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Background Inflammation is key in the pathogenesis of Nonalcoholic Fatty Liver Disease (NAFLD) – a common progressive liver disease. The soluble receptor for advanced glycation end products (sRAGE) attenuates inflammatory signaling; low levels of sRAGE are correlated with increased inflammation. Aim We sought to describe associations between sRAGE and NAFLD. Methods We conducted a cross-sectional analysis of 1088 Atherosclerosis Risk in Communities (ARIC) Study participants and used logistic regression to investigate the associations between sRAGE and NAFLD defined by elevated liver enzymes and fibrosis score. Results In this community-based sample (n = 1,088, mean age 56 years, 61% female, 78% Caucasian), persons in the lowest vs. highest quartile of sRAGE had significantly higher odds of elevated ALT (OR 2.82, 95% CI 1.18-6.76) but not elevated AST (OR 1.16, 95% CI 0.45-2.99); persons in the lowest vs. highest quartile had significantly lower odds of elevated FIB-4 index (OR 0.56, 95% CI 0.37-0.84). Conclusions We found an inverse cross-sectional association between sRAGE and liver inflammation; this is consistent with prior studies linking low sRAGE to inflammatory states. However, we observed a direct association between sRAGE and fibrosis. Our findings suggest that sRAGE is dynamic in NAFLD and patterns may vary with different stages of disease.

PURPOSE OF REVIEW: Comprehensive lifestyle programs are cornerstones of obesity management, but clinician referrals may be limited by program availability. Commercial weight-loss programs may be an alternative, but clinicians may be unaware of their efficacy and safety. This review describes the evidence for commercial programs, particularly 12-month weight loss, among individuals with obesity. RECENT FINDINGS: Several programs are concordant with evidence-based recommendations (i.e., lower-calorie diet, increased physical activity, and behavioral strategies). Among the guideline-concordant programs, National Diabetes Prevention Program, WW, Jenny Craig, Medifast and OPTIFAST have demonstrated 12-month weight-loss efficacy and safety. While other programs show promise, more evidence is needed before clinician referral may be recommended. SUMMARY: Clinical practice guidelines support referrals to commercial weight-loss programs that have peer-reviewed evidence to support their efficacy and safety. Clinicians should consider the available evidence, patient preference, and cost when considering referrals to these programs for weight management.

Neuroblastoma is a childhood cancer that is often fatal despite intense multimodality therapy. In an effort to identify therapeutic targets for this disease, we performed a comprehensive loss-of-function screen of the protein kinome. Thirty kinases showed significant cellular cytotoxicity when depleted, with loss of the cell cycle checkpoint kinase 1 (CHK1/CHEK1) being the most potent. CHK1 mRNA expression was higher in MYC–Neuroblastoma-related (MYCN)– amplified ( P < 0.0001) and high-risk ( P = 0.03) tumors. Western blotting revealed that CHK1 was constitutively phosphorylated at the ataxia telangiectasia response kinase target site Ser345 and the autophosphorylation site Ser296 in neuroblastoma cell lines. This pattern was also seen in six of eight high-risk primary tumors but not in control nonneuroblastoma cell lines or in seven of eight low-risk primary tumors. Neuroblastoma cells were sensitive to the two CHK1 inhibitors SB21807 and TCS2312, with median IC 50 values of 564 nM and 548 nM, respectively. In contrast, the control lines had high micromolar IC 50 values, indicating a strong correlation between CHK1 phosphorylation and CHK1 inhibitor sensitivity ( P = 0.0004). Furthermore, cell cycle analysis revealed that CHK1 inhibition in neuroblastoma cells caused apoptosis during S-phase, consistent with its role in replication fork progression. CHK1 inhibitor sensitivity correlated with total MYC(N) protein levels, and inducing MYCN in retinal pigmented epithelial cells resulted in CHK1 phosphorylation, which caused growth inhibition when inhibited. These data show the power of a functional RNAi screen to identify tractable therapeutical targets in neuroblastoma and support CHK1 inhibition strategies in this disease.

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 familial cases.

