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Purpose: Activating ALK mutations are present in almost 10% of primary neuroblastomas and mark patients for treatment with small-molecule ALK inhibitors in clinical trials. However, recent studies have shown that multiple mechanisms drive resistance to these molecular therapies. We anticipated that detailed mapping of the oncogenic ALK-driven signaling in neuroblastoma can aid to identify potential fragile nodes as additional targets for combination therapies.Experimental Design: To achieve this goal, transcriptome profiling was performed in neuroblastoma cell lines with the ALKF1174L or ALKR1275Q hotspot mutations, ALK amplification, or wild-type ALK following pharmacologic inhibition of ALK using four different compounds. Next, we performed cross-species genomic analyses to identify commonly transcriptionally perturbed genes in MYCN/ALKF1174L double transgenic versus MYCN transgenic mouse tumors as compared with the mutant ALK-driven transcriptome in human neuroblastomas.Results: A 77-gene ALK signature was established and successfully validated in primary neuroblastoma samples, in a neuroblastoma cell line with ALKF1174L and ALKR1275Q regulable overexpression constructs and in other ALKomas. In addition to the previously established PI3K/AKT/mTOR, MAPK/ERK, and MYC/MYCN signaling branches, we identified that mutant ALK drives a strong upregulation of MAPK negative feedback regulators and upregulates RET and RET-driven sympathetic neuronal markers of the cholinergic lineage.Conclusions: We provide important novel insights into the transcriptional consequences and the complexity of mutant ALK signaling in this aggressive pediatric tumor. The negative feedback loop of MAPK pathway inhibitors may affect novel ALK inhibition therapies, whereas mutant ALK induced RET signaling can offer novel opportunities for testing ALK-RET oriented molecular combination therapies. Clin Cancer Res; 21(14); 3327–39. ©2015 AACR.

The quassinoid analogue NBT-272 has been reported to inhibit MYC, thus warranting a further effort 7to better understand its preclinical properties in models of embryonal tumors (ET), a family of childhood malignancies sharing relevant biological and genetic features such as deregulated expression of MYC oncogenes. In our study, NBT-272 displayed a strong antiproliferative activity in vitro that resulted from the combination of diverse biological effects, ranging from G1/S arrest of the cell cycle to apoptosis and autophagy. The compound prevented the full activation of both eukaryotic translation initiation factor 4E (eIF4E) and its binding protein 4EBP-1, regulating cap-dependent protein translation. Interestingly, all responses induced by NBT-272 in ET could be attributed to interference with 2 main proproliferative signaling pathways, that is, the AKT and the MEK/extracellular signal-regulated kinase pathways. These findings also suggested that the depleting effect of NBT-272 on MYC protein expression occurred via indirect mechanisms, rather than selective inhibition. Finally, the ability of NBT-272 to arrest tumor growth in a xenograft model of neuroblastoma plays a role in the strong antitumor activity of this compound, both in vitro and in vivo, with its potential to target cell-survival pathways that are relevant for the development and progression of ET. Mol Cancer Ther; 9(12); 3145–57. ©2010 AACR.

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Supplemental data 9: Summary of Gene Set Enrichment analysis results performed on expression data upon ALK inhibition in 10 cell lines

Supplementary Data from The Quassinoid Derivative NBT-272 Targets Both the AKT and ERK Signaling Pathways in Embryonal Tumors

Purpose: Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic significance of ALK copy number and expression.Experimental Design: The frequency and type of ALK mutations, copy number gain, and expression were analyzed in a new series of 254 neuroblastoma tumors. Data from 455 published cases were used for further in-depth analysis.Results: ALK mutations were present in 6.9% of 709 investigated tumors, and mutations were found in similar frequencies in favorable [International Neuroblastoma Staging System (INSS) 1, 2, and 4S; 5.7%] and unfavorable (INSS 3 and 4; 7.5%) neuroblastomas (P = 0.087). Two hotspot mutations, at positions R1275 and F1174, were observed (49% and 34.7% of the mutated cases, respectively). Interestingly, the F1174 mutations occurred in a high proportion of MYCN-amplified cases (P = 0.001), and this combined occurrence was associated with a particular poor outcome, suggesting a positive cooperative effect between both aberrations. Furthermore, the F1174L mutant was characterized by a higher degree of autophosphorylation and a more potent transforming capacity as compared with the R1275Q mutant. Chromosome 2p gains, including the ALK locus (91.8%), were associated with a significantly increased ALK expression, which was also correlated with poor survival.Conclusions: ALK mutations occur in equal frequencies across all genomic subtypes, but F1174L mutants are observed in a higher frequency of MYCN-amplified tumors and show increased transforming capacity as compared with the R1275Q mutants. Clin Cancer Res; 16(17); 4353–62. ©2010 AACR.

