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

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

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

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