Testosterone-mediated activation of androgenic signalling sustains in vitro the transformed and radioresistant phenotype of rhabdomyosarcoma cell lines.

PURPOSE: Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in childhood, rarely affects adults, preferring male. RMS expresses the receptor for androgen (AR) and responds to androgen; however, the molecular action of androgens on RMS is unknown. METHODS: Herein, testosterone (T) effects were tested in embryonal (ERMS) and alveolar (ARMS) RMS cell lines, by performing luciferase reporter assay, RT-PCR, and western blotting experiments. RNA interference experiments or bicalutamide treatment was performed to assess the specific role of AR. Radiation treatment was delivered to characterise the effects of T treatment on RMS intrinsic radioresistance. RESULTS: Our study showed that RMS cells respond to sub-physiological levels of T stimulation, finally promoting AR-dependent genomic and non-genomic effects, such as the transcriptional regulation of several oncogenes, the phosphorylation-mediated post-transductional modifications of AR and the activation of ERK, p38 and AKT signal transduction pathway mediators that, by physically complexing or not with AR, participate in regulating its transcriptional activity and the expression of T-targeted genes. T chronic daily treatment, performed as for the hormone circadian rhythm, did not significantly affect RMS cell growth, but improved RMS clonogenic and radioresistant potential and increased AR mRNA both in ERMS and ARMS. AR protein accumulation was evident in ERMS, this further developing an intrinsic T-independent AR activity. CONCLUSIONS: Our results suggest that androgens sustain and improve RMS transformed and radioresistant phenotype, and therefore, their therapeutic application should be avoided in RMS post puberal patients.

Two-stage phase II study of imatinib mesylate in subjects with refractory or relapsing neuroblastoma.

BACKGROUND: Cure rate for subjects with refractory or relapsing metastatic neuroblastoma is <5%. In the search for a novel therapy, continuous daily oral administration of imatinib mesylate was evaluated. PATIENTS AND METHODS: Twenty-four subjects were enrolled in a two-stage study. Imatinib was administered for the first 4 weeks (cycle) at 170 mg/sqm b.i.d. If no major toxicity occurred, the dose was escalated to 300 mg/sqm b.i.d. for a maximum of 12 cycles. Clinical response and toxicity were evaluated according to international criteria. Pharmacokinetics (PK) profiles and tyrosine hydroxylase (TH) mRNA expression were also determined in a subset of subjects. RESULTS: Five (21%) complete responses, with one subject still alive at 68 months, and 2 (8%) partial responses lasting up to 29 months were obtained. No grade 4 toxicity was observed. At steady-state, PK exposure (69.7 µg h/ml) was similar to that of adults receiving 1000 mg/die. Responses appear to correlate with the absence or presence of metastasis in the bone marrow (BM) alone, with low TH expression levels at study entry and low imatinib exposure. CONCLUSIONS: Imatinib mesylate was well-tolerated and effective in the subset of subjects with low BM infiltration as only site of metastasis. Study identifier EudraCT: 2005-005778-63.

Synthesis of Ge-imogolite: influence of the hydrolysis ratio on the structure of the nanotubes.

The synthesis protocol for Ge-imogolite (aluminogermanate nanotubes) consists of 3 main steps: base hydrolysis of a solution of aluminum and germanium monomers, stabilization of the suspension and heating at 95 °C. The successful synthesis of these nanotubes was found to be sensitive to the hydrolysis step. The impact of the hydrolysis ratio (from n(OH)/n(Al) = 0.5 to 3) on the final product structure was examined using a combination of characterization tools. Thus, key hydrolysis ratios were identified: n(OH)/n(Al) = 1.5 for the formation of nanotubes with structural defects, n(OH)/n(Al) = 2 for the synthesis of a well crystallized Ge imogolite and n(OH)/n(Al) > 2.5 where nanotube formation is hindered. The capability of controlling the degree of the nanotube's crystallinity opens up interesting opportunities in regard to new potential applications.

Correlated TKD/EDS - TEM - APT analysis on selected interfaces of CoSi2 thin films.

