METTL3 drives telomere targeting of TERRA lncRNA through m6A-dependent R-loop formation: a therapeutic target for ALT-positive neuroblastoma.

Telomerase-negative tumors maintain telomere length by alternative lengthening of telomeres (ALT), but the underlying mechanism behind ALT remains poorly understood. A proportion of aggressive neuroblastoma (NB), particularly relapsed tumors, are positive for ALT (ALT+), suggesting that a better dissection of the ALT mechanism could lead to novel therapeutic opportunities. TERRA, a long non-coding RNA (lncRNA) derived from telomere ends, localizes to telomeres in a R-loop-dependent manner and plays a crucial role in telomere maintenance. Here we present evidence that RNA modification at the N6 position of internal adenosine (m6A) in TERRA by the methyltransferase METTL3 is essential for telomere maintenance in ALT+ cells, and the loss of TERRA m6A/METTL3 results in telomere damage. We observed that m6A modification is abundant in R-loop enriched TERRA, and the m6A-mediated recruitment of hnRNPA2B1 to TERRA is critical for R-loop formation. Our findings suggest that m6A drives telomere targeting of TERRA via R-loops, and this m6A-mediated R-loop formation could be a widespread mechanism employed by other chromatin-interacting lncRNAs. Furthermore, treatment of ALT+ NB cells with a METTL3 inhibitor resulted in compromised telomere targeting of TERRA and accumulation of DNA damage at telomeres, indicating that METTL3 inhibition may represent a therapeutic approach for ALT+ NB.

ALK ligand ALKAL2 potentiates MYCN-driven neuroblastoma in the absence of ALK mutation.

High-risk neuroblastoma (NB) is responsible for a disproportionate number of childhood deaths due to cancer. One indicator of high-risk NB is amplification of the neural MYC (MYCN) oncogene, which is currently therapeutically intractable. Identification of anaplastic lymphoma kinase (ALK) as an NB oncogene raised the possibility of using ALK tyrosine kinase inhibitors (TKIs) in treatment of patients with activating ALK mutations. 8-10% of primary NB patients are ALK-positive, a figure that increases in the relapsed population. ALK is activated by the ALKAL2 ligand located on chromosome 2p, along with ALK and MYCN, in the "2p-gain" region associated with NB. Dysregulation of ALK ligand in NB has not been addressed, although one of the first oncogenes described was v-sis that shares > 90% homology with PDGF. Therefore, we tested whether ALKAL2 ligand could potentiate NB progression in the absence of ALK mutation. We show that ALKAL2 overexpression in mice drives ALK TKI-sensitive NB in the absence of ALK mutation, suggesting that additional NB patients, such as those exhibiting 2p-gain, may benefit from ALK TKI-based therapeutic intervention.

Loss of Chromosome Y in Neuroblastoma Is Associated With High-Risk Disease, 11q-Deletion, and Telomere Maintenance.

Neuroblastoma (NB) is a heterogeneous childhood cancer with a slightly higher incidence in boys than girls, with the reason for this gender disparity unknown. Given the growing evidence for the involvement of loss of the Y chromosome (LoY) in male diseases including cancer, we investigated Y chromosome status in NB. Male NB tumor samples from a Swedish cohort, analyzed using Cytoscan HD SNP-microarray, were selected. Seventy NB tumors were analyzed for aneuploidy of the Y chromosome, and these data were correlated with other genetic, biological, and clinical parameters. LoY was found in 21% of the male NB tumors and it was almost exclusively found in those with high-risk genomic profiles. Furthermore, LoY was associated with increased age at diagnosis and enriched in tumors with 11q-deletion and activated telomere maintenance mechanisms. In contrast, tumors with an MYCN-amplified genomic profile retained their Y chromosome. The understanding of LoY in cancer is limited, making it difficult to conclude whether LoY is a driving event in NB or function of increased genomic instability. Gene expression analysis of Y chromosome genes in male NB tumors showed low expression of certain genes correlating with worse overall survival. KDM5D, encoding a histone demethylase stands out as an interesting candidate for further studies. LoY has been shown to impact the epigenomic layer of autosomal loci in nonreproductive tissues, and KDM5D has been reported as downregulated and/or associated with poor survival in different malignancies. Further studies are needed to explore the mechanisms and functional consequences of LoY in NB.