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Supplemental data 13: Summary of Gene Set Enrichment analysis results obtained on expression profiling data of CLB-­�GA on different time points (10' - 30'- 60' - 2h - 4h - 6h) after pharmacological inhibition using TAE-­�684.

Supplemental data 1: Cell line information: Summary of the characteristics of cell lines used in this study including origin, tumor type, ALK and MYCN status (wt = wild type, NA = non amplified, A = amplified). Supplemental data 2: GI50 concentration of TAE-­�684 in neuroblastoma cell lines Supplemental data 3: phospho-­�ALK and total ALK expression in a panel of 10 neuroblastoma cell lines Supplemental data 4: ALK expression upon ALK inhibition using shALK Supplemental data 6: Expression of 77 ALK regulated genes in a panel of 10 neuroblastoma cell lines treated with ALK inhibitor TAE-­�684 Supplemental data 7: Expression of 77 ALK regulated genes in SK-­�N-­�AS with induced ALK Supplemental Data 8: Progression-­�free survival versus ALK signature scors Supplemental data 11: ALK signature gene analysis upon inhibition of two ALK downstream pathways Supplemental data 14:Expression of MAPK genes in CLB-­�GA after ALK inhibition Supplemental data 15: A. RET mRNA expression levels upon FOXO3 activation (+Dox) and Pi3K/AKT inhibition (+PI) in cell line SY5Y (Santo et al., 2013, GEO-­�id: GSE42762); B. pFOXO3a levels upon ALK inhibition with the LDK-­�378 compound are downregulated Supplemental data 16: Cell growth upon treatment of neuroblastoma cell lines with different concentrations of Trametinib (MAPK/MEK inhibitor) is only modestly affected Supplemental Data 17: Scheme to clarify our hypothesis

Supplemental data 5: Differential expression analysis upon ALK inhibition

Supplemental data 10: Results of gene ontology analysis performed on the 77 ALK signature genes

Supplemental data 12: Results of Connectivity map analysis performed on ALK signature genes.

Background this study compares diuresis rate, sodium clearance and free water clearance (FWC) by age and time of day (nighttime vs. daytime) in subjects with and without nocturnal polyuria (NP) to determine whether these variables affect the phenotype of NP. Methods post hoc analysis of two prospective observational studies. Eight urine samples collected at 3-h intervals and a single blood sample were used to calculate daytime (10a/1p/4p/7p/10p) and nighttime (1a/4a/7a) diuresis rates, sodium clearance and FWC. Three mixed linear models were constructed for diuresis rate, sodium clearance and FWC using four predictor variables: NP status (present [nocturnal urine production >90 ml/h] vs. absent [≤90 ml/h]), time of day, age and study identification. Results subjects with NP experienced higher nighttime versus daytime diuresis rates, sodium clearance and FWC. Regardless of NP status, increased age was accompanied by an increase in the ratio of nighttime/daytime diuresis rate, nighttime sodium clearance and daytime sodium clearance. FWC showed a complex age effect, which was independent of time of day or NP status. Conclusions age-related increases in nighttime/daytime diuresis rate, 24-h sodium clearance and 24-h FWC are not specific to subjects with NP. The age-related surge in either nocturnal sodium clearance or nocturnal FWC may represent the relevant substrate for behavioural or pharmacologic interventions targeting sodium diuresis or free water diuresis, respectively. Increases in FWC in older age groups may reflect impaired circadian rhythmicity of endogenous AVP or changes in responsiveness of the aged nephron to water clearance.

IQSEC2 mutations are associated with IQSEC2-related intellectual disability (ID). Phenotypic spectrum has been better defined in the last few years by the increasing number of reported cases although the genotype-phenotype relationship for IQSEC2 remains overall complex. As for IQSEC2-related ID a wide phenotypic diversity has been described in Rett syndrome (RTT). Several patients harboring IQSEC2 mutations present with clinical symptoms similar to RTT and some cases meet most of the criteria for classic RTT. With the aim of establishing a genotype-phenotype correlation, we collected data of 16 patients harboring IQSEC2 point mutations (15 of them previously unreported) and of five novel patients carrying CNVs encompassing IQSEC2. Most of our patients surprisingly shared a moderate-to-mild phenotype. The similarities in the clinical course between our mild cases and patients with milder forms of atypical RTT reinforce the hypothesis that also IQSEC2 mutated patients may lay under the wide clinical spectrum of RTT and thus IQSEC2 should be considered in the differential diagnosis. Our data confirm that position, type of variant and gender are crucial for IQSEC2-associated phenotype delineation.