Correlative analysis is a powerful way to relate crystallographic and chemical information to the properties of materials. In this work, a procedure is proposed to select and analyze interfaces of polycrystalline thin film materials through correlative transmission Kikuchi diffraction/energy dispersive X-ray spectroscopy (TKD/EDS), transmission electron microscopy (TEM) and atom probe tomography (APT). TKD provides information on the crystallographic orientation. The EDS analysis performed together with TKD in the scanning electron microscope (SEM) makes chemical information available allowing phases of similar crystal structure, but with a different composition to be distinguished. The information of TKD/EDS can be correlated to successive TEM and APT analysis on selected interfaces for structural and chemical analysis at the atomic scale. An interface of an epitaxial orientated grain of a polycrystalline CoSi2 thin film on (111)Si is selected and analyzed. The selected interface has a twin character and shows facets of different orientation and area. Site-specific segregation of Ge to junctions of the facets is evidenced. The correlation between local strain from misfit (defects) at the interface and segregation is discussed.

Three-dimensional distribution of tyrosine hydroxylase, vasopressin and oxytocin neurones in the transparent postnatal mouse brain.

Over the years, advances in immunohistochemistry techniques have been a critical step in detecting and mapping neuromodulatory substances in the central nervous system. The better quality and specificity of primary antibodies, new staining procedures and the spectacular development of imaging technologies have allowed such progress. Very recently, new methods permitting tissue transparency have been successfully used on brain tissues. In the present study, we combined whole-mount immunostaining for tyrosine hydroxylase (TH), oxytocin (OXT) and arginine vasopressin (AVP), with the iDISCO+ clearing method, light-sheet microscopy and semi-automated counting of three-dimensionally-labelled neurones to obtain a (3D) distribution of these neuronal populations in a 5-day postnatal (P5) mouse brain. Segmentation procedure and 3D reconstruction allowed us, with high resolution, to map TH staining of the various catecholaminergic cell groups and their ascending and descending fibre pathways. We show that TH pathways are present in the whole P5 mouse brain, similar to that observed in the adult rat brain. We also provide new information on the postnatal distribution of OXT and AVP immunoreactive cells in the mouse hypothalamus, and show that, compared to AVP neurones, OXT neurones in the supraoptic (SON) and paraventricular (PVN) nuclei are not yet mature in the early postnatal period. 3D semi-automatic quantitative analysis of the PVN reveals that OXT cell bodies are more numerous than AVP neurones, although their immunoreactive soma have a volume half smaller. More AVP nerve fibres compared to OXT were observed in the PVN and the retrochiasmatic area. In conclusion, the results of the present study demonstrate the utility and the potency of imaging large brain tissues with clearing procedures coupled to novel 3D imaging technologies to study, localise and quantify neurotransmitter substances involved in brain and neuroendocrine functions.

[Limitations of active therapeutic and palliative care in chronic respiratory disease]. / Intensité des soins lors des exacerbations et des complications des pathologies respiratoires chroniques.

The issue of intensive and palliative care in patients with chronic disease frequently arises. This review aims to describe the prognostic factors of chronic respiratory diseases in stable and in acute situations in order to improve the management of these complex situations. The various laws on patients' rights provide a legal framework and define the concept of unreasonable obstinacy. For patients with chronic obstructive pulmonary disease, the most robust decision factors are good knowledge of the respiratory disease, the comorbidities, the history of previous exacerbations and patient preferences. In the case of idiopathic pulmonary fibrosis, it is necessary to know if there is a prospect of transplantation and to assess the reversibility of the respiratory distress. In the case of amyotrophic lateral sclerosis, treatment decisions depend on the presence of advance directives about the use of intubation and tracheostomy. For lung cancer patients, general condition, cancer history and the tumor treatment plan are important factors. A multidisciplinary discussion that takes into account the patient's medical history, wishes and the current state of knowledge permits the taking of a coherent decision.

Coexpression of messenger RNA for TRK protooncogene and low affinity nerve growth factor receptor in neuroblastoma with favorable prognosis.

