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

Cristiane de Sá Ferreira-Facio; Vitor Botafogo; Patrícia Mello Ferrão; Maria Clara Canellas; Cristiane B Milito; Sérgio Romano; Daiana V Lopes; Lisandra C Teixeira; Elen Oliveira; Enrico Bruno-Riscarolli; Fabiana V Mello; Patrícia F R Siqueira; Patrícia Moura; Francisco Nicanor Macedo; Danielle N Forny; Luíza Simião; Ana Luíza Pureza; Marcelo Gerardin Poirot Land; Carlos Eduardo Pedreira; Jacques J M van Dongen; Alberto Orfao; Elaine Sobral da Costa

doi:10.3390/cancers13194945

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

Cancers (Basel). 2021 Sep 30;13(19):4945. doi: 10.3390/cancers13194945.

Authors

Cristiane de Sá Ferreira-Facio^{1

2}, Vitor Botafogo^{1

2}, Patrícia Mello Ferrão², Maria Clara Canellas², Cristiane B Milito³, Sérgio Romano⁴, Daiana V Lopes², Lisandra C Teixeira^{1

2}, Elen Oliveira^{1

2}, Enrico Bruno-Riscarolli^{1

2}, Fabiana V Mello², Patrícia F R Siqueira^{1

2}, Patrícia Moura⁵, Francisco Nicanor Macedo⁵, Danielle N Forny⁶, Luíza Simião², Ana Luíza Pureza², Marcelo Gerardin Poirot Land¹, Carlos Eduardo Pedreira⁷, Jacques J M van Dongen⁸, Alberto Orfao⁹, Elaine Sobral da Costa^{1

2}

Affiliations

¹ Internal Medicine Postgraduate Program, Faculty of Medicine, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-617, Brazil.
² 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.
³ Department of Pathology, Faculty of Medicine, Clementino Fraga Filho University Hospital, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro 21941-617, Brazil.
⁴ Laboratory of Anatomical Pathology and Cytopathology, Instituto Nacional de Câncer (INCa), Rio de Janeiro 20220-400, Brazil.
⁵ I'Dor Institute, Hospital Estadual da Criança, Rio de Janeiro 21330-400, Brazil.
⁶ 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.
⁷ Systems and Computing Engineering Department (COPPE-PESC), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro 21941-972, Brazil.
⁸ Department of Immunohematology and Blood Transfusion (IHB), Leiden University Medical Center (LUMC), 2333 ZA Leiden, The Netherlands.
⁹ 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.

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.

Keywords: STOT; diagnosis; flow cytometry; immunophenotyping; pediatric cancer; pediatric solid tumors; standardization.

Abstract

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