Copy number variations (CNVs) and single nucleotide polymorphisms (SNPs) are two important potential sources of phenotypic variation in humans. Until now, only SNPs have been associated with cancer, but the increasing recognition that germline DNA dosage is a critical component of human diversity raises the possibility that CNVs might also influence susceptibility to this cancer. Diskin et al. now report that a common CNV at chromosome 1q21.1 is associated with the childhood cancer neuroblastoma, and that a transcript within this CNV, the previously unknown neuroblastoma breakpoint family gene NBPF23, is involved in the early stages of tumorigenesis. Copy number variations (CNVs) and single nucleotide polymorphisms (SNPs) are two important potential sources of phenotypic variation in humans; however, only SNPs have been associated with cancer. Here, a CNV at 1q21.1 is shown to be associated with neuroblastoma, and a transcript within this CNV, NBPF23, is implicated in early tumorigenesis of the disease. Common copy number variations (CNVs) represent a significant source of genetic diversity, yet their influence on phenotypic variability, including disease susceptibility, remains poorly understood. To address this problem in human cancer, we performed a genome-wide association study of CNVs in the childhood cancer neuroblastoma, a disease in which single nucleotide polymorphism variations are known to influence susceptibility1,2. We first genotyped 846 Caucasian neuroblastoma patients and 803 healthy Caucasian controls at ∼550,000 single nucleotide polymorphisms, and performed a CNV-based test for association. We then replicated significant observations in two independent sample sets comprised of a total of 595 cases and 3,357 controls. Here we describe the identification of a common CNV at chromosome 1q21.1 associated with neuroblastoma in the discovery set, which was confirmed in both replication sets. This CNV was validated by quantitative polymerase chain reaction, fluorescent in situ hybridization and analysis of matched tumour specimens, and was shown to be heritable in an independent set of 713 cancer-free parent–offspring trios. We identified a previously unknown transcript within the CNV that showed high sequence similarity to several neuroblastoma breakpoint family (NBPF) genes3,4 and represents a new member of this gene family (NBPF23). This transcript was preferentially expressed in fetal brain and fetal sympathetic nervous tissues, and the expression level was strictly correlated with CNV state in neuroblastoma cells. These data demonstrate that inherited copy number variation at 1q21.1 is associated with neuroblastoma and implicate a previously unknown neuroblastoma breakpoint family gene in early tumorigenesis of this childhood cancer.

We conducted a SNP-based genome-wide association study (GWAS) focused on the high-risk subset of neuroblastoma. As our previous unbiased GWAS showed strong association of common 6p22 SNP alleles with aggressive neuroblastoma, we restricted our analysis here to 397 high-risk cases compared to 2,043 controls. We detected new significant association of six SNPs at 2q35 within the BARD1 locus (P(allelic) = 2.35 x 10(-9)-2.25 x 10(-8)). We confirmed each SNP association in a second series of 189 high-risk cases and 1,178 controls (P(allelic) = 7.90 x 10(-7)-2.77 x 10(-4)). We also tested the two most significant SNPs (rs6435862, rs3768716) in two additional independent high-risk neuroblastoma case series, yielding combined allelic odds ratios of 1.68 each (P = 8.65 x 10(-18) and 2.74 x 10(-16), respectively). We also found significant association with known BARD1 nonsynonymous SNPs. These data show that common variation in BARD1 contributes to the etiology of the aggressive and most clinically relevant subset of human neuroblastoma.

Neuroblastoma is a malignant condition of the developing sympathetic nervous system that most commonly affects young children and is often lethal. Its cause is not known.We performed a genomewide association study by first genotyping blood DNA samples from 1032 patients with neuroblastoma and 2043 control subjects of European descent using the Illumina HumanHap550 BeadChip. Samples from three independent groups of patients with neuroblastoma (a total of 720 patients) and 2128 control subjects were then genotyped to replicate significant associations.We observed a significant association between neuroblastoma and the common minor alleles of three consecutive single-nucleotide polymorphisms (SNPs) at chromosome band 6p22 and containing the predicted genes FLJ22536 and FLJ44180 (P=1.71x10(-9) to 7.01x10(-10); allelic odds ratio, 1.39 to 1.40). Homozygosity for the at-risk G allele of the most significantly associated SNP, rs6939340, resulted in an increased likelihood of the development of neuroblastoma (odds ratio, 1.97; 95% confidence interval, 1.58 to 2.45). Subsequent genotyping of the three 6p22 SNPs in three independent case series confirmed our observation of an association (P=9.33x10(-15) at rs6939340 for joint analysis). Patients with neuroblastoma who were homozygous for the risk alleles at 6p22 were more likely to have metastatic (stage 4) disease (P=0.02), amplification of the MYCN oncogene in the tumor cells (P=0.006), and disease relapse (P=0.01).A common genetic variation at chromosome band 6p22 is associated with susceptibility to neuroblastoma.

