Multifocal Neuroblastoma and Central Hypoventilation in An Infant with Germline ALK F1174I Mutation.

A preterm infant with central hypoventilation was diagnosed with multifocal neuroblastoma. Congenital anomalies of the autonomic nervous system in association with neuroblastoma are commonly associated with germline mutations in PHOX2B. Further, the ALK gene is frequently mutated in both familial and sporadic neuroblastoma. Sanger sequencing of ALK and PHOX2B, SNP microarray of three tumor samples and whole genome sequencing of tumor and blood were performed. Genetic testing revealed a germline ALK F1174I mutation that was present in all tumor samples as well as in normal tissue samples from the patient. Neither of the patient's parents presented the ALK variant. Array profiling of the three tumor samples showed that two of them had only numerical aberrations, whereas one sample displayed segmental alterations, including a gain at chromosome 2p, resulting in two copies of the ALK-mutated allele. Whole genome sequencing confirmed the presence of the ALK variant and did not detect any aberrations in the coding or promotor region of PHOX2B. This study is to our knowledge the first to report a de novoALK F1174I germline mutation. This may not only predispose to congenital multifocal neuroblastoma but may also contribute to the respiratory dysfunction seen in this patient.

Neuroblastoma Heterogeneity, Plasticity, and Emerging Therapies.

PURPOSE OF REVIEW: The evolving information of the initiation, tumor cell heterogeneity, and plasticity of childhood neuroblastoma has opened up new perspectives for developing therapies based on detailed knowledge of the disease. RECENT FINDINGS: The cellular origin of neuroblastoma has begun to unravel and there have been several reports on tumor cell heterogeneity based on transcriptional core regulatory circuitries that have given us important information on the biology of neuroblastoma as a developmental disease. This together with new insight of the tumor microenvironment which acts as a support for neuroblastoma growth has given us the prospect for designing better treatment approaches for patients with high-risk neuroblastoma. Here, we discuss these new discoveries and highlight some emerging therapeutic options. Neuroblastoma is a disease with multiple facets. Detailed biological and molecular knowledge on neuroblastoma initiation, heterogeneity, and the communications between cells in the tumor microenvironment holds promise for better therapies.

PPM1D Is a Therapeutic Target in Childhood Neural Tumors.

Childhood medulloblastoma and high-risk neuroblastoma frequently present with segmental gain of chromosome 17q corresponding to aggressive tumors and poor patient prognosis. Located within the 17q-gained chromosomal segments is PPM1D at chromosome 17q23.2. PPM1D encodes a serine/threonine phosphatase, WIP1, that is a negative regulator of p53 activity as well as key proteins involved in cell cycle control, DNA repair and apoptosis. Here, we show that the level of PPM1D expression correlates with chromosome 17q gain in medulloblastoma and neuroblastoma cells, and both medulloblastoma and neuroblastoma cells are highly dependent on PPM1D expression for survival. Comparison of different inhibitors of WIP1 showed that SL-176 was the most potent compound inhibiting medulloblastoma and neuroblastoma growth and had similar or more potent effects on cell survival than the MDM2 inhibitor Nutlin-3 or the p53 activator RITA. SL-176 monotherapy significantly suppressed the growth of established medulloblastoma and neuroblastoma xenografts in nude mice. These results suggest that the development of clinically applicable compounds inhibiting the activity of WIP1 is of importance since PPM1D activating mutations, genetic gain or amplifications and/or overexpression of WIP1 are frequently detected in several different cancers.

High Expression of PPM1D Induces Tumors Phenotypically Similar to TP53 Loss-of-Function Mutations in Mice.

PPM1D is a negative regulator of p53 and genomic aberrations resulting in increased activity of PPM1D have been observed in cancers of different origins, indicating that PPM1D has oncogenic properties. We established a transgenic mouse model overexpressing PPM1D and showed that these mice developed a wide variety of cancers. PPM1D-expressing mice developed tumors phenotypically and genetically similar to tumors in mice with dysfunctional p53. T-cell lymphoblastic lymphoma was the most frequent cancer observed in these mice (55%) followed by adenocarcinomas (24%), leukemia (12%) and other solid tumors including neuroblastoma. Characterization of T-cell lymphomas in mice overexpressing PPM1D demonstrates Pten-deletion and p53-accumulation similar to mice with p53 loss-of-function. Also, Notch1 mutations which are recurrently observed in T-cell acute lymphoblastic lymphoma (T-ALL) were frequently detected in PPM1D-transgenic mice. Hence, PPM1D acts as an oncogenic driver in connection with cellular stress, suggesting that the PPM1D gene status and expression levels should be investigated in TP53 wild-type tumors.

Clinical response of the novel activating ALK-I1171T mutation in neuroblastoma to the ALK inhibitor ceritinib.