Neuroblastoma is an aggressive childhood cancer arising from sympatho-adrenergic neuronal progenitors. The low survival rates for high-risk disease point to an urgent need for novel targeted therapeutic approaches. Detailed molecular characterization of the neuroblastoma genomic landscape indicates that ALK-activating mutations are present in 10% of primary tumours. Together with other mutations causing RAS/MAPK pathway activation, ALK mutations are also enriched in relapsed cases and ALK activation was shown to accelerate MYCN-driven tumour formation through hitherto unknown ALK-driven target genes. To gain further insight into how ALK contributes to neuroblastoma aggressiveness, we searched for known oncogenes in our previously reported ALK-driven gene signature. We identified ETV5, a bona fide oncogene in prostate cancer, as robustly upregulated in neuroblastoma cells harbouring ALK mutations, and show high ETV5 levels downstream of the RAS/MAPK axis. Increased ETV5 expression significantly impacted migration, invasion and colony formation in vitro, and ETV5 knockdown reduced proliferation in a murine xenograft model. We also established a gene signature associated with ETV5 knockdown that correlates with poor patient survival. Taken together, our data highlight ETV5 as an intrinsic component of oncogenic ALK-driven signalling through the MAPK axis and propose that ETV5 upregulation in neuroblastoma may contribute to tumour aggressiveness.

Objectives: The objective of the study is to evaluate the use of an experimental smart diaper as an indicator of saturation for diaper change in persons with dementia living in nursing homes. Methods: A multicenter prospective study was conducted in 3 nursing homes amongst 18 residents with dementia. For each resident, a frequency-volume urine chart (FVUC) was kept for 24 h including voided volume and diaper weights, wearing smart diapers. A comparative study was set up between results obtained by smart diapers and data registered in FVUCs. Results: Analysis based on quantification of the agreement between saturation calculated by smart diaper and determined by FVUC indicates that measurements reported by sensor do not correspond with measurements based on FVUC. For the regular diaper, the saturation measured by sensor may be 26% below or 39% above saturation based on FVUC and for the super diaper, respectively, 34% below or 30% above. Discussion: This study indicates that the sensor detects and notifies wetness but is not sensitive enough for using it as an indicator for diaper change in people with severe dementia.

Aim Nocturia frequency has been used as a measure of treatment efficacy for nocturia even though fluctuation of the symptom over time has been well described in the literature. Additionally, given the multifactorial causal pathway and clinically relevant comorbidities, frequency alone may be an insufficient marker of treatment direction. The aim of this study was to investigate factors associated with nocturia-related bother to identify additional variables that may capture the impact of nocturia, direct clinical care and have potential to quantify treatment outcome. Methods Prospective data from tertiary hospital Urology and Continence cohorts were matched for identical variables to generate a sample of 204 datasets. Descriptive statistics were obtained to describe the two cohorts. Characteristics of patients were evaluated across levels of nocturia frequency and nocturia-related bother using nonparametric methods; statistically significant differences between groups in each cohort were established. Results Nocturia frequency alone does not comprehensively reflect attributable bother. Five sleep variables (poor quality sleep, short time to first awakening to void, less than 7 hours of total sleep, primary sleep latency, and daytime sleepiness) and daily urinary urgency were significantly associated with high nocturia-related bother. Attributable bother, despite high-frequency nocturia, was minimized by male gender, lack of daily urinary urgency and good sleep quality. Poor health status, urinary urgency and sleep latency were associated with nocturia frequency. Conclusions Items of importance to individuals with nocturia have been identified from patient data. These variables have the potential to sit alongside change in nocturia frequency as potential markers of treatment response.

