238

ALK

CD246|NBLST3;anaplastic lymphoma receptor tyrosine kinase;ALK;anaplastic lymphoma receptor tyrosine kinase

ALK tyrosine kinase receptor|CD246 antigen|mutant anaplastic lymphoma kinase

2p23

protein-coding

This report describes an unusual extramedullary hematologic malignancy in an 18-month-old child who presented with a capillary leak syndrome that evolved into hyperleukocytosis with malignant cells. The circulating tumor cells did not express an antigen profile typical of any subtype of leukemia commonly observed in children. tumor cells were CD3(-)/CD56(+); had germline TCR genes; and strongly expressed CD30, epithelial membrane antigen, and anaplastic lymphoma kinase (ALK) consistent with a null cell anaplastic large cell lymphoma (ALCL). The malignant cells contained a t(2;19)(p23;p13.1) that interrupted ALK and translocated it to the der(19). Reverse transcriptase-polymerase chain reaction and nucleotide sequence analysis revealed fusion of ALK to tropomyosin 4, an ALKfusion partner not described previously in hematologic malignancies. The clinical presentation and phenotypic features of this malignancy were not typical for ALCL because tumor cells expressed both myeloid (CD13, CD33, HLA-DR) and natural killer (NK) cell antigens. The neoplastic cells most resembled NK cells because in addition to being CD3(-)/CD56(+) with germline TCR genes, these cells were CD25(+)/CD122(+)/granzyme B(+) and possessed the functional properties of immature NK cells. The unusual clinical presentation, immunophenotype, and functional properties of these neoplastic cells suggest that this malignancy maybe derived from the putative myeloid-NK precursor cell. Furthermore co-expression of NK and ALCL features supports the concept that a minority of null-ALCL may bederived from NK cells and expands the spectrum of phenotypes that can be seen intumors produced by ALK fusion proteins. (Blood. 2001;98:1209-1216)

The use of effective combination chemotherapy for ALL stages and subtypes of non-Hodgkin"s lymphoma (NHL) in children has resulted in a striking improvement in cure rates. Event-free survival now ranges from 70% to 90%, depending on the stage of disease and the NHL subtype. Risk-adapted therapy has resulted in a dramatic improvement in outcome for high-risk patients, at the cost of significantly increased short-term toxicity, and a reduction of therapy and toxicity for the lower-risk patient, while maintaining the excellent cure rate. Successful risk allocation of patients is dependent on the identification and continual validation of prognostic factors. The specific treatment protocol is the single most important factor predicting outcome today. Traditional prognostic factors such as stage and tumor burden are useful in selecting the intensity andlength of therapy, rather than as a major indicator of likelihood of survival. In order to further improve cure rates and decrease toxicity, new biologic prognosticators need to be found and validated. Some promising avenues for studyappear to be the presence or absence of adhesion molecules and of aberrant proteins that are specific to subtypes of lymphomas, such as soluble CD30 and anaplastic lymphoma kinase (ALK), the molecular classification of lymphomas on the basis of gene expression, and the evaluation of biologic markers for measuring early response to therapy.

Neuroblastoma is a childhood cancer that can be inherited, but the genetic aetiology is largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase (ALK) gene explain most hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at chromosome bands 2p23-24 using a whole-genome scanin neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate germline missense mutations in the tyrosine kinase domain of ALK that segregated with the disease in eight separate families. Resequencing in 194high-risk neuroblastoma samples showed somatically acquired mutations in the tyrosine kinase domain in 12.4% of samples. Nine of the ten mutations map to critical regions of the kinase domain and were predicted, with high probability,to be oncogenic drivers. mutations resulted in constitutive phosphorylation, andtargeted knockdown of ALK messenger RNA resulted in profound inhibition of growth in ALL cell lines harbouring mutant or amplified ALK, as well as in two out of six wild-type cell lines for ALK. Our results demonstrate that heritable mutations of ALK are the main cause of familial neuroblastoma, and that germlineor acquired activation of this cell-surface kinase is a tractable therapeutic target for this lethal paediatric malignancy.

