Activating mutations in ALK provide a therapeutic target in neuroblastoma

Neuroblastoma, an embryonal tumour of the peripheral sympathetic nervous system, accounts for approximately 15% of all deaths due to childhood cancer. High-risk neuroblastomas are rapidly progressive; even with intensive myeloablative chemotherapy, relapse is common and almost uniformly fatal. Here we report the detection of previously unknown mutations in the ALK gene, which encodes a receptor tyrosine kinase, in 8% of primary neuroblastomas. Five non-synonymous sequence variations were identified in the kinase domain of ALK, of which three were somatic and two were germ line. The most frequent mutation, F1174L, was also identified in three different neuroblastoma cell lines. ALK complementary DNAs encoding the F1174L and R1275Q variants, but not the wild-type ALK cDNA, transformed interleukin-3-dependent murine haematopoietic Ba/F3 cells to cytokine-independent growth. Ba/F3 cells expressing these mutations were sensitive to the small-molecule inhibitor of ALK, TAE684 (ref. 4). Furthermore, two human neuroblastoma cell lines harbouring the F1174L mutation were also sensitive to the inhibitor. Cytotoxicity was associated with increased amounts of apoptosis as measured by TdT-mediated dUTP nick end labelling (TUNEL). Short hairpin RNA (shRNA)-mediated knockdown of ALK expression in neuroblastoma cell lines with the F1174L mutation also resulted in apoptosis and impaired cell proliferation. Thus, activating alleles of the ALK receptor tyrosine kinase are present in primary neuroblastoma tumours and in established neuroblastoma cell lines, and confer sensitivity to ALK inhibition with small molecules, providing a molecular rationale for targeted therapy of this disease.     

Identification of ALK as a major familial neuroblastoma predisposition gene

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 scan in 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 194 high-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, and targeted 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 germline or acquired activation of this cell-surface kinase is a tractable therapeutic target for this lethal paediatric malignancy.     

Oncogenic mutations of ALK kinase in neuroblastoma

Neuroblastoma in advanced stages is one of the most intractable paediatric cancers, even with recent therapeutic advances. Neuroblastoma harbours a variety of genetic changes, including a high frequency of MYCN amplification, loss of heterozygosity at 1p36 and 11q, and gain of genetic material from 17q, all of which have been implicated in the pathogenesis of neuroblastoma. However, the scarcity of reliable molecular targets has hampered the development of effective therapeutic agents targeting neuroblastoma. Here we show that the anaplastic lymphoma kinase (ALK), originally identified as a fusion kinase in a subtype of non-Hodgkin's lymphoma (NPM-ALK) and more recently in adenocarcinoma of lung (EML4-ALK), is also a frequent target of genetic alteration in advanced neuroblastoma. According to our genome-wide scans of genetic lesions in 215 primary neuroblastoma samples using high-density single-nucleotide polymorphism genotyping microarrays, the ALK locus, centromeric to the MYCN locus, was identified as a recurrent target of copy number gain and gene amplification. Furthermore, DNA sequencing of ALK revealed eight novel missense mutations in 13 out of 215 (6.1%) fresh tumours and 8 out of 24 (33%) neuroblastoma-derived cell lines. All but one mutation in the primary samples (12 out of 13) were found in stages 3-4 of the disease and were harboured in the kinase domain. The mutated kinases were autophosphorylated and displayed increased kinase activity compared with the wild-type kinase. They were able to transform NIH3T3 fibroblasts as shown by their colony formation ability in soft agar and their capacity to form tumours in nude mice. Furthermore, we demonstrate that downregulation of ALK through RNA interference suppresses proliferation of neuroblastoma cells harbouring mutated ALK. We anticipate that our findings will provide new insights into the pathogenesis of advanced neuroblastoma and that ALK-specific kinase inhibitors might improve its clinical outcome.     

