Principles of Minimal Residual Disease Detection for Hematopoietic Neoplasms by Flow Cytometry

Transcription

1 Cytometry Part B (Clinical Cytometry) 90B:47 53 (2016) Review Principles of Minimal Residual Disease Detection for Hematopoietic Neoplasms by Flow Cytometry Brent L. Wood 1,2 * 1 Department of Laboratory Medicine and Pathology, University of Washington, Seattle, Washington 2 Seattle Cancer Care Alliance, Seattle, Washington Flow cytometry has become an indispensible tool for the diagnosis and classification of hematopoietic neoplasms. The ability to rapidly distinguish cellular subpopulations via multiparametric assessment of quantitative differences in antigen expression on single cells and enumerate the relative sizes of the resulting subpopulations is a key feature of the technology. More recently, these capabilities have been expanded to include the identification and enumeration of rare subpopulations within complex cellular mixtures, for example, blood or bone marrow, leading to the application for post-therapeutic monitoring or minimal residual disease detection. This review will briefly present the principles to be considered in the construction and use of flow cytometric assays for minimal residual disease detection including the use of informative antibody combinations, the impact of immunophenotypic instability, enumeration, assay sensitivity, and reproducibility. VC 2015 International Clinical Cytometry Society Key terms: cytometry; leukemia; residual; minimal; monitoring How to cite this article: Wood BL. Principles of Minimal Residual Disease Detection for Hematopoietic Neoplasms by Flow Cytometry. Cytometry Part B 2016; 90B: Over the past two decades, flow cytometry has assumed a central role in the diagnosis and classification of hematopoietic neoplasms (1 3). Cellular subpopulations may be rapidly discriminated via multiparametric assessment of quantitative differences in antigen expression on single cells and the relative size of the subpopulations enumerated, a key feature of the technology. The capability of identifying and enumerating rare subpopulations within complex cellular mixtures, for example, blood or bone marrow, has led to the application in post-therapeutic monitoring or minimal residual disease (MRD) detection (4). The intent of MRD detection is to assess the response of patients to therapeutic interventions. This may include the identification of patients with good response to reduce therapy and avoid therapeutic side effects or toxicities, as well as patients with poor response to allow changes in therapeutic intensity or regimen to improve response. Most MRD assessment for hematopoietic neoplasms to date has focused on early time points after therapy in acute leukemia (5 13), particularly acute lymphoblastic leukemia (ALL), where therapeutic intervention can be altered to impact the induction of remission. The assessment of response at time points further from therapy has been of greater interest in more indolent neoplasms such as chronic lymphocytic leukemia (14) or multiple myeloma (15,16), where the durability of response is more relevant. This review will briefly present the principles to be considered in the construction and use of flow cytometric assays for the detection of MRD. An example of protocols for the assessment of MRD in acute leukemia may be found Ref. [17). SPECIMEN HANDLING AND PROCESSING The principal intent of specimen handling, processing, and acquisition is to produce data for interpretation that has high informational content (signal), is as free of artifact (noise) as possible, and is of consistent quality. Minimizing sample manipulation and standardizing Correspondence to: Brent L. Wood, Department of Laboratory Medicine and Pathology, University of Washington, Hematopathology Laboratory, Room G7-800, 825 Eastlake Ave. E, Seattle, WA woodbl@u.washington.edu Received 19 January 2015; Revised 9 March 2015; Accepted 18 March 2015 Published online 29 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: /cyto.b VC 2015 International Clinical Cytometry Society

2 48 WOOD FIG. 1. The detection of MRD for B-ALL. The post-therapy bone marrow aspirate was assayed with an informative antibody combination containing antibodies directed against CD45, CD19, CD20, CD34, CD38, CD58, and CD10. This combination allows the recognition of the relevant normal stages of immature B-cell maturation (cyan) and mature B cells (blue). In addition, it allows the identification of an abnormal immature B-cell population (red) that represents MRD characterized by abnormal expression of CD10 (increased), CD58 (increased), CD45 (absent), and CD34 (slightly decreased). The population is enumerated at 0.1% of white cells.