Joint single-cell genetic and transcriptomic analysis reveal pre-malignant SCP-like subclones in human neuroblastoma.

BACKGROUND: Neuroblastoma (NB) is a heterogeneous embryonal malignancy and the deadliest tumor of infancy. It is a complex disease that can result in diverse clinical outcomes. In some children, tumors regress spontaneously. Others respond well to existing treatments. But for the high-risk group, which constitutes approximately 40% of all patients, the prognosis remains dire despite collaborative efforts in basic and clinical research. While its exact cellular origin is still under debate, NB is assumed to arise from the neural crest cell lineage including multipotent Schwann cell precursors (SCPs), which differentiate into sympatho-adrenal cell states eventually producing chromaffin cells and sympathoblasts. METHODS: To investigate clonal development of neuroblastoma cell states, we performed haplotype-specific analysis of human tumor samples using single-cell multi-omics, including joint DNA/RNA sequencing of sorted single cells (DNTR-seq). Samples were also assessed using immunofluorescence stainings and fluorescence in-situ hybridization (FISH). RESULTS: Beyond adrenergic tumor cells, we identify subpopulations of aneuploid SCP-like cells, characterized by clonal expansion, whole-chromosome 17 gains, as well as expression programs of proliferation, apoptosis, and a non-immunomodulatory phenotype. CONCLUSION: Aneuploid pre-malignant SCP-like cells represent a novel feature of NB. Genetic evidence and tumor phylogeny suggest that these clones and malignant adrenergic populations originate from aneuploidy-prone cells of migrating neural crest or SCP origin, before lineage commitment to sympatho-adrenal cell states. Our findings expand the phenotypic spectrum of NB cell states. Considering the multipotency of SCPs in development, we suggest that the transformation of fetal SCPs may represent one possible mechanism of tumor initiation in NB with chromosome 17 aberrations as a characteristic element.

Altered methylation of imprinted genes in neuroblastoma: implications for prognostic refinement.

BACKGROUND: Neuroblastoma (NB) is a complex disease, and the current understanding of NB biology is limited. Deregulation in genomic imprinting is a common event in malignancy. Since imprinted genes play crucial roles in early fetal growth and development, their role in NB pathogenesis could be suggested. METHODS: We examined alterations in DNA methylation patterns of 369 NB tumours at 49 imprinted differentially methylated regions (DMRs) and assessed its association with overall survival probabilities and selected clinical and genomic features of the tumours. In addition, an integrated analysis of DNA methylation and allele-specific copy number alterations (CNAs) was performed, to understand the correlation between the two molecular events. RESULTS: Several imprinted regions with aberrant methylation patterns in NB were identified. Regions that underwent loss of methylation in > 30% of NB samples were DMRs annotated to the genes NDN, SNRPN, IGF2, MAGEL2 and HTR5A and regions with gain of methylation were NNAT, RB1 and GPR1. Methylation alterations at six of the 49 imprinted DMRs were statistically significantly associated with reduced overall survival: MIR886, RB1, NNAT/BLCAP, MAGEL2, MKRN3 and INPP5F. RB1, NNAT/BLCAP and MKRN3 were further able to stratify low-risk NB tumours i.e. tumours that lacked MYCN amplification and 11q deletion into risk groups. Methylation alterations at NNAT/BLCAP, MAGEL2 and MIR886 predicted risk independently of MYCN amplification or 11q deletion and age at diagnosis. Investigation of the allele-specific CNAs demonstrated that the imprinted regions that displayed most alterations in NB tumours harbor true epigenetic changes and are not result of the underlying CNAs. CONCLUSIONS: Aberrant methylation in imprinted regions is frequently occurring in NB tumours and several of these regions have independent prognostic value. Thus, these could serve as potentially important clinical epigenetic markers to identify individuals with adverse prognosis. Incorporation of methylation status of these regions together with the established risk predictors may further refine the prognostication of NB patients.

Occurrence of cancer in Marfan syndrome: Report of two patients with neuroblastoma and review of the literature.

