Staging and monitoring of childhood rhabdomyosarcoma with flow cytometry

Abstract

Patients with metastatic rhabdomyosarcoma (RMS) have a poor prognosis. The detection of contaminating RMS cells in the bone marrow (BM) is important in clinical staging and risk assessment. The cytological examination of the BM remains the gold standard for the diagnosis of RMS, but has a limited sensitivity. In the present study, 32 BM and two cerebrospinal fluid (CSF) samples from 11 patients with suspected metastasis were analyzed by flow cytometry (FCM) with ganglioside D2 (GD2) conjugated with fluorescein isothiocyanate, cluster of differentiation (CD)90-phycoerythrin, CD45-peridinin chlorophyll protein and CD56-allophycocyanin monoclonal antibody cocktail in parallel to morphological examination at diagnosis or during treatment. Five samples (14.7%) were positive for RMS onup morphological examination. By FCM, 16 samples (47.1%) were positive for RMS. A significant difference was identified between the two methods. The four-color FCM assay successfully detected RMS cells in BM samples to a level of 0.01% (1 per 104 cells). RMS cells demonstrated a phenotype with CD56+/CD90+/CD45−/GD2− expression, which is different from the CD56+/CD90+/CD45−/GD2+ expression phenotype in neuroblastoma cells. The follow-up of four patients by FCM demonstrated that two patients became minimal residual disease-negative following two and four cycles of chemotherapy, respectively, and survived. The other two cases remained FCM-positive despite receiving four courses of chemotherapy and consequently succumbed to progressive disease. In addition, FCM analysis of the CSF samples from one patient confirmed a diagnosis of CSF metastasis with RMS. In conclusion, FCM may have a role not only in staging and monitoring the effects of therapy, but also in providing diagnostic confirmation of CSF metastasis with RMS.

The important role of routine cytopathology in pediatric precision oncology

“…Flow cytometry was used to analyze tumor cell content in 11 cases using surface markers that have high expression in tumor cells. [27][28][29][30][31] The cell surface markers used in these experiments were selected based on the histopathologic diagnosis of the original patient tumor samples, as follows: R-phycoerythrin CD99 (PE) (clone TU12; Ewing sarcoma [EWS]; 1:20 dilution; BD Biosciences); PE ganglioside GD2 (clone 14G2a; NBL; 1:20 dilution; BD Biosciences); PE CD133 (clone AC133; CNS tumors; 1:20 dilution; Miltenyi Biotec), and fluorescein isothiocyanate CD90 (clone DG3; CNS tumors; 1:20 dilution; Miltenyi Biotec). Cells were stained according to the manufacturer's instructions before being acquired with the BD Biosciences LSRFortessa cell analyzer).…”

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

“…Interestingly, these studies already revealed different antigen expression profiles among BM metastatic non-hematopoietic tumor cells, some of which emerged as strongly associated with (or even specific for) some diagnostic subtypes of pediatric solid tumors [ 15 , 16 , 17 , 18 ]. Thus, co-expression of CD56 + , CD90 + , GD2 + , CD81 + , and CD9 + in the absence of CD45 is a typical immunophenotypic feature of neuroblastoma (NBL) cells [ 19 , 20 ], while a CD99 + CD45 − phenotype has been associated with Ewing sarcoma (ES) [ 14 , 18 ], and co-expression of CD90 + , CD56 + , and CD57 −/+ in the absence of CD45 is most frequently observed in rhabdomyosarcoma (RMS) tumor cells [ 16 , 21 ].…”

Fluorescence and antioxidant activity of heterologous expression of phycocyanin and allophycocyanin from Arthrospira platensis

“…Phycobiliproteins conjugated to biomolecules such as immunoglobulin or streptavidin have achieved great success in flow cytometry, fluorescence-activated cell sorting (FACS), histochemistry, imaging, and detection of reactive oxygen species. At present, phycoerythrin and allophycocyanin are commonly used in the market because of their fluorescent activity ( 7 – 9 ). Phycobiliproteins also have the properties of removing excessive reactive oxygen species in the body and increasing the activity of antioxidant-related enzymes ( 10 ).…”