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

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.

Routine flow cytometry approach for the evaluation of solid tumor neoplasms and immune cells in minimally invasive samples

“…Combining CC and flow cytometry a high diagnosis yield (81/96) was reached avoiding more invasive procedures. As previously reported (Béné et al, 2016;Ferreira-Facio et al, 2021;Gautam et al, 2008), this was not limited to small round cell tumors, but also in other tumors, such as ADC or SCC. FNA biopsy results can also be useful to establish an initial diagnosis or indicate the need to obtain additional samples (Leon et al, 2004).…”

“…MFC has a well-established role in the diagnosis, prognosis and follow up of hematolymphoid neoplasms and is successfully performed in peripheral blood, BM, body fluids, and tissue samples (Béné et al, 2016). Out of this context, MFC has been introduced in cancer research (Davidson et al, 2007;Davidson et al, 2012) but few studies are devoted to clinical applications for detection of malignant cells (Acosta et al, 2016;Ferreira-Facio et al, 2013;Ferreira-Facio et al, 2021;Gautam et al, 2008;Wong-Arteta et al, 2020).…”

“…In recent years, concurrently to the development of technology and novel antibodies and fluorochromes, multicolor techniques have been proposed to assist in the identification of non-hematopoyetic neoplasms (Ferreira-Facio et al, 2013). An 8-color tube for diagnostic screening and classification of cancer in pediatric populations based on multiparameter flow cytometry has been designed and validated showing high-diagnostic accuracy vs conventional histopathology (Ferreira-Facio et al, 2021).…”

Flow cytometry in acute leukemia diagnostics. Guidelines of Russian-Belarusian multicenter group for pediatric leukemia studies

“…Ключевым отличием бластов при ОЛ является экспрессия линейно-специфических антигенов, в то время как при солидных опухолях кроме отсутствия общелейкоцитарного антигена CD45 наблюдается экспрессия только неспецифических маркеров, встречающихся на гемопоэтических клетках (CD56, CD81, CD9, CD99 и др.) [12][13][14][15][16]. При выявлении в КМ только гемопоэтических клеток в ряде случаев может быть также актуальным вопрос подтверждения опухолевой природы выявленных в образце клеток-предшественников (особенно при их малом количестве).…”

The Value of Methods for Diagnosing Alveolar Rhabdomyosarcoma: A Clinical Case

“…It is mainly due to the need to obtain (fresh) suspensions of individual cells and that, unlike IHC, MFC does not provide information on the structure and location of tumor cells in tissues [7]. Therefore, early studies on the use of MFC in solid tumors mainly focused on detecting disseminated disease in the bone marrow [8,9]. Those studies revealed different antigen expression profiles among metastatic non-hematopoietic bone marrow tumor cells.…”