Quantification of nucleated cells, CD34-positive cells and CFU-GM colonies in single bone marrow samples and bone marrow harvests derived from healthy children.

Little is known regarding bone marrow (BM) cellularity, CD34+ fraction, and CFU-GM colony formation in relation to age and whether healthy children require a reference range distinct from healthy adults. We therefore analyzed a series of single BM aspirates from 45 healthy children who were evaluated as potential BM donors. Thirty-three of these children subsequently donated BM. We quantified the nucleated cell count, fraction of CD34+ cells, and number of CFU-GM colonies in single aspirates and BM harvests. Single aspirates displayed a mean nucleated cell count of 31.3 × 10(6) cells/mL, a mean fraction of 1.17% CD34+ cells, and a mean colony forming potential of 66.6 CFU-GM/10(5) cells. Harvests yielded the same number of nucleated cells but increased numbers of CD34+ cells and CFU-GM compared with single aspirates. The mean nucleated cell count in BM harvests was 31.1 × 10(6) /mL with a mean fraction of 1.95% CD34+ cells and a mean of 112.4 CFU-GM colonies/10(5) cells. The concentration of nucleated cells was elevated compared with reported adult counts, while CD34+ percentage and CFU-GM counts were similar. In this series of healthy children, the fraction of CD34+ cells, CFU-GM colonies, and nucleated cells decreased with age. We did not identify gender specific differences. To our knowledge, this represents the first comprehensive study of CD34+ cell fraction, CFU-GM counts, and nucleated cell numbers in the BM of healthy children. The findings provide valuable information for practical use for BM transplantation and contribute to the understanding of hematopoiesis from birth to adulthood.

De novo intraocular retinoblastoma development after chemotherapy in patients with hereditary retinoblastoma.

OBJECTIVE: Identification of incidence and risk factors for recurrence of de novo retinoblastomas after chemotherapy treatment in patients with hereditary retinoblastoma. METHODS: A retrospective, case-control study of 32 patients (50 eyes) with sporadic or familial bilateral retinoblastomas was conducted. Patients received a systemic chemotherapy regimen applying three courses of a combination of three drugs (including vincristine, etoposide, carboplatin, or cyclophosphamide) followed by additional local therapy. The primary outcome analyzed was the development of retinoblastomas, probably arising as the cause of a new mutational event (de novo) after completion of chemotherapy treatment. RESULTS: Patients were treated with an average of 5.8 +/- 1.8 chemotherapy courses (4.6 +/- 2.4-year follow-up time). Development of de novo tumors occurred in 48% of the treated eyes. These tumors occurred during chemotherapy treatment or within 7 months of chemotherapy completion. No de novo tumors developed in patients older than 3.2 years. Children who developed de novo tumors were significantly younger at the time of diagnosis (6.7 +/- 6.3 months vs 14.4 +/- 11.4 months, P < 0.001), and had a significantly lower number of tumors per eye at treatment begin (2.6 +/- 2.3 tumors vs 4.3 +/- 6.4 tumors, P < 0.001). The difference of the total numbers of retinoblastomas that developed per eye between the patients that developed de novo retinoblastomas during or after chemotherapy and patients who did not was not statistically significant (4.9 +/- 2.7 and 4.3 +/- 6.4, respectively, P = 0.8). No eye was lost because of de novo retinoblastoma development, and 92% of the eyes were preserved. CONCLUSIONS: De novo retinoblastomas developed both during and after completion of chemotherapy treatment. Younger children were at a significantly higher risk for developing de novo intraocular retinoblastomas. Good tumor control and eye preservation rates were achieved with regular and frequent control examinations in addition to the immediate treatment of de novo retinoblastomas.

Prediction of clinical outcome and biological characterization of neuroblastoma by expression profiling.

