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1. Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study

Author

Lhermitte, Ludovic, Barreau, Sylvain, Morf, Daniela, Fernandez, Paula, Grigore, Georgiana, Barrena, Susana, de Bie, Maaike, Flores-Montero, Juan, Brüggemann, Monika, Mejstrikova, Ester, Nierkens, Stefan, Burgos, Leire, Caetano, Joana, Gaipa, Giuseppe, Buracchi, Chiara, da Costa, Elaine Sobral, Sedek, Lukasz, Szczepański, Tomasz, Aanei, Carmen-Mariana, van der Sluijs-Gelling, Alita, Delgado, Alejandro Hernández, Fluxa, Rafael, Lecrevisse, Quentin, Pedreira, Carlos E., van Dongen, Jacques J.M., Orfao, Alberto, van der Velden, Vincent H.J., van Dongen, J. J.M., Bitter, W.M., Lubbers, B.R., Teodosio, C.I., Zlei, M., van der Sluijs-Gelling, A.J., de Bie, F., de Bruin-Versteeg, S., van der Burg, M., Schilham, M.W., van der Velden, V. H.J., Langerak, A.W., te Marvelde, J., Bras, A.E., Schilperoord-Vermeulen, J., Jugooa, R., Heezen, K.C., Orfao, A., Almeida, J., Vidriales, M.B., Flores-Montero, J., Pérez-Andrés, M., Matarraz, S., Martín, L., Lecrevisse, Q., Pérez-Morán, J.J., Puig, N., Almeida, A. Medina, Gomes da Silva, M., Faria, T., Brüggemann, M., Ritgen, M., Szczepanowski, M., Kohlscheen, S., Laqua, A., Harbst, E., Finke, J., Asnafi, V., Lhermitte, L., Duroyon, E., Trka, J., Hrusak, O., Kalina, T., Mejstrikova, E., Novakova, M., Thurner, D., Kanderova, V., Szczepanski, T., Sędek, L., Bulsa, J., Slota, L., Kulis, J., Pedreira, C.E., da Costa, E. Sobral, Nierkens, S., de Jong, A., de Koning, A., Lima, M., Santos, A.H., Böttcher, S., Lange, S., Engelmann, R., Paape, D., Machka, C., Gaipa, G., Burracchi, C., Bugarin, C., Lopez-Granados, E., del Pino Molina, L., Campos-Guyotat, L., Aanei, C., Miguel, J. F. San, Paiva, B., Burgos, L., Villamor-Casas, N., Magnano, L., Philippé, J., Bonroy, C., Denys, B., Willems, A., Breughe, P., de Wolf, J., Sousa, A.E., Silva, S.L., Fernandez, P., and Morf, D.

Published
2021
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2. Minimal residual disease assessment in B-cell precursor acute lymphoblastic leukemia by semi-automated identification of normal hematopoietic cells: A EuroFlow study

Author

Verbeek, M, Rodríguez, B, Sedek, L, Laqua, A, Buracchi, C, Buysse, M, Reiterová, M, Oliveira, E, Morf, D, Oude Alink, S, Barrena, S, Kohlscheen, S, Nierkens, S, Hofmans, M, Fernandez, P, de Costa, E, Mejstrikova, E, Szczepanski, T, Slota, L, Brüggemann, M, Gaipa, G, Grigore, G, van Dongen, J, Orfao, A, van der Velden, V, Verbeek M. W. C., Rodríguez B. S., Sedek L., Laqua A., Buracchi C., Buysse M., Reiterová M., Oliveira E., Morf D., Oude Alink S. R., Barrena S., Kohlscheen S., Nierkens S., Hofmans M., Fernandez P., de Costa E. S., Mejstrikova E., Szczepanski T., Slota L., Brüggemann M., Gaipa G., Grigore G., van Dongen J. J. M., Orfao A., van der Velden V. H. J., Verbeek, M, Rodríguez, B, Sedek, L, Laqua, A, Buracchi, C, Buysse, M, Reiterová, M, Oliveira, E, Morf, D, Oude Alink, S, Barrena, S, Kohlscheen, S, Nierkens, S, Hofmans, M, Fernandez, P, de Costa, E, Mejstrikova, E, Szczepanski, T, Slota, L, Brüggemann, M, Gaipa, G, Grigore, G, van Dongen, J, Orfao, A, van der Velden, V, Verbeek M. W. C., Rodríguez B. S., Sedek L., Laqua A., Buracchi C., Buysse M., Reiterová M., Oliveira E., Morf D., Oude Alink S. R., Barrena S., Kohlscheen S., Nierkens S., Hofmans M., Fernandez P., de Costa E. S., Mejstrikova E., Szczepanski T., Slota L., Brüggemann M., Gaipa G., Grigore G., van Dongen J. J. M., Orfao A., and van der Velden V. H. J.

Abstract

Presence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, data-analysis remains mainly expert-dependent. In this study, we designed and validated an Automated Gating & Identification (AGI) tool for MRD analysis in BCP-ALL patients using the two tubes of the EuroFlow 8-color MRD panel. The accuracy, repeatability, and reproducibility of the AGI tool was validated in a multicenter study using bone marrow follow-up samples from 174 BCP-ALL patients, stained with the EuroFlow BCP-ALL MRD panel. In these patients, MRD was assessed both by manual analysis and by AGI tool supported analysis. Comparison of MRD levels obtained between both approaches showed a concordance rate of 83%, with comparable concordances between MRD tubes (tube 1, 2 or both), treatment received (chemotherapy versus targeted therapy) and flow cytometers (FACSCanto versus FACSLyric). After review of discordant cases by additional experts, the concordance increased to 97%. Furthermore, the AGI tool showed excellent intra-expert concordance (100%) and good inter-expert concordance (90%). In addition to MRD levels, also percentages of normal cell populations showed excellent concordance between manual and AGI tool analysis. We conclude that the AGI tool may facilitate MRD analysis using the EuroFlow BCP-ALL MRD protocol and will contribute to a more standardized and objective MRD assessment. However, appropriate training is required for the correct analysis of MRD data.

Published
2023

4. Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies — a EuroFlow study

Author

Verbeek, M, Buracchi, C, Laqua, A, Nierkens, S, Sedek, L, Flores-Montero, J, Hofmans, M, Sobral de Costa, E, Novakova, M, Mejstrikova, E, Barrena, S, Kohlscheen, S, Szczepanowski, M, Kulis, J, Oliveira, E, Jugooa, R, de Jong, A, Szczepanski, T, Philippe, J, van Dongen, J, Orfao, A, Bruggemann, M, Gaipa, G, van der Velden, V, Verbeek M. W. C., Buracchi C., Laqua A., Nierkens S., Sedek L., Flores-Montero J., Hofmans M., Sobral de Costa E., Novakova M., Mejstrikova E., Barrena S., Kohlscheen S., Szczepanowski M., Kulis J., Oliveira E., Jugooa R., de Jong A. X., Szczepanski T., Philippe J., van Dongen J. J. M., Orfao A., Bruggemann M., Gaipa G., van der Velden V. H. J., Verbeek, M, Buracchi, C, Laqua, A, Nierkens, S, Sedek, L, Flores-Montero, J, Hofmans, M, Sobral de Costa, E, Novakova, M, Mejstrikova, E, Barrena, S, Kohlscheen, S, Szczepanowski, M, Kulis, J, Oliveira, E, Jugooa, R, de Jong, A, Szczepanski, T, Philippe, J, van Dongen, J, Orfao, A, Bruggemann, M, Gaipa, G, van der Velden, V, Verbeek M. W. C., Buracchi C., Laqua A., Nierkens S., Sedek L., Flores-Montero J., Hofmans M., Sobral de Costa E., Novakova M., Mejstrikova E., Barrena S., Kohlscheen S., Szczepanowski M., Kulis J., Oliveira E., Jugooa R., de Jong A. X., Szczepanski T., Philippe J., van Dongen J. J. M., Orfao A., Bruggemann M., Gaipa G., and van der Velden V. H. J.

