Flow cytometric phenotype of rhabdomyosarcoma bone marrow metastatic cells and its implication in differential diagnosis with neuroblastoma.

BACKGROUND: The goal of this study was to develop a flow cytometric (FCM) method for assessing the presence of metastatic cells in bone marrow (BM) and peripheral blood (PB) obtained from rhabdomysarcoma (RMS) patients. Myogenin (Myf4), a specific molecular RMS marker, was also investigated in the same samples. Since neuroblastoma (NB) metastasizes to the BM, the potential application of cytometry in differential diagnosis was explored. PATIENTS AND METHODS: CD45, CD56, CD90 and CD57 antibodies were used in 7 paired BM and PB samples (from 7 RMS stage IV patients at presentation), 23 BM samples (from 13 RMS stage I and II patients at presentation), and ten paired BM and PB samples taken at presentation and five BM samples taken at recurrence from 13 NB stage 4 patients. RESULTS: All seven BM samples from RMS stage IV (but not those from patients with localized disease) showed both the CD45- CD56+ phenotype and the Myf4 transcript. Four cases also showed CD90 and two CD57 positivity. Neither the CD45- CD56+ phenotype, nor Myf4 were recorded in the BM and PB samples from patients with localized disease. All the NB BM samples (15/15) showed the CD45- CD56+ CD90+ phenotype and 10/15 also showed CD57 positivity. Only 3/10 blood samples from the NB patients revealed tumor cells. CONCLUSION: CD45, CD56, CD90 and CD57 antibodies can be used in FCM for marrow metastasis detection in both, RMS and NB patients.

Detecting CD56+/NB84+/CD45- immunophenotype in the bone marrow of patients with metastatic neuroblastoma using flow cytometry.

BACKGROUND: In order to identify neuroblastoma cells infiltrating the bone marrow, a triple-color flow-cytometric assay was developed combining CD56 and CD45 with the intracellular anti-NB84 specific antibody. MATERIALS AND METHODS: The bilateral aspirates obtained from 27 consecutive children over the age of one year with stage 4 neuroblastoma were evaluated. RESULTS: Neuroblastoma cells were detected in the bone marrow of 17/27 (63%) and 19/27 (70%) cases using cytomorphology and triple-color flow-cytometry, respectively. Using cytometry, the percentage of CD56+/NB84+/CD45-cells infiltrating the bone marrow ranged from 0.02% to 65%. Five out of eight patients without bone marrow involvement according to cytometry are in continuous complete remission, while only 3 out of 19 patients whose bone marrow gave positive results are still alive. CONCLUSION: By combining CD45 and CD56 with the specific antibody, NB84, directed against neuroblastoma cells, we developed a rapid and reliable cytometric assay that can be associated with conventional cytomorphological bone marrow evaluation to detect infiltrating neuroblastoma cells, especially in cases of dubious positivity.

Molecular detection of dopamine decarboxylase expression by means of reverse transcriptase and polymerase chain reaction in bone marrow and peripheral blood: utility as a tumor marker for neuroblastoma.

A highly sensitive molecular method was used to evaluate the presence of dopamine decarboxylase (DDC) mRNA in the bone marrow and peripheral blood of patients with neuroblastoma (NB). DDC, like tyrosine hydroxylase (TH), is an enzyme involved in the catecholamine synthesis pathway and has recently been proposed as a specific marker of NB among pediatric malignancies. DDC transcript was detected in five of five NB cell lines, 10 of 10 NB primary tumors, 17 of 18 (94%) bone marrow samples, and 12 of 18 (66%) blood samples drawn at diagnosis in 18 patients affected by disseminated NB. In contrast, no PCR signal was found in 20 bone marrow samples obtained from patients with other malignancies or in eight of nine marrow and blood samples drawn from patients with localized NB (two stage 2 and seven stage 3). In addition, all marrow and blood samples obtained from NB patients at relapse revealed DDC mRNA. Furthermore, the percentage of DDC-positive samples was lower among the samples drawn from these patients during treatment. By comparison with conventional methods for disease evaluation, DDC transcript research can increase the sensitivity of NB cell detection in marrow and blood samples at diagnosis and during the treatment and follow-up of NB patients. These results suggest that finding DDC mRNA in NB patients could be a potential marker for minimal residual disease study.

Immunomodulation in a treatment program including pre- and post-operative interleukin-2 and chemotherapy for childhood osteosarcoma.

AIMS AND BACKGROUND: The treatment applied in our Institution to children with localized osteosarcoma between 1991 and 1999 consisted of four interleukin 2 (IL-2) courses (9 x 10(6) IU/mL/daily x 4), alternated with pre- and post-operative polichemotherapy. The aims of the present study were to quantify the modifications of some immunological parameters induced by IL-2 and to verify whether polychemotherapy could reduce them. An additional aim was to assess whether any correlation between the immune modifications and the clinical outcome could be found. PATIENTS AND METHODS: We evaluated in 18 consecutive patients the following changes, induced in blood by each IL-2 course: number of lymphocyte subpopulations and natural killer (NK) cells, lymphokine activated killer (LAK) and NK activities. RESULTS: Chemotherapy did not influence the modifications of the number of NK and CD4+ cells and of the LAK and NK activities, induced by each of the four courses of IL-2. The magnitudo of the NK activity and the peak of the NK absolute counts significantly correlated with the clinical outcome. CONCLUSIONS: The results show that the use of IL-2 permitted a repeated immune activation despite the intensive chemotherapy. Furthermore, although the limited number of cases precludes any definitive conclusion, the results suggest a possible role of the NK cells in the control of osteosarcoma.