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder caused by pathogenic variants in FBN1, with a hitherto unknown association with cancer. Here, we present two females with MFS who developed pediatric neuroblastoma. Patient 1 presented with neonatal MFS and developed an adrenal neuroblastoma with unfavorable tumor genetics at 10 months of age. Whole genome sequencing revealed a germline de novo missense FBN1 variant (NP_000129.3:p.(Asp1322Asn)), resulting in intron 32 inclusion and exon 32 retention. Patient 2 was diagnosed with classic MFS, caused by a germline de novo frameshift variant in FBN1 (NP_000129.3:p.(Cys805Ter)). At 18 years, she developed high-risk neuroblastoma with a somatic ALK pathogenic variant (NP_004295.2:p.(Arg1275Gln)). We identified 32 reported cases of MFS with cancer in PubMed, yet none with neuroblastoma. Among patients, we observed an early cancer onset and high frequency of MFS complications. We also queried cancer databases for somatic FBN1 variants, finding 49 alterations reported in PeCan, and variants in 2% of patients in cBioPortal. In conclusion, we report the first two patients with MFS and neuroblastoma and highlight an early age at cancer diagnosis in reported patients with MFS. Further epidemiological and functional studies are needed to clarify the growing evidence linking MFS and cancer.

TC-hunter: identification of the insertion site of a transgenic gene within the host genome.

BACKGROUND: Transgenic animal models are crucial for the study of gene function and disease, and are widely utilized in basic biological research, agriculture and pharma industries. Since the current methods for generating transgenic animals result in the random integration of the transgene under study, the phenotype may be compromised due to disruption of known genes or regulatory regions. Unfortunately, most of the tools that predict transgene insertion sites from high-throughput data are not publicly available or not properly maintained. RESULTS: We implemented TC-hunter, Transgene-Construct hunter, an open tool that identifies transgene insertion sites and provides simple reports and visualization aids. It relies on common tools used in the analysis of high-throughput data and makes use of chimeric reads and discordant read pairs to identify and support the transgenic insertion site. To demonstrate its applicability, we applied TC-hunter to four transgenic mice samples harboring the human PPM1D gene, a model used in the study of malignant tumor development. We identified the transgenic insertion site in each sample and experimentally validated them with Touchdown-polymerase chain reaction followed by Sanger sequencing. CONCLUSIONS: TC-hunter is an accessible bioinformatics tool that can automatically identify transgene insertion sites from DNA sequencing data with high sensitivity (98%) and precision (92.45%). TC-hunter is a valuable tool that can aid in evaluating any potential phenotypic complications due to the random integration of the transgene and can be accessed at https://github.com/bcfgothenburg/SSF .

Analysis of ALK, MYCN, and the ALK ligand ALKAL2 (FAM150B/AUGα) in neuroblastoma patient samples with chromosome arm 2p rearrangements.

Gain of chromosome arm 2p is a previously described entity in neuroblastoma (NB). This genomic address is home to two important oncogenes in NB-MYCN and anaplastic lymphoma kinase (ALK). MYCN amplification is a critical prognostic factor coupled with poor prognosis in NB. Mutation of the ALK receptor tyrosine kinase has been described in both somatic and familial NB. Here, ALK activation occurs in the context of the full-length receptor, exemplified by activating point mutations in NB. ALK overexpression and activation, in the absence of genetic mutation has also been described in NB. In addition, the recently identified ALK ligand ALKAL2 (previously described as FAM150B and AUGα) is also found on the distal portion of 2p, at 2p25. Here we analyze 356 NB tumor samples and discuss observations indicating that gain of 2p has implications for the development of NB. Finally, we put forward the hypothesis that the effect of 2p gain may result from a combination of MYCN, ALK, and the ALK ligand ALKAL2.

Rho-associated kinase is a therapeutic target in neuroblastoma.

Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3ß-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN-driven neuroblastoma growth in TH-MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma.

Familial intestinal degenerative neuropathy with chronic intestinal pseudo-obstruction linked to a gene locus with duplication in chromosome 9.