Neuroblastoma is a common childhood tumor comprising cases with rapid disease progression as well as spontaneous regression. Although numerous prognostic factors have been identified, risk evaluation in individual patients remains difficult. To define a reliable prognostic predictor and gene signatures characteristic of biological subgroups, we performed mRNA expression profiling of 68 neuroblastomas of all stages. Expression data were analysed using support vector machines (SVM-rbf), prediction analysis of microarrays (PAM), k-nearest neighbors (k-NN) algorithms and multiple decision trees. SVM-rbf performed best of all methods, and predicted recurrence of neuroblastoma with an accuracy of 85% (sensitivity 77%, specificity 94%). PAM identified a classifier of 39 genes reliably predicting outcome with an accuracy of 80%. In comparison, conventional risk stratification based on stage, age and MYCN-status only reached a predictive accuracy of 64%. Kaplan-Meier analysis using the PAM classifier indicated a 5-year survival of 20 versus 78% for patients with unfavorably versus favorably predicted neuroblastomas, respectively (P = 0.0001). Significance analysis of microarrays (SAM) identified additional genes differentially expressed among subgroups. MYCN-amplification and high expression of NTRK1/TrkA demonstrated a strong association with specific gene expression patterns. Our data suggest that microarray-derived data in addition to traditional clinical factors will be useful for risk assessment and defining biological properties of neuroblastoma.

Microarray analysis reveals differential gene expression patterns and regulation of single target genes contributing to the opposing phenotype of TrkA- and TrkB-expressing neuroblastomas.

Expression of neurotrophin receptors of the tyrosine kinase receptor (Trk) family is an important prognostic factor in solid tumors including neuroblastoma. High expression of TrkA (NTRK1) is associated with a favorable biology and outcome of neuroblastoma, whereas TrkB (NTRK2) is expressed on aggressive neuroblastomas with unfavorable outcome. To gain new insights into the global gene expression program resulting in these divergent biological phenotypes, we stably expressed either TrkA or TrkB in the human SH-SY5Y neuroblastoma cell line. Gene expression profiles were obtained from parental cells and transfectants activated by their ligands in a time course over 24 h using oligonucleotide microarrays. Basal activation of Trk receptors in the absence of exogenous ligand was sufficient to induce broad and divergent genetic changes. Global gene regulation following external ligand stimulation was surprisingly similar in SY5Y-TrkA and SY5Y-TrkB cells except for the differential expression of distinct novel target genes. Consistent with their divergent biological phenotype, SY5Y-TrkA cells were characterized by upregulation of proapoptotic genes and angiogenesis inhibitors, whereas SY5Y-TrkB cells demonstrated upregulation of genes involved in invasion or therapy resistance. We suggest that the transcriptional program of neuroblastoma cells is modulated by Trk-receptor expression and basal activation rather than by ligand-induced activation. Fine-tuning of the malignant phenotype may be achieved by additional ligand stimulation with subsequent activation of a few specific genes.

High activin A-expression in human neuroblastoma: suppression of malignant potential and correlation with favourable clinical outcome.

Amplification of the MYCN oncogene contributes to the malignant progression of human neuroblastomas, but the mechanisms have remained unclear. We have previously demonstrated that N-Myc facilitates angiogenesis by downregulating an angiogenesis inhibitor identified as the inhibin betaA homodimer activin A. Here, we have sought to define the molecular, biological and clinical consequences of activin A expression in human neuroblastoma. We report that enhanced activin A expression suppresses proliferation and colony formation of human neuroblastoma cells with amplified MYCN in vitro; that it inhibits neuroblastoma growth and angiogenesis in vivo; that it is highly expressed in differentiated, but not undifferentiated human neuroblastomas; and that it correlates with favourable outcome of neuroblastoma patients. Our results indicate that high activin A expression plays an important beneficial role in human neuroblastoma.

Hypoxia-induced erythropoietin expression in human neuroblastoma requires a methylation free HIF-1 binding site.

