Image analysis and polyphenol profiling unveil red-flesh apple phenotype complexity.

BACKGROUND: The genetic basis of colour development in red-flesh apples (Malus domestica Borkh) has been widely characterised; however, current models do not explain the observed variations in red pigmentation intensity and distribution. Available methods to evaluate the red-flesh trait rely on the estimation of an average overall colour using a discrete class notation index. However, colour variations among red-flesh cultivars are continuous while development of red colour is non-homogeneous and genotype-dependent. A robust estimation of red-flesh colour intensity and distribution is essential to fully capture the diversity among genotypes and provide a basis to enable identification of loci influencing the red-flesh trait. RESULTS: In this study, we developed a multivariable approach to evaluate the red-flesh trait in apple. This method was implemented to study the phenotypic diversity in a segregating hybrid F1 family (91 genotypes). We developed a Python pipeline based on image and colour analysis to quantitatively dissect the red-flesh pigmentation from RGB (Red Green Blue) images and compared the efficiency of RGB and CIEL*a*b* colour spaces in discriminating genotypes previously classified with a visual notation. Chemical destructive methods, including targeted-metabolite analysis using ultra-high performance liquid chromatography with ultraviolet detection (UPLC-UV), were performed to quantify major phenolic compounds in fruits' flesh, as well as pH and water contents. Multivariate analyses were performed to study covariations of biochemical factors in relation to colour expression in CIEL*a*b* colour space. Our results indicate that anthocyanin, flavonol and flavanol concentrations, as well as pH, are closely related to flesh pigmentation in apple. CONCLUSTION: Extraction of colour descriptors combined to chemical analyses helped in discriminating genotypes in relation to their flesh colour. These results suggest that the red-flesh trait in apple is a complex trait associated with several biochemical factors.

Cladribine and cytarabine in refractory multisystem Langerhans cell histiocytosis: results of an international phase 2 study.

An international phase 2 study combining cladribine and cytarabine (Ara-C) was initiated for patients with refractory, risk-organ-positive Langerhans cell histiocytosis (LCH) in 2005. The protocol, comprising at least two 5-day courses of Ara-C (1 g/m(2) per day) plus cladribine (9 mg/m(2) per day) followed by maintenance therapy, was administered to 27 patients (median age at diagnosis, 0.7 years; median follow-up, 5.3 years). At inclusion, all patients were refractory after at least 1 course of vinblastine (VBL) plus corticosteroid, all had liver and spleen involvement, and 25 patients had hematologic cytopenia. After 2 courses, disease status was nonactive (n = 2), better (n = 23), or stable (n = 2), with an overall response rate of 92%. Median disease activity scores decreased from 12 at the start of therapy to 3 after 2 courses (P < .0001). During maintenance therapy, 4 patients experienced reactivation in risk organs. There were 4 deaths; 2 were related to therapy toxicity and 2 were related to reactivation. All patients experienced severe toxicity, with World Health Organization grade 4 hematologic toxicity and 6 documented severe infections. The overall 5-year survival rate was 85% (95% confidence interval, 65.2%-94.2%). Thus, the combination of cladribine/Ara-C is effective therapy for refractory multisystem LCH but is associated with high toxicity.

Dissecting the mechanisms of Notch induced hyperplasia.

The outcome of the Notch pathway on proliferation depends on cellular context, being growth promotion in some, including several cancers, and growth inhibition in others. Such disparate outcomes are evident in Drosophila wing discs, where Notch overactivation causes hyperplasia despite having localized inhibitory effects on proliferation. To understand the underlying mechanisms, we have used genomic strategies to identify the Notch-CSL target genes directly activated during wing disc hyperplasia. Among them were genes involved in both autonomous and non-autonomous regulation of proliferation, growth and cell death, providing molecular explanations for many characteristics of Notch induced wing disc hyperplasia previously reported. The Notch targets exhibit different response patterns, which are shaped by both positive and negative feed-forward regulation between the Notch targets themselves. We propose, therefore, that both the characteristics of the direct Notch targets and their cross-regulatory relationships are important in coordinating the pattern of hyperplasia.

Role of transmembrane semaphorin Sema6A in oligodendrocyte differentiation and myelination.

