Supplementary Data 1. This supplementary data comprises a step-by-step description on how our gene-expression based classification models were generated and validated

Supplementary Data 2. This supplementary data comprises the r-algorithm scripts required to perform the both model selection and validation as described in the step-by-step protocol

Legends to Supplementaries. This documents comprises the legends to the supplementary figures and tables

Supplementary Table 3. This table summarizes the results of the Kaplan-Meier estimates for both EFS and OS of clinically relevant subgroups of neuroblastoma patients according to classification by the four remaining classifiers SVM_th22, SVM_th24, SVM_th26 and SVM_th44.

Supplementary Table 1. Clinical co-variates for the 709 patients who participated in the study. This supplementary table comprises detailed information on clinical co-variates for all 709 neuroblastoma patients who participated in the study.

Supplementary Figure 1. Highlights the EFS and OS for the cohort of neuroblastoma patients with MYCN-amplified disease (n=114, Fig 1a) and for th subcohort of patients >18 months of age with stage 4, MYCN non-amplified disease (n=102, fig. 1b). F, favorable; UF, unfavorable.

PDF file, 41K, Loss of DN-hTERT sequence of the transgene during the culture expansion of the transduced IGR-N-91 cells.

Supplementary Table 2. External performance validation of the top five classifiers. This supplementary table comprises the external performance metrics of the top five classifiers. Indicated are the values for classification accuracy, sensitivity , specificity and Matthew's correlation coefficient (MCC) in the prediction of 325 patients of the test set who fulfilled the criteria for classifier training (Favorable, n=187; Unfavorable, n=138).

Supplementary Table 4. Transcripts that contribute to the SVM_th10 classifier. This supplementary table highlights the transcripts that constitute the SVM_th10 classifier. Indicated for each feature are the Oligo-ID, the Gene symbols, the Entrez gene IDs, the RefSeq IDs, the Gene IDs and the Transcript IDs.

PURPOSE For localized, resectable neuroblastoma without MYCN amplification, surgery only is recommended even if incomplete. However, it is not known whether the genomic background of these tumors may influence outcome. PATIENTS AND METHODS Diagnostic samples were obtained from 317 tumors, International Neuroblastoma Staging System stages 1/2A/2B, from 3 cohorts: Localized Neuroblastoma European Study Group I/II and Children’s Oncology Group. Genomic data were analyzed using multi- and pangenomic techniques and fluorescence in-situ hybridization in 2 age groups (cutoff age, 18 months) and were quality controlled by the International Society of Pediatric Oncology European Neuroblastoma (SIOPEN) Biology Group. RESULTS Patients with stage 1 tumors had an excellent outcome (5-year event-free survival [EFS] ± standard deviation [SD], 95% ± 2%; 5-year overall survival [OS], 99% ± 1%). In contrast, patients with stage 2 tumors had a reduced EFS in both age groups (5-year EFS ± SD, 84% ± 3% in patients < 18 months of age and 75% ± 7% in patients ≥ 18 months of age). However, OS was significantly decreased only in the latter group (5-year OS ± SD in < 18months and ≥ 18months, 96% ± 2% and 81% ± 7%, respectively; P = .001). In < 18months, relapses occurred independent of segmental chromosome aberrations (SCAs); only 1p loss decreased EFS (5-year EFS ± SD in patients 1p loss and no 1p loss, 62% ± 13% and 87% ± 3%, respectively; P = .019) but not OS (5-year OS ± SD, 92% ± 8% and 97% ± 2%, respectively). In patients ≥ 18 months, only SCAs led to relapse and death, with 11q loss as the strongest marker (11q loss and no 11q loss: 5-year EFS ± SD, 48% ± 16% and 85% ± 7%, P = .033; 5-year OS ± SD, 46% ± 22% and 92% ± 6%, P = .038). CONCLUSION Genomic aberrations of resectable non– MYCN-amplified stage 2 neuroblastomas have a distinct age-dependent prognostic impact. Chromosome 1p loss is a risk factor for relapse but not for diminished OS in patients < 18 months, SCAs (especially 11q loss) are risk factors for reduced EFS and OS in those > 18months. In older patients with SCA, a randomized trial of postoperative chemotherapy compared with observation alone may be indicated.

Neuroblastoma (NB) is the most common extra cranial solid tumor in childhood and the most frequently diagnosed neoplasm during infancy. A striking feature of this tumor is its clinical heterogeneity. Several tumor progression markers have been delineated so far, among which MYCN amplification, which occurs in about 25% of total NB cases, with the percentage increasing to 30% in advanced stage NB. Although MYCN amplification is strongly correlated with NB of poor outcome, the MYCN status cannot alone predict all cases of poor survival in NB. Indeed NB without MYCN amplification (about 70–80% of NB) are not always favorable. WT1 was initially identified as a tumor suppressor gene involved in the development of a pediatric renal tumor (Wilms' tumor). Here, we describe an inverse correlation between WT1 expression and MYCN amplification and expression. However and most notably, our results show that WT1 gene expression is associated with a poor outcome for patients showing non‐MYCN‐amplified tumors. Thus WT1 expression is clinically significant in NB and may be a prognostic marker for better risk stratification and for an optimized therapeutic management of NB.