Wilms tumor and neuroblastoma are childhood tumors of the kidney and undifferentiated neural crest cells, respectively. Both disorders are primarily sporadic, but familial Wilms tumor pedigrees and familial neuroblastoma pedigrees are each well recognized and account for approximately 1-3% of each tumor type. Families with Wilms tumor and neuroblastoma in the same, or related individuals,have not been reported. Here, we present nine families with two or more individuals with Wilms tumor and/or neuroblastoma. The affected individuals wereotherwise well, without syndromic features. Although this co-occurrence might bedue to chance in some families, the coexistence of two rare embryonal tumors in related individuals of multiple families suggests an underlying genetic susceptibility to both tumors. We undertook mutational analysis of the genes known to predispose to non-syndromic familial Wilms tumor (WT1) or neuroblastoma(PHOX2B, ALK) which excluded these as the underlying predisposition genes in thenine families. We also excluded epigenetic and copy-number abnormalities at 11p15 which are known to predispose to embryonal tumors including Wilms tumor and neuroblastoma. Overall, these data suggest that families with both Wilms tumor and neuroblastoma represent a previously unrecognized familial cancer syndrome in which the underlying predisposition gene(s) remain to be determined.

Neuroblastoma (NB) is an embryonal cancer of the sympathetic nervous system observed in early childhood, characterized by a broad spectrum of clinical behaviors, ranging from spontaneous regression to fatal outcome despite aggressive therapies. NB accounts for 8-10% of pediatric cancers and 15% of the deaths attributable to malignant conditions in children. Interestingly, NB may occur in various contexts, being mostly sporadic but also familial or syndromic.This review focuses on recent advances in the identification of the genes and mechanisms implicated in NB pathogenesis. Although the extensive characterization of the genomic aberrations recurrently observed in sporadic NBs provides important insights into the understanding of the clinical heterogeneity of this neoplasm, analysis of familial and syndromic cases also unravels essential clueson the genetic bases of NB. Recently, the ALK gene emerged as an important NB gene, being implicated both in sporadic and familial cases. The identification of gene expression signatures associated with patient's outcome points out the potential of using gene expression profiling to improve clinical management of patients suffering from NB. Finally, based on recent observations integrating genomic analyses, biological data and clinical information, we discuss possible evolution/progression schemes in NB.

BTI - GeneReviews#. ALK-related neuroblastoma susceptibility occurs in individuals who are heterozygous for a germline ALK mutation and is characterized by an increased risk of developing neuroblastoma, ganglioneuroblastoma, or ganglioneuroma. The risk for tumor development is highest in infancy and decreases by late childhood. Individuals with familial neuroblastoma tend to develop tumors at a younger age (average 9 months) than those without familial predisposition (age 2-3 years). ALK is the only gene in which mutations are known to cause ALK-related neuroblastoma susceptibility. ALK mutations are found in equal frequencies across ALL neuroblastoma subtypes and risk groups. Oncogenic germline ALK mutations seem to occur only in families with high or moderate degrees of confidence for harboring a predisposing allele. In families with two or more first-degree relatives with neuroblastoma, the incidence of ALK germline mutations is high. In families in which two second-degree or more distant relatives have neuroblastoma, the incidence of ALK germline mutation is much lower. Treatment of manifestations: Treatment of neuroblastoma is best provided by a pediatric oncologist at a pediatric cancer center. Depending on the age of the child, stage of the tumor, and biologic characteristics of the tumor, treatment may involve observation or surgical resection. tumors with risk of metastatic spread requirechemotherapy and sometimes radiation therapy. Surveillance: Surveillance is at the discretion of the medical provider because no data are available as yet on the effect of screening in families with germline ALK mutations and surveillanceat the population level has not improved neuroblastoma outcome. However, screening with noninvasive techniques such as ultrasonography and measurement ofurinary catecholamine metabolites should probably be implemented for unaffected children who have a germline ALK mutation. Abdominal ultrasound examination and measurement of urine catecholamine metabolite levels have been performed every one to two months in infants up to age 12 months and every three to four months during childhood up to age ten years; less frequent intervals may also be appropriate. Evaluation of relatives at risk: In families with documented ALK-related neuroblastoma susceptibility, testing of ALL at-risk first-degree relatives including minors is indicated because germline mutations in ALK are highly penetrant and heterozygotes are at significant risk of developing cancer at a young age. Therapies under investigation: Small molecule inhibitors targeting the ALK tyrosine kinase, such as TAE684 and crizotinib, are currently in preclinical development and early-phase clinical trials. Crizotinib has demonstrated safety and tolerability in children, as well as promising activity in ALK-translocated tumors such as anaplastic large cell lymphoma. Activity in neuroblastoma is still under investigation. ALK-related neuroblastoma susceptibility is inherited in an autosomal dominant manner with incomplete penetrance. Some, but not all, individuals diagnosed with ALK-related neuroblastoma susceptibility have an affected parent; the proportion with de novo mutations is unknown. Each child of an individual with ALK-related neuroblastomasusceptibility has a 50% chance of inheriting the mutation. Prenatal diagnosis for pregnancies at increased risk for ALK-related neuroblastoma susceptibility is possible; however, such testing cannot predict if neuroblastoma will develop.#CI- Copyright (c) 1993-2013, University of Washington, Seattle. ALL rights reserved.#FED - Pagon, Roberta A#ED- Pagon RA#FED - Adam, Margaret P#ED- Adam MP#FED - Bird, Thomas D#ED- Bird TD#FED - Dolan, Cynthia R#ED- Dolan CR#FED - Fong, Chin-To#ED- Fong CT#FED - Stephens, Karen#ED- Stephens K#FAU - Johnson, Rebecca H