Abstract

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.

Published
2022

5. Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study

Author

Lhermitte, L, Barreau, S, Morf, D, Fernandez, P, Grigore, G, Barrena, S, de Bie, M, Flores-Montero, J, Bruggemann, M, Mejstrikova, E, Nierkens, S, Burgos, L, Caetano, J, Gaipa, G, Buracchi, C, da Costa, E, Sedek, L, Szczepanski, T, Aanei, C, van der Sluijs-Gelling, A, Delgado, A, Fluxa, R, Lecrevisse, Q, Pedreira, C, van Dongen, J, Orfao, A, van der Velden, V, Bitter, W, Lubbers, B, Teodosio, C, Zlei, M, de Bie, F, de Bruin-Versteeg, S, van der Burg, M, Schilham, M, Langerak, A, te Marvelde, J, Bras, A, Schilperoord-Vermeulen, J, Jugooa, R, Heezen, K, Almeida, J, Vidriales, M, Perez-Andres, M, Matarraz, S, Martin, L, Perez-Moran, J, Puig, N, Almeida, A, Gomes da Silva, M, Faria, T, Ritgen, M, Szczepanowski, M, Kohlscheen, S, Laqua, A, Harbst, E, Finke, J, Asnafi, V, Duroyon, E, Trka, J, Hrusak, O, Kalina, T, Novakova, M, Thurner, D, Kanderova, V, Bulsa, J, Slota, L, Kulis, J, de Jong, A, de Koning, A, Lima, M, Santos, A, Bottcher, S, Lange, S, Engelmann, R, Paape, D, Machka, C, Burracchi, C, Bugarin, C, Lopez-Granados, E, del Pino Molina, L, Campos-Guyotat, L, Miguel, J, Paiva, B, Villamor-Casas, N, Magnano, L, Philippe, J, Bonroy, C, Denys, B, Willems, A, Breughe, P, de Wolf, J, Sousa, A, Silva, S, Lhermitte L., Barreau S., Morf D., Fernandez P., Grigore G., Barrena S., de Bie M., Flores-Montero J., Bruggemann M., Mejstrikova E., Nierkens S., Burgos L., Caetano J., Gaipa G., Buracchi C., da Costa E. S., Sedek L., Szczepanski T., Aanei C. -M., van der Sluijs-Gelling A., Delgado A. H., Fluxa R., Lecrevisse Q., Pedreira C. E., van Dongen J. J. M., Orfao A., van der Velden V. H. J., Bitter W. M., Lubbers B. R., Teodosio C. I., Zlei M., van der Sluijs-Gelling A. J., de Bie F., de Bruin-Versteeg S., van der Burg M., Schilham M. W., Langerak A. W., te Marvelde J., Bras A. E., Schilperoord-Vermeulen J., Jugooa R., Heezen K. C., Almeida J., Vidriales M. B., Perez-Andres M., Matarraz S., Martin L., Perez-Moran J. J., Puig N., Almeida A. M., Gomes da Silva M., Faria T., Ritgen M., Szczepanowski M., Kohlscheen S., Laqua A., Harbst E., Finke J., Asnafi V., Duroyon E., Trka J., Hrusak O., Kalina T., Novakova M., Thurner D., Kanderova V., Bulsa J., Slota L., Kulis J., de Jong A., de Koning A., Lima M., Santos A. H., Bottcher S., Lange S., Engelmann R., Paape D., Machka C., Burracchi C., Bugarin C., Lopez-Granados E., del Pino Molina L., Campos-Guyotat L., Aanei C., Miguel J. F. S., Paiva B., Villamor-Casas N., Magnano L., Philippe J., Bonroy C., Denys B., Willems A., Breughe P., de Wolf J., Sousa A. E., Silva S. L., Lhermitte, L, Barreau, S, Morf, D, Fernandez, P, Grigore, G, Barrena, S, de Bie, M, Flores-Montero, J, Bruggemann, M, Mejstrikova, E, Nierkens, S, Burgos, L, Caetano, J, Gaipa, G, Buracchi, C, da Costa, E, Sedek, L, Szczepanski, T, Aanei, C, van der Sluijs-Gelling, A, Delgado, A, Fluxa, R, Lecrevisse, Q, Pedreira, C, van Dongen, J, Orfao, A, van der Velden, V, Bitter, W, Lubbers, B, Teodosio, C, Zlei, M, de Bie, F, de Bruin-Versteeg, S, van der Burg, M, Schilham, M, Langerak, A, te Marvelde, J, Bras, A, Schilperoord-Vermeulen, J, Jugooa, R, Heezen, K, Almeida, J, Vidriales, M, Perez-Andres, M, Matarraz, S, Martin, L, Perez-Moran, J, Puig, N, Almeida, A, Gomes da Silva, M, Faria, T, Ritgen, M, Szczepanowski, M, Kohlscheen, S, Laqua, A, Harbst, E, Finke, J, Asnafi, V, Duroyon, E, Trka, J, Hrusak, O, Kalina, T, Novakova, M, Thurner, D, Kanderova, V, Bulsa, J, Slota, L, Kulis, J, de Jong, A, de Koning, A, Lima, M, Santos, A, Bottcher, S, Lange, S, Engelmann, R, Paape, D, Machka, C, Burracchi, C, Bugarin, C, Lopez-Granados, E, del Pino Molina, L, Campos-Guyotat, L, Miguel, J, Paiva, B, Villamor-Casas, N, Magnano, L, Philippe, J, Bonroy, C, Denys, B, Willems, A, Breughe, P, de Wolf, J, Sousa, A, Silva, S, Lhermitte L., Barreau S., Morf D., Fernandez P., Grigore G., Barrena S., de Bie M., Flores-Montero J., Bruggemann M., Mejstrikova E., Nierkens S., Burgos L., Caetano J., Gaipa G., Buracchi C., da Costa E. S., Sedek L., Szczepanski T., Aanei C. -M., van der Sluijs-Gelling A., Delgado A. H., Fluxa R., Lecrevisse Q., Pedreira C. E., van Dongen J. J. M., Orfao A., van der Velden V. H. J., Bitter W. M., Lubbers B. R., Teodosio C. I., Zlei M., van der Sluijs-Gelling A. J., de Bie F., de Bruin-Versteeg S., van der Burg M., Schilham M. W., Langerak A. W., te Marvelde J., Bras A. E., Schilperoord-Vermeulen J., Jugooa R., Heezen K. C., Almeida J., Vidriales M. B., Perez-Andres M., Matarraz S., Martin L., Perez-Moran J. J., Puig N., Almeida A. M., Gomes da Silva M., Faria T., Ritgen M., Szczepanowski M., Kohlscheen S., Laqua A., Harbst E., Finke J., Asnafi V., Duroyon E., Trka J., Hrusak O., Kalina T., Novakova M., Thurner D., Kanderova V., Bulsa J., Slota L., Kulis J., de Jong A., de Koning A., Lima M., Santos A. H., Bottcher S., Lange S., Engelmann R., Paape D., Machka C., Burracchi C., Bugarin C., Lopez-Granados E., del Pino Molina L., Campos-Guyotat L., Aanei C., Miguel J. F. S., Paiva B., Villamor-Casas N., Magnano L., Philippe J., Bonroy C., Denys B., Willems A., Breughe P., de Wolf J., Sousa A. E., and Silva S. L.

