months (range, <1–52 months). Eighty-five (56.6%) patients subsequently developed relapse after a median of 4.1 months from detection of MRD re-emergence. The median re-emergent MRD level was significantly higher in the relapsed cohort than in the cohort with persistent CR (1.05% vs. 0.48%, p = 0.005). Of the 150 patients, 113 continued to receive chemotherapy and 37 underwent transplantation. The transplantation group demonstrated a significantly higher 2-year overall survival (88.7 ± 5.3% vs. 46.3 ± 4.8%, p < 0.001) and cumulative incidence of relapse (23.3 ± 7.4% vs. 64.0 ± 4.6%, p < 0.001) than the chemotherapy group.ConclusionsMRD re-emergence during treatment was associated with an adverse outcome in pediatric ALL patients. Transplantation could result in a significant survival advantage for these patients.

scholarly journals Intra-Tumoral Blast Heterogeneity and Implications for Minimal Residual Disease Detection in T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1076-1076
Author(s):  
Nina Friesgaard Öbro ◽  
Lars Peter Ryder ◽  
Hans Ole Madsen ◽  
Mette Klarskov Andersen ◽  
Birgitte Klug Albertsen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
T Cell Receptor ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Cell Receptor ◽  
Flow Sorting ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Minimal Residual

Abstract Introduction:The early treatment response, measured as minimal residual disease (MRD), is the most important tool for treatment stratification in T-cell acute lymphoblastic leukemia (T-ALL). Flow cytometry-based MRD (Flow-MRD) monitoring, in addition to the PCR-MRD method, is often important to ensure a sensitive MRD marker. Additionally, Flow-MRD investigation may add biological information to the MRD result itself, and allow cell sorting for biological and functional analyses. Flow-MRD in T-ALL consists of identification of cells with immature T-cell phenotype in bone marrow. However, important pitfalls in Flow-MRD, e.g. treatment-related marker modulation and intra-tumoral immunophenotypic heterogeneity, are poorly described. The aim of this study was to explore the implications of these pitfalls on T-ALL MRD detection and on the concordance between the two MRD methods. Potentially both PCR- and Flow-MRD methods might miss blast subpopulations, which is important if subpopulations have divergent chemosensitivity. Methods:The patient cohort included 49 Danish T-ALL patients (1-45 years of age) treated according to the NOPHO ALL2008 protocol. Standard PCR- and flow cytometry-based MRD data were obtained as part of routine MRD monitoring. We investigated intra-tumoral heterogeneity of the leukemia-associated immunophenotype by flow cytometry (diagnostic BM samples), including clonal T-cell receptor gene-rearrangements in flow-sorted blast subpopulations (22 patients). Immunophenotypic MRD markers (including assessment of modulation) were re-evaluated at follow-up in MRD-positive patients. Flow-MRD was validated by PCR-MRD analysis in flow-sorted cell populations (61 follow-up BM samples, 32 patients). Results:At diagnosis, more than 80% of the T-ALL patients had a heterogeneous immunophenotype, most often involving CD1a, CD4, and TdT. The degree of overall heterogeneity, as defined by the number of markers with heterogeneous expression showing distinct blast subpopulations, did not show association to day29 PCR-MRD. Except for one patient, the dominant T-cell receptor clonal gene rearrangements were conserved across phenotypically diverse blasts. Immunophenotypic changes in MRD-positive patients at early follow-up often included subpopulation-loss and/or marker down-modulation of CD1a, TdT and/or CD4. The marker modulations were frequently independent of each other in different subpopulations. Overall, flow cytometry-based identification of blasts and normal cells at Flow-MRD time points was verified by PCR in the flow-sorted cells: In patients where at least 90% of the blasts showed aberrant marker expression at diagnosis, the flow-sorted MRD cells were concordantly PCR-positive, and flow-sorted phenotypically normal cells were similarly PCR-negative in all but three samples that had very high MRD levels (>20%). However, many patients had only partly-informative immunophenotypes (less than 90% of blasts having aberrant marker). Three discrepant cases with Flow-MRD underestimation showed loss of CD1a- and TdT and down-modulation of CD99, verified in flow-sorting experiments. Conclusions and Discussion: We show that intra-tumoral immunophenotypic heterogeneity—a possible result of genetic instability—is common in T-ALL patients and involves several immaturity and T-linage markers commonly used in Flow-MRD. The dominant PCR-MRD targets are in most cases conserved across the diverse blast subpopulations at diagnosis, but in rare cases PCR-MRD might miss a subpopulation. The observed immunophenotypic changes in T-ALL blasts and blast subpopulations at early follow-up, including reduction of immaturity markers, represent important pitfalls in Flow-MRD. Flow-sorting experiments verified that, when all blasts of heterogeneous immunophenotypes were informative, MRD identified by flow cytometry at follow-up was highly concordant with PCR-MRD markers in sorted cells. The T-ALL blast heterogeneity and marker modulations, which are possibly treatment protocol-specific, are important to take into account to obtain reliable Flow-MRD and thus correct treatment stratification of T-ALL patients. Disclosures No relevant conflicts of interest to declare.