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.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.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.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 familial cases.

PHOX2B is a homeodomain-containing protein that is involved in the development of the peripheral nervous system and is the major disease gene for the rare congenital breathing disorder congenital central hypoventilation syndrome (CCHS). Germline PHOX2B alterations were also recently discovered in neuroblastoma cases with CCHS and/or Hirschsprung disease, but a comprehensive survey for mutational frequency and functional consequence has not been performed. We therefore studied a large panel of hereditary neuroblastomas to understand the frequency and functional effects of PHOX2B mutations. Three of 47 individuals with presumed genetic predisposition to neuroblastoma showed a germline PHOX2B mutation (6.4%). Mutations were also discovered in 2 of 30 human neuroblastoma-derived cell lines, but none of 86 primary tumors from patients with sporadically occurring neuroblastoma. The vast majority of primary tumors showed abundant PHOX2B mRNA expression relative to the remainder of the transcriptome. Consistent with its role as an important neurodevelopmental gene, forced overexpression of wild-type PHOX2B in neuroblastoma cell lines suppressed cell proliferation and synergized with all-trans retinoic acid to promote differentiation. Patient-derived mutant PHOX2B constructs retained the ability to suppress cellular proliferation, but were not able to promote differentiation or activate expression of a known PHOX2B target gene in vitro. These findings show that PHOX2B alterations are a rare cause of hereditary neuroblastoma, but disruption of this neurodevelopmental pathway can interfere with transcription-dependent terminal differentiation. These data also suggest that the genetics of neuroblastoma initiation are complex, and highlight genes involved in normal noradrenergic development as candidate predisposition genes.

