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

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.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 (CD56hi/GD2+/CD81hi), primitive neuroectodermal tumors (CD271hi/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.This work has been partially supported by the following grants: EO was partially supported by grant from CNPq- Brazilian National Research Council (Brasília, Brazil). MF was partially supported by a grant from FAPERJ- Research support foundation of Rio de Janeiro, (Rio de Janeiro, Brazil) and now she is partially supported by a grant from CAPES/Ministério da Educação (Brasília, Brazil). VB was partially supported by FAPERJ- Research support foundation of Rio de Janeiro, (Rio de Janeiro, Brazil). ESC was partially supported by grant from CNPq- Brazilian National Research Council (Brasília, Brazil) and FAPERJ- Research support foundation of Rio de Janeiro, (Rio de Janeiro, Brazil).Peer Reviewe

Flow cytometry immunophenotyping for diagnostic orientation and classification of pediatric cancer based on the euroflow solid tumor orientation tube (Stot)

© 2021 by the authors.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.This research was funded by the EuroFlow Consortium; Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro, Brazil (FAPERJ), numbers: E26/110.105/2014, E-26/010.101259/2018, and E26/102.191/2013; grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brasília, Brazil (CNPQ), Brasília, Brazil, numbers: 303765/2018-6, 409440/2016-7, and 400194/2014-7; and Instituto Desiderata/Chevron, Rio de Janeiro, Brazil, grant “Actions to improve pediatric cancer assistance in RJ”; the EuroFlow Consortium (grant LSHB-CT-2006-018708); Centro de Investigación Biomédica en Red de Cáncer (CIBER-ONC; Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain and FONDOS FEDER), numbers: CB16/12/00400, CB16/12/00233, CB16/12/00369, CB16/12/00489 and CB16/12/00480; grant from Bilateral Cooperation Program between Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES (Brasília/Brazil) and Dirección General de Políticas Universitárias (DGPU)-Ministério de Educación, Cultura y Deportes (Madrid/Spain) number DGPU 311/15

Immunophenotypic shifts during minimal residual evaluation in a case of leukemic form of anaplastic large cell lymphoma ALK+

Abstract Background This study aims to describe immunophenotypic explorations at diagnosis and follow up of a pediatric patient with leukemic phase of ALK+ anaplastic large cell lymphoma (ALCL) by multiparametric flow cytometry (MFC). Case An 8‐color MFC combination of antibodies allowed to identify neoplastic cells in concentrations until 0.02% during minimal residual disease (MRD) monitoring. Immunophenotypic shifts occurred in key markers as CD30, CD7, CD2, and CD5, however neoplastic cells were clearly discriminated from normal populations. Conclusion MFC can be a useful tool for ALCL diagnosis and MRD monitoring and may support therapeutic decisions

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

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

Immunophenotypic identification and chraracterization of pediatric tumor samples.

<p>In panel A, an illustrating example of the gating strategy and bivariate dot plot combinations used for the identification of CD45− tumor cells, CD45− residual stromal cells (e.g. endothelial cells and mesenquimal cells) and infiltrating hematopoietic cells (e.g. neutrophils, B and T cells) is shown. In turn, in panels B to J the immunophenotypic profile of CD45− tumor cells from a neuroblastoma (panels B and H), a PNET (panels C and I) and a rhabdomyossarcoma (panels D and J) tumor are shown together with representative pictures of the histophathological and immunohistochemical profiles of the same tumors stained with hematoxilin & eosin plus cromogranin (neuroblastoma cells in panel E), CD99 (PNET cells in panel F) and _(nu)myogenin (rhabdomyossarcoma cells in panel G).</p

Comparison between multiparameter flow cytometry (MFC) and histopathology plus immunohistochemistry (IH) as regards identification of infiltration by neoplastic cells versus normal/reactive cells.

<p>All 8 samples were obtained from pediatric cancer patients, but they were all negative for the presence of tumor cells by conventional diagnostic approaches. LL: lymphoblastic lymphoma; PNET- primitive neuroectodermal tumor.</p

Pattern of antigen expression by tumor cells from different diagnostic categories of pediatric solid tumors.

<p>−: negative; +lo :low expression levels/cells; +: positive; +hi: strong expression levels/cells.</p><p>Both CD7 and CD8 were systematically negative in all tumors analyzed.</p>*<p>The only ganglioneuroblastoma tumor analyzed showed a similar profile but it contained two distinct populations which differed on CD56, CD9 and CD81 expression, in the absence of CD117.</p> <p>CD271 was only partially present in one neuroblastoma tumor.</p>§<p>% of positive cells only among positive case.</p