Improved flow cytometric detection of minimal residual disease in childhood acute lymphoblastic leukemia

Denys, Barbara; Sluijs-Gelling, Alita J. van der; Homburg, Christa; Schoot, C. Ellen van der; Haas, Valérie de; Philippé, Jan; Pieters, Rob; Dongen, Jacques J. M. van; Velden, Vincent H.J. van der

doi:10.1038/leu.2012.231

Cited by 84 publications

(71 citation statements)

References 52 publications

(69 reference statements)

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“…Recent advances in leukemia indicate a concordance with PCR-based MRD data in up to 96% of cases. However, PCRs remain more sensitive at MRD levels below 10 3 leukemic cells in 10 7 blood MNC (34). This result concurs with those we obtained in RMS cells.…”

Section: Discussionsupporting

confidence: 83%

Optimization of rhabdomyosarcoma disseminated disease assessment by flow cytometry

et al. 2014

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Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. Circulating tumor cells in peripheral blood or disseminated to bone marrow, a concept commonly referred to as minimal residual disease (MRD), are thought to be key to the prediction of metastasis and treatment efficacy. To date, two MRD markers, MYOD and MYOGENIN, have been tested; however, MRD detection continues to be challenging mainly owing to the closeness of the detection limit and the discordance of both markers in some samples. Therefore, the addition of a third marker could be useful for more accurate MRD assessment. The PAX3 gene is expressed during embryo development in all myogenic precursor cells in the dermomyotome. As RMS cells are thought to originate from these muscle precursor cells, they are expected to be positive for PAX3. In this study, PAX3 expression was characterized in cancer cell lines and tumors, showing wide expression in RMS. Detection sensitivities by quantitative polymerase chain reaction (qPCR) of the previously proposed markers, MYOD and MYOGENIN, were similar to that of PAX3, thereby indicating the feasibility of its detection. Interestingly, the flow cytometry experiments supported the usefulness of this technique in the quantification of MRD in RMS using PAX3 as a marker. These results indicate that flow cytometry, albeit in some cases slightly less sensitive, can be considered a good approach for MRD assessment in RMS and more consistent than qPCR, especially owing to its greater specificity. Furthermore, fluorescence-activated cell sorting permits the recovery of cells, thereby providing material for further characterization of circulating or disseminated cancer cells.

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Section: Discussionsupporting

confidence: 83%

Optimization of rhabdomyosarcoma disseminated disease assessment by flow cytometry

et al. 2014

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“…10,[28][29][30] To evaluate which other markers could contribute to optimal separation of BCP-ALL cells from normal/ reactive BCP cells, the EuroFlow BCP-ALL diagnosis panel 18 was applied to 69 BCP-ALL patients as well as to normal/reactive BM samples. Based on principal component analysis (visualized through APS plots) 16,17,19 of the BCP-ALL cells vs normal/reactive BCP cells (analyzed per tube), CD9, CD123, CD66c, CD81, CD24, and CD10 appeared to be markers that were most frequently differentially expressed (see supplemental Figure 5).…”

Section: Resultsmentioning

confidence: 99%

“…Initial FCM-MRD data analysis was performed using 2-dimensional dot plots for sequential gating of BCP-ALL cells, comparable to previous studies using 4 to 6 color stainings. 10,12 For this study, we provisionally defined a minimum of 10 clustered events to consider a sample as MRD positive (lower limit of detection, LOD) and a minimum of 40 clustered events for accurate quantitation of the MRD level (lower limit of quantitation, LLOQ). 21 Interlaboratory variability in data analysis was evaluated as described in the supplemental Methods and supplemental Figure 4.…”

Section: Immunophenotyping Mrd Analysesmentioning

confidence: 99%

“…[1][2][3] Although flow cytometry (FCM) can be used for MRD detection as well, 4-9 studies so far indicate that the specificity and sensitivity of FCM-MRD diagnostics are inferior to PCR-based MRD diagnostics. [10][11][12][13] Nevertheless, we and others have recently shown that the use of 6-or 7-color immunostainings combined with the introduction of new markers and new marker combinations significantly improved FCM-MRD analysis in BCP-ALL patients. 10,12 These improvements were particularly related to specificity, whereas the sensitivity still appeared to be lower than for the PCR-based methods.…”