[color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] procedures for sample processing, reagent evaluation, and instrument performance best achieve these goals (18,19). The use of quality control and quality assurance programs promotes consistency. Peripheral blood and bone marrow aspirates yield the best results when anticoagulated with EDTA or heparin either with or without the addition of tissue culture media, transported at room temperature to the lab, and assayed within h of collection. Antibodies, titered and preferentially precocktailed, are incubated with aliquots of whole blood or marrow followed by erythrocyte lysis using a fixative-containing lysing reagent, for example, buffered NH 4 Cl containing 0.25% of formaldehyde, washed once, and acquired on the cytometer. Centrifugation steps should be optimized for recovery. Samples containing fewer cells than desired may be concentrated by prelysing a larger volume using buffered NH 4 Cl without fixative followed by centrifugation although preferential loss of certain leukemic populations may occur, particularly if already compromised by specimen age or degeneration. Preparation and pooling of multiple aliquots of sample is another approach to dealing with the samples of marginal cellularity. Sample acquisition on the cytometer is essentially similar to other applications, albeit often with longer acquisition times owing to the larger number of cells acquired to facilitate the detection of rare subpopulations (see below). However, particular attention should be paid to carryover between the aliquots of samples that are acquired. To minimize carryover, an aliquot of sheath fluid can be run between each sample aliquot until background returns to baseline, ideally zero. POPULATION IDENTIFICATION All immunophenotyping assays for the detection of disease, including MRD assays, rely on the recognition of features on cells in the sample that differ from those seen on normal cells of similar lineage and maturational stage. These differences can take a variety of forms including: alterations in the level of expression of antigens normally expressed by cells of similar type, abnormalities in the timing of expression within a maturational sequence, the appearance of antigens not normally expressed by cells of that type, or changes in the range of expression at a particular maturational stage. In contrast to the known variability in antigen expression within individual disease categories, normal populations of cells demonstrate consistent patterns of antigen expression regardless of patient age, sex, ethnicity, and so forth. This implies that what is most important for the recognition of disease is a detailed knowledge of normal maturation for cells of the relevant lineage and maturational stage, the details of which are beyond the scope of this review (20,21). The recognition of differences in antigen expression between disease and normal requires use of an informative antibody combination, that is, one that is able to demonstrate abnormality for the patient s disease (Fig. 1). Such antibody combinations invariably include reagents against antigens that are able to define the

3 PRINCIPLES OF MRD 49 FIG. 2. Immunophenotypic instability after therapy in leukemia-all. The diagnostic immunophenotype (red) for a case of B-ALL is compared with MRD detected at day 29 after induction therapy (blue). Normal mature B cells are also present with bright CD20 and the absence of CD10 (green). In comparison to diagnosis, the residual leukemic population shows decreased expression of CD10 and CD34 with increased expression of CD20 and CD45, a change in immunophenotype suggesting progressive maturation secondary to steroid therapy. The use of a predefined gate based on the immunophenotype seen at diagnosis is likely to entirely miss the MRD immunophenotype seen at the end of induction.[color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] relevant cell lineage and maturational stage. In addition, they include reagents that are known to be commonly abnormally expressed for the disease type of interest (22,23). In contrast to diagnostic immunophenotyping, reagent combinations informative for MRD assays require a higher signal-to-noise ratio to allow complete separation of abnormal populations of low frequency from normal cells of similar immunophenotype. One consequence is that diagnostic reagent panels are often not optimized or suitable for the detection of MRD. IMMUNOPHENOTYPIC STABILITY Immunophenotypic differences from normal may be termed the leukemia-associated immunophenotype (LAIP) and provide a description of immunophenotypic abnormality for a given sample. Using this information, it is tempting to use predefined gates in the areas devoid of normal cells at the time of diagnosis to assess for MRD after therapy, and a number of published studies use this LAIP approach for the detection of MRD (6,7,23,24). However, this approach assumes the stability of immunophenotype after therapy and it is now well documented that immunophenotypes commonly change after therapy for B-lymphoblastic leukemia (B- ALL), T-lymphoblastic leukemia (T-ALL) (25), and acute myeloid leukemia (AML) (26 28). In the case of B-ALL, the use of steroids during induction therapy appears to induce maturation such that immature antigens are reduced in intensity and mature antigens increased (Fig. 2) (29 31). The steroid effect also appears to be a likely explanation for immunophenotypic change in T- ALL. Such immunophenotypic change is perhaps not surprising for neoplasms composed of immature cells that likely retain some ability to differentiate under appropriate conditions. Neoplasms of more mature cell types would seem less likely to exhibit this behavior and this appears to be the case in chronic lymphocytic leukemia and multiple myeloma where post-therapy immunophenotypes appear more stable. The net result is that while immunophenotypic differences identified at diagnosis may serve as a useful starting point for the assessment of MRD, they should not be expected to persist after therapy, particularly for acute leukemia, and a broader evaluation for discrete populations different from normal should be routinely performed. Although the difference from normal approach is often contrasted with the use of LAIP, this is a false dichotomy as the latter is really a simplified subset of the former that in it strictest implementation does not take post-therapeutic changes in immunophenotype into account. The most robust approach to the detection of MRD incorporates difference from normal both at diagnosis (LAIP) and after therapy. In settings where the pretreatment immunophenotype is not known, difference from normal without knowledge of the diagnostic LAIP is the only option and MRD detection can still be successfully performed although it is not yet clear whether this approach results in a consistent preservation of assay sensitivity. A particularly problematic form of antigenic instability is the loss of an antigen as a result of targeted immunotherapy directed against the antigen. One well-described example is the loss of CD19 in B-lineage ALL treated with anti-cd19 chimeric antigen receptor-modified T cells (32). This immunophenotypic change both provides a mechanism for leukemic escape and ultimate relapse, as well as significantly compromising standard flow cytometric assays that almost uniformly have relied on CD19 to identify B-cell populations for further analysis. In these situations, novel approaches are needed for the detection of MRD. QUANTITATION The number of abnormal events necessary for the recognition of residual disease is ill-defined as it depends on the level and quality of background noise and the quality of the abnormal events including their distribution. The experience of others and ourselves suggests that frequently 10 events or less is sufficient for the recognition of MRD in a well-controlled assay within a

4 50 WOOD single laboratory. The determination of whether this level of performance is achievable between laboratories remains to be determined, but one study evaluating this issue in chronic lymphocytic leukemia suggests that 50 abnormal events may be required for reproducible MRD assessment using that assay in multi-institutional clinical trials (14). The number of events required for reproducible enumeration is a related but different issue governed largely by Poisson counting statistics when small numbers of events are involved. From Poisson counting statistics, the degree of variability for enumeration expressed as the p ffiffiffiffi coefficient of variation (CV) may be estimated as N /N where N is the number of events in the population. Thus, for a population identified consisting of 10 events, the CV for enumeration is estimated to be 31.7%. If a CV of 10% is desired, 100 events are required, and this is generally considered acceptable reproducibility for assays of this type. The total number of events to be acquired on the cytometer for a given assay can be determined if the lower limit of quantitation desired and the degree of reproducibility considered acceptable are known. For example, in the detection of B-ALL MRD, the current threshold for clinical decision making is 0.01% and provided a CV for enumeration of 10% is desired, the total number of events to be acquired would be 1,000,000 events. The collection of fewer events is unlikely to compromise the identification of MRD unless about 10- fold fewer events are collected, assuming recognition is possible with 10 events, but the variability of the enumeration will be progressively increased as fewer events are acquired. It should also be emphasized that the only events that are relevant are those that are included in the denominator for enumeration, and hence noise in the form of platelets, red cell fragments, tissue debris, or non-nucleated cells depending on the relevant specimen type and assay may artificially elevate the apparent number of events and reduce assay sensitivity unless more