Prognostic Value of DNA Ploidy By Flow Cytometry for Pediatric B-Cell Acute Lymphoblastic Leukemia: A Peruvian Cohort Study

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 4-5
Author(s):  
Elizabeth Cervantes ◽  
Daniel J Enriquez ◽  
Judith Vidal ◽  
Rosario Retamozo ◽  
Arturo Zapata ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Clinical Trials ◽  
Acute Lymphoblastic Leukemia ◽  
Prognostic Value ◽  
Dna Ploidy ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Minimal Residual

Background: B-cell Acute Lymphoblastic Leukemia (B-ALL) represents an aggressive malignancy but highly curable in children. Currently, pediatric collaborative clinical trials have reported survival rates that exceed 90%, and DNA ploidy by flow cytometry (FC) has been pointed for risk stratification and prognosis in these clinical trials. However, its generalized use remains controversial as few studies reported no impact in real-world populations. We aimed to evaluate prognostic value of DNA ploidy measured by FC in a large cohort of Peruvian children with B-ALL. Methods: We evaluated prospectively DNA-ploidy by FC in bone marrow diagnostic samples from newly diagnosed children (<15 years) with B-ALL treated at Instituto Nacional de Enfermedades Neoplasicas (Lima-Peru) between 2017-2019. DNA-ploidy was evaluated using Propidium Iodide and calculated as the mean ratio between fluorescence of pathologic B blasts and normal marrow cells. Ploidy categories were established based on previous reports of DNA-index(DI): Diploid + Low-Hyperdiploidy (DLH; DI: 0.95 - 1.15), hypo-diploid (HD; DI<0.95) and High-hyperdiploidy (HH; DI>1.15), and recalculated using maximally selected rank statistics. Samples were analyzed in a FacsCanto II flow cytometer (BD) and Infinicyt software (Cytognos). All patients received BFM-2009 protocol and had minimal residual disease evaluation at the end of IA and IB induction. Minimal residual disease (MRD) was evaluated with FC using a detection threshold of 0.0025% and considering positive ≥0.01%. Survival curves (event-free and overall survival) were estimated using the Kaplan-Meier method and compared with the Log-rank test. Results: A total of 192 children were included (2 HD, 141 DLH and 49 HH cases according to DNA ploidy by FC). Clinical characteristics and outcomes are shown in Table 1. Median age at diagnosis was 5 years (Range:1-14), 10 years for HD, 6 and 3 years for DLH and HH, respectively (p=0.002). F/M ratio was 1:1.3 for all cases, but 1:2 in HH group. Most karyotypes (62%) had unsatisfactory or poor-quality result, 29% were considered normal, and only 9 hyperdiploidy and 2 hypodiploidy cases were detected by conventional karyotyping. Regarding genetics, 21 TEL/AML, 19 E2A/PBX1 and 9 BCR/ABL cases were detected by multiplex-PCR and most balanced alterations had DLH subtype and only one HD case had TEL/AML. MRD positivity after Induction IA was 35% without difference between groups (50% HD, 36% DLH and 31% HH, p=0.77), however after Induction IB, MRD was positive in 50% of HD, 12% of DLH and 5% of HH (p=0.048). At eight-teen months of follow-up, one relapse was seen in HD cases, and 11% DLH and 10% in HH. Median EFS and OS was not reached, however one-year EFS and OS were 86% for both without significant differences between groups. Multivariate analysis showed that MRD positivity remains as the principal independent prognostic factor and ploidy by DNA did not show any impact in terms of MRD, EFS and OS. Conclusion: High-Hyperdiploidy by DNA ploidy was associated to better MRD negativity rate after induction IB but without impact in short-term EFS and OS. DNA ploidy did not represent a prognostic factor in our study cohort, however long-term follow-up is warranted. Additionally, a better genetic risk stratification is necessary to improve outcomes in Latino high-risk population. Disclosures No relevant conflicts of interest to declare.