Sequencing of neuroblastoma identifies chromothripsis and defects in neuritogenesis genes

Neuroblastoma is a childhood tumour of the peripheral sympathetic nervous system. The pathogenesis has for a long time been quite enigmatic, as only very few gene defects were identified in this often lethal tumour. Frequently detected gene alterations are limited to MYCN amplification (20%) and ALK activations (7%). Here we present a whole-genome sequence analysis of 87 neuroblastoma of all stages. Few recurrent amino-acid-changing mutations were found. In contrast, analysis of structural defects identified a local shredding of chromosomes, known as chromothripsis, in 18% of high-stage neuroblastoma. These tumours are associated with a poor outcome. Structural alterations recurrently affected ODZ3, PTPRD and CSMD1, which are involved in neuronal growth cone stabilization. In addition, ATRX, TIAM1 and a series of regulators of the Rac/Rho pathway were mutated, further implicating defects in neuritogenesis in neuroblastoma. Most tumours with defects in these genes were aggressive high-stage neuroblastomas, but did not carry MYCN amplifications. The genomic landscape of neuroblastoma therefore reveals two novel molecular defects, chromothripsis and neuritogenesis gene alterations, which frequently occur in high-risk tumours.     

Expression of N-myc in teratocarcinoma stem cells and mouse embryos

The N-myc gene, which is distantly related to the proto-oncogene c-myc, was first detected as an amplified sequence in human neuroblastoma cell lines and tumours. It has since been revealed that there is up to a 300-fold amplification of N-myc DNA in almost 50% of advanced metastatic human neuroblastomas, whereas amplification is not detected in less advanced tumours that have a better prognosis (ref.3 and M.S., unpublished data). Although expression of N-myc is detectable in all neuroblastoma cell lines and tumours examined, its level is greatly enhanced when the N-myc gene is amplified. Recently, it has been shown that on co-transfection with the c-Ha-ras (EJ) gene, N-myc can induce the malignant transformation of rat embryo fibroblasts. Taken together, these data imply a function for N-myc in the development and/or progression of human neuroblastomas. Surveys indicate that N-myc also may be amplified and/or expressed in two other types of human tumours and cell lines derived from them: retinoblastomas and small cell lung cancers. Here, we report that N-myc is expressed at high levels in mouse and human teratocarcinoma stem cells, thus identifying another tumour cell type that expresses the N-myc gene. In addition, we found that N-myc is abundantly expressed in mouse embryos at mid-gestation and that its expression appears to decrease as the embryo approaches term. In the adult mouse, N-myc is expressed at an approximately fivefold lower level in the brain than in teratocarcinoma stem cells and embryos, and at even lower levels in the adult testis and kidney. Our data represent the first demonstration of expression of the N-myc gene in normal cells, and suggest that N-myc may be involved in mammalian embryogenesis.     

全自动免疫组化筛查ALK基因融合非小细胞肺癌及其病理特征

目的探讨全自动免疫组化筛查间变性淋巴瘤激酶(ALK)基因融合非小细胞肺癌的临床特点及病理特征.方法选取经病理检查确诊的554例非小细胞肺癌组织,采用Ventana抗ALK试剂和全自动免疫组化(IHC)染色检测ALK状态,分析ALK基因融合非小细胞肺癌的临床特点和病理特征.结果本次研究的554例非小细胞肺癌患者组织中,共筛选出34例ALK阳性,占6.14%;年龄60岁的非小细胞肺癌患者ALK阳性率8.69%,明显高于年龄≥60岁的3.62%,差异具有显著统计学意义(P0.01);男性患者ALK阳性率6.71%高于女性5.21%,但差异无统计学意义(P0.05).组织形态学方面,34例ALK阳性非小细胞肺癌中28例为肺腺癌,6例为非肺腺癌.16例实体型为主腺癌合并黏液产生,7例腺泡型为主腺癌,1例为乳头型为主腺癌,4例为浸润性黏液腺癌,4例为鳞状细胞癌.EGFR基因突变检测显示:仅有1例合并该基因突变,其余均为野生型.9例IHC阳性样本,9例ALK基因融合非小细胞肺癌,9例IHC阴性样本经荧光原位杂交技术检测和RT-PCR检测均为阴性结果,6例IHC染色可能为阳性,经荧光原位杂交技术检测均显示为ALK融合阴性.结论 ALK基因融合肺癌是非小细胞肺癌一新的分子亚型,具有独特的临床表现和病理形态;Ventana抗ALK试剂和IHC染色是检测ALK阳性非小细胞肺癌首选方法,对提高该类型肺癌的检出率及个体化治疗具有重要意义.     