Background Resistance mechanisms in the androgen receptor (AR) signaling pathway remain key drivers in the progression to castration-resistant prostate cancer (CRPC) and relapse under antihormonal therapy. Materials and Methods We evaluated the circulating AR gene copy number (CN) gain using droplet digital polymerase chain reaction in 21 control and 91 prostate cancer serum samples and its prognostic and therapeutic implications in prostate cancer. Results In CRPC, AR CN gain was associated with faster progression to CRPC (P = .026), a greater number of previous treatments (P = .045), and previous chemotherapy (P = .016). Comparing patients with and without CN gain, the median progression-free survival (PFS) in the abiraterone subgroup was 1.7 months versus not reached (P = .004), and the median overall survival (OS) was 7 months versus 20.9 months (P = .020). In the enzalutamide subgroup, PFS was 1.7 versus 10.8 months (P = .006), and OS was 6.1 versus 16.5 months (P = .042). In the taxane subgroup, PFS was 3.2 versus 6.5 months (P = .093), and OS was 3.9 months versus not reached (P = .026). The presence of more AR copies correlated with shorter androgen deprivation (P = .002), abiraterone (P = .022), enzalutamide (P = .008), and taxane (P = .039) therapy. Conclusion Circulating AR CN gain predicts for a poor prognosis in CRPC. It is a promising biomarker predetermining rapid CRPC progression and predicting worse abiraterone and enzalutamide outcomes. Furthermore, it is associated with multiple previous treatments and previous chemotherapy.

Objectives To explore the mismatch between functional bladder capacity and nocturnal urine production, and to study the pathophysiology of an increased nocturnal urine production in older patients with urinary incontinence. Methods The present prospective observational study included adults aged ≥65 years with urinary incontinence. Participants completed questionnaires, frequency volume charts and renal function profiles. The nocturnal lower urinary tract symptom index was defined as nocturnal urine output/maximum voided volume; the nocturnal polyuria index as nocturnal/24 h urine output. Results The median age (n = 95) was 74 years (69–79), 87% were women and 73% had nocturnal lower urinary tract symptoms (nocturnal urinary incontinence or nocturia ≥2). Participants with nocturnal lower urinary tract symptoms had a significantly higher nocturnal urine output (809 mL vs 650 mL; P = 0.001) and no significant difference in maximum voided volume (350 mL vs 437 mL; P = 0.079) compared with participants without nocturnal lower urinary tract symptoms. Participants (nocturnal polyuria index >33% [n = 56], nocturnal polyuria index >40% [n = 42], nocturnal lower urinary tract symptom index >1.87 [n = 51]) showed higher night-time diuresis rates, free water and sodium clearance compared with during the daytime. Controls (nocturnal polyuria index ≤33% [n = 26], nocturnal polyuria index ≤40% [n = 40], nocturnal lower urinary tract symptom index ≤1.87 [n = 44]) had no circadian rhythm in their diuresis rate or sodium clearance, but more nocturnal free water clearance compared with during the daytime. Conclusions The majority of older adults with urinary incontinence present nocturnal lower urinary tract symptoms. An increased nocturnal sodium diuresis seems to be the only mechanism differentiating patients with nocturnal lower urinary tract symptoms from controls.

Purpose: Clean intermittent self-catheterization (CISC) is now considered the gold standard for the management of urinary retention. In the literature, several articles on patients' perspectives on CISC and adherence to this technique have been published. No studies have yet explored the points of view of professional caregivers, such as nurses and doctors. The aim of this study was to explore the opinions of urologists about CISC and to evaluate the need for dedicated nurses specialized in CISC through a self-administered questionnaire. Methods: A questionnaire was developed to explore the opinions of professional caregivers about self-catheterization and to evaluate the need to provide nurses with specialized education in CISC. Questionnaires were sent to 244 urologists through email. We received 101 completed questionnaires. The response rate was 41.4%. Results: Hand function, the presence or absence of tremor, and visual acuity were rated as the most important determinants for proposing CISC to a patient. Twenty-five percent of the urologists reported that financial remuneration would give them a greater incentive to propose CISC. The lack of dedicated nurses was reported by half of the urologists as a factor preventing them from proposing CISC. A meaningful number of urologists thought that patients perceive CISC as invasive and unpleasant. Although most urologists would choose CISC as a treatment option for themselves, almost 1 urologist out of 5 would prefer a permanent catheter. Conclusions: This questionnaire gave valuable insights into urologists' perceptions of CISC, and could serve as the basis for a subsequent broader international study. Further research should also focus on the opinions of nurses and other caregivers involved in incontinence management. Apart from financial remuneration, it is also clear that ensuring sufficient expertise and time for high-quality CISC care is important. This could be a potential role for dedicated nurses.

