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Background: Mutational processes generate unique patterns of base substitutions generally referred to as mutational signatures. Mutational signatures can give insights into fundamental mutational processes underlying somatic mutations in tumors and have been shown to provide a starting point from which to evaluate therapeutic options. Neuroblastoma is clinically heterogeneous ranging from tendency of spontaneous regression to frequent formation of metastases. Earlier studies have identified several recurrent genetic alterations, including mutations in genes such as ALK and ATRX, as well as chromosomal rearrangements such as MYCN amplification, 11q deletion and 17 gain. Despite recent progress in the treatment of high-risk neuroblastoma, patients in this subgroup still have a poor prognosis, underscoring the need to further stratify patients in order to help inform clinical decision making. Methods: Here, we sought to characterize high-risk patients and their underlying pattern of aberrations by conducting whole genome sequencing of 30 neuroblastoma patients. For each tumor/normal pair, the mutational landscape comprising single nucleotide and structural variants including copy number alterations was investigated. Non-negative matrix factorization was used to decompose single nucleotide variants to discover mutational signatures. Results: De novo mutational signature analysis identified two mutational profiles (signature A and signature B) that were compared to known signatures of the pan-cancer study by Alexandrov et al. (2013). The landscape of signature A is closest to their signature 18 (0.97 cosine similarity) and revealed a strong bias towards C>A substitutions mainly found in neuroblastoma, and signature B is closest to signature 5 (0.82 cosine similarity) identified in most of the cancers. Out of 13 patients with a clear dominance of signature A, 12 patients were classified with MYCN amplification or 11q deletion. Furthermore, 5 out of 6 patients with a main contribution of signature B were metastasis or relapse cases. Conclusions: Our results suggest that what is currently considered to be the typical neuroblastoma signature is mainly present in primary high-risk patients whereas the mutational landscape is altered in more advanced stages of the disease. To better understand the connection between the identified signatures and the high-risk subgroup, we are currently investigating the genome wide landscape of the observed C>A substitutions. Citation Format: Susanne E. Reinsbach, Malin Larsson, Angela Martinez-Monleon, Rose-Marie Sjöberg, Niloufar Javanmardi, Anna Djos, Per Kogner, Susanne Fransson, Erik Larsson Lekholm, Tommy Martinsson. Identification of mutational signatures in high-risk neuroblastoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2246.

Background. Despite major progress in treatment of pediatric cancer, aggressive neuroblastoma (NB) still constitutes a major clinical problem. Currently, the event free survival of high-risk NB is about 40% implicating that 60% either don’t go into full remission or falls into relapse. Most relapses have an acquired drug resistance meaning that conventional treatment options may not work. Therefore, further studies of this group are of great importance. Massive parallel sequencing has now become a valuable tool in both cancer research and in clinical evaluation as it provides valuable information regarding patient specific alterations that might be used in targeted therapy. Material and Methods. DNA from 54 patients in total have been subjects for massive parallel sequencing; 16 tumor/normal pairs and 27 single tumors have been analyzed using exome sequencing, 8 tumor/normal pairs with whole genome sequencing and three using both exome and whole genome sequencing. Exome sequences were mapped using BWA with GATK realignment followed by variant calling through SNPeff and copy number analysis trough ControlFreec. Whole genome data were analyzed using the CLC Genomic Workbench with annotation and filtering done in Ingenuity variant analysis software. Results. On average, 14 somatic protein changing single nucleotide variants were detected per patient (range 2-71) with recurrent alterations mainly detected mainly in ALK. Predicted deleterious germ line alterations in well-established cancer genes were detected in three patients. Structural variants included genes such as ATRX and TERT, as described by others previously, but also novel homozygous focal deletions at chromosome 19. Shortest regions of overlapping deletions of this chr19 region include seven gens whereof CIC (Capicua) might be the most interesting in a cancer context. Copy number analysis of both exome- and whole genome sequencing gives genomic profiles that are comparable to the genome profiles generated from the SNP-microarrays that current are in clinical use. Specific breakpoints for structural variants could be detected in all sample subjects for whole genome sequencing as well as for all for all MYCN amplified neuroblastomas that subjects for exome sequencing. Conclusions. Exome- and whole genome sequencing can be used for clinical evaluation of NB providing information regarding both constitutional and tumor specific alterations. The information can be used for clinical decision of patient specific therapeutic options such as ALK- or CDK4/6 inhibitors, but can also in rare cases give information regarding which treatments not to use. Furthermore, whole genome sequencing also gives the possibility to identify tumor specific structural alterations such as translocation breakpoints that can be used to monitor tumor burden in liquid biopsies. Citation Format: Angela Martinez Monleon, Susanne Reinsbach, Niloufar Javanmardi, Anna Djos, Rose-Marie Sjoberg, Per Kogner, Tommy Martinsson, Susanne Fransson. Exome- and whole genome sequencing for clinical evaluation and precision medicine in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4867. doi:10.1158/1538-7445.AM2017-4867