Background: Intestinal degenerative neuropathy without extra-intestinal involvement occurs as familial forms (FIDN) but the genetics behind is unknown. We studied a Swedish family with autosomal dominant disease and several cases of chronic intestinal pseudo-obstruction (CIP).Methods: We included 33 members of a family sharing a male ancestor. Chronic intestinal symptoms including diarrhoea occurred in 11, four had severe CIP. DNA was analysed with SNP-microarray (Affymetrix), linkage (Allegro Software) and gene dosage (CNAG 3.0).Results: Genetic linkage was found to the short arm of Ch9 to a 9.7 Mb region with 45 protein-coding genes, 22 of which were duplicated (1.2 Mb duplication) (dup(9)(p21.3) with breaking point in the FOCAD-gene. Lod score for the region was 3.4. Fourteen subjects were duplication carriers including all 11 subjects having severe chronic symptoms/CIP. Nineteen subjects had no duplication. The occurrence of gastrointestinal symptoms in the family was strongly linked to duplication carrier-ship (p = .0005). The two branches of the family had separate maternal ancestors (A and B). Including the previous generation, severe disease (overt CIP and/or death from intestinal failure) was assessed to occur in 100% (5/5) of duplication carriers in branch A and in 21% (3/14) in branch B (p = .005). In branch B the onset of symptoms was later (median 38 vs. 24 yrs) and three duplication carriers were symptom-free.Conclusions: In this family with autosomal dominant hereditary intestinal neuropathy, the disorder is linked to a 9.7 Mb region in Ch9 including a 1.2 Mb duplication. There is a significant difference in disease expressivity between family branches, seemingly related to separate maternal ancestors.

Molecular mechanisms and therapeutic targets in neuroblastoma.

Neuroblastoma is the most common extracranical tumor of childhood and the most deadly tumor of infancy. It is characterized by early age onset and high frequencies of metastatic disease but also the capacity to spontaneously regress. Despite intensive therapy, the survival for patients with high-risk neuroblastoma and those with recurrent or relapsed disease is low. Hence, there is an urgent need to develop new therapies for these patient groups. The molecular pathogenesis based on high-throughput omics technologies of neuroblastoma is beginning to be resolved which have given the opportunity to develop personalized therapies for high-risk patients. Here we discuss the potential of developing targeted therapies against aberrantly expressed molecules detected in sub-populations of neuroblastoma patients and how these selected targets can be drugged in order to overcome treatment resistance, improve survival and quality of life for these patients and also the possibilities to transfer preclinical research into clinical testing.

Intragenic anaplastic lymphoma kinase (ALK) rearrangements: translocations as a novel mechanism of ALK activation in neuroblastoma tumors.

Anaplastic lymphoma kinase (ALK) has been demonstrated to be deregulated in sporadic as well as in familiar cases of neuroblastoma (NB). Whereas ALK-fusion proteins are common in lymphoma and lung cancer, there are few reports of ALK rearrangements in NB indicating that ALK mainly exerts its oncogenic capacity via activating mutations and/or overexpression in this tumor type. In this study, 332 NB tumors and 13 cell lines were screened by high resolution single nucleotide polymorphism microarray. Gain of 2p was detected in 23% (60/332) of primary tumors and 46% (6/13) of cell lines, while breakpoints at the ALK locus were detected in four primary tumors and two cell lines. These were further analyzed by next generation sequencing and a targeted enrichment approach. Samples with both ALK and MYCN amplification displayed complex genomic rearrangements with multiple breakpoints within the amplicon. None of the translocations characterized in primary NB tumors are likely to result in a chimeric protein. However, immunohistochemical analysis reveals high levels of phosphorylated ALK in these samples despite lack of initial exons, possibly due to alternative transcription initiation sites. Both ALK proteins predicted to arise from such alterations and from the abnormal ALK exon 4-11 deletion observed in the CLB-BAR cell line show strong activation of downstream targets STAT3 and extracellular signal-regulated kinase (ERK) when expressed in PC12 cells. Taken together, our data indicate a novel, although rare, mechanism of ALK activation with implications for NB tumorigenesis.

Heterogeneous SSTR2 target expression and a novel KIAA1549::BRAF fusion clone in a progressive metastatic lesion following 177Lutetium-DOTATATE molecular radiotherapy in neuroblastoma: a case report.