scholarly journals Deep Sequencing Approach for Minimal Residual Disease Detection in Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1388-1388
Author(s):  
Malek Faham ◽  
Jianbiao Zheng ◽  
Martin Moorhead ◽  
Victoria Carlton ◽  
Patricia Lee Stow ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Research Funding ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Employment Equity ◽  
Equity Ownership ◽  
Minimal Residual

Abstract Abstract 1388 Background: The clinical management of patients with acute lymphoblastic leukemia (ALL) relies on accurate prediction of relapse hazard to determine the intensity of therapy and avoid over- or under-treatment.1 The measurement of minimal residual disease (MRD) during therapy has now emerged as the most important predictor of outcome in ALL.2 We developed the LymphoSIGHT platform, a high-throughput sequencing method, which universally amplifies antigen-receptor gene segments and can identify all leukemia-specific sequences at diagnosis, allowing monitoring of disease progression and clonal evolution during therapy. In this study, we determined the sensitivity and specificity of this method, delineated the extent of clonal evolution present at diagnosis, and compared its capacity to measure MRD to that of flow cytometry and allele-specific oligonucleotide PCR (ASO-PCR) in follow-up samples from >100 patients with ALL. Methods: Using the sequencing assay, we analyzed diagnostic bone marrow samples from 100 ALL patients for clonal rearrangements of immunoglobulin (IgH@) and T cell receptor (TRB@, TRD@, TRG@) genes, as well as the extent of clonal evolution present at diagnosis. We assessed the capacity of the sequencing assay to detect MRD using diagnostic samples from 12 ALL patients carrying 13 leukemic IgH clonal rearrangements. Serial dilutions were prepared in normal peripheral blood mononucleated cells, at a range between <1 in 1 million to >1 in 1,000 cells. We also assessed MRD in follow-up samples from 106 ALL patients and analyzed concordance between MRD results obtained by the sequencing assay, flow cytometry and ASO-PCR. Results: In diagnostic bone marrow samples, we detected the presence of a high-frequency clonal rearrangement of at least one receptor (“calibrating receptor”) in all the 100 ALL samples; 94 samples had at least 2 calibrating receptors at diagnosis, with 51 having 3 or more. We also detected a variable degree of clonal evolution: the number of evolved clones in each sample ranged from 0 to 6933, with 39 (37%) samples having 1–50 evolved clones and 17 (16%) >50 (Figure 1). In experiments with mixtures of normal and leukemic cells, the sequencing assay unequivocally and accurately detected leukemic signatures in all dilutions up to a concentration of at least one leukemic cell in 1 million leukocytes. In direct comparisons with established MRD assays performed on follow-up samples from patients with B-ALL, sequencing detected MRD in all 28 samples positive by flow cytometry, and in 35 of the 36 positive by ASO-PCR; it also revealed MRD in 10 and 3 additional samples that were negative by flow cytometry and ASO-PCR, respectively (Figure 2). Conclusions: The sequencing assay is precise, quantitative, and can detect MRD at levels below 1 in 1 million leukocytes (0.0001%), i.e., represents sensitivity 1–2 orders of magnitude higher than standard flow cytometric and ASO-PCR methods. Our assay also allows monitoring of all leukemic rearrangements regardless of their prevalence at diagnosis, which abrogates the risk of false-negative MRD results due to clonal evolution. Finally, the sequencing assay utilizes a set of universal primers and does not require development of patient-specific reagents. These data, together with the results of our comparison with standard MRD assays in clinical samples, strongly support the use of the sequencing assay as a next-generation MRD test for ALL. Disclosures: Faham: Sequenta: Employment, Equity Ownership, Research Funding. Zheng:Sequenta: Employment, Equity Ownership, Research Funding. Moorhead:Sequenta: Employment, Equity Ownership, Research Funding. Carlton:Sequenta: Employment, Equity Ownership, Research Funding.