The anaplastic lymphoma kinase gene (ALK) is a gene on chromosome 2p23 that has expression restricted to the brain, testis and small intestine but is not expressed in normal lymphoid tissue. It has similarity to the insulin receptor subfamily of kinases and is emerging as having increased pathologic and potential therapeutic importance in malignant disease. This gene was originally established as being implicated in the pathogenesis of rare diseases including inflammatory myofibroblastic tumour (IMT) and ALK-positive anaplastic large cell lymphoma, which is a subtype of non-Hodgkin's lymphoma. Recently the number of diseases inwhich ALK is implicated in their pathogenesis has increased. In 2007, an inversion of chromosome 2 involving ALK and a fusion partner gene in a subset ofnon-small cell lung cancer was discovered. In 2008, publications emerged implicating ALK in familial and sporadic cases of neuroblastoma, a childhood cancer of the sympatho-adrenal system. Chromosomal abnormalities involving ALK are translocations, amplifications or mutations. Chromosomal translocations are the longest recognised ALK genetic abnormality. When translocations occur a fusion gene is created between ALK and a gene partner. This has been described in ALK-positive anaplastic large cell lymphoma in which ALK is fused to NPM (nucleolar protein gene) and in non-small cell lung cancer where ALK is fused toEML4 (Echinoderm microtubule-associated protein 4). The most frequently described partner genes in inflammatory myofibroblastic tumour are tropomyosin 3/4 (TMP3/4), however in IMTs a diversity of ALK fusion partners have been found, with the ability to homodimerise a common characteristic. Point mutations and amplification of the ALK gene occur in the childhood cancer neuroblastoma. Therapeutic targeting of ALK fusion genes using tyrosine kinase inhibition, vaccination using an ALK specific antigen and treatment using viral vectors for RNAi are emerging potential therapeutic possibilities.#CI- Copyright 2010 Elsevier Ltd. ALL rights reserved.