To the Editor: We read with interest the study by Trochet and colleagues (2004), published in the April 2004 issue of The American Journal of Human Genetics, that described germline mutations of the paired-like homeobox 2B gene (PHOX2B [MIM 603851]) in neuroblastoma (MIM 256700). We have also considered PHOX2B as a candidate gene for predisposition to neuroblastoma, and we now report on a germline PHOX2B mutation in a pedigree with neuroblastoma. However, we also show that there is no evidence for mutation of this gene in eight other pedigrees with neuroblastoma screened to date. We think these data establish PHOX2B as the first bona fide gene that can predispose to neuroblastoma when mutated in the germline, and the findings further emphasize the complex genetics of this important pediatric malignancy. We previously demonstrated linkage of hereditary neuroblastoma to 16p12-13 by use of a genomewide screening strategy (Maris et al. 2002). Positional cloning of a putative 16p12-13 hereditary neuroblastoma-predisposition gene (HNB1) is ongoing, but the critical genomic region for this gene remains large. We had previously considered and excluded other genes known to be mutated in Hirschsprung disease (MIM 142623) and/or in congenital central hypoventilation syndrome (CCHS [MIM 209880]) as candidates for HNB1, because these disorders can occur coincident with both sporadic and hereditary neuroblastoma (Maris et al. 2002). Because of the recent reports that the vast majority of patients with CCHS harbor PHOX2B mutations, including two patients also affected with neuroblastoma (Amiel et al. 2003; Weese-Mayer et al. 2003), we initiated a screen for germline mutations in this gene in our series of pedigrees with neuroblastoma. Oligonucleotide primer pairs flanking the coding regions of exons 1, 2, and 3 of PHOX2B were designed by use of the program Primer 3.0; these primer pairs were used for PCR amplification and bidirectional sequencing of purified PCR products (primer sequences available on request). We screened germline DNA from the proband and an unaffected family member for each of the seven families that showed cosegregation of a 16p haplotype with disease, as well as for two pedigrees that consisted of cousins with neuroblastoma with no cosegregation of 16p marker haplotypes (see Maris et al. [2002] for details of pedigrees). We also sequenced 109 control DNA samples from the Coriell SNP500 Cancer Panel (Coriell Cell Repositories). All sequence aberrations were confirmed by repeat sequencing after cloning of purified PCR products (TOPO TA Cloning Kit [Invitrogen]), and DNA samples from the remaining available members of the pedigree were also screened for the variant. The Children’s Hospital of Philadelphia institutional review board approved this work. A heterozygous single-base deletion (676delG) was discovered in a complex pedigree with neuroblastoma (fig. 1) (see dbSNP Home Page). This family has seven members in three generations affected with neuroblastoma, and two of these individuals were also shown to have Hirschsprung disease. The proband was affected with neuroblastoma, Hirschsprung disease, and neurofibromatosis type 1 (MIM 162200). The putative nonsense mutation 676delG segregated with neuroblastoma through all three generations, and the frameshift was predicted to produce a slightly truncated protein that would no longer code for the second polyalanine tract. This family had previously been shown to cosegregate a 16p12-13 haplotype with neuroblastoma, and the proband was also shown to have an inactivating mutation in NF1 (3775delT) that was not present in either of her parents (Maris et al. 2002). Tumor material was available only for patient 1-001, and the tumor exon 3 sequence remained heterozygous for the 676delG mutation. In addition, loss-of-heterozygosity studies using microsatellite markers (D4S2912, D4S1587, D4S405, D4S2971, and D4S428) that are closely linked to the PHOX2B locus showed no evidence for allelic deletion. The only other sequence variant discovered in the remaining eight pedigrees was a putative SNP (C552T) in pedigree 12 that is not predicted to affect the resultant protein sequence (S184S) (see dbSNP Home Page). This sequence variant was present in the proband but was not detected in the patient’s affected father (no maternal DNA sample was available). It is important to note that neither sequence variant was identified in the bidirectional sequencing of 218 alleles from the control sample set. This strongly suggests that the 676delG sequence variant that segregates with the disease phenotype is a true mutation. The C552T sequence variant, which does not segregate with the disease, is more likely a very rare polymorphism, but we cannot formally exclude the possibility that there might be a functional effect of this presumably neutral polymorphism. Figure 1 Germline PHOX2B mutation in a pedigree segregating neuroblastoma and Hirschsprung disease. A, Family 1 pedigree structure. DNA samples from this family with neuroblastoma were available only for patients with a PHOX2B result shown (wt = wild type; 676delG ... Accumulated data strongly implicate PHOX2B as an essential regulator of normal autonomic nervous system development (Pattyn et al. 1999; Brunet and Pattyn 2002). The discovery of polyalanine-expansion mutations in the majority of patients with CCHS clearly defines a role for this gene in human disease (Amiel et al. 2003; Weese-Mayer et al. 2003), and there appears to be a correlation between the severity of the respiratory symptoms and the length of polyalanine expansion (Weese-Mayer et al. 2003; Matera et al. 2004). Neuroblastoma represents perhaps the most aberrant phenotype that results from abnormal adrenergic tissue development. The rare but well-described synchronous appearance of neuroblastoma with other disorders of the autonomic nervous system has suggested a common genetic etiology often referred to as a “neurocristopathy” (Gaisie et al. 1979; Nemecek et al. 2003). Although other genes implicated in Hirschsprung disease and/or CCHS have not been excluded as hereditary neuroblastoma-predisposition genes (Maris et al. 2002; Perri et al. 2002), our data further establish PHOX2B as an important gene involved in the initiation of neuroblastoma tumorigenesis. However, the fact that the majority of pedigrees studied here do not show PHOX2B mutations clearly implicates locus heterogeneity for hereditary predisposition to neuroblastoma. Assuming that our inferences of linkage to 16p are correct, and in light of the observation of two germline mutations in the proband of the family presented here, we suggest that an oligogenic mechanism for neuroblastoma initiation should be considered, as has been shown for other diseases of neural crest–derived tissues (Gabriel et al. 2002). It is not yet clear if the PHOX2B mutations discovered in patients with hereditary or sporadic neuroblastoma result in gain or loss of protein function. The hypothesis that PHOX2B functions as a tumor suppressor is supported by the potential predicted consequence of the five mutations described, to date, in patients with neuroblastoma. Weese-Mayer and colleagues discovered a nonsense mutation that predicts a significantly truncated protein that would miss most of exon 3, including all of the 20-alanine repeat motif (Weese-Mayer et al. 2003). The frameshift mutation described here is similar to that reported by Amiel and colleagues (2003) in a patient who also had CCHS, Hirschsprung disease, and neuroblastoma, and, in both cases, the changes in reading frame are predicted to abolish the polyalanine tract. Trochet and colleagues (2004) discovered two missense mutations, both of which map to a conserved portion of the homeodomain and thus may interfere with DNA binding. On the other hand, 4p12 is not a known site of frequent allelic deletion in neuroblastoma (Maris and Matthay 1999), and, to date, biallelic inactivation of PHOX2B has not been demonstrated, although far too few cases have been examined to assert this with confidence. Taken together, these data suggest that PHOX2B mutations are involved in the initiation of neuroblastoma tumorigenesis, especially in patients with associated disorders of the autonomic nervous system. Our data also indicate that germline mutational events in this gene are not involved in the majority of hereditary neuroblastoma cases and that alternative genetic events may predispose to tumorigenesis. Examination of additional patients will facilitate the definition of PHOX2B mutation frequency in the genetic and (apparently) sporadic forms of neuroblastoma and will help to clarify the role of PHOX2B mutations in tumor initiation and progression.