Polypeptide GalNAc-transferases (GalNAc-Ts) constitute a family of 20 human glycosyltransferases (comprising 9 subfamilies), which initiate mucin-type O-glycosylation. The O-glycoproteome is thought to be differentially regulated via the different substrate specificities and expression patterns of each GalNAc-T isoforms. Here, we present a comprehensive in vitro analysis of the peptide substrate specificity of GalNAc-T13, showing that it essentially overlaps with the ubiquitous expressed GalNAc-T1 isoform found in the same subfamily as T13. We have also identified and partially characterized nine splice variants of GalNAc-T13, which add further complexity to the GalNAc-T family. Two variants with changes in their lectin domains were characterized by in vitro glycosylation assays, and one (Δ39Ex9) was inactive while the second one (Ex10b) had essentially unaltered activity. We used reverse transcription-polymerase chain reaction analysis of human neuroblastoma cell lines, normal brain and a small panel of neuroblastoma tumors to demonstrate that several splice variants (Ex10b, ΔEx9, ΔEx2-7 and ΔEx6/8-39bpEx9) were highly expressed in tumor cell lines compared with normal brain, although the functional implications remain to be unveiled. In summary, the GalNAc-T13 isoform is predicted to function similarly to GalNAc-T1 against peptide substrates in vivo, in contrast to a prior report, but is unique by being selectively expressed in the brain.

Background: The prognostic impact of segmental chromosome alterations (SCAs) in children older than 1 year, diagnosed with localised unresectable neuroblastoma (NB) without MYCN amplification enrolled in the European Unresectable Neuroblastoma (EUNB) protocol is still to be clarified, while, for other group of patients, the presence of SCAs is associated with poor prognosis.Methods: To understand the role of SCAs we performed multilocus/pangenomic analysis of 98 tumour samples from patients enrolled in the EUNB protocol.Results: Age at diagnosis was categorised into two groups using 18 months as the age cutoff. Significant difference in the presence of SCAs was seen in tumours of patients between 12 and 18 months and over 18 months of age at diagnosis, respectively (P = 0.04). A significant correlation (P = 0.03) was observed between number of SCAs per tumour and age. Event-free (EFS) and overall survival (OS) were calculated in both age groups, according to both the presence and number of SCAs. In older patients, a poorer survival was associated with the presence of SCAs (EFS = 46% vs 75%, P = 0.023; OS = 66.8% vs 100%, P = 0.003). Moreover, OS of older patients inversely correlated with number of SCAs (P = 0.002). Finally, SCAs provided additional prognostic information beyond histoprognosis, as their presence was associated with poorer OS in patients over 18 months with unfavourable International Neuroblastoma Pathology Classification (INPC) histopathology (P = 0.018).Conclusions: The presence of SCAs is a negative prognostic marker that impairs outcome of patients over the age of 18 months with localised unresectable NB without MYCN amplification, especially when more than one SCA is present. Moreover, in older patients with unfavourable INPC tumour histoprognosis, the presence of SCAs significantly affects OS.

In neuroblastoma, MYCN amplification is associated with a worse prognosis and is a criterion used in the clinic to provide intensive treatments to children even with localized disease. In correlation with MYCN amplification, upregulation of TWIST1, a transcription factor playing a crucial role in inhibition of apoptosis and differentiation, was previously reported. Clinical data set analysis of MYCN, MYC and TWIST1 expression permits us to confirm that TWIST1 expression is upregulated in MYCN amplified neuroblastoma but also in a subset of neuroblastoma harboring high expression of MYCN or MYC without gene amplification. In silico analyses reveal the presence of several MYC regulatory motifs (E-Boxes and INR) within the TWIST1 promoter. Using gel shift assay and reporter activity assays, we demonstrate that both N-Myc and c-Myc proteins can bind and activate the TWIST1 promoter. Therefore, we propose TWIST1 as a direct MYC transcriptional target.