Nerve growth factor (NGF), essential for differentiation and survival of sympathetic neurons is suggested to play a role in differentiation or regression of neuroblastoma. Expression of mRNA for the trk protooncogene, encoding a tyrosine kinase receptor essential for functional NGF signal transduction, and mRNA for the low affinity NGF receptor (LNGFR) was examined in 45 neuroblastomas and 3 benign ganglioneuromas using Northern blot analysis. Expression of trk mRNA and LNGFR mRNA correlated with young age, favorable clinical stages, and absence of N-myc amplification. All children (n = 19) with neuroblastomas coexpressing mRNA for trk and LNGFR are alive 8-84 months from diagnosis, regardless of age and stage. In contrast, no child (n = 15) with tumor lacking trk mRNA is alive without disease. Three subsets of patients were distinguished, one favorable (trk+, LNGFR+, n = 19, 100% survival probability), one intermediate (trk+, LNGFR-, n = 11, 62.3% survival probability), and one unfavorable (trk-, LNGFR +/-, n = 15, 0% survival probability, P < 0.001). In widespread neuroblastoma stage IVS prone to spontaneous regression, three tumors coexpressing trk and LNGFR mRNAs regressed after no or minimal therapy while the remaining tumor expressing trk but not LNGFR mRNA progressed to a fatal outcome. It is concluded that neuroblastomas coexpressing mRNA for both NGF receptor subtypes are favorable tumors likely to differentiate or regress spontaneously or respond to conventional therapy. It is further hypothesized that loss of functional NGF receptors is an important step in tumorigenesis of undifferentiated malignant childhood neuroblastoma. For these unfavorable tumors current therapy remains futile and first-line innovative therapy is justified.

Effects of a single course of deferoxamine in neuroblastoma patients.

A phase II trial of a single 5-day course of deferoxamine in 9 patients with neuroblastomas was completed. Within 2 days of completion of treatment responses were observed in 7 of 9 patients and there was no drug toxicity. These responses were a decrease in bone marrow infiltration and, in one patient, a measurable reduction in her tumor mass. We conclude that deferoxamine given as an 8-h i.v. infusion daily for 5 days at 150 mg/kg/day has antitumor activity.

Lack of correlation between N-myc and MAX expression in neuroblastoma tumors and in cell lines: implication for N-myc-MAX complex formation.

Detectable levels of MAX messenger RNA were found in a set of human neuroblastoma tumors and established cell lines. MAX mRNA levels were independent of tumor stage and N-myc genomic amplification. By contrast, N-myc mRNA transcripts were detectable only in tumors with amplification of N-myc gene and in cell lines. Analysis by reverse transcriptase polymerase chain reaction and hybridization to specific oligodeoxynucleotide probes revealed approximately equal amounts of two MAX transcripts in all cases analyzed. Immunoprecipitations with a specific antibody to MAX detected two proteins of M(r) 21,000 and 22,000 in approximately equal amounts in all neuroblastoma lines regardless of N-myc amplification and/or expression. On the other hand, protein binding to the myc DNA consensus sequence correlated with N-myc expression in neuroblastoma cells. Thus, N-myc expression might be a limiting factor in the formation of the N-myc-MAX heterodimer in neuroblastomas.

A pharmacokinetic study of high-dose continuous infusion cisplatin in children with solid tumors.

The pharmacokinetics of cisplatin were investigated in 14 patients, aged 10 months through 13 years who were affected by solid malignant tumors. High-dose cisplatin (40 mg/m2/d) was administered with repeated courses for five days as a continuous intravenous (IV) infusion. Total platinum (Pt) levels in plasma and urine and free (protein-unbound) Pt levels in plasma ultrafiltrate were determined by inductively coupled plasma atomic emission spectrometry (ICP-AES). Areas under the concentration v time curve (AUCs) for mean total and free Pt levels were calculated for the 120-hour period of infusion and for the 384 and 120 hours following its completion, respectively. Half-lives of total and free Pt in plasma were calculated for the 216 hours following completion of infusion in five patients at their first course. The fraction of the administered Pt dose excreted in urine as Pt was determined for the five-day period of infusion and seven-day period after its completion. A total of 36 courses were studied. Maximum average Pt levels were reached after 120 hours of infusion: at the first course, 3.22 and 0.17 micrograms/mL for total and free Pt, respectively. Platinum levels declined according to a biexponential model, with initial half-lives of 18.3 and 16.9 minutes, and terminal half-lives of 81.9 and 59.0 hours as determined for total and free Pt, respectively. In the second and third courses studied there was a progressive increase in mean Pt plasma levels. Consequently, the free drug exposure as measured by AUC increased in all patients with repeated courses: 47.7% for the second and 124.4% for the third course, when compared with the first. At the same time, the mean fraction of the dose excreted in the urine for the 12-day period considered, was 44.1% for the first course, 36.2% for the second, and 28.4% for the third. The progressive enhancement of tissue exposure to the free cytotoxic drug, resulting from a reduced renal clearance of Pt with sequential courses of cisplatin, produced mainly increased toxicity while therapeutic effect progressively diminished.

