Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry: a multicentric study

Sebastian Bottcher; Robby Engelmann; Georgiana Grigore; Paula Fernandez; Joana Caetano; Juan Flores-Montero; Vincent H.J. van der Velden; Michaela Novakova; Jan Philippe; Matthias Ritgen; Leire Burgos; Quentin Lecrevisse; Sandra Lange; Tomas Kalina; Javier Verde Velasco; Rafael Fluxa Rodriguez; Jacques J.M. van Dongen; Carlos E. Pedreira; Alberto Orfao

doi:10.1182/bloodadvances.2021005725

Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry: a multicentric study

Sebastian Bottcher^*, Robby Engelmann, Georgiana Grigore, Paula Fernandez, Joana Caetano, Juan Flores-Montero, Vincent H.J. van der Velden, Michaela Novakova, Jan Philippe, Matthias Ritgen, Leire Burgos, Quentin Lecrevisse, Sandra Lange, Tomas Kalina, Javier Verde Velasco, Rafael Fluxa Rodriguez, Jacques J.M. van Dongen, Carlos E. Pedreira, Alberto Orfao

^*Corresponding author for this work

Immunology

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Abstract

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.

Original language	English
Pages (from-to)	976-992
Number of pages	17
Journal	Blood advances
Volume	6
Issue number	3
Early online date	4 Feb 2022
DOIs	https://doi.org/10.1182/bloodadvances.2021005725
Publication status	Published - 8 Feb 2022

Bibliographical note

Funding Information:
This work was supported by the EuroFlow Consortium, which received support from the FP6-2004-LIFESCIHEALTH-5 program of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP). The EuroFlow Consortium is part of the European Scientific Foundation for Hemato-Oncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA).

Publisher Copyright:
© 2022 by The American Society of Hematology.

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Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry, a multicentric studyFinal published version, 2.18 MBLicence: CC BY-NC-ND

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Bottcher, S., Engelmann, R., Grigore, G., Fernandez, P., Caetano, J., Flores-Montero, J., van der Velden, V. H. J., Novakova, M., Philippe, J., Ritgen, M., Burgos, L., Lecrevisse, Q., Lange, S., Kalina, T., Velasco, J. V., Rodriguez, R. F., van Dongen, J. J. M., Pedreira, C. E., & Orfao, A. (2022). Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry: a multicentric study. Blood advances, 6(3), 976-992. https://doi.org/10.1182/bloodadvances.2021005725

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title = "Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry: a multicentric study",

abstract = "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.",

author = "Sebastian Bottcher and Robby Engelmann and Georgiana Grigore and Paula Fernandez and Joana Caetano and Juan Flores-Montero and {van der Velden}, {Vincent H.J.} and Michaela Novakova and Jan Philippe and Matthias Ritgen and Leire Burgos and Quentin Lecrevisse and Sandra Lange and Tomas Kalina and Velasco, {Javier Verde} and Rodriguez, {Rafael Fluxa} and {van Dongen}, {Jacques J.M.} and Pedreira, {Carlos E.} and Alberto Orfao",

note = "Funding Information: This work was supported by the EuroFlow Consortium, which received support from the FP6-2004-LIFESCIHEALTH-5 program of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP). The EuroFlow Consortium is part of the European Scientific Foundation for Hemato-Oncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA). Publisher Copyright: {\textcopyright} 2022 by The American Society of Hematology.",

year = "2022",

month = feb,

day = "8",

doi = "10.1182/bloodadvances.2021005725",

language = "English",

volume = "6",

pages = "976--992",

journal = "Blood advances",

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publisher = "American Society of Hematology",

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Bottcher, S, Engelmann, R, Grigore, G, Fernandez, P, Caetano, J, Flores-Montero, J, van der Velden, VHJ, Novakova, M, Philippe, J, Ritgen, M, Burgos, L, Lecrevisse, Q, Lange, S, Kalina, T, Velasco, JV, Rodriguez, RF, van Dongen, JJM, Pedreira, CE & Orfao, A 2022, 'Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry: a multicentric study', Blood advances, vol. 6, no. 3, pp. 976-992. https://doi.org/10.1182/bloodadvances.2021005725

TY - JOUR

T1 - Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry

T2 - a multicentric study

AU - Bottcher, Sebastian

AU - Engelmann, Robby

AU - Grigore, Georgiana

AU - Fernandez, Paula

AU - Caetano, Joana

AU - Flores-Montero, Juan

AU - van der Velden, Vincent H.J.

AU - Novakova, Michaela

AU - Philippe, Jan

AU - Ritgen, Matthias

AU - Burgos, Leire

AU - Lecrevisse, Quentin

AU - Lange, Sandra

AU - Kalina, Tomas

AU - Velasco, Javier Verde

AU - Rodriguez, Rafael Fluxa

AU - van Dongen, Jacques J.M.

AU - Pedreira, Carlos E.

AU - Orfao, Alberto

N1 - Funding Information: This work was supported by the EuroFlow Consortium, which received support from the FP6-2004-LIFESCIHEALTH-5 program of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP). The EuroFlow Consortium is part of the European Scientific Foundation for Hemato-Oncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA). Publisher Copyright: © 2022 by The American Society of Hematology.

PY - 2022/2/8

Y1 - 2022/2/8

N2 - 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.

AB - 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.

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M3 - Article

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SN - 2473-9529

VL - 6

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EP - 992

JO - Blood advances

JF - Blood advances

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ER -

Expert-independent classification of mature B-cell neoplasms using standardized flow cytometry: a multicentric study

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