Orfao, A. (Alberto)
- Publications
- item.page.relationships.isContributorAdvisorOfPublication
- item.page.relationships.isContributorOfPublication
19 results
Search Results
Now showing 1 - 10 of 19
- 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.
- Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia(Springer Nature, 2018) Verde, J. (Javier); Marvelde, J. (Jeroen) te; Grigore, G. (Georgiana); Martin-Ayuso, M. (M.); Asnaf, V. (V.); Novakova, M. (Michaela); Buracchi, C. (Chiara); Fernandez, P. (P.); Bulsa, J. (J.); Orfao, A. (Alberto); Vidriales, M.B. (María Belén); Trinquand, A. (A.); Kalina, T. (Tomas); Mejstrikova, E. (Ester); Sedek, L. (Lukasz); Bras, A.E. (Anne E.); Matarraz, S. (Sergio); Lhermitte, L. (Ludovic); Sobral-da-Costa, E. (Elaine); Szczepanski, T. (Tomasz); Hrusak, O. (O.); Burgos, L. (Leire); Brüggemann, M. (Monika); López, A. (Andrés); Dongen, J.J.M. (Jacques J. M.) van; Paiva, B. (Bruno); Gaipa, G. (Giuseppe); Sonneveld, E. (E.); Sluijs-Gelling, A. (Alita) van der; Lecrevisse, Q. (Quentin); Sá-Bacelar, T. (T.) de; 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 towards the relevant classification panel (T-cell acute lymphoblastic leukemia (T-ALL), B-cell precursor (BCP)-ALL and/or acute myeloid leukemia (AML)) and final diagnosis. Now we built a reference database with 656 typical AL samples (145 T-ALL, 377 BCP-ALL, 134 AML), processed and analyzed via standardized protocols. Using principal component analysis (PCA)-based plots and automated classification algorithms for direct comparison of single-cells from individual patients against the database, another 783 cases were subsequently evaluated. Depending on the database-guided results, patients were categorized as: (i) typical T, B or Myeloid without or; (ii) with a transitional component to another lineage; (iii) atypical; or (iv) mixed-lineage. Using this automated algorithm, in 781/783 cases (99.7%) the right panel was selected, and data comparable to the final WHO-diagnosis was already provided in >93% of cases (85% T-ALL, 97% BCP-ALL, 95% AML and 87% mixed-phenotype AL patients), even without data on the full-characterization panels. Our results show that database-guided analysis facilitates standardized interpretation of ALOT results and allows accurate selection of the relevant classification panels, hence providing a solid basis for designing future WHO AL classifications.
- 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).
- Quality assessment of a large multi-center flow cytometric dataset of acute myeloid leukemia patients-a EuroFlow study(2022) Grigore, G. (Georgiana); Fernández, P. (Paula); Vieira-de-Mello, F. (Fabiana); Orfao, A. (Alberto); Nierkens, S. (Stefan); Bras, A.E. (Anne E.); Matarraz, S. (Sergio); Aanei, C.M. (Carmen Mariana); Burgos, L. (Leire); Dongen, J.J.M. (Jacques J. M.) van; Sluijs-Gelling, A. (Alita) van der; Philippé, J. (Jan); Velden, V.H.J. (Vicent H. J.) van derSimple Summary Flow cytometry allows detailed characterization of large numbers of cells and plays an important role in the diagnosis of acute myeloid leukemia. To facilitate analysis of flowcytometric data, reference databases of normal bone marrow samples and samples from acute myeloid leukemia patients, together with new software tools, are required. We here report on the building of a large database of acute myeloid leukemia patients (n = 1142) and 22 normal samples. We report on