In this case report, we present the treatment outcomes of the first patient enrolled in the LuDO-N trial. The patient is a 21-month-old girl diagnosed with high-risk neuroblastoma (NB) and widespread skeletal metastasis. The patient initially underwent first-line therapy according to SIOPEN HRNBL-1 but was switched to second-line treatments due to disease progression, and she was finally screened for enrollment in the LuDO-N trial due to refractory disease. Upon enrollment, the patient received two rounds of the radiolabeled somatostatin analogue lutetium-177 octreotate (177Lu-DOTATATE), which was well tolerated. A dosimetry analysis revealed a heterogeneous uptake across tumor lesions, resulting in a significant absorbed dose of 54 Gy in the primary tumor, but only 2 Gy at one of the metastatic sites in the distal femur. While the initial treatment response showed disease stabilization, the distal femoral metastasis continued to progress, leading to the eventual death of the patient. A tissue analysis of the biopsies collected throughout the course of the disease revealed heterogeneous drug target expression of somatostatin receptor 2 (SSTR2) across and within tumor lesions. Furthermore, genomic profiling revealed a novel KIAA1549::BRAF fusion oncogene amplification in the distal femoral metastasis at recurrence that might be related with resistance to radiation, possibly through the downregulation of SSTR2. This case report demonstrates a mixed response to molecular radiotherapy (MRT) with 177Lu-DOTATATE. The observed variation in SSTR2 expression between tumor lesions suggests that heterogeneous target expression may have been the reason for treatment failure in this patient's case. Further investigation within the LuDO-N trial will give a more comprehensive understanding of the correlation between SSTR2 expression levels and treatment outcomes, which will be important to advance treatment strategies based on MRT for children with high-risk NB.

Long-Lasting Response to Lorlatinib in Patients with ALK-Driven Relapsed or Refractory Neuroblastoma Monitored with Circulating Tumor DNA Analysis.

Patients with anaplastic lymphoma kinase (ALK)-driven neuroblastoma may respond to tyrosine kinase inhibitors, but resistance to treatment occurs and methods currently used for detection of residual disease have limited sensitivity. Here, we present a national unselected cohort of five patients with relapsed or refractory ALK-driven neuroblastoma treated with lorlatinib as monotherapy and test the potential of targeted circulating tumor DNA (ctDNA) analysis as a guide for treatment decisions in these patients. We developed a sequencing panel for ultrasensitive detection of ALK mutations associated with neuroblastoma or resistance to tyrosine kinase inhibitors and used it for ctDNA analysis in 83 plasma samples collected longitudinally from the four patients who harbored somatic ALK mutations. All four patients with ALK p.R1275Q experienced major responses and were alive 35 to 61 months after starting lorlatinib. A fifth patient with ALK p.F1174L initially had a partial response but relapsed after 10 months of treatment. In all cases, ctDNA was detected at the start of lorlatinib single-agent treatment and declined gradually, correlating with clinical responses. In the two patients exhibiting relapse, ctDNA increased 9 and 3 months, respectively, before clinical detection of disease progression. In one patient harboring HRAS p.Q61L in the relapsed tumor, retrospective ctDNA analysis showed that the mutation appeared de novo after 8 months of lorlatinib treatment. We conclude that some patients with relapsed or refractory high-risk neuroblastoma show durable responses to lorlatinib as monotherapy, and targeted ctDNA analysis is effective for evaluation of treatment and early detection of relapse in ALK-driven neuroblastoma. SIGNIFICANCE: We present five patients with ALK-driven relapsed or refractory neuroblastoma treated with lorlatinib as monotherapy. All patients responded to treatment, and four of them were alive after 3 to 5 years of follow-up. We performed longitudinal ctDNA analysis with ultra-deep sequencing of the ALK tyrosine kinase domain. We conclude that ctDNA analysis may guide treatment decisions in ALK-driven neuroblastoma, also when the disease is undetectable using standard clinical methods.

Diagnostic yield and clinical impact of germline sequencing in children with CNS and extracranial solid tumors-a nationwide, prospective Swedish study.