Background Despite the conflicting evidence about postvoid residual (PVR) and its variation in time and corresponding voided volume (VV), studies with urinary diaries and systematic measurements of PVR after each void have never been conducted in nursing home (NH) residents. Objective To describe the circadian rhythm of PVR and residual fraction (RF, the net quantity of PVR) and to identify the time window with the highest PVR and RF. Design, setting, and participants A multicentre prospective study conducted between 2014 and 2015 in 5 Belgian NHs. A convenience sample of cognitively intact residents completed a 24-hour frequency volume chart with PVR. Results Participants (n = 73) had a median age of 84 years (interquartile range 82-89) and moderate impairment of activities of daily living; 69% were women. In residents with nocturia, mean PVR was higher during the night [45 mL (26–80)] than during the day [36 mL (18–61)]. In residents without nocturia no difference was detected. In spite of the variation between diurnal and nocturnal VV and PVR in residents with nocturia, all residents emptied their bladder as effectively during daytime as during nighttime [mean RF = 20% (12–32)]. Maximum PVR and RF in residents with nocturia (n = 57) showed a circadian variation. The highest PVR and RF were found during the day. The PVR and RF of the first morning void were an indicator of the maximum nocturnal PVR and RF. Conclusions PVR and VV should be measured in NH residents during the waking hours (first morning void excepted) to detect the clinically relevant maximum PVR and RF.

Objectives: To evaluate the androgen receptor (AR) gene copy number in androgen deprivation therapy (ADT) treatment-naïve prostate cancer (PCa) patients and to evaluate the corresponding AR protein expression and assess the association between these features and prognostic factors. Materials and Methods: Chromosome X and AR gene copy number, using fluorescence-in-situ-hybridization, and epithelial-stromal AR expression, using AR immunohistochemistry, were analyzed in 62 ADT treatment-naïve PCa patients and 8 castration-refractory patients. Results: In ADT treatment-naïve PCa patients, the AR expression was higher in tumor epithelial cells versus surrounding stromal cells (p < 0.001) and versus normal epithelium in the same patient (p = 0.043). The difference between tumoral AR expression and expression in normal epithelium was higher in patients with ≥15% of tumor cells with increased AR copy number (p = 0.019). Peritumoral stroma had lower AR expression in patients with lymph-node or distant metastases compared to those without metastases (p = 0.038). Conclusions: This research evaluates the link between AR gene status, expression profile, and possible prognostic factors. Furthermore, it highlights the importance of the peritumoral environment in PCa. Additional research is needed to further clarify the role of stromal AR in PCa dissemination and identify possible therapeutic strategies to target this mechanism.

In neuroblastoma, tumor biopsies are used for prognostic evaluation and risk assessment by molecular genetic analyses such as fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (array CGH). Analysis of primary tumors by array CGH can be hampered by the lack of sufficient tumor cells due to small biopsy size or availability of invaded bone marrow only. Given the importance of accurate assessment of genetic alterations in the diagnostic work-up of patients with neuroblastoma, we evaluated the possibility to analyze bone marrow metastases in patients with disseminated disease. Disseminated neuroblastoma cells were isolated from bone marrow aspirates by using either laser capture microdissection (LCM) or magnetic activated cell sorting (MACS). The array CGH profiles of these isolated metastases were compared to array CGH profiles and/or FISH data of the corresponding primary tumor. Here, we show that the major recurrent DNA copy number alterations detected in primary neuroblastoma tumors (i.e., 1p, 3p and 11q deletion, 17q gain and MYCN amplification) can be detected, with high sensitivity and specificity, in the disseminated neuroblastoma cells isolated from the bone marrow aspirates, using an array platform with high coverage for these regions. Moreover, we demonstrate that for archived material, for example, for retrospective studies, LCM is the method of choice, while for fresh bone marrow aspirates, acquired at the time of diagnosis, MACS is superior.

Recent studies have unveiled copy number variants (CNVs) as an important source of genetic variation. Many of these CNVs contain coding sequences, which have been shown to be dosage sensitive. Evidence is accumulating that certain CNVs have impact on susceptibility to human diseases such as HIV infection and autoimmune diseases, as well as on adaptability to environmental conditions or nutrition. The possible role and impact of CNVs on cancer development and progression is only now emerging. In this review we look into the role of CNVs and their associated genomic structural features in relation to the formation of chromosome alterations in cancer cells and evolutionary genomic plasticity, as well as the de novo occurrence of known or putative CNVs as somatic events during oncogenesis. The role of germline CNVs in cancer predisposition is still largely unexplored. A number of observations seem to warrant the importance of further studies to elucidate the impact of these variants in the early steps of carcinogenesis.