Neuroblastoma is a pediatric cancer of the developing sympathetic nervous systemthat most often affects young children. It remains an important pediatric problem because it accounts for approximately 15% of childhood cancer mortality. The disease is clinically heterogeneous, with the likelihood of cure varying greatlyaccording to age at diagnosis, extent of disease, and tumor biology. This extreme clinical heterogeneity reflects the complexity of genetic and genomic events associated with development and progression of disease. Inherited genetic variants and mutations that initiate tumorigenesis have been identified in neuroblastoma and multiple somatically acquired genomic alterations have been described that are relevant to disease progression. This chapter focuses on recent genome-wide studies that have utilized high-density single nucleotide polymorphism (SNP) genotyping arrays to discover genetic factors predisposing totumor initiation such as rare mutations at locus 2p23 (in ALK gene) for familialneuroblastoma, common SNPs at 6p22 (FLJ22536 and FLJ44180) and 2q35 (BARD1), anda copy number polymorphism at 1q21.1 (NBPF23) for sporadic neuroblastoma. It also deals with well known and recently reported somatic changes in the tumor genome such as mutations, gain of alleles and activation of oncogenes, loss of alleles,or changes in tumor-cell ploidy leading to the diverse clinical behavior of neuroblastomas. Finally, this chapter reviews gene expression profiles of neuroblastoma associated with pathways of the signaling of neurotrophins and apoptotic factors that could have a role in neuroblastoma development and progression. Looking forward, a major challenge will be to understand how inherited genetic variation and acquired somatic alterations in the tumor genomeinteract to exact phenotypic differences in neuroblastoma, and cancer in general.

BACKGROUND: Neuroblastoma (NB) is a severe pediatric tumor originating from neural crest derivatives and accounting for 15% of childhood cancer mortality. The heterogeneous and complex genetic etiology has been confirmed with the identification of mutations in two genes, encoding for the receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK) and the transcription factor Paired-likeHomeobox 2B (PHOX2B), in a limited proportion of NB patients. Interestingly, these two genes are overexpressed in the great majority of primary NB samples and cell lines. These observations led us to test the hypothesis of a regulatory or functional relationship between ALK and PHOX2B underlying NB pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Following this possibility, we first confirmed astriking correlation between the transcription levels of ALK, PHOX2B and its direct target PHOX2A in a panel of NB cell lines. Then, we manipulated their expression in NB cell lines by siRNA-mediated knock-down and forced over-expression of each gene under analysis. Surprisingly, PHOX2B- and PHOX2A-directed siRNAs efficiently downregulated each other as well as ALK gene and, consistently, the enhanced expression of PHOX2B in NB cells yielded an increment of ALK protein. We finally demonstrated that PHOX2B drives ALK gene transcription by directly binding its promoter, which therefore represents a novel PHOX2B target. CONCLUSIONS/SIGNIFICANCE: These findings provide a compelling explanation of the concurrent involvement of these two genes in NB pathogenesis and are going to foster a better understanding of molecular interactions at the base of the disease. Moreover, this work opens new perspectives for NBs refractory to conventional therapies that may benefit from the design of novel therapeutic RNAi-based approaches for multiple gene targets.

Advances in the field of genomics have led to multiple recent discoveries in theunderstanding of genetic predisposition and molecular pathogenesis of the childhood cancer neuroblastoma. Neuroblastoma is the most common extracranial solid tumor of childhood and is responsible for 10% of childhood cancer related mortality. The genetic etiology of rare families with hereditary neuroblastoma is now largely understood, with the majority having activating mutations in the anaplastic lymphoma kinase (ALK) gene. Genome-wide association studies have identified multiple common, low penetrance genetic polymorphisms that are associated with a predisposition to sporadic neuroblastoma, and these associations are disease phenotype specific. While many of the discoveries related to variations in the host genome that predispose to neuroblastoma are recent, there is a long and robust history of investigation of tumor cell genomics, leading to the identification of multiple biomarkers of tumor aggressiveness. Current patient risk stratification algorithms utilize key genomic features for therapy assignment. Microarray-based tumor DNA and RNA profiling techniques and next generation sequencing efforts may further refine these risk groups and identify new tractable therapeutic targets. Moving forward, integrative genomics efforts will be needed to discover how the interaction of germline genetic variations influence oncogenesis in neuroblastoma-both initiation and progression. In this review, we summarize the recent advances in the understanding of germline predisposition and molecular pathogenesis of neuroblastoma.#CI- Copyright (c) 2011 Elsevier Inc. ALL rights reserved.