the quality assessment procedure used and its validation, discuss potential pitfalls, and provide possible solutions for avoiding such flaws in the construction of other databases. Our data show that obtaining and collecting reproducible flow cytometric data over time and across centers is feasible, but also that strict quality assessment remains crucial, even when standardized protocols for staining and instrument settings are being used in a multicenter setting. Flowcytometric analysis allows for detailed identification and characterization of large numbers of cells in blood, bone marrow, and other body fluids and tissue samples and therefore contributes to the diagnostics of hematological malignancies. Novel data analysis tools allow for multidimensional analysis and comparison of patient samples with reference databases of normal, reactive, and/or leukemia/lymphoma patient samples. Building such reference databases requires strict quality assessment (QA) procedures. Here, we compiled a datase
- Flow cytometry for fast screening and automated risk assessment in systemic light-chain amyloidosis(Nature, 2019) González, M.E. (María Esther); Cedena, M.T. (María Teresa); Verde, J. (Javier); Pérez, J.J. (José J.); Martínez-López, J. (Joaquín); Krsnik, I. (Isabel); Gironella, M. (Mercedes); Orfao, A. (Alberto); Vidriales, M.B. (María Belén); Ocio, E.M. (Enrique M.); Mateos, M.V. (María Victoria); Arriba, F. (Felipe) de; Puerta, J.E. (José Enrique) de la; Puig, N. (Noemí); Labrador, J. (Jorge); Burgos, L. (Leire); Lasa, M. (Marta); Palomera, L. (Luis); Lahuerta, J.J. (Juan José); Pérez-Montaña, A. (Albert); Gómez-Toboso, D. (Dolores); Paiva, B. (Bruno); Lecumberri, R. (Ramón); Oriol, A. (Albert); Rubia, J. (Javier) de la; Prosper-Cardoso, F. (Felipe); Casanova, M. (María); Lecrevisse, Q. (Quentin); Merino, J. (Juana); San-Miguel, J.F. (Jesús F.); Moreno, C. (Cristina); Cabañas, V. (Valentín); García-de-Coca, A. (Alfonso)Early diagnosis and risk stratification are key to improve outcomes in light-chain (AL) amyloidosis. Here we used multidimensional-flow-cytometry (MFC) to characterize bone marrow (BM) plasma cells (PCs) from a series of 166 patients including newly-diagnosed AL amyloidosis (N = 94), MGUS (N = 20) and multiple myeloma (MM, N = 52) vs. healthy adults (N = 30). MFC detected clonality in virtually all AL amyloidosis (99%) patients. Furthermore, we developed an automated risk-stratification system based on BMPCs features, with independent prognostic impact on progression-free and overall survival of AL amyloidosis patients (hazard ratio: ≥ 2.9;P ≤ .03). Simultaneous assessment of the clonal PCs immunophenotypic protein expression profile and the BM cellular composition, mapped AL amyloidosis in the crossroad between MGUS and MM; however, lack of homogenously-positive CD56 expression, reduction of B-cell precursors and a predominantly-clonal PC compartment in the absence of an MM-like tumor PC expansion, emerged as hallmarks of AL amyloidosis (ROC-AUC = 0.74;P < .001), and might potentially be used as biomarkers for the identification of MGUS and MM patients, who are candidates for monitoring pre-symptomatic organ damage related to AL amyloidosis. Altogether, this study addressed the need for consensus on how to use flow cytometry in AL amyloidosis, and proposes a standardized MFC-based automated risk classification ready for implementation in clinical practice.