Different Minimal Residual Disease Levels between Childhood Acute Lymphoblastic Leukemia and Acute Myeloid Leukemia Detected with Multi-Parameter Flow Cytometry.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4410-4410
Author(s):  
Yongmin Tang ◽  
Hongqiang Shen ◽  
Hua Song ◽  
Shilong Yang ◽  
Shuwen Shi ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Acute Leukemia ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Disease Recurrence ◽  
Free Survival ◽  
B Lineage ◽  
Childhood Acute Leukemia ◽  
Minimal Residual

Abstract Minimal residual diseases (MRD) are the problem of disease recurrence for childhood acute leukemia. Different levels of MRD determined with multi-parameter flow cytometry (MP-FCM) have been revealed prognostic for both adult and childhood acute leukemia in several recent studies. However, the MRD levels at the time of complete remission and the various check point during further treatment have not been reported. Methods: 122 cases (Male 73, Female 49, aged 2 month to 15yrs with a median of 6.5yrs) with childhood acute leukemia treated in our hospital from 2001.3 ~ 2004.7 were enrolled into this study. A total of 610 BM samples were detected with three or four color MP-FCM based on individualized leukemia-associated aberrant immunophenotypes (LAIP) in each patient according to their initial complete immunophenotypic profiles (B lineage ALL: 79 cases, T lineage ALL: 11 cases; AML: 32 cases). A total of 99 LAIPs (B lineage ALL: 48, T lineage ALL: 16, AML: 31, HAL: 4) were identified and single LAIP was used in most patients, but some patients needed more than one (2 or 3) LAIPs to detect their MRDs. At least 100,000 events were collected for each analysis. ALL and AML patients were treated with Chinese Childhood Cancer Study Group (CCCSG) ALL-protocol-1998 and AML-protocol-1998, respectively. Results: The CR rates for B lineage ALL, T lineage ALL and AML were 99.3%, 94.7% (overall ALL 98.8%) and 89.1%, respectively. The 1 yr/2 yrs of event free survival (EFS) for the three types of leukemia was 92.5%/85.2%, 78.3%/68.9% and 68.3%/56.4%, respectively. At the time of first CR after induction therapy, MRD level of B lineage ALL (79 cases) was 0.166%±0.057% (mean ± SE), significantly lower than those of T lineage ALL (11 cases, 0.783%±0.328%, t = 3.1926, P = 0.002) and AML (32 cases, 1.191%±0.257, t = 5.5177, P &lt; 0.00001), respectively. No significance was identified between the MRD levels of T lineage ALL and AML (t = 0.8488, P = 0.40). The MRD level of the overall ALL (T + B lineage: 90 cases) was also significantly lower than that of AML (32 cases) (t = 5.0249, P &lt; 0.00001). With further detection of MRD following the continuous consolidation and maintenance therapy, some patients relapsed and were excluded the study, and some had not reached the time for detection. At the time of 6 months after first CR, the MRD level of B lineage ALL (53 cases, 0.224%±0.065%) was not statistically different from that of T lineage ALL (7 cases, 0.627% ± 0.276%, t = 1.767, P = 0.083), while it was significantly higher than that of AML (16 cases, 0.966%±0.347%, t =3.334, P = 0.0014). At the time of 1 yr after CR, the MRD level in ALL (50 cases, 0.347%±0.090%) was not significantly different from that of AML (7 cases, 0.094%±0.072, t = 1.043, P = 0.31). At the time of 2 yrs after CR, the MRD level in ALL (49 cases, 0.302%±0.105%) was also not significantly different from that of AML (8 cases, 0.485%±0.246%, t = 0.6599, P = 0.51). Conclusions: MRD level of B lineage ALL is significantly lower than those of T lineage ALL and AML at the time of first CR, being true at the time of 6 months after CR. The differences are lost at 12 and 24 months after CR indicating that MRD detection with MP-FCM are well associated with the prognosis of childhood acute leukemia. Continuous follow-up to accumulate more cases is now underway.

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