total events are acquired. Consequently, it is desirable to monitor the number of denominator events during acquisition to ensure that the adequate total events are acquired. The denominator used for enumeration is poorly standardized between assays, even for a particular disease such as B-ALL. For instance, the ALL MRD assays used by the Children s Oncology Group has used a denominator of nucleated mononuclear cells to facilitate comparison with prior data collected using Ficolled cells and to minimize the impact of preferential neutrophil degeneration with sample age. This differs from ALL MRD collected by the BFM group where total nucleated cells are used as the denominator (7). Historically, clinical flow cytometric assays have also used total white cells or nonerythroid cells, for example, CD451 events, as a denominator for most assays. This is largely owing to the concerns about loss of nucleated erythroid cells in marrow specimens when red cell lysing reagents are used. More careful recent evaluation suggests that this concern is exaggerated and although nucleated red cells are compromised by erythrocyte lysing reagents and lose forward scatter, they are retained with the use of fixative-containing erythroid lysing reagents and can be accurately enumerated, provided the forward scatter is appropriately adjusted to collect low forward scatter events smaller than mature lymphocytes by either increasing the forward scatter voltage or lowering the threshold or discriminator (33,34). The presence of nonnucleated cells or debris, for example, platelets or red cell fragments, can also artificially expand the denominator, something easily corrected by the use of nucleic acid binding dyes, for example, Syto-16. Consequently, in the future the best denominator around which to standardize is likely to be total nucleated cells and may require the consistent use of a denominator tube containing a DNA-binding dye. Sample quality can have a significant impact on accurate MRD quantitation either through degeneration or hemodilution. Degenerative changes as a result of advanced sample age or improper collection or storage can result in preferential loss of cell populations, either background populations such as neutrophils or the neoplastic population of interest. In either case, quantitation is adversely affected and the impact can be quite variable between samples. There is no clear consensus on the use of viability data to exclude the samples from evaluation. Hemodilution during sample collection is another common and inevitable problem for bone marrow samples, principally when there are differences in the cellular composition and/or cell concentration between the peripheral blood and the marrow. The typical impact of hemodilution on MRD quantitation is an underestimation of MRD as hematopoietic neoplasms are often at higher numbers in marrow and generally cleared from blood more effectively than marrow by therapy, and hence the resulting marrow sample is variably diluted with proportionally more non-neoplastic cells. This problem can be minimized by adoption of good marrow collection technique where only 1 2 ml of marrow is aspirated during the first pull; however, these details are often out of the control of the laboratory evaluating the sample. The methods to estimate the degree of or normalize for hemodilution have been described, but are either not practical or not applicable to all situations (35). An inability to accurately define or identify the relevant neoplastic population can significantly impact the quantitation of MRD. This is particularly a problem for myeloid stem cell disorders where the neoplastic population in most cases originates from a relatively primitive cell that retains the ability to differentiate to a variety of more mature progeny, for example, myeloproliferative neoplasms or myelodysplastic syndromes, and where there may be significant overlap between normal and neoplastic immunophenotypes. Even in acute myeloid leukemia, it is now clear that apparently normal mature hematopoietic cells are frequently derived from the leukemic progenitor. Although it is likely that the ability to

5 PRINCIPLES OF MRD 51 FIG. 3. The effect of background populations on MRD sensitivity. The single most informative two-parameter antibody combinations are displayed (left) for two cases of B-ALL at diagnosis (red) and overlain with normal immature B cells (cyan). The antibody combination is more informative for the lower case in comparison to the upper case owing to the marked downregulation of CD38 in the former. The same data are displayed with fewer events in the leukemic B-cell populations to emulate MRD (center and right). When only small numbers of normal B cells precursors are also present (center), MRD can be confidently identified for both cases. However, in the presence of large numbers of normal B-cell precursors (right) MRD cannot be confidently identified for the first case (upper right) as the outliers of the normal distribution overlap with the leukemic events, whereas in the second case (lower right) MRD is still easily identified. The sensitivity of the MRD assay for the first leukemia (upper) is limited by the number of normal B-cell precursors in the sample. In contrast, the sensitivity of the MRD assay for the second leukemia (lower) is limited only by the number of events acquired.