Myelination is regulated by extracellular proteins, which control interactions between oligodendrocytes and axons. Semaphorins are repulsive axon guidance molecules, which control the migration of oligodendrocyte precursors during normal development and possibly in demyelinating diseases. We show here that the transmembrane semaphorin 6A (Sema6A) is highly expressed by myelinating oligodendrocytes in the postnatal mouse brain. In adult mice, Sema6A expression is upregulated in demyelinating lesions in cuprizone-treated mice. The analysis of the optic nerve and anterior commissure of Sema6A-deficient mice revealed a marked delay of oligodendrocyte differentiation. Accordingly, the development of the nodes of Ranvier is also transiently delayed. We also observed an arrest in the in vitro differentiation of purified oligodendrocytes lacking Sema6A, with a reduction of the expression level of Myelin Basic Protein. Their morphology is also abnormal, with less complex and ramified processes than wild-type oligodendrocytes. In myelinating co-cultures of dorsal root ganglion neurons and purified oligodendrocytes we found that myelination is perturbed in absence of Sema6A. These results suggest that Sema6A might have a role in myelination by controlling oligodendrocyte differentiation.

Imatinib is effective in children with previously untreated chronic myelogenous leukemia in early chronic phase: results of the French national phase IV trial.

PURPOSE: Imatinib is the standard of care in adults with chronic myeloid leukemia (CML) in chronic phase (CP). Only a few studies to assess efficacy in children have been performed. We report on the results of the French prospective trial (ClinicalTrials.gov identifier NCT00845221) conducted in children and adolescents with newly diagnosed CML in CP. PATIENTS AND METHODS: A total of 44 patients from age 10 months to 17 years with newly diagnosed CML in CP received daily imatinib 260 mg/m(2). Progression-free survival, responses, and tolerance were evaluated. RESULTS: With a median follow-up times of 31 months (range, 11 to 64 months), the estimated progression-free survival rate at 36 months was 98% (95% CI, 85% to 100%). A complete hematologic response was achieved in 98% of the patients. The rates of complete cytogenetic response (CCyR) and major molecular response (MMR) were 61% and 31% at 12 months, respectively. During follow-up, CCyR and MMR were achieved in 36 children (77%) and 25 children (57%), respectively. Overall, 30% of the patients discontinued imatinib, mainly because of unsatisfactory response. The most common adverse events were neutropenia and musculoskeletal events. CONCLUSION: Imatinib is effective in children with CML in CP with response rates similar to rates reported in adults. The adverse effects are acceptable, but longer follow-up studies are required to fully assess the long-term impact.

Revisiting the role of doxorubicin in the treatment of rhabdomyosarcoma: an up-front window study in newly diagnosed children with high-risk metastatic disease.

PURPOSE: Many cooperative groups have reported on the chemo-sensitivity of rhabdomyosarcoma (RMS). Doxorubicin has been tested but remains a controversial treatment option. We report here the results of the up-front evaluation of the efficacy of doxorubicin in children and adolescents with high-risk metastatic RMS. PATIENTS AND METHODS: Patients younger than 18 years of age (>6 months) with newly diagnosed, histologically confirmed high-risk metastatic RMS were required to have measurable disease, to have undergone no prior chemotherapy or radiation therapy and to have normal liver, renal and cardiac function before treatment. Doxorubicin was administered intravenously over 48h to a total dose of 60mg/m2. Two courses were given separated by a 21day interval. Response to therapy was assessed by diagnostic imaging after the second course. The study was designed as a two-stage procedure according to the multistep plan described by Fleming. RESULTS: Twenty patients were eligible for analysis. Median age at diagnosis was 9.8 years (range from 2 to 16). Thirteen of the 20 patients treated in the first step responded to treatment, corresponding to an overall response to doxorubicin of 65% [95% confidence interval (CI), 44-85%]. The rates of CR and PR were 5% [95% CI, 0-14%] and 60% [95% CI, 39-81%], respectively. Four (20%) patients had progressive disease, corresponding to a progression rate of 20% [95% CI, 2-38%]. CONCLUSION: This window study provides the definitive demonstration of the efficacy of doxorubicin in untreated RMS. Given the inconclusive results obtained from previous studies using differing schedules chemotherapy incorporating doxorubicin, the next step should be a randomised study testing dose intensity in high-risk localised RMS. This issue is being addressed in a current European study (EpSSG RMS 2005).

