Flow Cytometry Immunophenotyping for Diagnostic Orientation and Classification of Pediatric Cancer Based on the EuroFlow Solid Tumor Orientation Tube (STOT)

Cancers, vol. 13, pp. 4945

Abstract

Early diagnosis of pediatric cancer is key for adequate patient management and improved outcome. Although multiparameter flow cytometry (MFC) has proven of great utility in the diagnosis and classification of hematologic malignancies, its application to non-hematopoietic pediatric tumors remains limited. Here we designed and prospectively validated a new single eight-color antibody combination—solid tumor orientation tube, STOT—for diagnostic screening of pediatric cancer by MFC. A total of 476 samples (139 tumor mass, 138 bone marrow, 86 lymph node, 58 peripheral blood, and 55 other body fluid samples) from 296 patients with diagnostic suspicion of pediatric cancer were analyzed by MFC vs. conventional diagnostic procedures. STOT was designed after several design–test–evaluate–redesign cycles based on a large panel of monoclonal antibody combinations tested on 301 samples. In its final version, STOT consists of a single 8-color/12-marker antibody combination (CD99-CD8/numyogenin/CD4-EpCAM/CD56/GD2/smCD3-CD19/cyCD3-CD271/CD45). Prospective validation of STOT in 149 samples showed concordant results with the patient WHO/ICCC-3 diagnosis in 138/149 cases (92.6%). These included: 63/63 (100%) reactive/disease-free samples, 43/44 (98%) malignant and 4/4 (100%) benign non-hematopoietic tumors together with 28/38 (74%) leukemia/lymphoma cases; the only exception was Hodgkin lymphoma that required additional markers to be stained. In addition, STOT allowed accurate discrimination among the four most common subtypes of malignant CD45− CD56++ non-hematopoietic solid tumors: 13/13 (GD2++ numyogenin− CD271−/+ nuMyoD1− CD99− EpCAM−) neuroblastoma samples, 5/5 (GD2− numyogenin++ CD271++ nuMyoD1++ CD99−/+ EpCAM−) rhabdomyosarcomas, 2/2 (GD2−/+ numyogenin− CD271+ nuMyoD1− CD99+ EpCAM−) Ewing sarcoma family of tumors, and 7/7 (GD2− numyogenin− CD271+ nuMyoD1− CD99− EpCAM+) Wilms tumors. In summary, here we designed and validated a new standardized antibody combination and MFC assay for diagnostic screening of pediatric solid tumors that might contribute to fast and accurate diagnostic orientation and classification of pediatric cancer in routine clinical practice.

Authors 22

1. Cristiane de Sá Ferreira-Facio

   Universidade Federal do Rio de Janeiro

   Affiliation as printed

   Cytometry Service, Institute of Paediatrics and Puericultura Martagão Gesteira (IPPMG), Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-612, Brazil

   Internal Medicine Postgraduate Program, Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-617, Brazil

2. Vitor Botafogo

   Universidade Federal do Rio de Janeiro

   Affiliation as printed

   Cytometry Service, Institute of Paediatrics and Puericultura Martagão Gesteira (IPPMG), Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-612, Brazil

   Internal Medicine Postgraduate Program, Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-617, Brazil

3. Patrícia Mello Ferrão

   Universidade Federal do Rio de Janeiro

   Affiliation as printed

   Cytometry Service, Institute of Paediatrics and Puericultura Martagão Gesteira (IPPMG), Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-612, Brazil

4. Maria Clara Canellas

   Universidade Federal do Rio de Janeiro

   Affiliation as printed

   Cytometry Service, Institute of Paediatrics and Puericultura Martagão Gesteira (IPPMG), Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-612, Brazil

5. Cristiane Bedran Milito

   Universidade Federal do Rio de Janeiro · Hospital Universitário Clementino Fraga Filho

   Affiliation as printed

   Department of Pathology, Faculty of Medicine, Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-617, Brazil

6. Sérgio de Oliveira Romano

   Instituto Nacional de Câncer - INCA

   Affiliation as printed

   Laboratory of Anatomical Pathology and Cytopathology, Instituto Nacional de Câncer (INCa), Rio de Janeiro 20220-400, Brazil

7. Daiana V. Lopes

   Universidade Federal do Rio de Janeiro

   Affiliation as printed

   Cytometry Service, Institute of Paediatrics and Puericultura Martagão Gesteira (IPPMG), Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-612, Brazil