[color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] regenerate and propagate neoplasia is restricted to the more immature component of the neoplasm, it is less clear exactly which component(s) along the maturational continuum should be quantitated, and hence the most immature component of the neoplasm, often expressing CD34 or CD117, is commonly used as a surrogate for the estimation of MRD. This results in variation in quantitation and is probably one reason for the lack of consensus on the significance of MRD quantitation in clinical decision making for acute myeloid leukemia. This is less of an issue for relatively mature neoplasms such as lymphoma, chronic lymphocytic leukemia, plasma cell neoplasms, and even ALL where the neoplastic population has a relatively more restricted maturational range and can be more discretely identified and quantitated. SENSITIVITY The sensitivity of an MRD assay is a direct result of interplay between the degree of immunophenotypic abnormality of the MRD population (signal) and the number of cells present of similar type (noise). For markedly abnormal populations, an assay can be made arbitrarily sensitive by controlling the total number of events acquired, that is, is independent of other populations in the sample. However, for most MRD assays, the abnormal populations are at best moderately abnormal and the character and number of background populations are what limit sensitivity. This further emphasizes the importance of using highly informative antibody combinations if assays with consistently high sensitivity are desired. A corollary is that assay sensitivity is not constant, but often varies depending on the presence of populations of similar immunophenotype. For example, in B-ALL after induction therapy, the number of normal B-cell precursors (hematogones) is markedly reduced to absent and assay sensitivity is as high as 0.001%, whereas after consolidation therapy the number of normal B-cell precursors may be markedly increased as part of marrow regeneration, particularly in children, limiting sensitivity to 0.1% or lower depending on the leukemic immunophenotype in question (Fig. 3). At the present time, sensitivities of 0.01% can be routinely achieved for the very large majority of B-ALL and T-ALL at the end of

6 52 WOOD induction therapy, 0.1% of AML with a significant subset having higher sensitivity, and 0.001% or higher for chronic lymphocytic leukemia and multiple myeloma. REPRODUCIBILITY Reproducibility remains a major concern with the use of flow cytometry for the assessment of MRD, particularly between institutions in both clinical trial and routine clinical settings. To a large degree, this concern arises owing to an essential lack of standardization of assays between the laboratories and the somewhat subjective interpretive component inherent to flow cytometric assays of this type. In principle, the use of a standardized protocol should greatly improve reproducibility and the limited data published to date in CLL (14), myeloma (36) and B-ALL (37,38)) suggest that this is true with the most difficult component to standardize being the human analysis and interpretation of flow cytometric data. The determination of whether harmonized assays, that is, assays standardized around a common set of objectives rather than a common analytical protocol, can provide a similar degree of reproducibility is a matter of debate. Our own experience in the Children s Oncology Group using a largely standardized assay for B-ALL MRD detection in two expert laboratories demonstrates a very high degree of concordance for exchanged samples, frequencies of MRD detected on very large patient cohorts, and identical clinical outcome data (manuscript submitted). These data suggest that standardization of flow cytometric MRD assays is entirely possible and is not an inherent limitation of the technology, rather it is the lack of consensus and commitment on the part of its practitioners that is the major roadblock to progress in this area. The Foundation of the National Institute of Health (FNIH) has recently launched an initiative to standardize B-ALL flow cytometric MRD detection among the US cooperative oncology group laboratories and a limited number of other laboratories with the intent of promulgating a standardized assay for use in both clinical trial and clinical settings throughout North America, and similar efforts are underway in Europe. FUTURE The future of flow