Four-color flow cytometry bypasses limitations of IG/TCR polymerase chain reaction for minimal residual disease detection in certain subsets of children with acute lymphoblastic leukemia.

International audienceBACKGROUND AND OBJECTIVES: Competitive immunoglobulin/T-cell receptor polymerase-chain reaction (PCR) analysis with fluorescent detection is a rapid, cheap and reproducible method for quantifying minimal residual disease (MRD), which is well adapted to the recognition of high-risk childhood acute lymphoblastic leukemia (ALL). We aimed at defining whether flow cytometry (FC) techniques can bypass limitations of PCR for MRD determination. DESIGN AND METHODS: We analyzed 140 remission samples from 91 patients using both competitive PCR amplification of antigen-receptor genes and four-color FC identification of leukemia immunophenotype. These methods were chosen with the aim of detecting at least 0.1% blasts. RESULTS: MRD was measured using both PCR and FC methods in 123 samples and the two methods provided concordant results in 119 of them (97%). Moreover, three out of the four discordant results appeared minor since MRD was detectable by both methods, but at different levels. In 12 of 13 samples from nine patients, mainly infants with early CD10- and/or t(4;11) B-cell ALL and children with immature T-cell ALL, MRD could be determined using FC whereas PCR failed. Conversely, FC methods were unfeasible due to inappropriate leukemia immunophenotype in three additional children (including two with T-cell ALL) for whom PCR successfully provided MRD results. INTERPRETATION AND CONCLUSIONS: The MRD results provided by FC techniques were highly concordant with those of competitive PCR. Moreover, the applicability of FC appeared higher in certain ALL subsets, although the appropriateness of this technique in terms of outcome prediction remains to be demonstrated

Detection of t(11;14) using interphase molecular cytogenetics in mantle cell lymphoma and atypical chronic lymphocytic leukemia

The chromosomal translocation t(11;14)(q13;q32) fuses the IGH and CCND1 genes and leads to cyclin D1 overexpression. This genetic abnormality is the hallmark of mantle cell lymphoma (MCL), but is also found in some cases of atypical chronic lymphocytic leukemia (CLL), characterized by a poor outcome. For an unequivocal assessment of this specific chromosomal rearrangement on interphase cells, we developed a set of probes for fluorescence in situ hybridization (FISH). Northern blotting was performed for analysis of the cyclin D1 expression in 18 patients. Thirty-eight patients, with either a typical MCL leukemic phase (17 patients) or atypical CLL with an MCL-type immunophenotype, i.e., CD19+, CD5+, CD23(-/low), CD79b/sIgM(D)++, and FMC7+ (21 patients), were analyzed by dual-color interphase FISH. We selected an IGH-specific BAC probe (covering the JH and first constant regions) and a commercially available CCND1 probe. An IGH-CCND1 fusion was detected in 28 of the 38 patients (17 typical MCL and 11 cases with CLL). Cyclin D1 was not overexpressed in two patients with typical MCL and an IGH- CCND1 fusion. In view of the poor prognosis associated with MCL and t(11;14)- positive CLL, we conclude that this set of probes is a valuable and reliable tool for a rapid diagnosis of these entities

Etude du polynucleaire neutrophile humain : distribution des recepteurs membranaires et recherche d'une implication de l'AMPc dans la regulation des phenomenes phagocytaires

SIGLECNRS T 55542 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

The Coexpression of CD11a and CD45bright Is the Hallmark of Proliferating Myeloma Cells.

Abstract To identify new potential therapeutical targets in multiple myeloma (MM), we have defined the phenotype of the subset of proliferative myeloma cells (n=66) in comparison with that of normal PC (n=25). Proliferation was evaluated by ex vivo incorporation of BrdU (labeling index, LI). Surface PC phenotype was performed in a four-color assay with CD38, CD45, CD138 and the mAb indicated. For intracellular BrdU staining, cells were first labeled with CD38, CD45 and CD138, fixed and permeabilized before BdrU staining. At least 1000 normal PC and 10000 myeloma cells were analyzed. We show that all bone marrow PC, either malignant or normal, always included a subset of proliferative PC (BrdU+) that was always located within the CD45++subpopulation. Indeed, CD45++ myeloma cells (median 12%) had a labeling index 7.5-fold higher of that of CD45+/− myeloma cells (7.1% versus 0.94%). Actually, in all cases of MM, CD45++ myeloma cells were always the most proliferative myeloma cells. As observed for myeloma cells, LI of normal PC was heterogeneous i.e., higher in the CD45++ population of PC: CD45++ PC (median 65%) had a LI 5.7-fold higher of that of CD45+/− PC. Compared to myeloma cells, LI of PC were higher in both subsets, of 20.5% and 3.6% for CD45++ and CD45+/−, respectively. Non-malignant PC from blood or tonsil were homogeneously CD45++ and did proliferate (LI&gt; 10% and up to 45% for reactive PC). In all PC (normal, reactive, malignant), we found an inverse correlation between CD45 and Bcl-2, confirming a known inverse correlation between proliferation and Bcl-2 expression. Our data suggest that a minor cycling Bcl2lowCD45++ population of myeloma cells differentiate into a no more cycling major Bcl2high CD45+/− population of myelom a cells that accumulates. We further characterized the phenotype of the CD45++ myeloma cells population: we found that CD11a and to a less extend HLA-DR were expressed by CD45++ myeloma cells only in contrast to CD40 and CXCR4 that were expressed by all myeloma cells. Moreover, all CD45++ myeloma cells coexpressed CD11a. Thus, the-to-be-killed population of myeloma cells could be targeted through CD45 or CD11a.</jats:p