Neuroblastoma (NB) is a childhood tumor originating from sympathetic nervous system cells. Although recently new insights into genes involved in NB have emerged, the molecular basis of neuroblastoma development and progression still remains poorly understood. The best-characterized genetic alterations include amplification of the proto-oncogene MYCN, ALK activating mutations or amplification, gain of chromosome arm 17q and losses of 1p, 3p, and 11q. Epigenetic alterations have been described as well: caspase-8 (CASP8) and RAS-association domain family 1 isoform A (RASSF1A) DNA-methylation are important events for the development and progression of neuroblastoma. In total, there areabout 75 genes described as epigenetically affected in NB cell lines and/or NB primary samples. These epigenetic alterations were either found using a candidate gene approach or based on the analysis of genome-wide screening techniques. Thisreview gives an extensive overview of ALL epigenetic changes described in NB as of today, with a main focus on both prognostic use and the potential of genome-wide techniques to find epigenetic prognostic biomarkers in NB. We summarize the key findings so far and the state-of-the-art of the upcoming methods at a unique time frame in the transition towards combined genome wide chromatin immune-precipitation (ChIP) and DNA sequencing techniques.

BACKGROUND: Neuroblastoma (NB) is an important childhood cancer with a strong genetic component related to disease susceptibility. Approximately 1% of NB cases have a positive family history. Following a genome-wide linkage analysis and sequencing of candidate genes in the critical region, we identified ALK as the major familial NB gene. Dominant mutations in ALK are found in more than 50% of familial NB cases. However, in the families used for the linkage study, only about 50% of carriers of ALK mutations are affected by NB. METHODS: To test whether genetic variation may explain the reduced penetrance of the disease phenotype, we analyzed genome-wide genotype data in ALK mutation-positive families using a model-based linkage approach with different liability classes for carriers and non-carriers of ALK mutations. RESULTS: The region with the highest LOD score was located at chromosome 2p23-p24 and included the ALK locus under models of dominant and recessive inheritance. CONCLUSIONS: This finding suggests that variants in the non-mutated ALK gene or another gene linked to it may affect penetrance of the ALK mutations and risk of developing NB in familialcases.#CI- Copyright (c) 2011 S. Karger AG, Basel.

Neuroblastoma is a pediatric solid tumor that can be stratified into stroma-richand stroma-poor histological subgroups. The stromal compartment of neuroblastomais composed mostly of Schwann cells, and they play critical roles in the differentiation, survival, and angiogenic responses of tumor cells. In certain neuroblastoma cell lines, the coexistence of neuroblastic N-type and substrate-adherent S-type is frequently observed. One such cell line, SK-N-SH, harbors a F1174L oncogenic mutation in the anaplastic lymphoma kinase (ALK) gene. Treatment of SK-N-SH with an ALK chemical inhibitor, TAE684, resulted in the outgrowth of S-type cells that expressed the Schwann cell marker, S100alpha6. Nucleotide sequencing analysis of these TAE684-resistant (TR) sublines revealed the presence of the ALK F1174L mutation, suggesting their tumor origin, althoughALK protein was not detected. Consistent with these findings, TR cells displayedapproximately 9-fold higher IC(50) values than N-type cells. Also, unlike N-typecells, TR cells have readily detectable phosphorylated STAT3 but weaker phosphorylated AKT. Under coculture conditions, TR cells conferred survival to N-type cells against the apoptotic effect of TAE684. Cocultivation also greatly enhanced the overall phosphorylation of STAT3 and its transcriptional activity in N-type cells. Finally, conditioned medium from TR clones enhanced cell viabilityof N-type cells, and this effect was phosphatidylinositol 3-kinase dependent. Taken together, these results demonstrate the ability of tumor-derived S-type cells in protecting N-type cells against the apoptotic effect of an ALK kinase inhibitor through upregulating prosurvival signaling.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and arises from cells of the developing sympathoadrenergic lineage. Activating mutations in the gene encoding the ALK tyrosine kinase receptor predispose for NB. Here, we focus on the normal function of Alk signaling in the control of sympathetic neuron proliferation, as well as on the effects of mutant ALK. Forced expression of wild-type ALK and NB-related constitutively active ALK mutants in cultures of proliferating immature sympathetic neurons results in a strong proliferation increase, whereas Alk knockdown and pharmacological inhibition of Alk activity decrease proliferation. Alk activation upregulates NMyc and trkB and maintains Alk expression by an autoregulatory mechanism involving Hand2. The Alk-ligand Midkine (Mk) is expressed in immature sympathetic neurons and in vivoinhibition of Alk signaling by virus-mediated shRNA knockdown of Alk and Mk leads to strongly reduced sympathetic neuron proliferation. Taken together, these results demonstrate that the extent and timing of sympathetic neurogenesis is controlled by Mk/Alk signaling. The predisposition for NB caused by activating ALK mutations may thus be explained by aberrations of normal neurogenesis, i.e. elevated and sustained Alk signaling and increased NMyc expression.