cytometric MRD detection is uncertain. Although the technology has clear advantages in comparison to molecular PCR-based assays in terms of speed, applicability and cost, the poor standardization to date has made it difficult to convince sponsors and regulators that consistent results can be obtained outside of a few expert laboratories. In addition, the advent of next-generation sequencing assays that overcome some of the technical limitations of PCR-based MRD assays and that are capable of both easier standardization and higher sensitivity suggest that this may be a preferred technology in the future for the assessment of MRD (39 42), a case in point being the successful use of rearranged immunoglobulin heavy-chain sequences as a clonal MRD marker for patients with myeloma (43). LITERATURE CITED 1. Swerdlow SH. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues.Lyon, France: IARC Press; Cherian S, Wood BL. Flow Cytometry in Evaluation of Hematopoietic Neoplasms: A Case Based Approach. CAP Press: Northfield, IL; Craig FE, Foon KA. Flow cytometric immunophenotyping for hematologic neoplasms. Blood 2008;111: Campana D, Coustan-Smith E. Detection of minimal residual disease in acute leukemia by flow cytometry. Cytometry 1999;38: Borowitz MJ, Devidas M, Hunger SP, Bowman WP, Carroll AJ, Carroll WL, Linda S, Martin PL, Pullen DJ, Viswanatha D, et al. Clinical significance of minimal residual disease in childhood acute lymphoblastic leukemia and its relationship to other prognostic factors: A children s oncology group study. Blood 2008;111: Stow P, Key L, Chen X, Pan Q, Neale GA, Coustan-Smith E, Mullighan CG, Zhou Y, Pui CH, Campana D. Clinical significance of low levels of minimal residual disease at the end of remission induction therapy in childhood acute lymphoblastic leukemia. Blood 2010;115: Gaipa G, Basso G, Biondi A, Campana D. Detection of minimal residual disease in pediatric acute lymphoblastic leukemia. Cytometry B Clin Cytom 2013;84B: Schrappe M, Valsecchi MG, Bartram CR, Schrauder A, Panzer- Grumayer R, Moricke A, Parasole R, Zimmermann M, Dworzak M, Buldini B, et al. Late MRD response determines relapse risk overall and in subsets of childhood T-cell ALL: Results of the AIEOP-BFM- ALL 2000 study. Blood 2011;118: Terwijn M, van Putten WL, Kelder A, van der Velden VH, Brooimans RA, Pabst T, Maertens J, Boeckx N, de Greef GE, Valk PJ, et al. High prognostic impact of flow cytometric minimal residual disease detection in acute myeloid leukemia: Data from the HOVON/SAKK AML 42A study. J Clin Oncol 2013;31: Walter RB, Buckley SA, Pagel JM, Wood BL, Storer BE, Sandmaier BM, Fang M, Gyurkocza B, Delaney C, Radich JP, et al. Significance of minimal residual disease before myeloablative allogeneic hematopoietic cell transplantation for AML in first and second complete remission. Blood 2013;122: Kern W, Voskova D, Schoch C, Hiddemann W, Schnittger S, Haferlach T. Determination of relapse risk based on assessment of minimal residual disease during complete remission by multiparameter flow cytometry in unselected patients with acute myeloid leukemia. Blood 2004;104: Loken MR, Alonzo TA, Pardo L, Gerbing RB, Raimondi SC, Hirsch BA, Ho PA, Franklin J, Cooper TM, Gamis AS, et al. Residual disease detected by multidimensional flow cytometry signifies high relapse risk in patients with de novo acute myeloid leukemia: A report from children s oncology group. Blood 2012;120: San Miguel JF, Vidriales MB, Lopez-Berges C, Diaz-Mediavilla J, Gutierrez N, Canizo C, Ramos F, Calmuntia MJ, Perez JJ, Gonzalez M, et al. Early immunophenotypical evaluation of minimal residual disease in acute myeloid leukemia identifies different patient risk groups and may contribute to postinduction treatment stratification. Blood 2001;98: Rawstron AC, Bottcher S, Letestu R, Villamor N, Fazi C, Kartsios H, de Tute RM, Shingles J, Ritgen M, Moreno C, et al. Improving efficiency and sensitivity: European Research Initiative in CLL (ERIC) update on the international harmonised approach for flow cytometric residual disease monitoring in CLL. Leukemia 2013;27: Rawstron AC, Child JA, de Tute RM, Davies FE, Gregory WM, Bell SE, Szubert AJ, Navarro-Coy N, Drayson MT, Feyler S, et al. Minimal residual disease assessed by multiparameter flow cytometry in multiple myeloma: Impact on outcome in the medical research council myeloma IX study. J Clin Oncol 2013;31: Paiva B, Vidriales MB, Cervero J, Mateo G, Perez JJ, Montalban MA, Sureda A, Montejano L, Gutierrez NC, Garcia de Coca A, et al. Multiparameter flow cytometric remission is the most relevant prognostic factor for multiple myeloma patients who undergo autologous stem cell transplantation. Blood 2008;112: Wood BL. Flow cytometric monitoring of residual disease in acute leukemia. Methods Mol Biol 2013;999: Clinical and Laboratory Standards Institute. Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline, 2nd ed. CLSI Document H43-A p 27.

