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

SIMPLE SUMMARY: Pediatric solid tumors are a heterogenous group of diseases that comprise ≈ 40% of all pediatric cancers, early diagnosis being key for improved survival. Here we designed, tested, and validated a single eight-color tube for the diagnostic screening of pediatric cancer—solid tumor orientation tube (STOT)—based on multiparameter flow cytometry vs. conventional diagnostic procedures. Prospective clinical validation of STOT in 149 samples (63 tumor mass, 38 bone marrow, 30 lymph node, and 18 body fluid samples) screened for pediatric cancer, apart from 26 blood specimens that were excluded from analysis, showed concordant results with the final WHO/ICCC-3 diagnosis in 138/149 cases (92.6%). This included correct diagnostic orientation by STOT in 43/44 (98%) malignant and 4/4 (100%) benign non-hematopoietic tumors, together with 28/38 (74%) leukemia/lymphoma cases. The only recurrently missed diagnosis was Hodgkin lymphoma (0/8), which would require additional markers. These results support the use of STOT as a complementary tool for fast and accurate diagnostic screening, orientation, and classification of pediatric cancer in suspicious patients. 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/(nu)myogenin/CD4-EpCAM/CD56/GD2/(sm)CD3-CD19/(cy)CD3-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(++) (nu)myogenin(−) CD271(−/+) (nu)MyoD1(−) CD99(−) EpCAM(−)) neuroblastoma samples, 5/5 (GD2(−) (nu)myogenin(++) CD271(++) (nu)MyoD1(++) CD99(−/+) EpCAM(−)) rhabdomyosarcomas, 2/2 (GD2(−/+) (nu)myogenin(−) CD271(+) (nu)MyoD1(−) CD99(+) EpCAM(−)) Ewing sarcoma family of tumors, and 7/7 (GD2(−) (nu)myogenin(−) CD271(+) (nu)MyoD1(−) 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.