Comparison of the Performance of Surface Alone or Surface Plus Cytoplasmic Approaches for the Assessment of Minimal Residual Disease in Multiparameter Flow Cytometry in Multiple Myeloma

Background. During the follow-up of treated myeloma patients, the assessment of minimal residual disease (MRD) is gaining an increasing importance. The detection of remaining abnormal plasma-cells (PC) may rely on molecular techniques investigating immunoglobulin rearrangements of the malignant clone or on multiparameter flow cytometry (MFC). The latter allows to obtain a rapid response by dealing with fresh cells. It also focuses on cells still alive, since dead cells are discarded as debris. Numerous publications have reported that the most reliable markers of PC in MFC are CD38 and CD138 their co-expression being a good way to select the population of PC in a bone marrow (BM) or, more rarely tested, blood sample. Malignant PC often but not always differ from normal PC by the loss of CD19 expression and the acquisition of CD56. Other immunophenotypic alterations are related, among others, to the expression of CD20, CD27, CD28, CD33, CD45, CD81 or CD117. Malignant PC also display the monotypic usage of light chains by the myelomatous immunoglobulin, which can readily be assessed in MFC after permeabilization of the PC, although this induces an additional technical step that could induce some cell loss. Here we compared the two panels proposed by the Euroflow consortium (Flores Montero, 2018) which use the same backbone of antibodies with a "surface" strategy associating CD81 and CD117 or a "cytoplasmic" strategy investigating for the expression of kappa and lambda immunoglobulin light chains. Methods. From a cohort of patients for whom MRD had been assessed in our MFC platform, 100 samples were retrospectively selected as displaying detectable MRD in the cytoplasmic strategy. All BM samples had first been submitted to bulk lysis to increase the PC concentration. Between 5 and 10x106 nucleated cells were used for surface staining, premeabilization and intracytoplasmic staining. Another aliquot of the same suspension, with 3 to 5x106 nucleated cells, was used for the "surface" tube. Briefly, both samples were surface stained with antibodies to CD45 (Ozyme), CD19 (Beckman Coulter), CD38 (Cytognos), CD138 (BD Biosciences) and CD27 (Ozyme). The "surface tube" also contained antibodies to CD81 (Clinisciences) and CD117 (BD Biosciences). After this incubation, the "cytoplasmic tube" was submitted to permeabilization (Intrastain® Dako) and cells further incubated with antibodies to kappa and lambda chains (Dako and Clinisciences). All samples were acquired on the same day. Listmodes of the "cytoplasmic tubes" were analyzed and data provided to the clinician within 24 hours. For this study, the listmodes of the "surface tube" were analyzed blindly using the Kaluza® software. Data were then compared to those of the "cytoplasmic tube" Results. A good linear correlation was observed between the two results, with a R2 coefficient of 0.73. The global difference between both tubes was usually a lower MRD level detected with the "cytoplasmic tube", seen in 68% of the cases (median -0.0113; range -0.0001 to -1.4). Of note higher levels (0.0007 to 1.44) were observed in 32%, ruling out a systematic loss of cells that could have been responsible for this difference. The gating strategy adopted (Robillard 2013) delineated four populations on a CD19/CD56 bivariate histogram. Monotypy was then investigated in each of the four subsets thus identified. The same strategy was applied for the "surface tube" looking at the coexpression profile of CD81 and CD117 in each subset. Globally, 58% of the samples were CD56 positive among which 43 were CD19-. CD19 was also absent in 40 CD56- samples. All configurations of CD117 and CD81 coexpression were seen, making each patient a challenging case. In about 10% of the cases, two suspect subsets were seen in the "surface tube" while monotypy was seen in only one in the "cytoplasmic tube". Conclusion. Although this study shows a good correlation between the two panels, it was found that a greater confidence could be attributed to the "cytoplasmic tube", where data are comforted by identification of a monotypic population with the same light chain as the monoclonal peak. Moreover, although confirmation of the abnormal subset was required in numerous cases with the "surface tube", the reverse was never observed. Single use of the "cytoplasmic" combination can thus be recommended as a robust method of MRD assessment in multiple myeloma. Disclosures Moreau: AbbVie: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. </jats:sec

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

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. </jats:sec