7 PRINCIPLES OF MRD Kalina T, Flores-Montero J, van der Velden VH, Martin-Ayuso M, Bottcher S, Ritgen M, Almeida J, Lhermitte L, Asnafi V, Mendonca A, et al. EuroFlow standardization of flow cytometer instrument settings and immunophenotyping protocols. Leukemia 2012;26: Wood BL, Borowitz MJ. The flow cytometric evaluation of hematopoietic neoplasia. In: Clinical Diagnosis and Management by Laboratory Methods (Henry). Philadelphia, PA: W.B. Saunders; Wood B. Multicolor immunophenotyping: Human immune system hematopoiesis. Methods Cell Biol 2004;75: Krampera M, Perbellini O, Vincenzi C, Zampieri F, Pasini A, Scupoli MT, Guarini A, Propris D, Coustan-Smith MSE. Campana D, et al. Methodological approach to minimal residual disease detection by flow cytometry in adult B-lineage acute lymphoblastic leukemia. Haematologica 2006;91: Kern W, Schoch C, Haferlach T, Schnittger S. Monitoring of minimal residual disease in acute myeloid leukemia. Crit Rev Oncol Hematol 2005;56: Feller N, van der Velden VH, Brooimans RA, Boeckx N, Preijers F, Kelder A, de Greef I, Westra G, Te Marvelde JG, Aerts P, et al. Defining consensus leukemia-associated immunophenotypes for detection of minimal residual disease in acute myeloid leukemia in a multicenter setting. Blood Cancer J 2013;3:e Roshal M, Fromm JR, Winter S, Dunsmore K, Wood BL. Immaturity associated antigens are lost during induction for T cell lymphoblastic leukemia: Implications for minimal residual disease detection. Cytometry B Clin Cytom 2010;78B: Langebrake C, Brinkmann I, Teigler-Schlegel A, Creutzig U, Griesinger F, Puhlmann U, Reinhardt D. Immunophenotypic differences between diagnosis and relapse in childhood AML: Implications for MRD monitoring. Cytometry B Clin Cytom 2005;63B: Voskova D, Schoch C, Schnittger S, Hiddemann W, Haferlach T, Kern W. Stability of leukemia-associated aberrant immunophenotypes in patients with acute myeloid leukemia between diagnosis and relapse: Comparison with cytomorphologic, cytogenetic, and molecular genetic findings. Cytometry B Clin Cytom 2004;62B: Baer MR, Stewart CC, Dodge RK, Leget G, Sule N, Mrozek K, Schiffer CA, Powell BL, Kolitz JE, Moore JO, et al. High frequency of immunophenotype changes in acute myeloid leukemia at relapse: Implications for residual disease detection (cancer and leukemia group B study 8361). Blood 2001;97: Dworzak MN, Gaipa G, Schumich A, Maglia O, Ratei R, Veltroni M, Husak Z, Basso G, Karawajew L, Gadner H, et al. Modulation of antigen expression in B-cell precursor acute lymphoblastic leukemia during induction therapy is partly transient: Evidence for a druginduced regulatory phenomenon. Results of the AIEOP-BFM-ALL- FLOW-MRD-study group. Cytometry B Clin Cytom 2010;78B: Gaipa G, Basso G, Aliprandi S, Migliavacca M, Vallinoto C, Maglia O, Faini A, Veltroni M, Husak D, Schumich A, et al. Prednisone induces immunophenotypic modulation of cd10 and cd34 in nonapoptotic B-cell precursor acute lymphoblastic leukemia cells. Cytometry B Clin Cytom 2008;74B: Gaipa G, Basso G, Maglia