Abstract

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 (r2 > 0.95 for all cell types in PB and r2 > 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). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a “sample-in and result-out” approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal p

Published
2021

6. An extensive quality control and quality assurance (QC/QA) program significantly improves inter-laboratory concordance rates of flow-cytometric minimal residual disease assessment in acute lymphoblastic leukemia: An I-BFM-FLOW-network report

Author

Maurer-Granofszky, M, Schumich, A, Buldini, B, Gaipa, G, Kappelmayer, J, Mejstrikova, E, Karawajew, L, Rossi, J, Suzan, A, Agriello, E, Anastasiou-Grenzelia, T, Barcala, V, Barna, G, Batinic, D, Bourquin, J, Bruggemann, M, Bukowska-Strakova, K, Burnusuzov, H, Carelli, D, Deniz, G, Dubravcic, K, Feuerstein, T, Gaillard, M, Galeano, A, Giordano, H, Gonzalez, A, Groeneveld-Krentz, S, Hevessy, Z, Hrusak, O, Iarossi, M, Jakso, P, Prevodnik, V, Kohlscheen, S, Kreminska, E, Maglia, O, Malusardi, C, Marinov, N, Martin, B, Moller, C, Nikulshin, S, Palazzi, J, Paterakis, G, Popov, A, Ratei, R, Rodriguez, C, Sajaroff, E, Sala, S, Samardzija, G, Sartor, M, Scarparo, P, Sedek, L, Slavkovic, B, Solari, L, Svec, P, Szczepanski, T, Taparkou, A, Torrebadell, M, Tzanoudaki, M, Varotto, E, Vernitsky, H, Attarbaschi, A, Schrappe, M, Conter, V, Biondi, A, Felice, M, Campbell, M, Kiss, C, Basso, G, Dworzak, M, Maurer-Granofszky M., Schumich A., Buldini B., Gaipa G., Kappelmayer J., Mejstrikova E., Karawajew L., Rossi J., Suzan A. C., Agriello E., Anastasiou-Grenzelia T., Barcala V., Barna G., Batinic D., Bourquin J. -P., Bruggemann M., Bukowska-Strakova K., Burnusuzov H., Carelli D., Deniz G., Dubravcic K., Feuerstein T., Gaillard M. I., Galeano A., Giordano H., Gonzalez A., Groeneveld-Krentz S., Hevessy Z., Hrusak O., Iarossi M. B., Jakso P., Prevodnik V. K., Kohlscheen S., Kreminska E., Maglia O., Malusardi C., Marinov N., Martin B. M., Moller C., Nikulshin S., Palazzi J., Paterakis G., Popov A., Ratei R., Rodriguez C., Sajaroff E. O., Sala S., Samardzija G., Sartor M., Scarparo P., Sedek L., Slavkovic B., Solari L., Svec P., Szczepanski T., Taparkou A., Torrebadell M., Tzanoudaki M., Varotto E., Vernitsky H., Attarbaschi A., Schrappe M., Conter V., Biondi A., Felice M., Campbell M., Kiss C., Basso G., Dworzak M. N., Maurer-Granofszky, M, Schumich, A, Buldini, B, Gaipa, G, Kappelmayer, J, Mejstrikova, E, Karawajew, L, Rossi, J, Suzan, A, Agriello, E, Anastasiou-Grenzelia, T, Barcala, V, Barna, G, Batinic, D, Bourquin, J, Bruggemann, M, Bukowska-Strakova, K, Burnusuzov, H, Carelli, D, Deniz, G, Dubravcic, K, Feuerstein, T, Gaillard, M, Galeano, A, Giordano, H, Gonzalez, A, Groeneveld-Krentz, S, Hevessy, Z, Hrusak, O, Iarossi, M, Jakso, P, Prevodnik, V, Kohlscheen, S, Kreminska, E, Maglia, O, Malusardi, C, Marinov, N, Martin, B, Moller, C, Nikulshin, S, Palazzi, J, Paterakis, G, Popov, A, Ratei, R, Rodriguez, C, Sajaroff, E, Sala, S, Samardzija, G, Sartor, M, Scarparo, P, Sedek, L, Slavkovic, B, Solari, L, Svec, P, Szczepanski, T, Taparkou, A, Torrebadell, M, Tzanoudaki, M, Varotto, E, Vernitsky, H, Attarbaschi, A, Schrappe, M, Conter, V, Biondi, A, Felice, M, Campbell, M, Kiss, C, Basso, G, Dworzak, M, Maurer-Granofszky M., Schumich A., Buldini B., Gaipa G., Kappelmayer J., Mejstrikova E., Karawajew L., Rossi J., Suzan A. C., Agriello E., Anastasiou-Grenzelia T., Barcala V., Barna G., Batinic D., Bourquin J. -P., Bruggemann M., Bukowska-Strakova K., Burnusuzov H., Carelli D., Deniz G., Dubravcic K., Feuerstein T., Gaillard M. I., Galeano A., Giordano H., Gonzalez A., Groeneveld-Krentz S., Hevessy Z., Hrusak O., Iarossi M. B., Jakso P., Prevodnik V. K., Kohlscheen S., Kreminska E., Maglia O., Malusardi C., Marinov N., Martin B. M., Moller C., Nikulshin S., Palazzi J., Paterakis G., Popov A., Ratei R., Rodriguez C., Sajaroff E. O., Sala S., Samardzija G., Sartor M., Scarparo P., Sedek L., Slavkovic B., Solari L., Svec P., Szczepanski T., Taparkou A., Torrebadell M., Tzanoudaki M., Varotto E., Vernitsky H., Attarbaschi A., Schrappe M., Conter V., Biondi A., Felice M., Campbell M., Kiss C., Basso G., and Dworzak M. N.

Abstract

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

Published
2021

7. Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies — a EuroFlow study

Author

Verbeek, M. W. C., Buracchi, C., Laqua, A., Nierkens, S., Sedek, L., Flores-Montero, J., Hofmans, M., Sobral de Costa, E., Nováková, M., Mejstrikova, E., Barrena, S., Kohlscheen, S., Szczepanowski, M., Kulis, J., Oliveira, E., Jugooa, R., de Jong, A. X., Szczepanski, T., Philippé, J., van Dongen, J. J. M., Orfao, A., Brüggemann, M., Gaipa, G., van der Velden, V. H. J., Verbeek, M. W. C., Buracchi, C., Laqua, A., Nierkens, S., Sedek, L., Flores-Montero, J., Hofmans, M., Sobral de Costa, E., Nováková, M., Mejstrikova, E., Barrena, S., Kohlscheen, S., Szczepanowski, M., Kulis, J., Oliveira, E., Jugooa, R., de Jong, A. X., Szczepanski, T., Philippé, J., van Dongen, J. J. M., Orfao, A., Brüggemann, M., Gaipa, G., and van der Velden, V. H. J.