Age at diagnosis, stage, and MYCN amplification are the cornerstones of the risk-stratification score of neuroblastoma that enables defining patients at low- and high risk. Refinement of this stratification is needed to optimize standard treatment and to plan future clinical trials. We investigated whether two parental imprinted miRNAs (miR-487b and miR-516a-5p) may lead to a risk score with a better discrimination. Expression levels of maternal miR-487b and paternal miR-516a-5p were determined using quantitative RT-PCR both for 231 neuroblastoma tumors (derivation set) and 101 independent neuroblastoma tumors (validation set). Survival outcomes were overall survival (OS) and disease-free survival (DFS). Multivariable Cox models were developed from derivation set and their performance evaluated using Akaike's information criterion (AIC) (goodness-of-fit) and time-dependent area under curves (discrimination). The selected model was validated using internal and external validation. The prognostic model including current prognostic factors plus miR-487b, miR-516a-5p, and interaction between two miRNAs was selected. Performance of this model was better in terms of both predictive ability (smallest AIC) and discrimination power (AUC close to 0.70). This model identifies three risk groups: high (3), intermediate (2), and low (1). Hazard ratios (HR) across risk groups were HR2/1 = 6.3 (2.7-14.6), HR3/1 = 14.8 (7.2-30.2) for OS and HR2/1 = 2.8 (1.5-5.4), HR3/1 = 7.2 (3.9-13.4) for DFS. The rank between these three risk groups was maintained and validated when performing internal and external validation. Expression of maternal miR-487b and paternal miR-516a-5p improves the risk stratification. This better discrimination at diagnosis is of clinical utility both for current and future treatments of neuroblastoma patients.

Les tumeurs de la granulosa de l’ovaire (TGO) sont des tumeurs rares. Elles prennent naissance à partir des cellules des cordons sexuels et du stroma de l’ovaire. Elles se caractérisent par leur histoire naturelle lente, et leur tendance à la récidive longtemps après le diagnostic initial. La chirurgie complète avec Staging de la maladie est la pierre angulaire du traitement. La chimiothérapie est indiquée en cas de tumeurs localisées avec un haut risque de récidive, et en cas de tumeurs avancées ou récidivantes. Une surveillance prolongée est recommandée.Ovarian granulosa cell tumors (TGO) are rare neoplasms. They arise from sex cord stromal cells of the ovaries. They are characterized by their slow natural history, and their tendency to relapse long time after the initial diagnosis. Complete staging surgery of the disease is the cornerstone of treatment. Chemotherapy is indicated for localized tumors with a high risk of recurrence, and for recurrent or advanced tumors. Prolonged follow-up is recommended.

BACKGROUNDThe Sonic Hedgehog (SHH) signaling pathway plays an important role in neural crest cell fate during embryonic development and has been implicated in the progression of multiple cancers that include neuroblastoma, a neural crest cell-derived disease. While most of the SHH signaling is mediated by the well-described canonical pathway leading to the activation of Smoothened and Gli, it has recently been shown that cell-adhesion molecule-related/downregulated by oncogenes (CDON) serves as a receptor for SHH and contributes to SHH-induced signaling. CDON has also been recently described as a dependence receptor, triggering apoptosis in the absence of SHH. This CDON proapoptotic activity has been suggested to constrain tumor progression.METHODSCDON expression was analyzed by quantitative-reverse transcription-polymerase chain reaction in a panel of 226 neuroblastoma patients and associated with stages, overall survival, and expression of miR181 family members using Kaplan Meier and Pearson correlation methods. Cell death assays were performed in neuroblastoma cell lines and tumor growth was investigated in the chick chorioallantoic model. All statistical tests were two-sided.RESULTSCDON expression was inversely associated with neuroblastoma aggressiveness (P < .001). Moreover, re-expression of CDON in neuroblastoma cell lines was associated with apoptosis in vitro and tumor growth inhibition in vivo. We show that CDON expression is regulated by the miR181 miRNA family, whose expression is directly associated with neuroblastoma aggressiveness (survival: high miR181-b 73.2% vs low miR181-b 54.6%; P = .03).CONCLUSIONSTogether, these data support the view that CDON acts as a tumor suppressor in neuroblastomas, and that CDON is tightly regulated by miRNAs.

La seconde moitié du xxe siècle a été dominée par la génétique du cancer, qui fournissait des outils conceptuels capables d'expliquer la genèse d'une tumeur et son évolution. D'autres domaines – épigénétique, métabolisme – sont apparus, qui donnent une image infiniment plus complexe de la physiopathologie tumorale. Par ailleurs, la découverte de cellules souches tumorales et l'hétérogénéité intratumorale peuvent expliquer la récurrence des tumeurs. Une difficulté majeure est que chaque tumeur se comporte comme un organe qui évolue en fonction de son microenvironnement. L'intégration de l'ensemble des progrès dans des modèles de plus en plus sophistiqués pourrait ouvrir la voie à de nouveaux marqueurs diagnostiques et pronostiques et sélectionner des cibles thérapeutiques pertinentes et efficaces.