9514 Background: Mutations in the ALK proto-oncogene are the major cause of hereditary neuroblastoma (NB), can be somatically acquired, and may offer a tractable therapeutic target for a subset of patients. Methods: We identified 1,604 paired constitutional and tumor DNA samples from patients representative of the natural spectrum of NB for comprehensive evaluation of ALK genomic status. This set has complete annotation of clinical covariates and patient outcome, and provides sufficient power to identify clinically relevant ALK aberrations within each NB risk group. We performed DNA sequencing of the ALK kinase domain and defined ALK copy number (CN) status using quantitative PCR. For each tumor where a somatic mutation was identified, we sequenced matched germline DNA, allowing us to understand the frequency of hereditary predisposition to NB. Results: Mutations were discovered in 126/1,604 (7.9%) primary NB tumor samples distributed across the spectrum of phenotypes. Mutations at R1275 were most common ...

Genome-wide association studies have proven to be a powerful tool to identify susceptibility loci in cancer, however the molecular basis of a majority of these association signals remain undefined. We have previously reported nine common single nucleotide polymorphism (SNP) alleles at chromosome 2q35, within and 5′ upstream of the BRCA1-associated RING domain (BARD1) gene, to be significantly associated with high-risk neuroblastoma (Capasso, M et al., Nature Genetics 2009). Here, we focus on understanding the biological underpinnings of this genetic association by investigating how common variation at this locus correlates with changes in BARD1 expression in both the germline and in neuroblastoma samples, and further how these BARD1 alterations are involved in driving neuroblastoma tumorigenesis. We first showed that the neuroblastoma associated SNP variation at 2q35 is in linkage disequilibrium (LD) with multiple genomic areas dense with regulatory domains, including many putative binding sites for the neuronal splicing regulator, FOX2. We next demonstrated that in peripheral blood mononuclear cells, these SNPs correlate both with a trend towards increased overall BARD1 expression and with differential BARD1 exon expression. Specifically, a majority of the neuroblastoma associated SNPs were significantly associated with decreased expression of the RING domain forming BARD1 exons 2 and 3 (P = 0.0008-0.034). We have identified 16 unique BARD1 isoforms in the neuroblastoma tissue of origin, sympathetic fetal ganglia, with the β-BARD1 isoform specifically splicing out exons 2 and 3. β-BARD1 was found to be ubiquitously expressed in a panel of fetal sympathetic ganglia, neuroblastoma cell lines, and high-risk primary tumors, with its expression being significantly increased in cell lines harboring a homozygous BARD1 risk allele genotype at the most significantly associated 2q35 SNP, rs6435862 (N = 22; P = 0.04). Transient transfection of a specific siRNA targeting the alternatively spliced boundary of β-BARD1 in a panel of neuroblastoma cell lines (N = 12) showed that neuroblastoma cells harboring at least one risk allele at SNP rs6435862 and the highest β-BARD1 expression showed a significant inhibition of cell proliferation upon β-BARD1 knockdown (% growth inhibition = 45-81%, P = 0.0004-0.034), while six of seven cell lines with a homozygous major allele genotype at rs6435862 and lower β-BARD1 expression did not show significant growth inhibition. These data are consistent with β-BARD1 acting as an oncogene in neuroblastoma and we hypothesize that common genomic variation drives formation of this isoform, underlying neuroblastoma predisposition on 2q35. Ongoing work will focus on further defining the role of common variation in BARD1's RNA splicing patterns and identifying the molecular basis of β-BARD1's oncogenicity in neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3867.

