Prognostic value of initial bone marrow disease detection by multiparameter flow cytometry in children with neuroblastoma.

PURPOSE: Multicolor flow cytometry (MFC) is widely available, fast and has an easy-to perform approach for finding neuroblastoma (NB) cells among normal bone marrow (BM) hematopoietic cells. Aim of the study was to investigate prognostic significance of initial MFC tumor cells' detection in BM of children with NB. METHODS: 51 patients (24 boys and 27 girls) aged from 6 days to 15 years (median age 1 year 3 months) with NB were included in the study. BM samples at the time of diagnosis were obtained from 2 to 5 aspiration sites per patient. CD45(-)CD56(+)CD81(+)GD2(+)-cells were evaluated by MFC. RESULTS: NB cells were detected in BM by FC more frequently compared to conventional cytomorphology (49.0% and 29.4% patients, respectively, р = 0.043). Patients with NB cells detected in BM by MFC had significantly worse event-free survival and cumulative incidence of relapse/progression [0.24(0.08) and 0.60(0.10), respectively] compared to children with negative result of immunophenotyping [0.85(0.07) and 0.12(0.06), respectively, p < 0.001 in both cases]. BM involvement detection by MFC maintained its prognostic significance in various patients groups. In multivariate analysis, immunophenotyping proved to be an independent prognostic factor when analyzed jointly with other NB risk factors. In 42 patients BM involvement was also studied by RQ-PCR for PHOX2B and TH genes expression. Within groups of patients divided by RQ-PCR positivity, MFC-positivity retained prognostic significance. CONCLUSIONS: Thus flow cytometric BM involvement detection has very strong prognostic impact even stronger than RQ-PCR. It could be used in combination with other parameters for the treatment strategy choice in patients with NB.

Prospective investigation of applicability and the prognostic significance of bone marrow involvement in patients with neuroblastoma detected by quantitative reverse transcription PCR.

BACKGROUND: Detection of bone marrow (BM) involvement in patients with neuroblastoma is crucial for staging and defining prognosis. Furthermore, the persistence of residual tumor cells in the BM is associated with an unfavorable outcome. METHODS: Expression of PHOX2B, TH, ELAVL4, and B4GALNT1 (GD2-synthase) was analyzed by quantitative polymerase chain reaction in neuroblastoma cell lines, control BM samples, and in BM samples from patients. The threshold level of expression for each gene was established through receiver operator characteristic analysis and used to determine the diagnostic test performance. The prognostic significance of BM involvement was assessed by survival rates calculations. The median of follow-up time was 36.1 months. RESULTS: Neither PHOX2B nor TH expression was detected in control BM, while expression of ELAVL4 was found in 20 (76.9%) and GD2-synthase in 15 (57.7%) of 26 samples. The overall correct predictive value for TH, ELAVL4, and GD2-synthase, based on thresholds levels, was 0.952, 0.828, and 0.767, respectively, whereas the overall correct predictive value for PHOX2B was 0.994. The PHOX2B/TH expression in diagnostic BM of patients with neuroblastoma corresponded with a decreased survival rate (P < 0.001) in the total cohort and in different risk groups. Predominance of normalized expression of PHOX2B over TH > 1.68 in the diagnostic BM samples demonstrated an adverse prognostic effect (P = 0.006). Persistence of PHOX2B/TH expression in the BM during and after induction chemotherapy resulted in dismal outcome (P = 0.022 and P = 0.012). CONCLUSION: PHOX2B and TH are the most optimal markers for detection of BM involvement, allowing identification of high-risk patients. Predominance of PHOX2B expression over TH has a strong adverse prognostic impact.

An inter-laboratory comparison of PNH clone detection by high-sensitivity flow cytometry in a Russian cohort.

OBJECTIVES: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by partial or absolute deficiency of glycophosphatidyl-inositol (GPI) anchor-linked surface proteins on blood cells. A lack of precise diagnostic standards for flow cytometry has hampered useful comparisons of data between laboratories. We report data from the first study evaluating the reproducibility of high-sensitivity flow cytometry for PNH in Russia. METHODS: PNH clone sizes were determined at diagnosis in PNH patients at a central laboratory and compared with follow-up measurements in six laboratories across the country. Analyses in each laboratory were performed according to recommendations from the International Clinical Cytometry Society (ICCS) and the more recent 'practical guidelines'. Follow-up measurements were compared with each other and with the values determined at diagnosis. RESULTS: PNH clone size measurements were determined in seven diagnosed PNH patients (five females, two males: mean age 37 years); five had a history of aplastic anemia and three (one with and two without aplastic anemia) had severe hemolytic PNH and elevated plasma lactate dehydrogenase. PNH clone sizes at diagnosis were low in patients with less severe clinical symptoms (0.41-9.7% of granulocytes) and high in patients with severe symptoms (58-99%). There were only minimal differences in the follow-up clone size measurement for each patient between the six laboratories, particularly in those with high values at diagnosis. CONCLUSIONS: The ICCS-recommended high-sensitivity flow cytometry protocol was effective for detecting major and minor PNH clones in Russian PNH patients, and showed high reproducibility between laboratories.