Decreased expression of N-myc precedes retinoic acid-induced morphological differentiation of human neuroblastoma

Proto-oncogenes may be important in the cellular processes central for the growth and differentiation of normal cells. N-myc is a DNA sequence which shares limited homology to the proto-oncogene c-myc and has been found to be amplified in both primary tissue and cell lines from neuroblastoma, a childhood tumour of neuroectodermal origin. Differentiation of this embryonal tumour is of clinical importance, since occasional tumours have been noted to differentiate in vivo to benign ganglioneuroma. In vitro, many human neuroblastoma cell lines can be induced to differentiate morphologically and biochemically by a variety of agents. Retinoic acid (RA), an analogue of vitamin A, has been shown to inhibit neuroblastoma cell growth and clonability in soft agar, and to induce extensive neurite outgrowth. Therefore we examined the relationship of N-myc expression to the in vitro differentiation of these cells. We report here that in the case of RA-induced differentiation, a decreased level of expression is detected within 6 h of treatment and precedes both cell-cycle changes and morphological differentiation.     

Inactivation of the apoptosis effector Apaf-1 in malignant melanoma

Metastatic melanoma is a deadly cancer that fails to respond to conventional chemotherapy and is poorly understood at the molecular level. p53 mutations often occur in aggressive and chemoresistant cancers but are rarely observed in melanoma. Here we show that metastatic melanomas often lose Apaf-1, a cell-death effector that acts with cytochrome c and caspase-9 to mediate p53-dependent apoptosis. Loss of Apaf-1 expression is accompanied by allelic loss in metastatic melanomas, but can be recovered in melanoma cell lines by treatment with the methylation inhibitor 5-aza-2'-deoxycytidine (5aza2dC). Apaf-1-negative melanomas are invariably chemoresistant and are unable to execute a typical apoptotic programme in response to p53 activation. Restoring physiological levels of Apaf-1 through gene transfer or 5aza2dC treatment markedly enhances chemosensitivity and rescues the apoptotic defects associated with Apaf-1 loss. We conclude that Apaf-1 is inactivated in metastatic melanomas, which leads to defects in the execution of apoptotic cell death. Apaf-1 loss may contribute to the low frequency of p53 mutations observed in this highly chemoresistant tumour type.     

Human N-myc gene contributes to neoplastic transformation of mammalian cells in culture

Proto-oncogenes represent a group of eukaryotic genes whose activated forms are implicated in the development of cancer. We have recently identified a human gene, N-myc, that is distantly related to the proto-oncogene c-myc. N-myc is expressed at abnormally high levels consequent to amplification in numerous human neuroblastoma cell lines and metastatic neuroblastoma tumours. In addition, enhanced expression of N-myc, often a result of amplification, has been found in retinoblastoma cell lines and tumours (refs 5, 7 and M.S., unpublished data) and in cell lines derived from small-cell carcinomas of the lung. Here, we show that enhanced expression of N-myc subsequent to co-transfections of an N-myc expression vector and the mutant c-Ha-ras-1(EJ) (from the human bladder carcinoma cell line EJ) is a factor in tumorigenic conversion of secondary rat embryo cells. The transformed cells elicit tumours in athymic mice and isogeneic rats. The ability of N-myc to contribute to neoplastic transformation of cultured mammalian cells raises the possibility that enhanced expression consequent to amplification of N-myc may be a factor in the aetiology of human neuroblastoma.     