BACKGROUND: The Anaplastic Lymphoma Kinase (ALK) is an orphan receptor tyrosine kinase, which undergoes post-translational N-linked glycosylation. The catalyticdomain of ALK was originally identified in the t(2;5) translocation that produces the unglycosylated oncogenic protein NPM-ALK, which occurs in Anaplastic Large Cell Lymphoma (ALCL). Recently, both germline and somatic activating missense mutations of ALK have been identified in neuroblastoma (NB), a pediatric cancer arising from neural crest cells. Moreover, we previously reported that ALK expression is significantly upregulated in advanced/metastatic NB. We hypothesized that ALK function may depend on N-linked glycosylation and that disruption of this post-translational modification would impair ALK activation, regardless the presence of either gene mutations or overexpression. METHODS: We employed tunicamycin to inhibit N-linked glycosylation. The following ALK-positive NB cell lines were used: SH-SY5Y and KELLY (ALK mutation F1174L), UKF-NB3 (ALK mutation R1275Q) and NB1 (ALK amplification). As a control, we usedthe NB cell lines LA1-5S and NB5 (no ALK expression), and the ALCL cell line SU-DHL1 (NPM-ALK). RESULTS: Tunicamycin treatment of ALK-positive NB cells resulted in a hypoglycosylated ALK band and in decreased amounts of mature full size receptor. Concomitantly, we observed a marked reduction of mature ALK phosphorylation. On the contrary, tunicamycin had no effects on NPM-ALK phosphorylation in SU-DHL1 cells. Moreover, phosphorylation levels of ALK downstream effectors (AKT, ERK1/2, STAT3) were clearly impaired only in ALK mutated/amplified NB cell lines, whereas no significant reduction was observed in both ALK-negative and NPM-ALK-positive cell lines. Furthermore, inhibition of N-linked glycosylation considerably impaired cell viability only of ALK mutated/amplified NB cells. Finally, the cleavage of the Poly-ADP-ribose-polymerase (PARP) suggested that apoptotic pathways may be involved in cell death. CONCLUSIONS: In this study we showed that inhibition of N-linked glycosylation affects ALK phosphorylation and disrupts downstream pro-survival signaling, indicating that inhibition of this post-translational modification may be a promising therapeutic approach. However, as tunicamycin isnot a likely candidate for clinical use other approaches to alter N-linked glycosylation need to be explored. Future studies will assess whether the efficacy in inhibiting ALK activity might be enhanced by the combination of ALK specific small molecule and N-linked glycosylation inhibitors.

Anaplastic lymphoma kinase-(ALK-) positive large B-cell lymphoma (ALK+ LBCL) is a rare, aggressive tumor characterized by an immunoblastic or plasmablastic morphologic appearance, expression of ALK, CD138, CD45, EMA, and often IgA by immunohistochemistry, and characteristic chromosomal translocations or rearrangements involving the ALK locus. The morphologic and immunophenotypic overlap of this tumor with other hematologic and nonhematologic malignancies mayresult in misdiagnosis. The tumor has been identified in both pediatric and adult populations and demonstrates a male predominance. Presentation is most often nodal, particularly cervical. No association with immunocompromise or geographiclocation has been recognized. The most common gene rearrangement is between clathrin and ALK (t(2;17)(p23;q23)), resulting in the CLTC-ALK chimeric protein,although other fusions have been described. Response to conventional chemotherapy is poor. The recent introduction of the small molecule ALK inhibitor, crizotinib, may provide a potential new therapeutic option for patients with this disease.