Birth-related characteristics, congenital malformation, maternal reproductive history and neuroblastoma: the ESCALE study (SFCE).

Since neuroblastoma occurs very early in children's lives, it has been hypothesized that pre- and perinatal factors may play a role in its etiology. This study investigated the role of birth characteristics, congenital malformation and maternal reproductive history in neuroblastoma. The data used were generated by the national population-based case-control study, ESCALE, conducted in France in 2003-2004. The mothers of 191 neuroblastoma cases and 1,681 controls, frequency-matched by age and gender, were interviewed by telephone, using a standardized questionnaire, on several factors including pregnancy, medical history, lifestyle, childhood medical conditions and exposures. A positive association between congenital malformation and all neuroblastoma cases was observed [Odds ratio (OR) = 2.2, 95% confidence interval (95% CI): 1.1-4.5]. Congenital malformations were highly associated to neuroblastoma in children aged less than 1 year (OR = 16.8, 95% CI: 3.1-90), while no association was observed in children aged 1 year or more (OR = 1.0, 95% CI: 0.3-2.9). A negative association with a maternal history of spontaneous abortions was also found (OR = 0.6, 95% CI: 0.4-0.9). The results strongly support the hypothesis that congenital anomalies may be associated with neuroblastoma, particularly in infant (less than 1 year of age).

A TAT-DEF-Elk-1 peptide regulates the cytonuclear trafficking of Elk-1 and controls cytoskeleton dynamics.

The transcription factor Elk-1 plays a key role in cell differentiation, proliferation and apoptosis. This role is thought to arise from its phosphorylation by activated extracellular signal-regulated kinases (ERKs), a critical posttranslational event for the transcriptional activity of the ternary complex composed of Elk-1 and a dimer of serum response factor (SRF) at the serum response element (SRE) regulatory site of transcription. In addition to its nuclear localization, Elk-1 is found in the dendrites and soma of neuronal cells and recent evidence implicate a cytoplasmic proapoptotic function of Elk-1, via its association with the mitochondrial permeability transition pore complex. Thus, the nuclear versus cytoplasmic localization of Elk-1 seems to be crucial for its biological function. In this study we show that the excitatory neurotransmitter, glutamate, induces an ERK-dependent Elk-1 activation and nuclear relocalization. We demonstrate that Elk-1 phosphorylation on Ser383/389 has a dual function and triggers both Elk-1 nuclear translocation and SRE-dependent gene expression. Mutating these sites into inactive residues or using a synthetic penetrating peptide (TAT-DEF-Elk-1), which specifically interferes with the DEF docking domain of Elk-1, prevents Elk-1 nuclear translocation without interfering with ERK nor MSK1 (mitogen- and stress-activated protein kinase 1), a CREB kinase downstream from ERK- activation. This results in a differential regulation of glutamate-induced IEG regulation when compared with classical inhibitors of the ERK pathway. Using the TAT-DEF-Elk-1 peptide or the dominant-negative version of Elk-1, we show that Elk-1 phosphorylation controls dendritic elongation, SRF and Actin expression levels as well as cytoskeleton dynamics.

Liposomal amphotericin B in combination with caspofungin for invasive aspergillosis in patients with hematologic malignancies: a randomized pilot study (Combistrat trial).