Background: Childhood cancer predisposition (ChiCaP) syndromes are increasingly recognized as contributing factors to childhood cancer development. Yet, due to variable availability of germline testing, many children with ChiCaP might go undetected today. We report results from the nationwide and prospective ChiCaP study that investigated diagnostic yield and clinical impact of integrating germline whole-genome sequencing (gWGS) with tumor sequencing and systematic phenotyping in children with solid tumors. Methods: gWGS was performed in 309 children at diagnosis of CNS (n = 123, 40%) or extracranial (n = 186, 60%) solid tumors and analyzed for disease-causing variants in 189 known cancer predisposing genes. Tumor sequencing data were available for 74% (227/309) of patients. In addition, a standardized clinical assessment for underlying predisposition was performed in 95% (293/309) of patients. Findings: The prevalence of ChiCaP diagnoses was 11% (35/309), of which 69% (24/35) were unknown at inclusion (diagnostic yield 8%, 24/298). A second-hit and/or relevant mutational signature was observed in 19/21 (90%) tumors with informative data. ChiCaP diagnoses were more prevalent among patients with retinoblastomas (50%, 6/12) and high-grade astrocytomas (37%, 6/16), and in those with non-cancer related features (23%, 20/88), and ≥2 positive ChiCaP criteria (28%, 22/79). ChiCaP diagnoses were autosomal dominant in 80% (28/35) of patients, yet confirmed de novo in 64% (18/28). The 35 ChiCaP findings resulted in tailored surveillance (86%, 30/35) and treatment recommendations (31%, 11/35). Interpretation: Overall, our results demonstrate that systematic phenotyping, combined with genomics-based diagnostics of ChiCaP in children with solid tumors is feasible in large-scale clinical practice and critically guides personalized care in a sizable proportion of patients. Funding: The study was supported by the Swedish Childhood Cancer Fund and the Ministry of Health and Social Affairs.

Aneuploidy in neuroblastoma tumors is not associated with inactivating point mutations in the STAG2 gene.

BACKGROUND: Chromosomal instability is a hallmark of human cancer caused by errors in mitotic control and chromosome segregation. STAG2 encodes a subunit of the cohesion complex that participates in mitotic chromatid separation and was recently found to show low expression and inactivating mutations in Ewing's sarcoma, melanoma and glioblastoma.In the childhood tumor neuroblastoma (NB) segmental chromosomal alterations are associated with poor prognosis whereas tumors displaying whole chromosome gains and losses have a much better prognosis. METHOD: As the genetic contribution to aneuploidy is unknown in NB, we investigated the presence of STAG2 mutations through sequence analysis of all 33 coding exons in 37 primary NB tumors. RESULTS AND CONCLUSION: As no STAG2 mutation was detected in this study, we conclude that inactivating mutation of STAG2 is not likely causative to neuroblastoma aneuploidy.

Identification of recurrent 3q13.31 chromosomal rearrangement indicates LSAMP as a tumor suppressor gene in neuroblastoma.

Neuroblastoma (NB) is a childhood malignancy of the sympathetic nervous system. NB is mainly driven by copy number alterations, such as MYCN amplification, large deletions of chromosome arm 11q and gain of chromosome arm 17q, which are all markers of high­risk disease. Genes targeted by recurrent, smaller, focal alterations include CDKN2A/B, TERT, PTPRD and ATRX. Our previous study on relapsed NB detected recurrent structural alterations centered at limbic system­associated membrane protein (LSAMP; HUGO Gene Nomenclature Committee: 6705; chromosomal location 3q13.31), which is a gene frequently reported to be deleted or downregulated in other types of cancer. Notably, in cancer, LSAMP has been shown to have tumor­suppressing functions. The present study performed an expanded investigation using whole genome sequencing of tumors from 35 patients, mainly with high­risk NB. Focal duplications or deletions targeting LSAMP were detected in six cases (17%), whereas single nucleotide polymorphism­microarray analysis of 16 NB cell lines detected segmental alterations at 3q13.31 in seven out of the 16 NB cell lines (44%). Furthermore, low expression of LSAMP in NB tumors was significantly associated with poor overall and event­free survival. In vitro, knockdown of LSAMP in NB cell lines increased cell proliferation, whereas overexpression decreased proliferation and viability. These findings supported a tumor suppressor role for LSAMP in NB. However, the higher incidence of LSAMP aberrations in cell lines and in relapsed NB tumors suggested that these alterations were a late event predominantly in advanced NB with a poor prognosis, indicating a role of LSAMP in tumor progression rather than in tumor initiation. In conclusion, the present study demonstrated recurrent genomic aberrations of chromosomal region 3q13.31 that targeted the LSAMP gene, which encodes a membrane protein involved in cell adhesion, central nervous system development and neurite outgrowth. The frequent aberrations affecting LSAMP, together with functional evidence, suggested an anti­proliferative role of LSAMP in NB.

Telomere Maintenance Mechanisms in a Cohort of High-Risk Neuroblastoma Tumors and Its Relation to Genomic Variants in the TERT and ATRX Genes.