Objectives To describe the effects of cytoreductive nephrectomy (CN) on the natural course of metastatic renal cell carcinoma (mRCC). CN appears to stabilize metastatic lesions in mRCC in a subgroup of patients and we hypothesize that systemic treatment might be deferred in these patients with stable disease after CN. Subjects and methods Overall, 45 patients with mRCC who underwent CN and subsequent oncologic follow-up were included in this retrospective, single-center analysis. After CN, patients were followed at least every 3 months with clinical evaluation, contrast-enhanced computerized tomography scan of chest and abdomen, with additional imaging if needed. At 3 months, patients were radiographically evaluated and categorized into nonresponders (death or progression) or responders (stable disease or remission). Kaplan-Meier and Cox proportional hazards regression statistics were used to describe prognostic factors for overall survival (OS) and systemic therapy–free survival (STFS). Results Median OS was 31(3–121) months. Further, 24 (53.3%) and 21 (46.7%) patients were classified as responders and nonresponders at 3 months, respectively. Responders had a significant better 2-year OS compared with nonresponders (81.7% vs. 26.5%, P = 0.005). Responders also had a better 2-year STFS (40.3% vs. 6.3%, P = 0.005). On Cox regression analysis, worse OS was found to be associated with low preoperative hemoglobin levels, the absence of postoperative radiographical response, and the presence of non–clear cell pathology. The presence of postoperative radiographical response, normal preoperative lactate dehydrogenase levels, the presence of a single metastasis, and performing metastasis-directed therapy was found to be associated with a longer systemic therapy-free period. Conclusion A beneficial oncologic response is observed in approximately half of the patients undergoing CN. Absence of radiographic progression at 3 months is an important marker for OS and STFS. Therefore, systemic treatment might be postponed in selected patients.

Purpose: Activating ALK mutations are present in almost 10% of primary neuroblastomas and mark patients for treatment with small-molecule ALK inhibitors in clinical trials. However, recent studies have shown that multiple mechanisms drive resistance to these molecular therapies. We anticipated that detailed mapping of the oncogenic ALK-driven signaling in neuroblastoma can aid to identify potential fragile nodes as additional targets for combination therapies. Experimental Design: To achieve this goal, transcriptome profiling was performed in neuroblastoma cell lines with the ALKF1174L or ALKR1275Q hotspot mutations, ALK amplification, or wild-type ALK following pharmacologic inhibition of ALK using four different compounds. Next, we performed cross-species genomic analyses to identify commonly transcriptionally perturbed genes in MYCN/ALKF1174L double transgenic versus MYCN transgenic mouse tumors as compared with the mutant ALK-driven transcriptome in human neuroblastomas. Results: A 77-gene ALK signature was established and successfully validated in primary neuroblastoma samples, in a neuroblastoma cell line with ALKF1174L and ALKR1275Q regulable overexpression constructs and in other ALKomas. In addition to the previously established PI3K/AKT/mTOR, MAPK/ERK, and MYC/MYCN signaling branches, we identified that mutant ALK drives a strong upregulation of MAPK negative feedback regulators and upregulates RET and RET-driven sympathetic neuronal markers of the cholinergic lineage. Conclusions: We provide important novel insights into the transcriptional consequences and the complexity of mutant ALK signaling in this aggressive pediatric tumor. The negative feedback loop of MAPK pathway inhibitors may affect novel ALK inhibition therapies, whereas mutant ALK induced RET signaling can offer novel opportunities for testing ALK-RET oriented molecular combination therapies. Clin Cancer Res; 21(14); 3327–39. ©2015 AACR.