Abstract

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.

Published
2022

8. P681: EUROFLOW STANDARDIZATION TECHNIQUE AND NORMALIZATION PROCEDURES IN LONGITUDINAL FLOW CYTOMETRIC EXPRESSION ANALYSIS OF CD20 IN CLL PATIENTS RECEIVING ANTI-CD20 DIRECTED THERAPY

Author

Walter, P. J., primary, Schilhabel, A., additional, Cramer, P., additional, von Tresckow, J., additional, Kohlscheen, S., additional, Fischer, K., additional, Eichhorst, B., additional, Böttcher, S., additional, Brüggemann, M., additional, Kneba, M., additional, Hallek, M., additional, and Ritgen, M., additional

Published
2022
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9. Blood monitoring of circulating tumor plasma cells by next generation flow in multiple myeloma after therapy

Author

Sanoja-Flores, L., Flores-Montero, J., Puig, N., Contreras-Sanfeliciano, T., Pontes, R., Corral-Mateos, A., Garcia-Sanchez, O., Diez-Campelo, M., Magalhaes, R.J.P. de, Garcia-Martin, L., Alonso-Alonso, J.M., Garcia-Mateo, A., Aguilar-Franco, C., Labrador, J., Barez-Garcia, A., Maiolino, A., Paiva, B., San Miguel, J., Costa, E.S. da, Gonzalez, M., Mateos, M.V., Durie, B., Dongen, J.J.M. van, Orfao, A., Bitter, W.M., Lubbers, B.R., Meij, A.S.M. van der, Teodosio, C.I., Zlei, M., Sluijs-Gelling, A.J. van der, Burg, M. van der, Velden, V.H.J. van der, Langerak, A.W., Marvelde, J. te, Schilperoord-Vermeulen, J., Blijkerk, A., Heezen, K.C., Almeida, J., Vidriales, M.B., Perez-Andres, J.F.M.M., Matarraz, S., Blanco, E., Martin, L., Lecrevisse, Q., Perez-Moran, J.J., Almeida, A.M., Silva, M.G. da, Faria, T., Bruggemann, M., Ritgen, M., Szczepanowski, M., Kohlscheen, S., Steinert, A., Harbst, E., Finke, J., Asnafi, V., Lhermitte, L., Duroyon, E., Trka, J., Hrusak, O., Kalina, T., Mejstrikova, E., Novakova, M., Thurner, D., Kanderova, V., Szczepanski, T., Sedek, L., Bulsa, J., Slota, L., Kulis, J., Pedreira, C.E., Nierkens, S., Jong, A. de, Koning, A. de, Lima, M., Santos, A.H., Bottcher, S., Lange, S., Engelmann, R., Paape, D., Machka, C., Gaipa, G., Burracchi, C., Bugarin, C., Lopez-Granados, E., Molina, L.D., Vlkova, M., Nechvatalova, J., Roussel, M., Campos-Guyotat, L., Aanei, C., Burgos, L., Villamor-Casas, N., Magnano, L., Philippe, J., Bonroy, C., Denys, B., Willems, A., Breughe, P., Wolf, J. de, Sousa, A.E., Silva, S.L., Fernandez, P., Morf, D., EuroFlow Consortium, and Immunology

Subjects
Pathology, medicine.medical_specialty, medicine.diagnostic_test, business.industry, Immunology, Plasma Cells, Cell Biology, Hematology, medicine.disease, Flow Cytometry, Neoplastic Cells, Circulating, Prognosis, Biochemistry, Flow cytometry, Immunophenotyping, Text mining, Flow (mathematics), medicine, Humans, business, Multiple Myeloma, Letter to Blood, Multiple myeloma
Published
2019

10. Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies

Author

Martijn W. C. Verbeek, Chiara Buracchi, Anna Laqua, Stefan Nierkens, Lukasz Sedek, Juan Flores‐Montero, Mattias Hofmans, Elaine Sobral de Costa, Michaela Nováková, Ester Mejstrikova, Susana Barrena, Saskia Kohlscheen, Monika Szczepanowski, Jan Kulis, Elen Oliveira, Romana Jugooa, Anja X. Jong, Tomasz Szczepanski, Jan Philippé, Jacques J. M. Dongen, Alberto Orfao, Monika Brüggemann, Giuseppe Gaipa, Vincent H. J. Velden, Immunology, European Commission, European Hematology Association, Silesian University of Technology, Verbeek, M, Buracchi, C, Laqua, A, Nierkens, S, Sedek, L, Flores-Montero, J, Hofmans, M, Sobral de Costa, E, Novakova, M, Mejstrikova, E, Barrena, S, Kohlscheen, S, Szczepanowski, M, Kulis, J, Oliveira, E, Jugooa, R, de Jong, A, Szczepanski, T, Philippe, J, van Dongen, J, Orfao, A, Bruggemann, M, Gaipa, G, and van der Velden, V

Subjects
acute leukaemia, Neoplasm, Residual, Minimal residual disease, flow cytometry, Antigens, CD19, hemic and immune systems, Hematology, diagnostic haematology, Precursor Cell Lymphoblastic Leukemia-Lymphoma, Burkitt Lymphoma, Acute leukaemia, body regions, Diagnostic haematology, hemic and lymphatic diseases, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma, Medicine and Health Sciences, minimal residual disease, Humans, Flow cytometry, Adaptor Proteins, Signal Transducing
Abstract

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct., The EuroFlow Consortium received support from the FP6-2004-LIFESCIHEALTH-5 programme 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). TS and LS were supported by a Scientific Grant from the Medical University of Silesia Nr. PCN-1-050/K/0/K.

Published
2021

11. Automated identification of leukocyte subsets improves standardization of database-guided expert-supervised diagnostic orientation in acute leukemia: a EuroFlow study