Tumor cells are hallmarked by their capacity to undergo unlimited cell divisions, commonly accomplished either by mechanisms that activate TERT or through the alternative lengthening of telomeres pathway. Neuroblastoma is a heterogeneous pediatric cancer, and the aim of this study was to characterize telomere maintenance mechanisms in a high-risk neuroblastoma cohort. All tumor samples were profiled with SNP microarrays and, when material was available, subjected to whole genome sequencing (WGS). Telomere length was estimated from WGS data, samples were assayed for the ALT biomarker c-circles, and selected samples were subjected to methylation array analysis. Samples with ATRX aberration in this study were positive for c-circles, whereas samples with either MYCN amplification or TERT re-arrangement were negative for c-circles. Both ATRX aberrations and TERT re-arrangement were enriched in 11q-deleted samples. An association between older age at diagnosis and 1q-deletion was found in the ALT-positive group. TERT was frequently placed in juxtaposition to a previously established gene in neuroblastoma tumorigenesis or cancer in general. Given the importance of high-risk neuroblastoma, means for mitigating active telomere maintenance must be therapeutically explored.

Diagnostic Yield From a Nationwide Implementation of Precision Medicine for all Children With Cancer.

PURPOSE: Several studies have indicated that broad genomic characterization of childhood cancer provides diagnostically and/or therapeutically relevant information in selected high-risk cases. However, the extent to which such characterization offers clinically actionable data in a prospective broadly inclusive setting remains largely unexplored. METHODS: We implemented prospective whole-genome sequencing (WGS) of tumor and germline, complemented by whole-transcriptome sequencing (RNA-Seq) for all children diagnosed with a primary or relapsed solid malignancy in Sweden. Multidisciplinary molecular tumor boards were set up to integrate genomic data in the clinical decision process along with a medicolegal framework enabling secondary use of sequencing data for research purposes. RESULTS: During the study's first 14 months, 118 solid tumors from 117 patients were subjected to WGS, with complementary RNA-Seq for fusion gene detection in 52 tumors. There was no significant geographic bias in patient enrollment, and the included tumor types reflected the annual national incidence of pediatric solid tumor types. Of the 112 tumors with somatic mutations, 106 (95%) exhibited alterations with a clear clinical correlation. In 46 of 118 tumors (39%), sequencing only corroborated histopathological diagnoses, while in 59 cases (50%), it contributed to additional subclassification or detection of prognostic markers. Potential treatment targets were found in 31 patients (26%), most commonly ALK mutations/fusions (n = 4), RAS/RAF/MEK/ERK pathway mutations (n = 14), FGFR1 mutations/fusions (n = 5), IDH1 mutations (n = 2), and NTRK2 gene fusions (n = 2). In one patient, the tumor diagnosis was revised based on sequencing. Clinically relevant germline variants were detected in 8 of 94 patients (8.5%). CONCLUSION: Up-front, large-scale genomic characterization of pediatric solid malignancies provides diagnostically valuable data in the majority of patients also in a largely unselected cohort.

Whole exome sequencing identifies KIF26B, LIFR and LAMC1 mutations in familial vesicoureteral reflux.

Vesicoureteral reflux (VUR) is a common urological problem in children and its hereditary nature is well recognised. However, despite decades of research, the aetiological factors are poorly understood and the genetic background has been elucidated in only a minority of cases. To explore the molecular aetiology of primary hereditary VUR, we performed whole-exome sequencing in 13 large families with at least three affected cases. A large proportion of our study cohort had congenital renal hypodysplasia in addition to VUR. This high-throughput screening revealed 23 deleterious heterozygous variants in 19 candidate genes associated with VUR or nephrogenesis. Sanger sequencing and segregation analysis in the entire families confirmed the following findings in three genes in three families: frameshift LAMC1 variant and missense variants of KIF26B and LIFR genes. Rare variants were also found in SALL1, ROBO2 and UPK3A. These gene variants were present in individual cases but did not segregate with disease in families. In all, we demonstrate a likely causal gene variant in 23% of the families. Whole-exome sequencing technology in combination with a segregation study of the whole family is a useful tool when it comes to understanding pathogenesis and improving molecular diagnostics of this highly heterogeneous malformation.