10008b Background: Mutations in the ALK oncogene are the major cause of hereditary neuroblastoma (NB), and may offer a tractable therapeutic target. Methods: We resequenced the ALK tyrosine kinase domain in 496 primary NBs. We integrated data from 76 primary tumors analyzed using Illumina HumanHap550 SNP and Human-6 mRNA expression arrays. We stained a tissue microarray (TMA) of 134 NBs with anti-ALK and anti-pALK antibodies. We evaluated the in vitro and in vivo activity of PF-02341066, a small molecule inhibitor of ALK. Computational chemistry was used to homology map ALK interaction with PF-02341066. Results: Mutations were discovered in 38/496 NBs (8%), across the spectrum of phenotypes. R1275Q substitutions were most common (45%), followed by F1174L (15%). Evaluation of matched constitutional DNAs demonstrated 10% carried occult germline mutations. Integration of primary tumor microarrays from 76 samples showed that ALK mRNA expression was highly correlated with ALK DNA copy number (p=0.0005). ALK protein was nearly universally expressed (129/134; 93%), and there was evidence for ALK activation in 15% of tumors (pALK moderate or strong staining in 20/134), suggesting an additional and alternative pathway to ALK activation. A panel of 18 NB cell lines showed differential in vitro sensitivity to PF-2341066 dependent on ALK mutation and copy number status. A wild type (WT) amplified line and 2 lines harboring R1275Q mutations were sensitive (IC50 145 - 301 nM). Lines harboring F1174L mutations were more resistant (IC50 >950nM) as were lines with no ALK aberrations. Inhibition of proliferation was associated with abolishing phosphorylation of ALK, STAT3 and AKT. PF-02341066 caused complete regression of R1275Q xenografts, but caused only minimal growth delay in F1174L and WT xenografts. Based on homology mapping to a crystal structure of PF-02341066 bound to c-Met, the F1174L mutation is predicted to destabilize the specific conformation of the kinase activation loop required for binding PF-02341066. Conclusions: ALK pathway activation occurs in at least 15% of human neuroblastomas. PF-02341066 is a potent inhibitor of the most common ALK mutation, but there is a structural basis for resistance to F1174L mutations. Co-crystallization studies are ongoing and a phase 1 study is planned. No significant financial relationships to disclose.

10010 Background: A subset of neuroblastomas show inheritance as an autosomal dominant trait with incomplete penetrance. Familial neuroblastoma is often lethal during childhood, resulting in very few pedigrees of sufficient size for linkage analysis. Low-resolution genetic approaches have not been successful in identifying a narrow genomic region consistent with linkage, a critical first step in identifying the causal gene. Methods: We performed a genome-wide scan for linkage in eighteen neuroblastoma pedigrees at ∼6000 single nucleotide polymorphisms. Parametric and non-parametric analyses were used to identify and refine candidate regions. Candidate genes were resequenced in a panel of 15 probands using Sanger-based or 454 technology. Results: We discovered a highly significant linkage signal covering a 34 Mb region on chromosome 2p with a maximum non-parametric LOD score of 4.23 (p=0.00001), and 13/18 families screened were consistent with linkage to this locus. No other genomic region was suggestive o...

Genetic studies have established anaplastic lymphoma kinase (ALK), a cell surface receptor tyrosine kinase, as a tractable molecular target in neuroblastoma. We describe comprehensive genomic, biochemical, and computational analyses of ALK mutations across 1596 diagnostic neuroblastoma samples. ALK tyrosine kinase domain mutations occurred in 8% of samples; at three hotspots plus 13 minor sites – and correlated significantly with poorer survival in high- and intermediate-risk neuroblastoma. Biochemical and computational studies distinguished oncogenic (constitutively activating) from non-oncogenic mutations and allowed robust computational prediction of their effects. We also established differential in vitro crizotinib sensitivity of mutated variants. Our studies identify ALK genomic status as a clinically important therapeutic stratification tool in neuroblastoma, and will allow tailoring of ALK-targeted therapy to specific mutations.