N-myc genomic content and DNA ploidy in stage IVS neuroblastoma.

DNA ploidy and N-myc genomic content were analyzed in a series of stage IVS neuroblastomas by flow cytometry and Southern blot hybridization, respectively. Of the 12 stage IVS neuroblastomas studied, nine were aneuploid (DNA index [DI] greater than 1), two were diploid (DI = 1), and one was not assessable for DNA content due to insufficient tumor material. N-myc gene amplification was present in two of 12 tumors. None of the aneuploid tumors exhibited N-myc amplification. Among the aneuploid neuroblastomas, the DIs were between 1.27 and 1.60, ie, in the near-triploid range. The follow-up from diagnosis ranged from 1 to 41 months (mean, 20 months). The nine neuroblastomas with near-triploid DNA content were free of disease at the end of the follow-up period. In contrast, a rapid and fatal tumor progression was observed for the three neuroblastomas with N-myc amplification and/or diploidy. Although involving only a limited series, these results strongly suggest that the combined analysis of DNA ploidy and N-myc genomic content could predict clinical outcome in stage IVS neuroblastoma and should help to identify patients for whom a more aggressive therapy is required.

Clinical relevance of DNA ploidy and proliferative activity in childhood rhabdomyosarcoma: a retrospective analysis of patients enrolled onto the Italian Cooperative Rhabdomyosarcoma Study RMS88.

PURPOSE: Evaluation of the possible clinical relevance of DNA ploidy and proliferative activity assessed as S-phase fraction (SPF) in childhood rhabdomyosarcoma (RMS). PATIENTS AND METHODS: We conducted a retrospective study on 59 RMS patients enrolled onto the ICS-RMS88 protocol (seven botryoid, 35 embryonal, and 17 alveolar RMS), for which formalin-fixed paraffin-embedded (FFPE) tissue was available. Nuclear suspensions for cytometric investigation were obtained using a mechanical disaggregation. Tumors were distinguished according to their DNA index (DI) value as follows: diploid (0.9 < DI < 1.1), hyperdiploid (1.1 < or = DI < 1.8 or DI > or = 2.2), and tetraploid (1.8 < or = DI < 2.2); for analysis of SPF, a cutoff value of 14% was used. RESULTS: DNA histograms were diploid in 19 (33%) cases, hyperdiploid in 29 (49%), and tetraploid in 10 (32%). One patient showed both a hyperdiploid and a tetraploid peak. The 5-year overall survival (OS) rate by ploidy status was 73% in hyperdiploid patients as compared with 33% and 25% in diploid and tetraploid patients, respectively (P = .0012). A striking difference emerged when the 5-year OS for the combined diploid and tetraploid RMS groups was compared with survival of the hyperdiploid RMS group: 30% versus 73%, respectively (P = .0006). In addition, the SPF was prognostically relevant: 5-year OS by SPF less than or greater than 14% was 70% and 36%, respectively (P = .009). Multivariate analysis confirmed the importance of DNA content (P = .0006) and SPF (P = .034) in predicting survival. CONCLUSION: These findings confirm that ploidy and SPF are important new prognostic factors that are able to identify selected groups of patients at high risk of treatment failure, even if the tumor's presentation is favorable according to standard criteria.

Association of near-diploid DNA content and N-myc amplification in neuroblastomas.