8. Lisandra Antônia Castro Teixeira

   Universidade Federal do Rio de Janeiro

   Affiliation as printed

   Cytometry Service, Institute of Paediatrics and Puericultura Martagão Gesteira (IPPMG), Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-612, Brazil

   Internal Medicine Postgraduate Program, Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-617, Brazil

9. Elen Oliveira

   Universidade Federal do Rio de Janeiro

   Affiliation as printed

   Cytometry Service, Institute of Paediatrics and Puericultura Martagão Gesteira (IPPMG), Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-612, Brazil

   Internal Medicine Postgraduate Program, Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-617, Brazil

10. Enrico Bruno-Riscarolli

    Universidade Federal do Rio de Janeiro

    Affiliation as printed

    Cytometry Service, Institute of Paediatrics and Puericultura Martagão Gesteira (IPPMG), Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-612, Brazil

    Internal Medicine Postgraduate Program, Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-617, Brazil

11. Fabiana V. Mello

    Universidade Federal do Rio de Janeiro

    Affiliation as printed

    Cytometry Service, Institute of Paediatrics and Puericultura Martagão Gesteira (IPPMG), Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-612, Brazil

12. Patrícia F. R. Siqueira

    Universidade Federal do Rio de Janeiro

    Affiliation as printed

    Cytometry Service, Institute of Paediatrics and Puericultura Martagão Gesteira (IPPMG), Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-612, Brazil

    Internal Medicine Postgraduate Program, Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-617, Brazil

13. Patrícia Moura

    D’Or Institute for Research and Education

    Affiliation as printed

    I’Dor Institute, Hospital Estadual da Criança, Rio de Janeiro 21330-400, Brazil

14. Francisco Nicanor Macedo

    D’Or Institute for Research and Education

    Affiliation as printed

    I’Dor Institute, Hospital Estadual da Criança, Rio de Janeiro 21330-400, Brazil

15. Danielle Nunes Forny

    Universidade Federal do Rio de Janeiro

    Affiliation as printed

    Department of Pediatric Surgery, Institute of Paediatrics and Puericultura Martagão Gesteira (IPPMG), Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-612, Brazil

16. Luíza Simião

    Universidade Federal do Rio de Janeiro

    Affiliation as printed

    Cytometry Service, Institute of Paediatrics and Puericultura Martagão Gesteira (IPPMG), Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-612, Brazil

17. Ana Luíza Pureza

    Universidade Federal do Rio de Janeiro

    Affiliation as printed

    Cytometry Service, Institute of Paediatrics and Puericultura Martagão Gesteira (IPPMG), Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-612, Brazil

18. Marcelo Gerardin Poirot Land

    Universidade Federal do Rio de Janeiro

    Affiliation as printed

    Internal Medicine Postgraduate Program, Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-617, Brazil

19. Carlos Eduardo Pedreira

    Universidade Federal do Rio de Janeiro

    Affiliation as printed

    Systems and Computing Engineering Department (COPPE-PESC), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro 21941-972, Brazil

20. Jacques J. M. van Dongen Aachen Department of Immunohematology and Blood Transfusion

    Leiden University Medical Center

    Affiliation as printed

    Department of Immunohematology and Blood Transfusion (IHB), Leiden University Medical Center (LUMC), 2333 ZA Leiden, The Netherlands

21. Alberto Órfão corresponding

    Consejo Superior de Investigaciones Científicas · Universidad de Salamanca · Instituto de Investigación Biomédica de Salamanca · Centro de Investigación del Cáncer · Instituto de Biología Molecular y Celular del Cáncer de Salamanca

    Affiliation as printed

    Translational and Clinical Research Program, Centro de Investigación del Cáncer and IBMCC (CSIC-University of Salamanca), Cytometry Service, NUCLEUS, Department of Medicine, University of Salamanca (USAL), Institute of Biomedical Research of Salamanca (IBSAL), 37007 Salamanca, Spain

22. Elaine Sobral da Costa corresponding

    Universidade Federal do Rio de Janeiro

    Affiliation as printed

    Cytometry Service, Institute of Paediatrics and Puericultura Martagão Gesteira (IPPMG), Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-612, Brazil

    Internal Medicine Postgraduate Program, Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-617, Brazil