Author

Ludovic Lhermitte, Sylvain Barreau, Daniela Morf, Paula Fernandez, Georgiana Grigore, Susana Barrena, Maaike de Bie, Juan Flores-Montero, Monika Brüggemann, Ester Mejstrikova, Stefan Nierkens, Leire Burgos, Joana Caetano, Giuseppe Gaipa, Chiara Buracchi, Elaine Sobral da Costa, Lukasz Sedek, Tomasz Szczepański, Carmen-Mariana Aanei, Alita van der Sluijs-Gelling, Alejandro Hernández Delgado, Rafael Fluxa, Quentin Lecrevisse, Carlos E. Pedreira, Jacques J.M. van Dongen, Alberto Orfao, Vincent H.J. van der Velden, J. J.M. van Dongen, W.M. Bitter, B.R. Lubbers, C.I. Teodosio, M. Zlei, A.J. van der Sluijs-Gelling, F. de Bie, S. de Bruin-Versteeg, M. van der Burg, M.W. Schilham, V. H.J. van der Velden, A.W. Langerak, J. te Marvelde, A.E. Bras, J. Schilperoord-Vermeulen, R. Jugooa, K.C. Heezen, A. Orfao, J. Almeida, M.B. Vidriales, J. Flores-Montero, M. Pérez-Andrés, S. Matarraz, L. Martín, Q. Lecrevisse, J.J. Pérez-Morán, N. Puig, A. Medina Almeida, M. Gomes da Silva, T. Faria, M. Brüggemann, M. Ritgen, M. Szczepanowski, S. Kohlscheen, A. Laqua, E. Harbst, J. Finke, V. Asnafi, L. Lhermitte, E. Duroyon, J. Trka, O. Hrusak, T. Kalina, E. Mejstrikova, M. Novakova, D. Thurner, V. Kanderova, T. Szczepanski, L. Sędek, J. Bulsa, L. Slota, J. Kulis, C.E. Pedreira, E. Sobral da Costa, S. Nierkens, A. de Jong, A. de Koning, M. Lima, A.H. Santos, S. Böttcher, S. Lange, R. Engelmann, D. Paape, C. Machka, G. Gaipa, C. Burracchi, C. Bugarin, E. Lopez-Granados, L. del Pino Molina, L. Campos-Guyotat, C. Aanei, J. F. San Miguel, B. Paiva, L. Burgos, N. Villamor-Casas, L. Magnano, J. Philippé, C. Bonroy, B. Denys, A. Willems, P. Breughe, J. de Wolf, A.E. Sousa, S.L. Silva, P. Fernandez, D. Morf, European Commission, Instituto de Salud Carlos III, Ministerio de Economía y Competitividad (España), Agencia Estatal de Investigación (España), Ministerio de Ciencia, Innovación y Universidades (España), Silesian University of Technology, Lhermitte, L, Barreau, S, Morf, D, Fernandez, P, Grigore, G, Barrena, S, de Bie, M, Flores-Montero, J, Bruggemann, M, Mejstrikova, E, Nierkens, S, Burgos, L, Caetano, J, Gaipa, G, Buracchi, C, da Costa, E, Sedek, L, Szczepanski, T, Aanei, C, van der Sluijs-Gelling, A, Delgado, A, Fluxa, R, Lecrevisse, Q, Pedreira, C, van Dongen, J, Orfao, A, van der Velden, V, Bitter, W, Lubbers, B, Teodosio, C, Zlei, M, de Bie, F, de Bruin-Versteeg, S, van der Burg, M, Schilham, M, Langerak, A, te Marvelde, J, Bras, A, Schilperoord-Vermeulen, J, Jugooa, R, Heezen, K, Almeida, J, Vidriales, M, Perez-Andres, M, Matarraz, S, Martin, L, Perez-Moran, J, Puig, N, Almeida, A, Gomes da Silva, M, Faria, T, Ritgen, M, Szczepanowski, M, Kohlscheen, S, Laqua, A, Harbst, E, Finke, J, Asnafi, V, Duroyon, E, Trka, J, Hrusak, O, Kalina, T, Novakova, M, Thurner, D, Kanderova, V, Bulsa, J, Slota, L, Kulis, J, de Jong, A, de Koning, A, Lima, M, Santos, A, Bottcher, S, Lange, S, Engelmann, R, Paape, D, Machka, C, Burracchi, C, Bugarin, C, Lopez-Granados, E, del Pino Molina, L, Campos-Guyotat, L, Miguel, J, Paiva, B, Villamor-Casas, N, Magnano, L, Philippe, J, Bonroy, C, Denys, B, Willems, A, Breughe, P, de Wolf, J, Sousa, A, Silva, S, and Immunology

Subjects
0301 basic medicine, Pathology, medicine.medical_specialty, Standardization, Computer science, Leukaemia, Laboratory techniques and procedures, Article, Immunophenotyping, Pathology and Forensic Medicine, 03 medical and health sciences, 0302 clinical medicine, EuroFlow, Leukocytes, medicine, Humans, Leukaemia, Flow cytometry, Future, Acute leukemia, Orientation (computer vision), business.industry, Laboratory techniques and procedures, Pattern recognition, Flow Cytometry, Peripheral blood, Leukemia, Myeloid, Acute, Identification (information), 030104 developmental biology, Área de Biomedicina, T cell subset, Artificial intelligence, business, Algorithms, 030215 immunology
Abstract

© The Author(s) 2020., 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 ( 0.95 for all cell types in PB and r2 > 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). Similar data were obtained using alternative, commercially available tubes, confirming the robustness of the developed tools. The AGI tool represents an innovative step in minimizing human intervention and requirements in expertise, toward a “sample-in and result-out” approach which may result in more objective and reproducible data analysis and diagnostics. The AGI tool may improve quality of immunophenotyping in individual laboratories, since high percentages of checks in normal samples are an alert on the quality of the internal procedures., The EuroFlow Consortium 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 Prague team received support from the grant number NV18-03-00343. The Salamanca team received support from the Instituto de Salud Carlos III (ISCIII) (PI16/00787-FEDER) and from Agencia Estatal de Investigación (RTC-2016-4865-1-FEDER), Ministerio de Economía y Competitividad, Madrid, Spain. AHD is supported from the program DI-17-09591 from Agencia Estatal de Investigación, Ministerio de Ciencia, Innovación y Universidades, Madrid, Spain. SB is supported from the program PTQ16-08364 from Agencia Estatal de Investigación, Ministerio de Ciencia, Innovación y Universidades, Madrid, Spain. Medical University of Silesia in Katowice team was supported by the Strategmed III PersonALL grant [No. 304586/5/NCBR/2017] from the Polish National Center of Research and Development. 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).

Published
2021

12. Minimal residual disease assessment in B-cell precursor acute lymphoblastic leukemia by semi-automated identification of normal hematopoietic cells: A EuroFlow study.

Author

Verbeek MWC, Rodríguez BS, Sedek L, Laqua A, Buracchi C, Buysse M, Reiterová M, Oliveira E, Morf D, Oude Alink SR, Barrena S, Kohlscheen S, Nierkens S, Hofmans M, Fernandez P, de Costa ES, Mejstrikova E, Szczepanski T, Slota L, Brüggemann M, Gaipa G, Grigore G, van Dongen JJM, Orfao A, and van der Velden VHJ

Subjects
Humans, Child, Male, Female, Adolescent, Child, Preschool, Adult, Middle Aged, Young Adult, Reproducibility of Results, Immunophenotyping methods, Aged, Infant, Neoplasm, Residual pathology, Neoplasm, Residual diagnosis, Flow Cytometry methods, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma pathology, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma diagnosis
Abstract

Presence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, data-analysis remains mainly expert-dependent. In this study, we designed and validated an Automated Gating & Identification (AGI) tool for MRD analysis in BCP-ALL patients using the two tubes of the EuroFlow 8-color MRD panel. The accuracy, repeatability, and reproducibility of the AGI tool was validated in a multicenter study using bone marrow follow-up samples from 174 BCP-ALL patients, stained with the EuroFlow BCP-ALL MRD panel. In these patients, MRD was assessed both by manual analysis and by AGI tool supported analysis. Comparison of MRD levels obtained between both approaches showed a concordance rate of 83%, with comparable concordances between MRD tubes (tube 1, 2 or both), treatment received (chemotherapy versus targeted therapy) and flow cytometers (FACSCanto versus FACSLyric). After review of discordant cases by additional experts, the concordance increased to 97%. Furthermore, the AGI tool showed excellent intra-expert concordance (100%) and good inter-expert concordance (90%). In addition to MRD levels, also percentages of normal cell populations showed excellent concordance between manual and AGI tool analysis. We conclude that the AGI tool may facilitate MRD analysis using the EuroFlow BCP-ALL MRD protocol and will contribute to a more standardized and objective MRD assessment. However, appropriate training is required for the correct analysis of MRD data., (© 2023 The Authors. Cytometry Part B: Clinical Cytometry published by Wiley Periodicals LLC on behalf of International Clinical Cytometry Society.)