Preferential mutation of paternally derived RB gene as the initial event in sporadic osteosarcoma

Successive loss of function of both alleles of the retinoblastoma susceptibility gene (RB) on human chromosome 13 seems to be critical in the development of retinoblastoma and osteosarcoma. In cases where the tumour is familial and susceptibility is inherited, a mutation in one of the alleles is carried in the germline. We have recently shown that cytogenetically visible germline mutations are usually in the paternally derived gene. Such a bias would not be expected for sporadic (non-familial) tumours, where both mutations occur in somatic tissue, but there has been some indication of a bias towards initial somatic mutation in the paternally derived gene on chromosome 11 in sporadic Wilms tumour. We have now examined 13 sporadic osteosarcomas and find evidence which indicates that in 12 cases the initial mutation was in the paternal gene, suggesting the involvement of germinal imprinting in producing the differential susceptibility of the two genes to mutation.     

Amplified DNA with limited homology to myc cellular oncogene is shared by human neuroblastoma cell lines and a neuroblastoma tumour

Amplified cellular genes in mammalian cells frequently manifest themselves as double minute chromosomes (DMs) and homogeneously staining regions of chromosomes (HSRs). With few exceptions both karyotypic abnormalities appear to be confined to tumour cells. All vertebrates possess a set of cellular genes homologous to the transforming genes of RNA tumour viruses, and there is circumstantial evidence that these cellular oncogenes are involved in tumorigenesis. We have recently shown that DMs and HSRs in cells of the mouse adrenocortical tumour Y1 and an HSR in the human colon carcinoma COLO320 contain amplified copies of the cellular oncogenes c-Ki-ras and c-myc, respectively. Both DMs and HSRs are found with remarkable frequency in cells of human neuroblastomas. We show here that a DNA domain detectable by partial homology to the myc oncogene is amplified up to 140-fold in cell lines derived from different human neuroblastomas and in a neuroblastoma tumour, but not in other tumour cells showing cytological evidence for gene amplification. By in situ hybridization we found that HSRs are the chromosomal sites of the amplified DNA. The frequency with which this amplification appears in cells from neuroblastomas and its apparent specificity raise the possibility that one or more of the genes contained within the amplified domain contribute to tumorigenesis.     

Somatic misexpression of germline P granules and enhanced RNA interference in retinoblastoma pathway mutants

Caenorhabditis elegans homologues of the retinoblastoma (Rb) tumour suppressor complex specify cell lineage during development. Here we show that mutations in Rb pathway components enhance RNA interference (RNAi) and cause somatic cells to express genes and elaborate perinuclear structures normally limited to germline-specific P granules. Furthermore, particular gene inactivations that disrupt RNAi reverse the cell lineage transformations of Rb pathway mutants. These findings suggest that mutations in Rb pathway components cause cells to revert to patterns of gene expression normally restricted to germ cells. Rb may act by a similar mechanism to transform mammalian cells.     

Mutations of the BRAF gene in human cancer

Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS RAF MEK ERK MAP kinase pathway mediates cellular responses to growth signals. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.     

Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-Fraumeni syndrome

Tumour suppressor genes, whose usual function seems to be controlling normal cell proliferation, have been implicated in many inherited and sporadic forms of malignancies Much evidence supports the concept of tumour formation by loss-of-function mutations in suppressor genes, as predicted by the two-hit model of Knudson and DeMars. The suppressor gene, p53, is affected in such a manner by numerous mutations, which occur in a variety of human tumours. These mutations usually represent the loss of one allele and the substitution of a single base in the other. We have now analysed the p53 gene in a family affected by Li-Fraumeni syndrome, a rare autosomal dominant syndrome characterized by the occurrence of diverse mesenchymal and epithelial neoplasms at multiple sites. In some instances the neoplasms seem to be related to exposure to carcinogens, including ionizing radiation. The Li-Fraumeni family that we studied had noncancerous skin fibroblasts (NSF) with an unusual radiation-resistant phenotype. DNA derived from the NSF cells of four family members, spanning two generations, had the same point mutation in codon 245 (GGC----GAC) of the p53 gene. This mutation leads to substitution of aspartic acid for glycine in one of the regions identified as a frequent target of point mutations in p53. The NSF cell lines with the mutation also retained the normal p53 allele. This inherited p53 mutation may predispose the members of this family to increased susceptibility to cancer.