Seventeen neuroblastomas at different clinical stages were analysed for their N-myc copy number and flow cytometrically determined DNA content. Aneuploidy was found in 11 patients (65 per cent), whereas the remaining were near-diploid. N-myc amplification was found significantly (P less than 0.05) confined to near-diploid tumors (3 out of 6 cases). This finding indicates a very selective mechanism of oncogene amplification which is independent of gross chromosomal imbalance and limited to specific loci in the human genome. Association of near-diploidy and age at diagnosis older than 24 months was also demonstrated (P less than 0.05). Thus, flow cytometric analysis of DNA content together with N-myc gene dosage allowed us to distinguish two different subsets of neuroblastoma tumors: the first one aneuploid, with single-copy N-myc, usually observed in patients younger than 24 months with localized or IV-S clinical stages; the second one near-diploid, with frequent N-myc amplification, usually observed in patients older than 24 months with advanced clinical stages.

Coexpression of mRNA for the full-length neurotrophin receptor trk-C and trk-A in favourable neuroblastoma.

Neuroblastoma, a childhood tumour of the sympathetic nervous system, may sometimes regress spontaneously in infants, or progress to a poor clinical outcome despite intensive therapy. Neuroblastomas express neurotrophin receptors and high levels of mRNA for trk-A correlates with favourable outcome, whereas trk-B mRNA is expressed by more unfavourable tumours. Using a sensitive RNase protection assay, mRNA expression for the neurotrophin receptor trk-C was investigated in 50 tumour samples from 45 children at different stages including metastatic and relapsing tumour tissue, out of which 22 were also investigated for trk-A mRNA. Thirty-seven of 43 primary tumours (86%) showed trk-C mRNA with more than 300-fold difference between the highest and the lowest values. A higher trk-C index (trk-C mRNA/GAPDH mRNA) was associated with favourable features such as younger age (P = 0.009-0.003), favourable tumour stage (1, 2 or 4S; P < 0.001) and favourable prognosis (P = 0.044). Better survival probability was shown in children with intermediate or high trk-C index compared with patients with low or undetectable levels (P = 0.031). All localised tumours co-expressed mRNA for trk-A and trk-C receptors. RT-PCR analysis detected mRNA encoding the cytoplasmic trk-C tyrosine kinase region only in favourable neuroblastomas. We conclude that favourable neuroblastoma may express the full-length trk-C receptor while unfavourable tumours, especially those with MYCN amplification, seem to either express no trk-C or truncated trk-C receptors with unknown biological function. Trk-C and possibly its preferred ligand NT-3 may be involved in the biology of favourable neuroblastomas showing apoptosis or differentiation.

Immunohistochemical detection of high-affinity nerve growth factor receptor in neuroblastoma.

High levels of mRNA (as assessed by northern blot) for the high-affinity nerve growth factor receptor (p140TRK) are predictive of favourable outcome in neuroblastoma. The feasibility of determining p140trk on frozen sections using a recently developed monoclonal antibody was evaluated, and immunohistochemical findings were compared to those obtained from northern blot analysis. Primary tumour samples from 28 untreated patients were quick frozen and an indirect immunofluorescence assay was performed on 4-microns acetone-fixed cryostat sections. 9 cases were positive with immunohistochemistry, and these were among the 15 cases also positive by northern blot. None of the cases negative by northern blot were positive with immunohistochemistry. The concordance rate was 79% (P < 0.03), with a sensitivity of 60% and a specificity of 100%. Immunohistochemistry can thus be rather reliable for assessing p140trk expression, even when only very small amounts of tissue are available, such as with needle biopsy.

Somatostatin in neuroblastoma and ganglioneuroma.