Published
2024
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13. CD20 Expression as a Possible Novel Prognostic Marker in CLL: Application of EuroFlow Standardization Technique and Normalization Procedures in Flow Cytometric Expression Analysis.

Author

Schilhabel A, Walter PJ, Cramer P, von Tresckow J, Kohlscheen S, Szczepanowski M, Laqua A, Fischer K, Eichhorst B, Böttcher S, Schneider C, Tausch E, Brüggemann M, Kneba M, Hallek M, and Ritgen M

Abstract

Background: CD20 expression is a controversial issue regarding response prediction to anti-CD20 therapy in chronic lymphocytic leukemia (CLL)., Methods: Median fluorescence intensities (MFIs) of standard fluorescence beads from the daily calibration of flow cytometers according to EuroFlow protocols were used to establish a normalization approach to study CD20 expression on CLL cells. CD20 MFI was retrospectively assessed prior to and during treatment from flow cytometric measurements of peripheral blood in patients with different depths of molecular response in the four phase-II CLL2-BXX trials (BIG; BAG; BIO; BCG; N = 194) administering either Obinutuzumab or Ofatumumab in combination with targeted agents., Results: No significant difference was observed between the normalized and measured MFIs of CD19 and CD20 on CLL cells. During treatment, CD20 expression levels on CLL cells did not significantly differ between the four investigated different treatment schemes, but a strong molecular response to Ofatumumab seemed to correlate with higher CD20 expression prior to therapy., Conclusions: Standardized staining and instrument monitoring enable a robust assessment of longitudinal biological variations of marker expression based on MFI values. Obinutuzumab showed a higher proportion of patients with a strong MRD response independent from initial CD20 expression, whereas high pre-therapeutic CD20 expression levels seem to correlate with a profound response to Ofatumumab.

Published
2022
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14. Influenza A virus infection instructs hematopoiesis to megakaryocyte-lineage output.

Author

Rommel MGE, Walz L, Fotopoulou F, Kohlscheen S, Schenk F, Miskey C, Botezatu L, Krebs Y, Voelker IM, Wittwer K, Holland-Letz T, Ivics Z, von Messling V, Essers MAG, Milsom MD, Pfaller CK, and Modlich U

Subjects
Animals, Antiviral Agents metabolism, Cytokines metabolism, Endothelial Protein C Receptor metabolism, Hematopoiesis, Humans, Megakaryocytes metabolism, Mice, Receptors, Interleukin-1 metabolism, Thrombopoietin metabolism, Influenza A virus, Influenza, Human metabolism
Abstract

Respiratory tract infections are among the deadliest communicable diseases worldwide. Severe cases of viral lung infections are often associated with a cytokine storm and alternating platelet numbers. We report that hematopoietic stem and progenitor cells (HSPCs) sense a non-systemic influenza A virus (IAV) infection via inflammatory cytokines. Irrespective of antiviral treatment or vaccination, at a certain threshold of IAV titer in the lung, CD41-positive hematopoietic stem cells (HSCs) enter the cell cycle while endothelial protein C receptor-positive CD41-negative HSCs remain quiescent. Active CD41-positive HSCs represent the source of megakaryocytes, while their multi-lineage reconstitution potential is reduced. This emergency megakaryopoiesis is thrombopoietin independent and attenuated in IAV-infected interleukin-1 receptor-deficient mice. Newly produced platelets during IAV infection are immature and hyper-reactive. After viral clearance, HSC quiescence is re-established. Our study reveals that non-systemic viral respiratory infection has an acute impact on HSCs via inflammatory cytokines to counteract IAV-induced thrombocytopenia., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Published
2022
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15. Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies - a EuroFlow study.

Author

Verbeek MWC, Buracchi C, Laqua A, Nierkens S, Sedek L, Flores-Montero J, Hofmans M, Sobral de Costa E, Nováková M, Mejstrikova E, Barrena S, Kohlscheen S, Szczepanowski M, Kulis J, Oliveira E, Jugooa R, de Jong AX, Szczepanski T, Philippé J, van Dongen JJM, Orfao A, Brüggemann M, Gaipa G, and van der Velden VHJ

Subjects
Adaptor Proteins, Signal Transducing, Antigens, CD19 therapeutic use, Flow Cytometry methods, Humans, Neoplasm, Residual, Burkitt Lymphoma, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma diagnosis, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma drug therapy, Precursor Cell Lymphoblastic Leukemia-Lymphoma
Abstract

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct., (© 2021 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Published
2022
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16. An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: An I-BFM-FLOW-Network Report.

Author

Maurer-Granofszky M, Schumich A, Buldini B, Gaipa G, Kappelmayer J, Mejstrikova E, Karawajew L, Rossi J, Suzan AÇ, Agriello E, Anastasiou-Grenzelia T, Barcala V, Barna G, Batinić D, Bourquin JP, Brüggemann M, Bukowska-Strakova K, Burnusuzov H, Carelli D, Deniz G, Dubravčić K, Feuerstein T, Gaillard MI, Galeano A, Giordano H, Gonzalez A, Groeneveld-Krentz S, Hevessy Z, Hrusak O, Iarossi MB, Jáksó P, Kloboves Prevodnik V, Kohlscheen S, Kreminska E, Maglia O, Malusardi C, Marinov N, Martin BM, Möller C, Nikulshin S, Palazzi J, Paterakis G, Popov A, Ratei R, Rodríguez C, Sajaroff EO, Sala S, Samardzija G, Sartor M, Scarparo P, Sędek Ł, Slavkovic B, Solari L, Svec P, Szczepanski T, Taparkou A, Torrebadell M, Tzanoudaki M, Varotto E, Vernitsky H, Attarbaschi A, Schrappe M, Conter V, Biondi A, Felice M, Campbell M, Kiss C, Basso G, Dworzak MN, and On Behalf Of I-Bfm-Flow-Network

Abstract

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts.

Published
2021
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17. Signaling properties of murine MPL and MPL mutants after stimulation with thrombopoietin and romiplostim.

Author

Rommel MGE, Hoerster K, Milde C, Schenk F, Roser L, Kohlscheen S, Heinz N, and Modlich U

Subjects
Amino Acid Substitution, Animals, Cell Line, MAP Kinase Signaling System genetics, Mice, Mice, Knockout, Receptors, Fc, Receptors, Thrombopoietin genetics, Thrombopoiesis genetics, MAP Kinase Signaling System drug effects, Mutation, Missense, Receptors, Thrombopoietin metabolism, Recombinant Fusion Proteins pharmacology, Thrombopoiesis drug effects, Thrombopoietin pharmacology
Abstract

