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Contribution of multiparameter flow cytometry immunophenotyping to the diagnostic screening and classification of pediatric cancer.

ABSTRACT: Pediatric cancer is a relatively rare and heterogeneous group of hematological and non-hematological malignancies which require multiple procedures for its diagnostic screening and classification. Until now, flow cytometry (FC) has not been systematically applied to the diagnostic work-up of such malignancies, particularly for solid tumors. Here we evaluated a FC panel of markers for the diagnostic screening of pediatric cancer and further classification of pediatric solid tumors. The proposed strategy aims at the differential diagnosis between tumoral vs. reactive samples, and hematological vs. non-hematological malignancies, and the subclassification of solid tumors. In total, 52 samples from 40 patients suspicious of containing tumor cells were analyzed by FC in parallel to conventional diagnostic procedures. The overall concordance rate between both approaches was of 96% (50/52 diagnostic samples), with 100% agreement for all reactive/inflammatory and non-infiltrated samples as well as for those corresponding to solid tumors (n?=?35), with only two false negative cases diagnosed with Hodgkin lymphoma and anaplastic lymphoma, respectively. Moreover, clear discrimination between samples infiltrated by hematopoietic vs. non-hematopoietic tumor cells was systematically achieved. Distinct subtypes of solid tumors showed different protein expression profiles, allowing for the differential diagnosis of neuroblastoma (CD56(hi)/GD2(+)/CD81(hi)), primitive neuroectodermal tumors (CD271(hi)/CD99(+)), Wilms tumors (>1 cell population), rhabdomyosarcoma (nuMYOD1(+)/numyogenin(+)), carcinomas (CD45(-)/EpCAM(+)), germ cell tumors (CD56(+)/CD45(-)/NG2(+)/CD10(+)) and eventually also hemangiopericytomas (CD45(-)/CD34(+)). In summary, our results show that multiparameter FC provides fast and useful complementary data to routine histopathology for the diagnostic screening and classification of pediatric cancer.

SUBMITTER: Ferreira-Facio CS

PROVIDER: S-EPMC3589426 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Contribution of multiparameter flow cytometry immunophenotyping to the diagnostic screening and classification of pediatric cancer.

Ferreira-Facio Cristiane S CS Milito Cristiane C Botafogo Vitor V Fontana Marcela M Thiago Leandro S LS Oliveira Elen E da Rocha-Filho Ariovaldo S AS Werneck Fernando F Forny Danielle N DN Dekermacher Samuel S de Azambuja Ana Paula AP Ferman Sima Esther SE de Faria Paulo Antônio Silvestre PA Land Marcelo G P MG Orfao Alberto A Costa Elaine S ES

PloS one 20130305 3

Pediatric cancer is a relatively rare and heterogeneous group of hematological and non-hematological malignancies which require multiple procedures for its diagnostic screening and classification. Until now, flow cytometry (FC) has not been systematically applied to the diagnostic work-up of such malignancies, particularly for solid tumors. Here we evaluated a FC panel of markers for the diagnostic screening of pediatric cancer and further classification of pediatric solid tumors. The proposed s ...[more]

PMID: 23472067

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Project description: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.

Project description:Understanding the immunological phenotype of transplant recipients is important to improve outcomes and develop new therapies. Immunophenotyping of whole peripheral blood (WPB) by flow cytometry is a rapid method to obtain large amounts of data relating to the outcomes of different transplant treatments with limited patient impact. Healthy individuals and patients with type 1 diabetes (T1D) enrolled in islet transplantation were recruited and WPB was collected. 46 fluorochrome-conjugated mouse-anti-human antibodies were used (43 of 46 antibodies were titrated). BD cytometer setup and tracking beads were used to characterize and adjust for cytometer performance. Antibody cocktails were pre-mixed <60 minutes before staining. Multicolour panels were designed based on fluorochrome brightness, antigen density, co-expression, and fluorochrome spillover into non-primary detectors in each panel on a 5 laser flow cytometer. WPB sample staining used 50-300 μl WPB for each panel and was performed within 2 hours of blood sample collection. Samples were acquired on a BD-LSRFortessa. The operating procedures, including specimen collection, antibody cocktails, staining protocol, flow-cytometer setup and data analysis, were standardized. The staining index of 43 antibodies and the spillover spreading matrix for each panel was calculated. The final concentrations for the 46 antibodies used was determined for staining of WPB samples. Absolute cell-count and 7 leukocyte profiling panels consisting of subsets and/or status of granulocytes, monocytes, dendritic, B, NK, and T cells including regulatory T cells (Tregs) and NKT were designed and established on a 5 laser BD-LSR Fortessa. 13 T1D patients, including 4 islet transplant recipients and 8 healthy controls, were evaluated. The ability to reproducibly measure immune subsets and immune-profiles of islet transplant patients up to 18 months post transplantation has been established as a tool to measure immune cell reconstitution after transplantation.

Dataset Information

Contribution of multiparameter flow cytometry immunophenotyping to the diagnostic screening and classification of pediatric cancer.

Publications

Contribution of multiparameter flow cytometry immunophenotyping to the diagnostic screening and classification of pediatric cancer.

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