BACKGROUND: Invasive aspergillosis (IA) has a poor prognosis in immunocompromised patients. Combinations of drugs that act on different targets are expected to improve the clinical efficacy of separate compounds. METHODS: Patients with proven or probable IA were randomized in a prospective, open pilot study to receive either a combination of liposomal amphotericin B (AmB) at the standard dose (3 mg/kg daily) and caspofungin at the standard dose or monotherapy with a high-dose AmB regimen (10 mg/kg daily). RESULTS: Thirty patients (21 men and 9 women) with hematologic malignancies were analyzed, and there were 15 patients in each arm. The median duration of treatment was 18 days for the combination group and 17 days for the high-dose monotherapy group. At the end of treatment, there were significantly more favorable overall responses (partial or complete responses; P = .028) in the combination group (10 of 15 patients; 67%) compared with the high-dose monotherapy group (4 of 15 patients; 27%). Survival rates at 12 weeks after inclusion were 100% and 80%, respectively. Infusion-related reactions occurred in 3 patients in the high-dose monotherapy group. A 2-fold increase in serum creatinine occurred in 4 of 17 patients (23%) who received high-dose monotherapy and 1 of 15 patient (7%) who received combination therapy; hypokalemia <3 mmol/L occurred in 3 patients and 2 patients, respectively. CONCLUSIONS: The combination of liposomal AmB and caspofungin was promising as therapy for IA compared with monotherapy. A trial that includes more patients will be required next to confirm the results of this pilot study.

Results of a phase II trial testing interferon-alpha 2b and cytarabine in children and adolescents with chronic myelogenous leukemia.

BACKGROUND: Chronic myelogenous leukemia (CML) is a rare disease in children and only few data are available concerning the results of interferon based therapy in this age group. Before the imatinib mesylate era, a prospective phase II trial was conducted to assess the efficacy and tolerance of a combination of interferon-alpha 2b (IFN) and cytarabine in children with CML in first chronic phase without a suitable HLA-identical donor. PROCEDURE: Fourteen consecutive children were recruited from 12 pediatric centers. Children received daily IFN (5 million U/m2) and subcutaneous cytarabine (20 mg/m2) for 10 days every month. RESULTS: The median duration of follow-up is 13 months (range 2-32 months). Seven children achieved a complete hematologic response after a median time of treatment of 3 months (range 1 week-4 months). Three children were not evaluable for the cytogenetic response. A major cytogenetic response was achieved in seven patients (including complete cytogenetic response in two patient) within 12 months. The median time to major cytogenetic response was 7 months (range 3-12 months). Thirteen patients discontinued the treatment protocol after a median time of 11 months. Probability of progression free survival at 11 months was 83% (95% CI, 61%-100%). Grade 3 and 4 toxicity was observed in eight patients. The most frequently reported drug-related events were fever, mucositis, neutropenia, and thrombocytopenia. CONCLUSIONS: The combination of IFN and cytarabine provides hematologic and cytogenetic responses in children and adolescents with CML. In the imatinib mesylate era, the role of this combination as second line therapy in children with CML remains to be determined.

Results of induction chemotherapy in children older than 1 year with a stage 4 neuroblastoma treated with the NB 97 French Society of Pediatric Oncology (SFOP) protocol.

PURPOSE: To test the metastatic response rate in stage 4 neuroblastoma, using dose-intensive induction chemotherapy in a multi-institutional setting. PATIENTS AND METHODS: From 1998 to 1999, 47 consecutive children were treated according to N7 protocol. Children received cyclophosphamide 140 mg/kg, doxorubicin 75 mg/m(2), and vincristine 0.066 mg/kg (CAV) in cycles 1, 2, 4, and 6, and cisplatinum 200 mg/m(2) and etoposide 600 mg/m(2) (P/VP) in cycles 3, 5, and 7. The International Neuroblastoma Staging system was used with an emphasis on skeletal evaluation by 123-iodine-metaiodobenzylguanidine (MIBG) scintigraphy. A phase II study evaluating the metastasis complete response rate after induction chemotherapy was conducted in patients who had positive metastatic sites on MIBG scans at diagnosis. RESULTS: Forty-six patients were assessable for toxicity. Hematologic toxicity was the main toxicity observed. Neutropenia was more frequent after CAV than after P/VP (P < .001). A higher rate of thrombocytopenia was observed after P/VP (P = .03). Forty patients with positive MIBG were assessable for metastatic response, and complete regression of metastases was achieved in 17 patients (ie, 43%; 95% CI, 27% to 59%). Of all 47 patients, 21 achieved complete metastatic response. CONCLUSION: The N7 induction chemotherapy protocol was feasible in a multicentric setting. The observed metastasis complete response rate was similar to that obtained in our previous studies and significantly lower than that published in a previous series using the same regimen. In our hands, escalating doses of cyclophosphamide and prolonging conventional chemotherapy with the same drugs failed to improve the metastasis complete response rate.