Thrombopoietin (THPO) and its receptor myeloproliferative leukemia virus oncogene (MPL) regulate hematopoietic stem cell (HSC) quiescence and maintenance, but also megakaryopoiesis. Thrombocytopenias or aplastic anemias can be treated today with THPO peptide mimetics (romiplostim) or small-molecule THPO receptor agonists (e.g., eltrombopag). These THPO mimetics were designed for human application; however, many preclinical studies are performed in murine models. We investigated the activation of wild-type murine MPL (mMPL) by romiplostim. Romiplostim stimulated AKT, ERK1/2, and STAT5 phosphorylation without preference for one of these pathways, however, with a four- to fivefold lower phosphorylation intensity at high concentration. Faster internalization of mMPL after romiplostim binding could be one explanation of reduced signaling. In vitro megakaryocyte differentiation, proliferation, and maturation by romiplostim was less efficient compared with stimulation with mTHPO. We further dissected mMPL signaling by lentiviral overexpression of mMPL mutants with tyrosine (Y)-to-phenylalanine (F) substitutions in the distal cytoplasmic tyrosines 582 (Y582F), 616 (Y616F), and 621 (Y621F) individually and in combination (Y616F&#95;Y621F) and in truncated receptors lacking 53 (Δ53) or 69 (Δ69) C-terminal amino acids. Mutation at tyrosine residue Y582F caused a gain-of-function with baseline activation and increased ERK1/2 phosphorylation upon stimulation. In agreement with this, proliferation in Y582F-32D cells was increased, yet did not rescue in vitro megakaryopoiesis from Mpl-deficient cells. Y616F and Y621F mutated receptors exhibited strongly impaired ERK1/2 and decreased AKT signaling and conferred reduced proliferation to 32D cells upon mTHPO stimulation but a partial correction of immature megakaryopoiesis in vitro., (Copyright © 2020 ISEH -- Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Published
2020
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18. Endothelial protein C receptor supports hematopoietic stem cell engraftment and expansion in Mpl-deficient mice.

Author

Kohlscheen S, Schenk F, Rommel MGE, Cullmann K, and Modlich U

Subjects
Animals, Cell Proliferation, Gene Deletion, Hematopoiesis, Hematopoietic Stem Cells metabolism, Humans, Mice, Mice, Inbred C57BL, Endothelial Protein C Receptor metabolism, Hematopoietic Stem Cell Transplantation, Hematopoietic Stem Cells cytology, Receptors, Thrombopoietin genetics
Abstract

Thrombopoietin (Thpo)/myeloproliferative leukemia virus oncogene (Mpl) signaling controls hematopoietic stem cell (HSC) self-renewal and quiescence; however, how these 2 seemingly opposing functions are controlled is not well understood. By transplantation of lentiviral-transduced hematopoietic cells in the Mpl-deficient mouse model, we addressed whether known or predicted Thpo target genes were able to rescue the Mpl-deficient phenotype of the mice. Among the tested genes, we identified endothelial protein C receptor (Epcr) to expand HSCs with the long-term (LT)-HSC surface phenotype in Mpl -/- mice and to enable secondary transplantation of Mpl-deficient bone marrow (BM). Epcr-transduced Mpl -/- HSCs enter quiescence earlier after transplantation than control-transduced Mpl -/- cells, and upregulated expression of the anti-apoptotic gene Bcl-xL. Also, in the wild-type background, Epcr expression marked the engrafting population in the BM. Furthermore, Epcr expression in Mpl -/- hematopoiesis increased the number of megakaryocytes in the BM. In vitro Thpo supported the surface expression of Epcr on primary murine hematopoietic stem and progenitor cells. With these data, we add new insights into Thpo-dependent influence on HSC engraftment after transplantation. This may be of use for the in vitro manipulation of HSCs, also in the context of gene therapy., (© 2019 by The American Society of Hematology.)

Published
2019
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19. Promises and Challenges in Hematopoietic Stem Cell Gene Therapy.

Author

Kohlscheen S, Bonig H, and Modlich U

Subjects
Animals, Drug Evaluation, Preclinical, Gene Transfer Techniques, Genetic Diseases, Inborn genetics, Genetic Diseases, Inborn metabolism, Genetic Diseases, Inborn therapy, Genetic Vectors, Hematopoietic Stem Cell Transplantation methods, Humans, Mutagenicity Tests, Transgenes, Genetic Therapy adverse effects, Genetic Therapy methods, Hematopoietic Stem Cells cytology, Hematopoietic Stem Cells metabolism
Abstract

Hematopoietic stem cell-directed gene therapy (HSC-GT) provides an innovative treatment option for hematological disorders. Gene therapy promises to cure the disease "at the root" and is therefore exceptional in its potential, but also formidable in its challenges, as long-term side effects are hard to predict and clinical experience remains limited. Many excellent reviews on the topic by designated experts in the field of HSC-GT have come forth, elucidating the successes and pitfalls in the various clinical studies. This review attempts to discuss what we understand from those studies to represent current state of the art with respect to vectors, stem cell transduction, and pretransplant preparatory regimes, what limitations may remain, and which types of diseases may be more suited for HSC-GT than others (targets). We thus discuss the available vector platforms (tools) and preclinical/clinical and basic research (tricks) that contribute to our current understanding of HSC-GT, as well as some overarching principles we can conclude from these. The field has also learned from previous shortcomings, although some of the major concerns of the past, specifically insertional mutagenesis, may not be of relevance for future trials. This very positive development in HSC-GT, however, has to compete with the improvements in hematopoietic stem cell transplantation or enzyme-replacement therapy, leaving a narrow margin for gene therapy.

Published
2017
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20. Inhibition of Thrombopoietin/Mpl Signaling in Adult Hematopoiesis Identifies New Candidates for Hematopoietic Stem Cell Maintenance.

Author

Kohlscheen S, Wintterle S, Schwarzer A, Kamp C, Brugman MH, Breuer DC, Büsche G, Baum C, and Modlich U

Subjects
Animals, Bone Marrow Cells metabolism, Cell Membrane metabolism, Mice, Mice, Transgenic, Receptors, Thrombopoietin genetics, Thrombocytopenia metabolism, Hematopoiesis physiology, Hematopoietic Stem Cells metabolism, Receptors, Thrombopoietin metabolism, Signal Transduction physiology, Thrombopoietin metabolism
Abstract

Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, hematopoietic stem cell (HSC) maintenance and post-transplant expansion. Mpl expression is tightly controlled and deregulation of Thpo/Mpl-signaling is linked to hematological disorders. Here, we constructed an intracellular-truncated, signaling-deficient Mpl protein which is presented on the cell surface (dnMpl). The transplantation of bone marrow cells retrovirally transduced to express dnMpl into wildtype mice induced thrombocytopenia, and a progressive loss of HSC. The aplastic BM allowed the engraftment of a second BM transplant without further conditioning. Functional analysis of the truncated Mpl in vitro and in vivo demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding. Intracellular inhibition of Mpl could be excluded as the major mechanism by the use of a constitutive-dimerized dnMpl. To further elucidate the molecular changes induced by Thpo/Mpl-inhibition on the HSC-enriched cell population in the BM, we performed gene expression analysis of Lin-Sca1+cKit+ (LSK) cells isolated from mice transplanted with dnMpl transduced BM cells. The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population). We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations. In summary, we established a novel way of Thpo/Mpl inhibition in the adult mouse and performed in depth analysis of the phenotype including gene expression profiling.

Published
2015
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21. A ubiquitous chromatin opening element prevents transgene silencing in pluripotent stem cells and their differentiated progeny.