The glycoprotein hormone Erythropoietin (EPO) stimulates red cell production and maturation. EPO is produced by the kidneys and the fetal liver in response to hypoxia (HOX). Recently, EPO expression has also been observed in the central nervous system where it may be neuroprotective. It remained unclear, however, whether EPO is expressed in the peripheral nervous system and, if so, whether a neuronal phenotype is required for its regulation. Herein, we report that EPO expression was induced by HOX and a HOX mimetic in two cell lines derived from neuroblastoma (NB), a tumor of the peripheral nervous system. Both cell lines with inducible EPO expression, SH-SY5Y and Kelly cells, expressed typical neuronal markers like neuropeptide Y (NPY), growth-associated protein-43 (GAP-43), and neuron-specific enolase (ENO). NB cells with a more epithelial phenotype like SH-SHEP and LAN-5 did not show HOX inducible EPO gene regulation. Still, oxygen sensing and up-regulation of hypoxia-inducible factor-1 (HIF-1) were intact in all cell lines. We found that CpG methylation of the HIF binding site (HBS) in the EPO gene 3' enhancer was only present in the SH-SHEP and LAN-5 cells but not in SH-SY5Y and Kelly cells with regulated EPO expression. The addition of recombinant EPO to all NB cells, both under normoxic and hypoxic conditions, had no effect on cell proliferation. We conclude that the ability to respond to HOX with an increase in EPO expression in human NB may depend on CpG methylation and the differentiation status of these embryonic tumor cells but does not affect the proliferative characteristics of the cells.

Higher vessel densities in retinoblastoma with local invasive growth and metastasis.

In this study, the importance of angiogenesis (the growth of new blood vessels from existing ones) for the growth of retinoblastoma was investigated by a retrospective immunohistochemical analysis. An individual vessel index for each tumor was determined using the endothelial-specific antibody CD 31 for vessel staining. The obtained data were correlated with clinical features, pathohistological characteristics, and the presence of metastasis. In 107 retinoblastomas collected between 1980 and 1990, we found no difference in the vessel densities between uni- and bilateral retinoblastomas (P = 0.41). However, tumors that had invaded the chorioid and/or the optic nerve statistically showed higher vessel densities than tumors without local invasive growth (P = 0.05 and P = 0.024). A tendency of higher vessel densities in retinoblastomas presenting with metastasis at the time of diagnosis was observed (P = 0.11). Based on this observation, we proceeded to examine all retinoblastomas presenting with metastasis at the time of diagnosis. These included patients that were treated between 1968 and 1993. The 18 investigated retinoblastomas had significantly higher vessel densities than all other retinoblastomas presenting without metastasis (P = 0.025). Our data indicate that in retinoblastoma, blood vessels are essential for local and systemic invasive growth. Therefore, an anti-angiogenic therapy could be considered in the multimodal therapy concept for retinoblastomas with invasive growth, both locally and systemically.

Arginine deiminase and other antiangiogenic agents inhibit unfavorable neuroblastoma growth: potentiation by irradiation.

In spite of aggressive therapy, children suffering from neuroblastoma have a poor prognosis. Therapeutic failure is most often observed in neuroblastomas with unfavorable features, including amplification/over-expression of the N-myc oncogene, rapid growth, effective angiogenesis and/or the tendency to metastasize. Here, we have used cultured human neuroblastoma cells with such features and we have examined whether antiangiogenic agents alone or in combination with tumor irradiation inhibit their angiogenesis and growth in vivo. We report that antiangiogenic agents (arginine deiminase, SU5416 and DC101) inhibit in vivo growth of neuroblastomas with unfavorable properties and that these effects are potentiated by simultaneous irradiation. Combination of either agent with irradiation leads to a reduction in the absolute number of tumor vessels and of perfused tumor vessels. Combination of arginine deiminase or DC101 with irradiation does not increase tumor hypoxia. Our data demonstrate for the first time that arginine deiminase suppresses the growth of unfavorable experimental neuroblastomas and that this effect is potentiated by irradiation. We suggest that antiangiogenesis alone or in combination with established therapeutic regimen may improve the outcome of unfavorable neuroblastomas in a clinical setting.