Tumors with anaplastic lymphoma kinase (ALK) fusion rearrangements, including non-small-cell lung cancer and anaplastic large cell lymphoma, are highly sensitive to ALK tyrosine kinase inhibitors (TKIs), underscoring the notion that such cancers are addicted to ALK activity. Although mutations in ALK are heavily implicated in childhood neuroblastoma, response to the ALK TKI crizotinib has been disappointing. Embryonal tumors in patients with DNA repair defects such as Fanconi anemia (FA) often have a poor prognosis, because of lack of therapeutic options. Here we report a child with underlying FA and ALK mutant high-risk neuroblastoma responding strongly to precision therapy with the ALK TKI ceritinib. Conventional chemotherapy treatment caused severe, life-threatening toxicity. Genomic analysis of the initial biopsy identified germline FANCA mutations as well as a novel ALK-I1171T variant. ALK-I1171T generates a potent gain-of-function mutant, as measured in PC12 cell neurite outgrowth and NIH3T3 transformation. Pharmacological inhibition profiling of ALK-I1171T in response to various ALK TKIs identified an 11-fold improved inhibition of ALK-I1171T with ceritinib when compared with crizotinib. Immunoaffinity-coupled LC-MS/MS phosphoproteomics analysis indicated a decrease in ALK signaling in response to ceritinib. Ceritinib was therefore selected for treatment in this child. Monotherapy with ceritinib was well tolerated and resulted in normalized catecholamine markers and tumor shrinkage. After 7.5 mo treatment, the residual primary tumor shrunk, was surgically removed, and exhibited hallmarks of differentiation together with reduced Ki67 levels. Clinical follow-up after 21 mo treatment revealed complete clinical remission including all metastatic sites. Therefore, ceritinib presents a viable therapeutic option for ALK-positive neuroblastoma.

The ENaC-overexpressing mouse as a model of cystic fibrosis lung disease.

Chronic lung disease remains the major cause of morbidity and mortality of cystic fibrosis (CF) patients. Cftr mutant mice developed severe intestinal obstruction, but did not exhibit the characteristic CF ion transport defects (i.e. deficient cAMP-dependent Cl(-) secretion and increased Na(+) absorption) in the lower airways, and failed to develop CF-like lung disease. These observations led to the generation of transgenic mice with airway-specific overexpression of the epithelial Na(+) channel (ENaC) as an alternative approach to mimic CF ion transport pathophysiology in the lung. Studies of the phenotype of ßENaC-transgenic mice demonstrated that increased airway Na(+) absorption causes airway surface liquid (ASL) depletion, reduced mucus transport and a spontaneous CF-like lung disease with airway mucus obstruction and chronic airway inflammation. Here, we summarize approaches that can be applied for studies of the complex in vivo pathogenesis and preclinical evaluation of novel therapeutic strategies in this model of CF lung disease.

Preventive but not late amiloride therapy reduces morbidity and mortality of lung disease in betaENaC-overexpressing mice.

RATIONALE: Increased airway Na(+) absorption mediated by epithelial Na(+) channels (ENaC) is a characteristic abnormality in the pathogenesis of cystic fibrosis (CF) lung disease. However, inhalation therapy with the ENaC blocker amiloride did not have therapeutic benefits in patients with CF with established lung disease. OBJECTIVES: We hypothesized that preventive inhibition of increased Na(+) absorption in a structurally normal lung may be required for effective therapy of CF lung disease in vivo, and that therapeutic effects of late amiloride intervention may be impeded by the chronic disease process. METHODS: To test this hypothesis in vivo, we used the betaENaC-overexpression mouse as a model of CF lung disease and determined therapeutic effects of preventive versus late amiloride therapy on survival, airway mucus plugging, chronic bronchitis, and airway remodeling. MEASUREMENTS AND MAIN RESULTS: We show that early intervention, i.e., from the first day of life, with the intranasal administration of amiloride significantly reduced pulmonary mortality, airway mucus obstruction, epithelial necrosis, goblet cell metaplasia, and airway inflammation in betaENaC-overexpressing mice. In contrast, consistent with previous human trials in patients with CF, amiloride administration did not have benefits if treatment was started after the development of CF-like lung disease in betaENaC-overexpressing mice. CONCLUSIONS: We conclude that preventive inhibition of increased airway Na(+) absorption provides an effective therapy for CF-like lung disease in vivo. These results suggest that amiloride therapy may be an effective preventive therapy for patients with CF if initiated early in life before the onset of lung disease.

Development of chronic bronchitis and emphysema in beta-epithelial Na+ channel-overexpressing mice.

RATIONALE: Chronic obstructive pulmonary disease is a leading cause of death worldwide, but its pathogenesis is not well understood. Previous studies have shown that airway surface dehydration in beta-epithelial Na(+) channel (betaENaC)-overexpressing mice caused a chronic lung disease with high neonatal pulmonary mortality and chronic bronchitis in adult survivors. OBJECTIVES: The aim of this study was to identify the initiating lesions and investigate the natural progression of lung disease caused by airway surface dehydration. METHODS: Lung morphology, gene expression, bronchoalveolar lavage, and lung mechanics were studied at different ages in betaENaC-overexpressing mice. MEASUREMENTS AND MAIN RESULTS: Mucus obstruction in betaENaC-overexpressing mice originated in the trachea in the first days of life and was associated with hypoxia, airway epithelial necrosis, and death. In surviving betaENaC-overexpressing mice, mucus obstruction extended into the lungs and was accompanied by goblet cell metaplasia, increased mucin expression, and airway inflammation with transient perinatal increases in tumor necrosis factor-alpha and macrophages, IL-13 and eosinophils, and persistent increases in keratinocyte-derived cytokine (KC), neutrophils, and chitinases in the lung. betaENaC-overexpressing mice also developed emphysema with increased lung volumes, distal airspace enlargement, and increased lung compliance. CONCLUSIONS: Our studies demonstrate that airway surface dehydration is sufficient to initiate persistent neutrophilic airway inflammation with chronic airways mucus obstruction and to cause transient eosinophilic airway inflammation and emphysema. These results suggest that deficient airway surface hydration may play a critical role in the pathogenesis of chronic obstructive pulmonary diseases of different etiologies and serve as a target for novel therapies.