O, Leoni V, Faini A, Cazzaniga G, Bugarin C, Veltroni M, Michelotto B., Ratei R, et al. Drug-induced immunophenotypic modulation in childhood ALL: Implications for minimal residual disease detection. Leukemia 2005;19: Grupp SA, Kalos M, Barrett D, Aplenc R, Porter DL, Rheingold SR, Teachey DT, Chew A, Hauck B, Wright JF, et al. Chimeric antigen receptor-modified T cells for acute lymphoid leukemia. N Engl J Med 2013;368: Sandes AF, Kerbauy DM, Matarraz S, Chauffaille Mde L, Lopez A, Orfao A, Yamamoto M. Combined flow cytometric assessment of cd45, HLA-DR, cd34, and cd117 expression is a useful approach for reliable quantification of blast cells in myelodysplastic syndromes. Cytometry B Clin Cytom 2013;84B: Wangen JR, Eidenschink Brodersen L, Stolk TT, Wells DA, Loken MR. Assessment of normal erythropoiesis by flow cytometry: Important considerations for specimen preparation. Int J Lab Hematol 2014;36: Loken MR, Chu SC, Fritschle W, Kalnoski M, Wells DA. Normalization of bone marrow aspirates for hemodilution in flow cytometric analyses. Cytometry B Clin Cytom 2009;76B: Mathis S, Chapuis N, Borgeot J, Maynadie M, Fontenay M, Bene MC, Guy J, Bardet V. Comparison of cross-platform flow cytometry minimal residual disease evaluation in multiple myeloma using a common antibody combination and analysis strategy. Cytometry B Clin Cytom 2015;88B: Bjorklund E, Matinlauri I, Tierens A, Axelsson S, Forestier E, Jacobsson S, Ahlberg AJ, Kauric G, Mantymaa P, Osnes L, et al. Quality control of flow cytometry data analysis for evaluation of minimal residual disease in bone marrow from acute leukemia patients during treatment. J Pediatr Hematol Oncol 2009;31: Dworzak MN, Gaipa G, Ratei R, Veltroni M, Schumich A, Maglia O, Karawajew L, Benetello A, Potschger U, Husak Z, et al. Standardization of flow cytometric minimal residual disease evaluation in acute lymphoblastic leukemia: Multicentric assessment is feasible. Cytometry B Clin Cytom 2008;74B: Faham M, Zheng J, Moorhead M, Carlton VE, Stow P, Coustan-Smith E, Pui CH, Campana D. Deep-sequencing approach for minimal residual disease detection in acute lymphoblastic leukemia. Blood 2012;120: Salipante SJ, Fromm JR, Shendure J, Wood BL, Wu D. Detection of minimal residual disease in NPM1-mutated acute myeloid leukemia by next-generation sequencing. Mod Pathol 2014;27: Wu D, Emerson RO, Sherwood A, Loh ML, Angiolillo A, Howie B, Vogt J, Rieder M, Kirsch I, Carlson C, et al. Detection of minimal residual disease in B lymphoblastic leukemia by high-throughput sequencing of IGH. Clin Cancer Res 2014;20: Wu D, Sherwood A, Fromm JR, Winter SS, Dunsmore KP, Loh ML, Greisman HA, Sabath DE, Wood BL, Robins H. High-throughput sequencing detects minimal residual disease in acute T lymphoblastic leukemia. Sci Transl Med 2012;4:134ra Martinez-Lopez J, Lahuerta JJ, Pepin F, Gonzalez M, Barrio S, Ayala R, Puig N, Montalban MA, Paiva B, Weng L, et al. Prognostic value of deep sequencing method for minimal residual disease detection in multiple myeloma. Blood 2014;123: