Montero, J. (Juan)
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- EuroFlow Lymphoid Screening Tube (LST) data base for automated identification of blood lymphocyte subsets(Elsevier BV, 2019) Böttcher, S. (Sebastian); Muñoz, N. (Noemí); Fluxá, R. (Rafael); Montero, J. (Juan); Grigore, G. (Georgiana); Santos, A.H. (Ana Helena); Fernández, P. (Paula); Orfao, A. (Alberto); Hernandez, J.M. (J. M.); Barrena, S. (Susana); Sedek, L. (Lukasz); Almeida, J. (J.); Hernández, A. (Alejando); Dongen, J.J.M. (Jacques J. M.) van; Paiva, B. (Bruno); Lima, M. (Margarida); Lecrevisse, Q. (Quentin); Velden, V.H.J. (Vicent H. J.) van derIn recent years the volume and complexity of flow cytometry data has increased substantially. This has led to a greater number of identifiable cell populations in a single measurement. Consequently, new gating strategies and new approaches for cell population definition are required. Here we describe how the EuroFlow Lymphoid Screening Tube (LST) reference data base for peripheral blood (PB) samples was designed, constructed and validated for automated gating of the distinct lymphoid (and myeloid) subsets in PB of patients with chronic lymphoproliferative disorders (CLPD). A total of 46 healthy/reactive PB samples which fulfilled predefined technical requirements, were used to construct the LST-PB reference data base. In addition, another set of 92 PB samples (corresponding to 10 healthy subjects, 51 B-cell CLPD and 31 T/NK-cell CLPD patients), were used to validate the automated gating and cell-population labeling tools with the Infinicyt software. An overall high performance of the LST-PB data base was observed with a median percentage of alarmed cellular events of 0.8% in 10 healthy donor samples and of 44.4% in CLPD data files containing 49.8% (range: 1.3–96%) tumor cells. The higher percent of alarmed cellular events in every CLPD sample was due to aberrant phenotypes (75.6% cases) and/or to abnormally increased cell counts (86.6% samples). All 18 (22%) data files that only displayed numerical alterations, corresponded to T/NK-cell CLPD cases which showed a lower incidence of aberrant phenotypes (41%) vs B-cell CLPD cases (100%). Comparison between automated vs expert-bases manual classification of normal (r2 = 0.96) and tumor cell populations (rho = 0.99) showed a high degree of correlation. In summary, our results show that automated gating of cell populations based on the EuroFlow LST-PB data base provides an innovative, reliable and reproducible tool for fast and simplified identification of normal vs pathological B and T/NK lymphocytes in PB of CLPD patients.
- Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study(2021) Fluxá, R. (Rafael); Montero, J. (Juan); Grigore, G. (Georgiana); Buracchi, C. (Chiara); Fernández, P. (Paula); Morf, D. (Daniela); Orfao, A. (Alberto); Nierkens, S. (Stefan); Mejstrikova, E. (Ester); Barrena, S. (Susana); Sedek, L. (Lukasz); Bie, M. (Maaike) de; Lhermitte, L. (Ludovic); Sobral-da-Costa, E. (Elaine); Szczepanski, T. (Tomasz); Barreau, S. (Sylvain); Aanei, C.M. (Carmen Mariana); Burgos, L. (Leire); Brüggemann, M. (Monika); Dongen, J.J.M. (Jacques J. M.) van; Caetano, J. (Joana); Gaipa, G. (Giuseppe); Hernández-Delgado, A. (Alejandro); Sluijs-Gelling, A. (Alita) van der; Lecrevisse, Q. (Quentin); Velden, V.H.J. (Vicent H. J.) van der; Pedreira, C.E. (Carlos E.)Precise classification of acute leukemia (AL) is crucial for adequate treatment. EuroFlow has previously designed an AL orientation tube (ALOT) to guide toward the relevant classification panel and final diagnosis. In this study, we designed and validated an algorithm for automated (database-supported) gating and identification (AGI tool) of cell subsets within samples stained with ALOT. A reference database of normal peripheral blood (PB,n = 41) and bone marrow (BM;n = 45) samples analyzed with the ALOT was constructed, and served as a reference for the AGI tool to automatically identify normal cells. Populations not unequivocally identified as normal cells were labeled as checks and were classified by an expert. Additional normal BM (n = 25) and PB (n = 43) and leukemic samples (n = 109), analyzed in parallel by experts and the AGI tool, were used to evaluate the AGI tool. Analysis of normal PB and BM samples showed low percentages of checks (<3% in PB, <10% in BM), with variations between different laboratories. Manual analysis and AGI analysis of normal and leukemic samples showed high levels of correlation between cell numbers (r(2) > 0.95 for all cell types in PB andr(2) > 0.75 in BM) and resulted in highly concordant classification of leukemic cells by our previously published automated database-guided expert-supervised orientation tool for immunophenotypic diagnosis and classification of acute leukemia (Compass tool).
- Next generation flow for minimally-invasive blood characterization of MGUS and multiple myeloma at diagnosis based on circulating tumor plasma cells (CTPC)(2018) Durie, B. (B.); Montero, J. (Juan); Gonzalez, M. (Marcos); Pontes, R. (R.); Hernández-Martín, J. (J.); Diez-Campelo, M. (M.); García-Mateo, A. (A.); Corral-Mateos, A. (A.); García-Sanz, R. (Ramón); Orfao, A. (Alberto); Vidriales, M.B. (María Belén); Mateos, M.V. (María Victoria); Romero, A. (A.); Puig, N. (Noemí); Millacoy, P. (P.); Blanco, J.F. (J. F.); Garcés-Latre, J.J. (Juan José); Burgos, L. (Leire); García-Sanchez, O. (O.); Palomera, L. (Luis); Rodriguez-Otero, P. (Paula); Ríos-Tamayo, R. (Rafael); Dongen, J.J.M. (Jacques J. M.) van; Blanco, E. (E.); Paiva, B. (Bruno); EuroFlow-Consortium; Sanoja-Flores, L. (L.); Prosper-Cardoso, F. (Felipe); Merino, J. (Juana); San-Miguel, J.F. (Jesús F.); Pérez-Andrés, M. (M.)Here, we investigated for the first time the frequency and number of circulating tumor plasma cells (CTPC) in peripheral blood (PB) of newly diagnosed patients with localized and systemic plasma cell neoplasms (PCN) using next-generation flow cytometry (NGF) and correlated our findings with the distinct diagnostic and prognostic categories of the disease. Overall, 508 samples from 264 newly diagnosed PCN patients, were studied. CTPC were detected in PB of all active multiple myeloma (MM; 100%), and smoldering MM (SMM) patients (100%), and in more than half (59%) monoclonal gammopathy of undetermined significance (MGUS) cases (p < 0.0001); in contrast, CTPC were present in a small fraction of solitary plasmacytoma patients (18%). Higher numbers of CTPC in PB were associated with higher levels of BM infiltration and more adverse prognostic features, together with shorter time to progression from MGUS to MM (p < 0.0001) and a shorter survival in MM patients with active disease requiring treatment (p <= 0.03). In summary, the presence of CTPC in PB as assessed by NGF at diagnosis, emerges as a hallmark of disseminated PCN, higher numbers of PB CTPC being strongly associated with a malignant disease behavior and a poorer outcome of both MGUS and MM.
- B-cell regeneration profile and minimal residual disease status in bone marrow of treated multiple myeloma patients(2021) Aguilera-Sanz, C. (Carmen); Durie, B. (B.); Montero, J. (Juan); Marvelde, J. (Jeroen) te; Aguilar, C. (Carlos); Pérez-Morán, J. (José); García-Mateo, A. (A.); Mendonca-de-Pontes, R. (Robéria); Corral-Mateos, A. (A.); Orfao, A. (Alberto); Maiolino, A. (Angelo); Mateos, M.V. (María Victoria); Barez, A. (A.); Leoz, P. (Pilar); Sobral-da-Costa, E. (Elaine); Puig, N. (Noemí); Labrador, J. (Jorge); Burgos, L. (Leire); García-Sanchez, O. (O.); Dongen, J.J.M. (Jacques J. M.) van; Paiva, B. (Bruno); EuroFlow-Consortium; Sanoja-Flores, L. (L.); Salgado, A.B. (Anna Beatriz); Pessoa-de-Magalhaes, R.J. (Roberto J.); Velden, V.H.J. (Vicent H. J.) van derSimple Summary B-cell regeneration during therapy has been associated with the outcome of multiple myeloma (MM) patients. However, the effects of therapy and hemodilution in bone marrow (BM) B-cell recovery have not been systematically evaluated. Here, we show that hemodilution is present in a significant fraction of MM BM samples, leading to lower total B-cell, B-cell precursor (BCP), and normal plasma cell (nPC) counts. Among MM BM samples, decreased percentages (vs. healthy donors) of BCP, transitional/naive B-cell (TBC/NBC) and nPC populations were observed at diagnosis. BM BCP, but not TBC/NBC, increased after induction therapy. At day+100 post-autolo-gous stem cell transplantation, a greater increase in BCP with recovered TBC/NBC numbers but persistently low memory B-cell and nPC counts were found. At the end of therapy, complete response (CR) BM samples showed higher CD19(-) nPC counts vs. non-CR specimens with no clear association between BM B-cell regeneration profiles and patient outcomes. B-cell regeneration during therapy has been considered as a strong prognostic factor in multiple myeloma (MM). However, the effects of therapy and hemodilution in bone marrow (BM) B-cell recovery have not been systematically evaluated during follow-up. MM (n = 177) and adult (>= 50y) healthy donor (HD; n = 14) BM samples were studied by next-generation flow (NGF) to simultaneously assess measurable residual disease (MRD) and residual normal B-cell populations. BM hemodilution was detected in 41 out of 177 (23%) patient samples, leading to lower total B-cell, B-cell precursor (BCP) and normal plasma cell (nPC) counts. Among MM BM, decreased percentages (vs. HD) of BCP, transitional/naive B-cell (TBC/NBC) and nPC populations were observed at diagnosis. BM BCP increased after induction therapy, whereas TBC/NBC counts remained abnormally low. At day+100 postautologous stem cell transplantation, a greater increase in BCP with recovered TBC/NBC cell numbers but persistently low memory B-cell and nPC counts were found. At the end of therapy, complete response (CR) BM samples showed higher CD19(-) nPC counts vs. non-CR specimens. MRD positivity was associated with higher BCP and nPC percentages. Hemodilution showed a negative impact on BM B-cell distribution. Different BM B-cell regeneration profiles are present in MM at diagnosis and after therapy with no significant association with patient outcome.