// Alex Cazes 1,2 , Lucille Lopez-Delisle 1,2 , Konstantina Tsarovina 3 , Cecile Pierre-Eugene 1,2 , Katleen De Preter 4 , Michel Peuchmaur 5,6 , Andre Nicolas 7 , Claire Provost 8 , Caroline Louis-Brennetot 1,2 , Romain Daveau 1,2 , Candy Kumps 4 , Ilaria Cascone 9 , Gudrun Schleiermacher 1,2,10 , Aurelie Prignon 8 , Frank Speleman 4 , Hermann Rohrer 3 , Olivier Delattre 1,2 and Isabelle Janoueix-Lerosey 1,2 1 Inserm U830, 26 rue d’Ulm, 75005 Paris, France. 2 Institut Curie, Centre de Recherche, 26 rue d’Ulm, 75005 Paris, France. 3 Research Group Developmental Neurobiology, Max Planck Institute for Brain Research, Max-von-Laue-Str. 4, 60438 Frankfurt/M, Germany. 4 Center for Medical Genetics, Ghent University Hospital, De Pintelaan 185, B-9000 Ghent, Belgium. 5 Departement of Pathology, AP-HP, Hopital Universitaire Robert Debre, 48 boulevard Serurier, 75019 Paris, France. 6 Universite Diderot Paris 7, Paris Sorbonne Cite, Paris, France. 7 Platform of Experimental Pathology, Institut Curie, 26 rue d’Ulm, 75005 Paris, France. 8 LIMP (Laboratoire d’Imagerie Moleculaire Positonique), Hopital Tenon, 4 rue de la Chine, 75020 Paris, France. 9 Laboratoire CRRET, EAC CNRS 7149, Universite Paris 12-Val de Marne, 61, avenue du General de Gaulle, 94010 Creteil, France. 10 Institut Curie, Departement de Pediatrie, 26 rue d’Ulm, 75005 Paris, France. Correspondence: Isabelle Janoueix-Lerosey, email: // Keywords : Neuroblastoma, ALK, neurogenesis, therapeutic target, RET Received : March 24, 2014 Accepted : April 01, 2014 Published : April 02, 2014 Abstract Activating mutations of the ALK (Anaplastic lymphoma Kinase) gene have been identified in sporadic and familial cases of neuroblastoma, a cancer of early childhood arising from the sympathetic nervous system (SNS). To decipher ALK function in neuroblastoma predisposition and oncogenesis, we have characterized knock-in (KI) mice bearing the two most frequent mutations observed in neuroblastoma patients. A dramatic enlargement of sympathetic ganglia is observed in Alk F1178L mice from embryonic to adult stages associated with an increased proliferation of sympathetic neuroblasts from E14.5 to birth. In a MYCN transgenic context, the F1178L mutation displays a higher oncogenic potential than the R1279Q mutation as evident from a shorter latency of tumor onset. We show that tumors expressing the R1279Q mutation are sensitive to ALK inhibition upon crizotinib treatment. Furthermore, our data provide evidence that activated ALK triggers RET upregulation in mouse sympathetic ganglia at birth as well as in murine and human neuroblastoma. Using vandetanib, we show that RET inhibition strongly impairs tumor growth in vivo in both MYCN /KI Alk R1279Q and MYCN /KI Alk F1178L mice. Altogether, our findings demonstrate the critical role of activated ALK in SNS development and pathogenesis and identify RET as a therapeutic target in ALK mutated neuroblastoma.

Neuroblastoma, a childhood cancer that originates from neural crest-derived cells, is the most common deadly solid tumor of infancy. Amplification of the MYCN oncogene, which occurs in approximately 20-25% of human neuroblastomas, is the most prominent genetic marker of high-stage disease. The availability of valid preclinical in vivo models is a prerequisite to develop novel targeted therapies. We here report on the generation of transgenic mice with Cre-conditional induction of MYCN in dopamine ?-hydroxylase-expressing cells, termed LSL-MYCN;Dbh-iCre. These mice develop neuroblastic tumors with an incidence of >75%, regardless of strain background. Molecular profiling of tumors revealed upregulation of the MYCN-dependent miR-17-92 cluster as well as expression of neuroblastoma marker genes, including tyrosine hydroxylase and the neural cell adhesion molecule 1. Gene set enrichment analyses demonstrated significant correlation with MYC-associated expression patterns. Array comparative genome hybridization showed that chromosomal aberrations in LSL-MYCN;Dbh-iCre tumors were syntenic to those observed in human neuroblastomas. Treatment of a cell line established from a tumor derived from a LSL-MYCN;Dbh-iCre mouse with JQ1 or MLN8237 reduced cell viability and demonstrated oncogene addiction to MYCN. Here we report establishment of the first Cre-conditional human MYCN-driven mouse model for neuroblastoma that closely recapitulates the human disease with respect to tumor localization, histology, marker expression and genomic make up. This mouse model is a valuable tool for further functional studies and to assess the effect of targeted therapies. OA hybrid

Activating anaplastic lymphoma kinase (ALK) mutations were recently detected in most familial and 10% of sporadic neuroblastomas. However, the role of mutated ALK in tumorigenesis remains elusive. We demonstrate that targeted expression of the most frequent and aggressive variant, ALK(F1174L), is tumorigenic in mice. Tumors resembled human neuroblastomas in morphology, metastasis pattern, gene expression, and the presence of neurosecretory vesicles as well as synaptic structures. This ALK-driven neuroblastoma mouse model precisely recapitulated the genetic spectrum of the disease. Chromosomal aberrations were syntenic to those in human neuroblastoma, including 17q gain and MYCN oncogene amplification. Targeted ALK(F1174L) and MYCN coexpression revealed a strong synergism in inducing neuroblastoma with minimal chromosomal aberrations, suggesting that fewer secondary hits are required for tumor induction if both oncoproteins are targeted. Treatment of ALK(F1174L) transgenic mice with the ALK inhibitor TAE-684 induced complete tumor regression, indicating that tumor cells were addicted to ALK(F1174L) activity. We conclude that an activating mutation within the ALK kinase domain is sufficient to induce neuroblastoma development, and ALK inhibitors show promise for treating human neuroblastomas harboring ALK mutations.