- Clinical benefit associated with idiotypic vaccination in patients with follicular lymphoma.(Oxford University Press, 2006) Garcia-Muñoz, R. (R.); Pastor, F. (Fernando); Orfao, A. (Alberto); Lopez-Diaz-de-Cerio, A. (Ascensión); Rodriguez-Caballero, A. (Arancha); Rocha, E. (Eduardo); Panizo, C. (Carlos); Inoges, S. (Susana); Perez-Calvo, J. (Javier); Villanueva, H. (Helena); Melero, I. (Ignacio); Bendandi, M. (Maurizio); Suarez, L. (Lilia); Soria, E. (Elena); Zabalegui, N. (Natalia); Rodriguez-Calvillo, M. (Mercedes)BACKGROUND: Follicular lymphoma is considered incurable, although cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy can induce sequential remissions. A patient's second complete response is typically shorter than that patient's first complete response. Idiotype vaccines can elicit specific immune responses and molecular remissions in patients with follicular lymphoma. However, a clinical benefit has never been formally proven. METHODS: Thirty-three consecutive follicular lymphoma patients in first relapse received six monthly cycles of CHOP-like chemotherapy. Patients who achieved a second complete response were vaccinated periodically for more than 2 years with autologous lymphoma-derived idiotype protein vaccine. Specific humoral and cellular responses were assessed, and patients were followed for disease recurrence. Statistical tests were two-sided. RESULTS: Idiotype vaccine could be produced for 25 patients who had a second complete response. In 20 patients (80%), a humoral (13/20) and/or a cellular (18/20) idiotype-specific response was detected. The median duration of the second complete response has not been reached, but it exceeds 33 months (range = 20+ to 51+ months). None of the 20 responders relapsed while undergoing active vaccination. All responders with enough follow-up for the comparison to be made experienced a second complete response that was statistically significantly (P<.0001) longer than both their first complete response (18 of 18 patients) and than the median duration of a CHOP-induced second complete response, i.e., 13 months (20 of 20 patients). The five nonresponders all had a second complete response that was shorter (median = 10 months; range = 8-13 months) than their first complete response (median = 17 months; range = 10-39 months). CONCLUSIONS: Idiotypic vaccination induced a specific immune response in the majority of patients with follicular lymphoma. Specific immune response was associated with a dramatic and highly statistically significant increase in disease-free survival. This is the first formal demonstration of clinical benefit associated with the use of a human cancer vaccine.
- Phenotypic and genomic analysis of multiple myeloma minimal residual disease tumor cells: a new model to understand chemoresistance(American Society of Hematology, 2016) Gironella, M. (Mercedes); Bladé, J. (Joan); Barlogie, B. (Bart); García-Sanz, R. (Ramón); Orfao, A. (Alberto); Vidriales, M.B. (María Belén); Sanchez, M.L. (Maria Luz); Ocio, E.M. (Enrique M.); Gonzalez, Y. (Yolanda); Mateos, M.V. (María Victoria); Maiso, P. (Patricia); Arriba, F. (Felipe) de; Epstein, J. (Joshua); Rodriguez, I. (Idoia); Hernandez, M.T. (Miguel Teodoro); Puig, N. (Noemí); Burgos, L. (Leire); Alignani, D. (Diego); Palomera, L. (Luis); Lahuerta, J.J. (Juan José); Paiva, B. (Bruno); Oriol, A. (Albert); Corchete, L.A. (Luis A.); Barcena, P. (Paloma); San-Miguel, J.F. (Jesús F.); Johnson, S.K. (Sarah K.); Echeveste, M.A. (Maria Asuncion)Persistence of chemoresistant minimal residual disease (MRD) plasma cells (PCs) is associated with inferior survival in multiple myeloma (MM). Thus, characterization of the minor MRD subclone may represent a unique model to understand chemoresistance, but to our knowledge, the phenotypic and genetic features of the MRD subclone have never been investigated. Here, we compared the antigenic profile of MRD vs diagnostic clonal PCs in 40 elderly MM patients enrolled in the GEM2010MAS65 study and showed that the MRD subclone is enriched in cells overexpressing integrins (CD11a/CD11c/CD29/CD49d/CD49e), chemokine receptors (CXCR4), and adhesion molecules (CD44/CD54). Genetic profiling of MRD vs diagnostic PCs was performed in 12 patients; 3 of them showed identical copy number alterations (CNAs), in another 3 cases, MRD clonal PCs displayed all genetic alterations detected at diagnosis plus additional CNAs that emerged at the MRD stage, whereas in the remaining 6 patients, there were CNAs present at diagnosis that were undetectable in MRD clonal PCs, but also a selected number of genetic alterations that became apparent only at the MRD stage. The MRD subclone showed significant downregulation of genes related to protein processing in endoplasmic reticulum, as well as novel deregulated genes such as ALCAM that is prognostically relevant in MM and may identify chemoresistant PCs in vitro. Altogether, our results suggest that therapy-induced clonal selection could be already present at the MRD stage, where chemoresistant PCs show a singular phenotypic signature that may result from the persistence of clones with different genetic and gene expression profiles. This trial was registered at www.clinicaltrials.gov as #NCT01237249.