- Minimal residual disease monitoring and immune profiling using second generation flow cytometry in elderly multiple myeloma(American Society of Hematology, 2016) Cedena, M.T. (María Teresa); Montero, J. (Juan); Martínez-López, J. (Joaquín); Gironella, M. (Mercedes); Martin, J. (Jesus); Bladé, J. (Joan); Orfao, A. (Alberto); Vidriales, M.B. (María Belén); Ocio, E.M. (Enrique M.); Gonzalez, Y. (Yolanda); Martin-Ramos, M.L. (Maria Luisa); Mateos, M.V. (María Victoria); Encinas, C. (Cristina); Hernandez, M.T. (Miguel Teodoro); Rosiñol, L. (Laura); Puig, N. (Noemí); Arana, P. (Paula); Cabrera, C. (Carmen); Cordón, L. (Lourdes); Lahuerta, J.J. (Juan José); Gutierrez, N.C. (Norma C.); Dongen, J.J.M. (Jacques J. M.) van; Teruel, A.I. (Ana Isabel); Paiva, B. (Bruno); Oriol, A. (Albert); Bargay, J. (Joan); Martínez, R. (Rafael); San-Miguel, J.F. (Jesús F.); Echeveste, M.A. (Maria Asuncion)The value of minimal residual disease (MRD) in multiple myeloma (MM) has been more frequently investigated in transplant-eligible than elderly patients. Since an optimal balance between treatment efficacy and toxicity is of utmost importance in elderly MM, sensitive MRD monitoring might be particularly valuable in this patient population. Here, we used 2nd generation 8-color multiparameter-flow-cytometry (MFC) to monitor MRD in 162 transplant-ineligible MM patients enrolled in the PETHEMA/GEM2010MAS65 study, The transition from 1st to 2nd generation MFC resulted in increased sensitivity, and allowed to identify three patient groups according to MRD levels: MRD-negative (<10-5; n=54, 34%), MRD-positive between <10-4 and ≥10-5 (n=20, 12%), and MRD-positive ≥10-4 (n=88, 54%). MRD status was an independent prognostic factor for time-to progression (-TTP- HR:2.7; P=.007) and overall survival (-OS- HR:3.1; P=.04) with significant benefit for MRD-negative patients (median TTP not reached, 70% OS at 3-years), and similar poorer outcomes for cases with MRD levels between <10-4 and ≥10-5 vs ≥10-4 (both median TTP of 15 months; 63% and 55% OS at 3-years). Furthermore, MRD-negativity significantly improved TTP of patients >75-years (HR:4.8; P<.001), and those with high-risk cytogenetics (HR:12.6; P=.01). Using 2nd generation MFC, immune profiling concomitant to MRD monitoring also contributed to identify patients with poor, intermediate and favorable outcome (25%, 61% and 100% OS at 3-years; P=.01); the later patients being characterized by an increased compartment of mature B-cells. Our results show that similarly to transplant-candidates, MRD monitoring is one of the most relevant prognostic factors in elderly MM, irrespectively of patients’ age and cytogenetic risk.
- Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry: a multicentric study(2022) Böttcher, S. (Sebastian); Fluxá, R. (Rafael); Verde, J. (Javier); Montero, J. (Juan); Grigore, G. (Georgiana); Novakova, M. (Michaela); Fernández, P. (Paula); Orfao, A. (Alberto); Kalina, T. (Tomas); Engelmann, R. (Robby); Ritgen, M. (Matthias); Burgos, L. (Leire); Lange, S. (Sandra); Dongen, J.J.M. (Jacques J. M.) van; Caetano, J. (Joana); Lecrevisse, Q. (Quentin); Philippé, J. (Jan); Velden, V.H.J. (Vicent H. J.) van der; Pedreira, C.E. (Carlos E.)Reproducible expert-independent flow-cytometric criteria for the differential diagnoses between mature B-cell neoplasms are lacking. We developed an algorithm-driven classification for these lymphomas by flow cytometry and compared it to the WHO gold standard diagnosis. Overall, 662 samples from 662 patients representing 9 disease categories were analyzed at 9 laboratories using the previously published EuroFlow 5-tube-8-color B-cell chronic lymphoproliferative disease antibody panel. Expression levels of all 26 markers from the panel were plotted by B-cell entity to construct a univariate, fully standardized diagnostic reference library. For multivariate data analysis, we subsequently used canonical correlation analysis of 176 training cases to project the multidimensional space of all 26 immunophenotypic parameters into 36 2-dimensional plots for each possible pairwise differential diagnosis. Diagnostic boundaries were fitted according to the distribution of the immunophenotypes of a given differential diagnosis. A diagnostic algorithm based on these projections was developed and subsequently validated using 486 independent cases. Negative predictive values exceeding 92.1% were observed for all disease categories except for follicular lymphoma. Particularly high positive predictive values were returned in chronic lymphocytic leukemia (99.1%), hairy cell leukemia (97.2%), follicular lymphoma (97.2%), and mantle cell lymphoma (95.4%). Burkitt and CD101 diffuse large B-cell lymphomas were difficult to distinguish by the algorithm. A similar ambiguity was observed between marginal zone, lymphoplasmacytic, and CD102 diffuse large B-cell lymphomas. The specificity of the approach exceeded 98% for all entities. The univariate immunophenotypic library and the multivariate expert-independent diagnostic algorithm might contribute to increased reproducibility of future diagnostics in mature B-cell neoplasms.
- Comparison of next-generation sequencing (NGS) and next-generation flow (NGF) for minimal residual disease (MRD) assessment in multiple myeloma(2020) Gonzalez-Calle, V. (Veronica); Cedena, M.T. (María Teresa); Montero, J. (Juan); Martínez-López, J. (Joaquín); Garcia-Alvarez, M. (María); Gonzalez, M. (Marcos); Gironella, M. (Mercedes); Bladé, J. (Joan); Prieto-Conde, M.I. (María Isabel); Sarasquete, M.E. (María E.); García-Sanz, R. (Ramón); Orfao, A. (Alberto); Alcoceba, M. (Miguel); Medina, A. (Alejandro); Mateos, M.V. (María Victoria); Hernandez, M.T. (Miguel Teodoro); Rosiñol, L. (Laura); Puig, N. (Noemí); Lahuerta, J.J. (Juan José); Gutierrez, N.C. (Norma C.); Paiva, B. (Bruno); Oriol, A. (Albert); Chillón, M.C. (María del Carmen); San-Miguel, J.F. (Jesús F.); Jiménez, C. (Cristina)
- Reference values to assess hemodilution and warn of potential false-negative minimal residual disease results in myeloma(2021) Cedena, M.T. (María Teresa); Montero, J. (Juan); Pérez, J.J. (José J.); Martínez-López, J. (Joaquín); Bladé, J. (Joan); Orfao, A. (Alberto); Mateos, M.V. (María Victoria); Rosiñol, L. (Laura); Puig, N. (Noemí); Manrique, I. (Irene); Burgos, L. (Leire); Cordón, L. (Lourdes); Lahuerta, J.J. (Juan José); Rodriguez-Otero, P. (Paula); Paiva, B. (Bruno); Sanoja-Flores, L. (L.); San-Miguel, J.F. (Jesús F.)Although the majority of patients with myeloma who achieve undetectable minimal residual disease show prolonged survival, some of them relapse shortly afterwards. False-negative results due to hemodiluted bone marrow samples could explain this inconsistency, but there is no guidance on how to evaluate them. We analyzed three cell populations normally absent in peripheral blood in 1404 aspirates obtained in numerous disease settings and in 85 healthy adults. Pairwise comparisons according to age and treatment showed significant variability, thus suggesting that hemodilution should be preferably evaluated with references obtained after receiving identical regimens. Leveraging the minimal residual disease results from 118 patients, we showed that a comparison with age-matched healthy adults could also inform on potential hemodilution. Our study supports the routine assessment of bone marrow cellularity to evaluate hemodilution, using as reference values either treatment-specific or from healthy adults if the former are unavailable.