[Osteopetrosis, from mouse to man]. / L'ostéopétrose, de la souris à l'homme.

The osteoclast is the main effector of bone resorption. Failure in osteoclast differentiation or function leads to osteopetrosis, a bone disease characterized by an impaired bone resorption. Analysis of mouse models developing osteopetrosis as a consequence of naturally occurring mutations or gene knockouts allowed to establish the osteoclast differentiation pathway. Among these models, the oc/oc, the gl/gl and the Clcn7(-/-) mice present a phenotype similar to the one displayed by patients with infantile malignant osteopetrosis, the most severe form of osteopetrosis in human. Analysis of these models led to the identification of different mutations in the corresponding human genes TCIRG1, GL and CLCN7, in osteopetrotic patients. Mutations in the TCIRG1 gene seem the most frequent cause of malignant osteopetrosis and mutations in the CLCN7 gene seem the most frequent cause of type II osteopetrosis. Therefore, these three mouse models appear to be particularly well suited for the study of the osteoclast function in order to provide new insights in the therapy of osteopetrosis.

Significance of molecular quantification of minimal residual disease in metastatic neuroblastoma.

Molecular detection of tumor cells is the most sensitive approach to study residual disease in bone marrow (BM), peripheral blood (PB), and peripheral blood stem cell (PBSC) autografts from children with metastatic neuroblastoma (NB). We have developed a real-time PCR assay that allows the quantification of tyrosine hydroxylase (TH) mRNA, a tissue-specific marker of neuroblasts. We investigated a total of 165 BM, PB, and PBSC samples from 30 children over 1 year of age with stage IV NB and correlated the findings with disease status and patient survival. The levels of TH mRNA agreed well with clinical status and were significantly different across the groups that included samples obtained from patients at diagnosis, after three cycles of chemotherapy, in complete or very good partial remission and at relapse. We found that overall survival was significantly worse for patients with >1000 TH copies in BM after initial chemotherapy (p=0.0075). In 57% of cases, autologous PBSC harvests were found to be contaminated by neuroblasts, the level of TH >500 copies being associated with a decreased survival (p=0.003). In addition, molecular quantification enabled an estimation of tumor depletion in contaminated autografts using CD34 selection (median, 3 logs). In conclusion, quantification of minimal residual disease in metastatic NB using real-time RT-PCR for TH mRNA appears to be of potential clinical value. Further studies are needed to ascertain prognostic implications of molecular analysis of residual disease.

A 22-year French experience with solid tumors in children with Down syndrome.

Malignant solid tumors have rarely been reported in children with Down syndrome (DS) and are not well known. The authors collected from 1980 to 2001 all cases of solid tumors observed in DS patients aged from birth to 19 years within the network of the Société Française d'Oncologie Pédiatrique (SFOP). Only 21 cases were observed, with a peculiar distribution: a lack of intracranial tumors and embryonal neoplasms combined with an overrepresentation of lymphomas and germ cell tumors. The treatment of solid tumors in DS is difficult, due to physical and psychological impairments, different pharmacogenetic profile, and associated malformations.

Transient hematologic and clinical effect of E21R in a child with end-stage juvenile myelomonocytic leukemia.

E21R is a modified granulocyte macrophage-colony-stimulating factor (GM-CSF) protein which results in antagonism of GM-CSF function via selective binding to the GM-CSF receptor complex. Juvenile chronic myelomonocytic leukemia (JMML) is a rare leukemia where spontaneous proliferation of myeloid and monocytic precursors in patients' bone marrow cultures is dependent on GM-CSF. For patients who progress after systemic chemotherapy, there are no effective therapies. In vitro and in vivo studies in an animal model demonstrating that E21R exerts an antileukemic action prompted us to consider its potential utility in a child with end-stage JMML. E21R was well-tolerated during the 3 courses of subcutaneous treatment. A clear in vivo efficacy was observed after 2 courses of E21R but the disease appeared completely refractory during the third course. This novel therapeutic approach clearly deserves further evaluation in JMML.