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 DNA Methylation Adds Prognostic Value to Minimal Residual Disease Status in Pediatric T‐Cell Acute Lymphoblastic Leukemia

2016 ◽  
Vol 63 (7) ◽  
pp. 1185-1192 ◽  
Author(s):  
Magnus Borssén ◽  
Zahra Haider ◽  
Mattias Landfors ◽  
Ulrika Norén‐Nyström ◽  
Kjeld Schmiegelow ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Minimal Residual Disease ◽  
Prognostic Value ◽  
Residual Disease ◽  
Lymphoblastic Leukemia ◽  
Disease Status ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Minimal Residual

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.

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