Neuroblastoma, a childhood tumour of the sympathetic nervous system, may in some cases differentiate to a benign ganglioneuroma or regress due to apoptosis. Somatostatin may inhibit neuroblastoma growth and induce apoptosis in vitro and was therefore investigated. Using a radioimmunoassay, we found that all ganglioneuromas contained high somatostatin concentrations (> 16 pmol/g), significantly higher than neuroblastomas (n = 117, median 2.8 pmol/g), healthy adrenals, Wilms' tumours, phaeochromocytomas and other neuroendocrine tumours (P < 0.001). Neuroblastomas contained more somatostatin than control tumours (P < 0.001-0.05). Neuroblastomas amplified for the MYCN oncogene contained less somatostatin than non-amplified tumours (1.2 pmol/g versus 4.0 pmol/g, respectively; P = 0.026). In a clinically unfavourable neuroblastoma subset (age > 12 months, stage 3 or 4) 16 children with high concentrations of somatostatin in primary tumours had a better prognosis than 23 with low somatostatin (46.7% versus 0% survival at 5 years, P < 0.005). Scintigraphy using 111In-pentetreotide identified tumours expressing high-affinity somatostatin receptors in vivo. However, no significant correlation was found between somatostatin receptor expression and peptide content in 15 tumours. Similarly, human SH-SY5Y neuroblastoma xenografts grown in nude rats showed low somatostatin concentrations, but were positive for somatostatin receptor scintigraphy. Treatment of these rats with the somatostatin analogue octreotide seemed to upregulate in vivo receptor expression of somatostatin and vasoactive intestinal peptide more effectively than 13-cis retinoic acid. In conclusion, somatostatin in neuroblastoma is associated with differentiation to benign ganglioneuromas in vivo and favourable outcome in advanced tumours. Furthermore, somatostatin receptor scintigraphy may identify tumours with high-affinity receptors in children that might benefit from targeted therapy using synthetic somatostatin analogues.

Transcription of N-myc and proliferation-related genes is linked in human neuroblastoma.

A definite association between the transcription of N-myc oncogene and proliferation-related genes, histone H3, c-myc and p53, was found in a set of 12 primary untreated neuroblastomas and a metastasis of one of these at relapse. Molecular analysis allowed us to discriminate between actually proliferating and non-proliferating tumors, and suggested a link between N-myc and proliferation. Flow cytometric analysis of DNA distribution was less reliable for assessing tumor proliferative activity. Our data also seem to indicate a down-regulation of c-myc by N-myc in human neuroblastoma.

Chemotherapeutic agent cisplatin monitoring in biological fluids by means of inductively-coupled plasma emission spectrometry (ICP-AES).

The antitumoral agent cis-diamminedichloroplatinum (II) was administered at doses of 40 mg m-2 body surface area daily for 5 days via continuous i.v. infusion in association with etoposide (VP-16-213). The Pt concentration in serum up to 30 days from the beginning of the therapy was monitored by inductively-coupled plasma emission spectrometry. Results lead to two main conclusions: the analytical technique employed is suitable for measurements of Pt in biological fluids with the necessary precision (0.95-2.5%), accuracy (recovery 98.5-101.7%) and detection power (0.002-0.004 mg/l); there were effective Pt plasma concentrations for a greater length of time (with peak value 2.0 mg/l towards the end of treatment) than those achieved by other therapies so far adopted. On the other hand, toxic side effects, in particular gastrointestinal toxicity, myelosuppression and nephrotoxicity, were found to be not worse than those generally caused by the administration of the chemotherapeutic compound at lower doses. Both aspects were deeded to be essential prerequisites for better exploiting the drug's effectiveness.

Multidrug resistance gene transcript level, and P-glycoprotein expression in paediatric malignant mesenchymal tumours.

Twenty-four malignant mesenchymal tumour specimens were analysed for human multidrug resistance (MDRI) gene transcript levels using Northern and slot blot techniques. The presence of P-glycoprotein was assessed in 12 of the 24 samples by immunohistochemistry using the monoclonal antibody (MAb) C219. Increased MDRI transcript levels were found in 2 (8.3%), while, using immunohistochemistry, 2 samples were positive and 3 faintly positive (41.6%). Overall, elevated P-glycoprotein or MDRI transcript levels were found in tumours of 6 patients, 3 of whom are dead. The relationship to MDRI expression and subsequent resistance to chemotherapy has to be established.

Peripheral blood stem cells in children with solid tumors. Part II. Immunocytologic detection of tumor cells in bone marrow and peripheral blood stem cell harvests.

Simultaneous comparative assessment of tumor contamination in bone marrow aspirates and peripheral blood stem cell collections using immunocytochemical techniques was done in 6 children with neuroectodermal tumors. In 3 neuroblastoma patients tumor cells were detected in 5 of 16 marrow samples but not in peripheral blood stem cells. Clonogenic tumor cell reinfusion in the autologous support setting may be avoided in neuroblastoma patients by using peripheral blood stem cells.