Neuroblastic tumors (NBT) are composed by neuroblasts and Schwannian-like stroma. The origin of these two cell subtypes remains unclear. In this study, we describe, a neuroblastic-like subpopulation in neuroblastoma (NB) coexpressing GD2 and S100A6, neuroblastic and glial lineage markers respectively. The GD2(+)/S100A6(+) neuroblastic subpopulation was found to be enriched in low risk NB, distributed around the perivascular niche. Some stromal bundles showed GD2(+)/S100A6 costaining. Metastatic bone marrow specimens also showed GD2(+)/S100A6(+) cells. During in vitro retinoic acid induced differentiation of NB cell lines, rare GD2(+)/S100A6 neuroblatic cells appeared. We conclude that GD2(+)/S100A6(+) neuroblasts may represent a tumoral glial precursor subpopulation in NBT.

Purpose: Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic significance of ALK copy number and expression. Experimental Design: The frequency and type of ALK mutations, copy number gain, and expression were analyzed in a new series of 254 neuroblastoma tumors. Data from 455 published cases were used for further in-depth analysis. Results: ALK mutations were present in 6.9% of 709 investigated tumors, and mutations were found in similar frequencies in favorable [International Neuroblastoma Staging System (INSS) 1, 2, and 4S; 5.7%] and unfavorable (INSS 3 and 4; 7.5%) neuroblastomas (P = 0.087). Two hotspot mutations, at positions R1275 and F1174, were observed (49% and 34.7% of the mutated cases, respectively). Interestingly, the F1174 mutations occurred in a high proportion of MYCN-amplified cases (P = 0.001), and this combined occurrence was associated with a particular poor outcome, suggesting a positive cooperative effect between both aberrations. Furthermore, the F1174L mutant was characterized by a higher degree of autophosphorylation and a more potent transforming capacity as compared with the R1275Q mutant. Chromosome 2p gains, including the ALK locus (91.8%), were associated with a significantly increased ALK expression, which was also correlated with poor survival. Conclusions: ALK mutations occur in equal frequencies across all genomic subtypes, but F1174L mutants are observed in a higher frequency of MYCN-amplified tumors and show increased transforming capacity as compared with the R1275Q mutants. Clin Cancer Res; 16(17); 4353–62. ©2010 AACR.

Increased activity of MYC protein-family members is a common feature in many cancers. Using neuroblastoma as a tumor model, we established a microRNA (miRNA) signature for activated MYCN/c-MYC signaling in two independent primary neuroblastoma tumor cohorts and provide evidence that c-MYC and MYCN have overlapping functions. On the basis of an integrated approach including miRNA and messenger RNA (mRNA) gene expression data we show that miRNA activation contributes to widespread mRNA repression, both in c-MYC- and MYCN-activated tumors. c-MYC/MYCN-induced miRNA activation was shown to be dependent on c-MYC/MYCN promoter binding as evidenced by chromatin immunoprecipitation. Finally, we show that pathways, repressed through c-MYC/MYCN miRNA activation, are highly correlated to tumor aggressiveness and are conserved across different tumor entities suggesting that c-MYC/MYCN activate a core set of miRNAs for cooperative repression of common transcriptional programs related to disease aggressiveness. Our results uncover a widespread correlation between miRNA activation and c-MYC/MYCN-mediated coding gene expression modulation and further substantiate the overlapping functions of c-MYC and MYCN in the process of tumorigenesis.

Background: Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL), acute myeloid leukemia (AML), prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations. Results: The geNorm algorithm in the software package qbase+ was used to rank the candidate reference genes according to their expression stability. We observed that the stability of most of the candidate reference genes varies greatly in perturbation experiments. Expressed Alu repeats show relatively stable expression regardless of experimental condition. These Alu repeats are ranked among the best reference assays in all perturbation experiments and display acceptable average expression stability values (M