Neuroblastoma is the most common cancer in childhood. The majority of patients with neuroblastoma are assigned to the high-risk group based on age at diagnosis, stage, histology, MYCN status, and DNA ploidy. Their prognosis remains unsatisfactory; the 5-year event-free survival (EFS) rate is generally 40 %. During the past 20 years, much effort has been made to reinforce chemotherapy, including the introduction of high-dose chemotherapy with autologous stem cell rescue, resulting in a 5-year EFS rate of around 30 %. Subsequently, maintenancetherapy aimed at eradicating residual tumors after induction and consolidation therapies was introduced, consisting of differentiation-inducing agents, retinoids, and immunotherapy using anti-GD2 antibodies combined with cytokines. However, such additional treatment provided benefit to only 10-20 % of patients,while the prognosis of about half the patients remains poor. Currently, novel targeted agents are under development. Among them, anaplastic lymphoma kinase (ALK) inhibitors and aurora kinase A inhibitors are promising. ALK somatic mutation or gene amplification predisposing neuroblastoma development occurs in up to 15 % of neuroblastomas. Crizotinib is a dual-specific inhibitor of ALK/Metand inhibits proliferation of neuroblastoma cells harboring R1275Q-mutated ALK or amplified wild-type ALK, but not cells harboring F1174L. Instead, cells with F1174L are sensitive to another small molecule ALK inhibitor, TAE684. Aurora kinase A plays a pivotal role in centrosome maturation and spindle formation during mitosis. MLN8237 (alisertib) is a small molecule inhibitor of aurora kinase A that is currently in early-phase clinical testing. Future treatment will be individually planned, adapting targeted agents based on personal biological tumor characteristics.

The anaplastic lymphoma kinase (ALK) gene has been found either rearranged or mutated in several neoplasms such as anaplastic large-cell lymphoma, non-small-cell lung cancer, neuroblastoma and anaplastic thyroid cancer. Medulloblastoma (MB) is an embryonic pediatric cancer arising from nervous system, a tissue in which ALK is expressed during embryonic development. We performed an ALK mutation screening in 52 MBs and we found a novel heterozygous germline deletion of a single base in exon 23 (3605delG) in a case with marked anaplasia. This G deletion results in a frameshift mutation producing a premature stop codon in exon 25 of ALK tyrosine kinase domain. We also screened three human MB cell lines without finding any mutation of ALK gene. Quantitative expression analysis of 16 out of 52 samples showed overexpression of ALK mRNA in three MBs.In the present study, we report the first mutation of ALK found in MB. Moreover,a deletion of ALK gene producing a stop codon has not been detected in human tumors up to now. Further investigations are now required to elucidate whether the truncated form of ALK may have a role in signal transduction.

OBJECTIVE: To investigate mutations of anaplastic lymphoma kinase (ALK) in Chinese children with neuroblastoma (NB). METHODS: Genomic DNA was extracted from 22 cases of paraffin-embedding NB tumor tissues. Gene mutations in the exons 20-26 which were mutational hotspots of ALK were analyzed by PCR-DNA direct sequencing. RESULTS: A novel synonymous mutation C3586T (Leu1196Leu) and a knownsynonymous mutation C3375A (Gly1125Gly) were found and located at exon 23 and exon 21 of ALK respectively. There were 10 cases (46%) of known synonymous mutation C3375A in 22 cases of NB. The C3375A allelic frequency was 27%. No statistically significant correlation was found between mutation C3375A and clinical parameters of NB such as age, sex, metastasis and tumor differentiation. mutation was not found in the other 5 exons. CONCLUSIONS: A novel ALK gene synonymous mutation C3586T was identified using PCR-DNA sequencing. A known mutation C3375A in ALK was successfully identified in children, and its incidence is not influenced by the clinical features of childhood NB.