Author

Pfaff N, Lachmann N, Ackermann M, Kohlscheen S, Brendel C, Maetzig T, Niemann H, Antoniou MN, Grez M, Schambach A, Cantz T, and Moritz T

Subjects
Animals, Cell Differentiation genetics, Chromatin metabolism, Genetic Vectors genetics, Genetic Vectors metabolism, Humans, Induced Pluripotent Stem Cells cytology, Induced Pluripotent Stem Cells metabolism, Mice, Transgenes, Chromatin genetics, Gene Silencing, Induced Pluripotent Stem Cells physiology
Abstract

Methylation-induced gene silencing represents a major obstacle to efficient transgene expression in pluripotent cells and thereof derived tissues. As ubiquitous chromatin opening elements (UCOE) have been shown to prevent transgene silencing in cell lines and primary hematopoietic cells, we hypothesized a similar activity in pluripotent cells. This concept was investigated in the context of cytidine deaminase (CDD) gene transfer, an approach to render hematopoietic cells resistant to the chemotherapeutic agent Ara-C. When murine induced pluripotent stem cells (iPSC)/embryonic stem cells (ESCs) were transduced with self-inactivating lentiviral vectors using housekeeping (truncated elongation factor 1α; EFS) or viral (spleen focus-forming virus; SFFV) promoters, incorporation of an heterogeneous nuclear ribonucleoproteins A2 B1/chromobox protein homolog 3 locus-derived UCOE (A2UCOE) significantly increased transgene expression and Ara-C resistance and effectively prevented silencing of the SFFV-promoter. The EFS promoter showed relatively stable transgene expression in naïve iPSCs, but rapid transgene silencing was observed upon hematopoietic differentiation. When combined with the A2UCOE, however, the EFS promoter yielded stable transgene expression in 73% ± 6% of CD41(+) hematopoietic progeny, markedly increased CDD expression levels, and significantly enhanced Ara-C resistance in clonogenic cells. Bisulfite sequencing revealed protection from differentiation-induced promoter CpG methylation to be associated with these effects. Similar transgene promoting activities of the A2UCOE were observed during murine neurogenic differentiation, in naïve human pluripotent cells, and during nondirected multilineage differentiation of these cells. Thus, our data provide strong evidence that UCOEs can efficiently prevent transgene silencing in iPS/ESCs and their differentiated progeny and thereby introduce a generalized concept to circumvent differentiation-induced transgene silencing during the generation of advanced iPSC/ESC-based gene and cell therapy products., (Copyright © 2013 AlphaMed Press.)

Published
2013
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22. Efficient hematopoietic redifferentiation of induced pluripotent stem cells derived from primitive murine bone marrow cells.

Author

Pfaff N, Lachmann N, Kohlscheen S, Sgodda M, Araúzo-Bravo MJ, Greber B, Kues W, Glage S, Baum C, Niemann H, Schambach A, Cantz T, and Moritz T

Subjects
Animals, Blotting, Western, Bone Marrow Cells metabolism, Cells, Cultured, DNA Methylation, Embryoid Bodies cytology, Embryoid Bodies metabolism, Embryonic Stem Cells cytology, Embryonic Stem Cells metabolism, Gene Expression Profiling, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, HEK293 Cells, Hematopoietic Stem Cells metabolism, Homeodomain Proteins genetics, Homeodomain Proteins metabolism, Humans, Induced Pluripotent Stem Cells metabolism, Mice, Mice, Inbred C3H, Mice, Inbred NOD, Mice, SCID, Mice, Transgenic, Nanog Homeobox Protein, Octamer Transcription Factor-3 genetics, Octamer Transcription Factor-3 metabolism, Promoter Regions, Genetic genetics, Receptors, CXCR4 genetics, Receptors, CXCR4 metabolism, Reverse Transcriptase Polymerase Chain Reaction, SOXB1 Transcription Factors genetics, SOXB1 Transcription Factors metabolism, Bone Marrow Cells cytology, Cell Differentiation, Hematopoietic Stem Cells cytology, Induced Pluripotent Stem Cells cytology
Abstract

Heterogeneity among induced pluripotent stem cell (iPSC) lines with regard to their gene expression profile and differentiation potential has been described and at least partly linked to the tissue of origin. Here, we generated iPSCs from primitive [lineage negative (Lin(neg))] and nonadherent differentiated [lineage positive (Lin(pos))] bone marrow cells (BM-iPSC), and compared their differentiation potential to that of fibroblast-derived iPSCs (Fib-iPSC) and embryonic stem cells (ESC). In the undifferentiated state, individual iPSC clones but also ESCs proved remarkably similar when analyzed for alkaline phosphatase and SSEA-1 staining, endogenous expression of the pluripotency genes Nanog, Oct4, and Sox2, or global gene expression profiles. However, substantial differences between iPSC clones were observed after induction of differentiation, which became most obvious upon cytokine-mediated instruction toward the hematopoietic lineage. All 3 BM-iPSC lines derived from undifferentiated Lin(neg) cells yielded high proportions of cells expressing the hematopoietic differentiation marker CD41 and in 2 of these lines high proportions of CD41+/ CD45+ cells were detected. In contrast, little hematopoiesis-specific surface marker expression was detected in 4 Lin(pos) BM-iPSC and 3 Fib-iPSC lines. These results were corroborated by functional studies demonstrating robust colony outgrowth from hematopoietic progenitors in 2 of the Lin(neg) BM-iPSCs only. Thus, in conclusion, our data demonstrate efficient generation of iPSCs from primitive hematopoietic tissue as well as efficient hematopoietic redifferentiation for Lin(neg) BM-iPSC lines, thereby supporting the notion of an epigenetic memory in iPSCs.

Published
2012
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23. Release of TcdA and TcdB from Clostridium difficile cdi 630 is not affected by functional inactivation of the tcdE gene.

Author

Olling A, Seehase S, Minton NP, Tatge H, Schröter S, Kohlscheen S, Pich A, Just I, and Gerhard R

Subjects
Bacterial Toxins genetics, Cell Line, Clostridioides difficile genetics, Enterotoxins genetics, Gene Expression Regulation, Bacterial, Humans, Protein Transport, Bacterial Proteins genetics, Bacterial Proteins metabolism, Bacterial Toxins metabolism, Clostridioides difficile metabolism, Enterotoxins metabolism, Gene Silencing
Abstract

The small open reading frame tcdE is located between the genes tcdA and tcdB which encode toxin A (TcdA) and B (TcdB), respectively, within the pathogenicity locus of Clostridium difficile. Sequence and structure similarities to bacteriophage-encoded holins have led to the assumption that TcdE mediates the release of the toxins from C. difficile into the extracellular environment. A TcdE-deficient C. difficile 630 strain was generated by insertional inactivation of the tcdE gene. Data revealed that TcdE does not regulate or affect growth or sporogenesis. TcdE-deficiency was accompanied by a moderately increased accumulation of TcdA and TcdB prior to sporulation in this microorganism. Interestingly, this observation did not correlate with a delayed or inhibited toxin release: inactivation of TcdE neither significantly altered kinetics of release nor the absolute level of secreted TcdA and TcdB, indicating that TcdE does not account for the pathogenicity of C. difficile strain 630. Furthermore, mass spectrometry analysis could not reveal differences in the secretome of wild type and TcdE-deficient C. difficile, indicating that TcdE did not function as a secretion system for protein release. TcdE was expressed as a 19 kDa protein in C. difficile, whereas TcdE expressed in Escherichia coli appeared as a 19 and 16 kDa protein. Expression of the short 16 kDa TcdE correlated with bacterial cell death. We conclude that TcdE does not exhibit pore-forming function in C. difficile since in these cells only the non-lytic full length 19 kDa protein is expressed., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Published
2012
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