Section: Introductionmentioning

confidence: 99%

“…[10][11][12][13] Nevertheless, we and others have recently shown that the use of 6-or 7-color immunostainings combined with the introduction of new markers and new marker combinations significantly improved FCM-MRD analysis in BCP-ALL patients. 10,12 These improvements were particularly related to specificity, whereas the sensitivity still appeared to be lower than for the PCR-based methods. To further improve FCM-based MRD diagnostics, more objective and efficient discrimination of BCP-ALL cells from normal BCP cells and improved sample preparation procedures for acquisition of larger numbers of cells are a prerequisite.…”

Section: Introductionmentioning

confidence: 99%

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Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia

Theunissen

Mejstříková

Sędek

et al. 2017

Blood

338

296

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Key Points• Standardized flow cytometry allows highly sensitive MRD measurements in virtually all BCP-ALL patients.• If sufficient cells are measured (.4 million), flow cytometric MRD analysis is at least as sensitive as current PCR-based MRD methods.A fully-standardized EuroFlow 8-color antibody panel and laboratory procedure was stepwise designed to measure minimal residual disease (MRD) in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) patients with a sensitivity of £10 25, comparable to real-time quantitative polymerase chain reaction (RQ-PCR)-based MRD detection via antigen-receptor rearrangements. Leukocyte markers and the corresponding antibodies and fluorochromes were selected based on their contribution in separating BCP-ALL cells from normal/regenerating BCP cells in multidimensional principal component analyses. After 5 multicenter design-test-evaluate-redesign phases with a total of 319 BCP-ALL patients at diagnosis, two 8-color antibody tubes were selected, which allowed separation between normal and malignant BCP cells in 99% of studied patients. These 2 tubes were tested with a new erythrocyte bulk-lysis protocol allowing acquisition of high cell numbers in 377 bone marrow follow-up samples of 178 BCP-ALL patients.Comparison with RQ-PCR-based MRD data showed a clear positive relation between the percentage concordant cases and the number of cells acquired. For those samples with >4 million cells acquired, concordant results were obtained in 93% of samples. Most discordances were clarified upon high-throughput sequencing of antigen-receptor rearrangements and blind multicenter reanalysis of flow cytometric data, resulting in an unprecedented concordance of 98% (97% for samples with MRD < 0.01%). In conclusion, the fully standardized EuroFlow BCP-ALL MRD strategy is applicable in >98% of patients with sensitivities at least similar to RQ-PCR (£10 25 ), if sufficient cells (>4 3 10 6 , preferably more) are evaluated. (Blood. 2017;129(3):347-357)

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Method for DNA Ploidy Analysis Along with Immunophenotyping for Rare Populations in a Sample using FxCycle Violet

et al. 2017

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The clinical use of flow cytometric DNA ploidy assay has been extended towards stratifying the risk of diseases, such as monoclonal gammopathies or B cell acute lymphoblastic leukemia, and to detect circulating tumor cells, both of which require detection of minute cell populations. This unit describes a protocol for determining DNA ploidy in fixed samples with simultaneous surface immunophenotyping. It is an easy method for simultaneous 6- to 8-color immunophenotyping and DNA content analysis using FxCycle Violet (FCV; DAPI) dye. This protocol is a one-step modification of routine multicolor immunophenotyping that includes surface staining followed by fixation and then DNA staining with FCV. It utilizes mature lymphocytes from the sample as an internal control for determination of DNA index. It is a sensitive method that allows DNA-ploidy determination and cell cycle analysis in a rare tumor population as low as 100 events, as well as DNA ploidy determination in various subsets of hematopoietic cells in the same sample based on their immunophenotype. © 2017 by John Wiley & Sons, Inc.

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Improved flow cytometric detection of minimal residual disease in childhood acute lymphoblastic leukemia

Cited by 84 publications

References 52 publications

Optimization of rhabdomyosarcoma disseminated disease assessment by flow cytometry

Optimization of rhabdomyosarcoma disseminated disease assessment by flow cytometry

Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia

Method for DNA Ploidy Analysis Along with Immunophenotyping for Rare Populations in a Sample using FxCycle Violet

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