Activating mutations of the ALK gene have been identified in sporadic and familial cases of neuroblastoma (NB), a cancer of the peripheral nervous system,and are thought to be the primary mechanism of oncogenic activation of this receptor in this pediatric neoplasm. To address the possibility that ALK activation may occur through genomic rearrangements as detected in other cancers, we first took advantage of high-resolution array-comparative genomic hybridization to search for ALK rearrangements in NB samples. Using complementary experiments by capture/paired-end sequencing and FISH experiments, various typesof rearrangements were fully characterized, including partial gains or amplifications, in several NB cell lines and primary tumors. In the CLB-Bar cellline, we described a genomic rearrangement associated with an amplification of the ALK locus, leading to the expression of a 170 kDa protein lacking part of the extracellular domain encoded by exons 4 to 11, named ALK(Delta4-11). Analysis ofgenomic DNA from the tumor at diagnosis and relapse revealed that the ALK gene was amplified at diagnosis but that the rearranged ALK allele was observed at the relapse stage only, suggesting that it may be implicated in tumor aggressiveness. Consistently, oncogenic and tumorigenic properties of the ALK(Delta4-11) variantwere shown after stable expression in NIH3T3 cells. Moreover, we documented an increased constitutive kinase activity of this variant, as well as an impaired maturation and retention into intracellular compartments. These results indicatethat genomic rearrangements constitute an alternative mechanism to ALK point mutations resulting in receptor activation.

Neuroblastoma is the most common solid tumor of infancy, and mutations in several genes have been implicated as playing a role in tumor development. Here, we describe a pediatric patient with a constitutional microduplication of 2p24.3 who developed Stage 4 neuroblastoma at age 11 months. He represents the sixth patient described in the literature with partial trisomy 2p and neuroblastoma. ALL previous cases had duplication events spanning two genes implicated in neuroblastoma, MYCN and ALK. Our patient is unique because his duplicated regionincludes the MYCN gene only; the ALK gene is unaffected. These data, combined with the relatively high incidence of neuroblastoma reported in partial trisomy 2p patients, support the notion that MYCN duplication should be added to the growing list of genetic factors associated with an increased risk of neuroblastoma. The mechanism of increased risk is unclear, but the fact that ourpatient had dramatic amplification of MYCN in his tumor suggests that a germlineduplication might predispose to further amplification. Additionally, our patienthas several morphologic features common to patients with partial trisomy 2p including high forehead, hypertelorism, postaxial polydactyly, and developmentaldelay despite having a microduplication spanning approximately 1 Mb and including just three intact genes. This case may therefore help further delineate the genotype-phenotype correlations associated with partial trisomy 2p.#CI- Copyright (c) 2013 Wiley Periodicals, Inc.

Anaplastic lymphoma kinase (ALK) and MYC are oncogenes often dysregulated in pediatric lymphomas. NPM-ALK/t(2;5)(p23;q35) is a genetic hallmark of ALK anaplastic large cell lymphoma (ALCL). MYC gene translocations are frequently detected in high-grade B-cell lymphomas. ALKALCL cases with concurrent MYC translocation are exceedingly rare and are more aggressive and chemoresistent compared with other ALKALCL. We report a patient who presented with ALKALCL possessing coexistent MYC rearrangement, massive tumor dissemination, and early widespread relapse. This case underscores the importance of recognition of closecorrelation between dual ALK and MYC rearrangements and the characteristic clinical features in this unusual ALCL variant.

Anaplastic lymphoma kinase (ALK) is an oncogenic tyrosine kinase that is deregulated due to a variety of molecular mechanisms in pediatric cancer. They include chromosomal translocations, activation mutations, and gene amplifications. Since the initial discovery of ALK as an oncogenic tyrosine kinase involved in the chromosomal translocation t(2, 5)(p23;q35) in 1994, more than 20 translocation partners of ALK have been identified in various cancers. Furthermore, deregulation of ALK tyrosine kinase activity is critical for the pathogenesis of several other pediatric tumors, including neuroblastomas and inflammatory myofibroblastic tumors. The recent discovery of ALK translocations in adult lung cancer patients (non-small cell lung cancer) has accelerated the development of inhibitors of ALK tyrosine kinase as therapeutic agents. While excellent clinical response has been observed in many patients, the acquisition of clinical resistance to ALK inhibition highlights the need for development of second-generation ALK kinase inhibitors and/or combination therapies that targetdownstream signaling mediators or antibody drug conjugates. This article provides an update on the spectrum of ALK-driven tumors in the pediatric population and the potential therapies which target these tumors.