- Prognostic value of antigen expression in multiple myeloma: a PETHEMA/GEM study on 1,265 patients enrolled in four consecutive clinical trials(2018) Chiodi, F. (Francesca); Cedena, M.T. (María Teresa); Martínez-López, J. (Joaquín); Gironella, M. (Mercedes); Bladé, J. (Joan); Orfao, A. (Alberto); Vidriales, M.B. (María Belén); Mateos, M.V. (María Victoria); Hernandez, M.T. (Miguel Teodoro); Rosiñol, L. (Laura); Puig, N. (Noemí); Lopez-Anglada, L. (Lucia); Burgos, L. (Leire); Cordón, L. (Lourdes); Lahuerta, J.J. (Juan José); Gutierrez, N.C. (Norma C.); Teruel, A.I. (Ana Isabel); Paiva, B. (Bruno); Aranaz-Oroz, P. (Paula); Oriol, A. (Albert); Rubia, J. (Javier) de la; Martínez, R. (Rafael); San-Miguel, J.F. (Jesús F.); Echeveste, M.A. (Maria Asuncion)Persistence of minimal residual disease (MRD) after treatment for myeloma predicts inferior outcomes, but within MRD-positive patients there is great heterogeneity with both early and very late relapses. Among different MRD techniques, flow cytometry provides additional information about antigen expression on tumor cells, which could potentially contribute to stratify MRD-positive patients. We investigated the prognostic value of those antigens required to monitor MRD in 1265 newly diagnosed patients enrolled in the GEM2000, GEM2005MENOS65, GEM2005MAS65 and GEM2010MAS65 protocols. Overall, CD19pos, CD27neg, CD38lo, CD45pos, CD81pos, CD117neg and CD138lo expression predicted inferior outcomes. Through principal component analysis, we found that simultaneous CD38lowCD81posCD117neg expression emerged as the most powerful combination with independent prognostic value for progression-free survival (HR:1.69; P=0.002). This unique phenotypic profile retained prognostic value among MRD-positive patients. We then used next-generation flow to determine antigen stability throughout the course of the disease, and found that the expression of antigens required to monitor MRD is mostly stable from diagnosis to MRD stages, except for CD81 whose expression progressively increased from baseline to chemoresistant tumor cells (14 vs 28%). Altogether, we showed that the phenotypic profile of tumor cells provides additional prognostic information, and could be used to further predict risk of relapse among MRD-positive patients.
- 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.
- In-depth proteomic characterization of classical and non-classical monocyte subsets(MDPI AG, 2018) García, F. (Fernando); Zarzuela, E. (Eduardo); Paradela, A. (Alberto); Corrales, F.J. (Fernando José); Fuentes, M. (Manuel); Garin-Muga, A. (Alba); Díez, P. (Paula); Orfao, A. (Alberto); Sanchez-del-Pino, M.M. (Manuel M.); Garcia-Montero, A. (A.); Beaskoetxea, J. (Javier); Cantero, L. (Laura); Arizmendi, J.M. (Jesús M.); Dégano, R.M. (Rosa M.); Navajas, R. (Rosana); Aloria, K. (Kerman); Muñoz, J. (Javier); Segura, V. (Víctor); Valero, M.L. (María L.)Monocytes are bone marrow-derived leukocytes that are part of the innate immune system. Monocytes are divided into three subsets: classical, intermediate and non-classical, which can be differentiated by their expression of some surface antigens, mainly CD14 and CD16. These cells are key players in the inflammation process underlying the mechanism of many diseases. Thus, the molecular characterization of these cells may provide very useful information for understanding their biology in health and disease. We performed a multicentric proteomic study with pure classical and non-classical populations derived from 12 healthy donors. The robust workflow used provided reproducible results among the five participating laboratories. Over 5000 proteins were identified, and about half of them were quantified using a spectral counting approach. The results represent the protein abundance catalogue of pure classical and enriched non-classical blood peripheral monocytes, and could serve as a reference dataset of the healthy population. The functional analysis of the differences between cell subsets supports the consensus roles assigned to human monocytes.