Background The pediatric cancer neuroblastoma (NB) is heterogeneous in terms of both genotype and clinical behavior. NB patients show complex patterns of genetic abnormalities, which may include amplification, translocation or oncogenic mutations of ALK kinase in sporadic and familial cases. Three hot spot residues (F1174, F1245, and R1275) localized within the kinase domain of ALK, accounts for 85% of mutant ALK in NB. In this study we used ultra deep sequencing for sensitive detection of ALK mutations. Methods To detect the sub-clonal ALK mutations in 105 neuroblastoma tumors that can remain undetected using Sanger chemistry, we used two different approaches; hybridization-based Haloplex kit (Agilent) and PCR-based amplicon sequencing, both sequenced on the MiSeq Illumina platform. Coverage, uniformity and variant calling ability showed similar performance between both methods. Findings ALK mutations were detected in 14/105 patients (13%) whereof six with allele frequency below 20%. At the I1171 locus an ALK mutation was detected in one case with variant allele frequency as low as 2.6%, the lowest level detected in our cohort. Mutations at the F1174 hotspot were observed in nine cases: six cases with F1174L, while the remaining three showed F1174C, F1174S and F1174I substitutions, with mutated allele fractions ranging from 14% to 60%. Interestingly the patient harboring F1174S mutation with 58% frequency, in parallel contained a small sub-clone of F1174I mutation with 8% frequency of the mutated allele. F1245-codon alterations were detected in three cases: two F1245I mutations and one F1245C mutation, with frequencies of 14%, 51% and 52%, respectively. A single case showed a L1240V variant with a mutated allele fraction at 57%. Sanger sequencing of all ALK mutated samples could confirm ALK status for all samples with variant allele fraction above 15%. Because of the limits of detection, 4 out of 6 samples with a mutated allele fraction below 15% would have gone undetected relying on Sanger sequencing. Our results fail to reveal a distinct mutation spectrum in relation to neuroblastoma genomic subtypes. Moreover, a variety of neuroblastic tumours, from benign ganglioneuroma to very aggressive metastatic neuroblastoma, could be observed in the context of ALK mutation. Interpretation These results indicate that, in neuroblastoma, ALK mutations can be present at sub-clonal level with the possibility of subsequent clonal expansion at the time of progression. Tyrosine kinase inhibitors have become the gold standard therapy in treating ALK positive NBs, making diagnostic high sensitive detection of ALK mutations a necessary step in identifying optimal treatment modalities. This study is of utmost importance in clinical practice, highlighting the potential of NGS and the importance of serial samplings for therapeutic decisions. Citation Format: Susanne M. Fransson, Niloufar Javanmardi, Rose-Marie Sjoberg, Per Kogner, Tommy Martinsson. Low frequent ALK hotspot mutations in neuroblastoma tumors detected by ultra deep sequencing: Implications for ALK inhibitor treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4876. doi:10.1158/1538-7445.AM2017-4876

Neuroblastoma (NB) is a highly malignant pediatric tumor of the sympathetic nervous system that commonly displays low overall mutation frequency. We searched for new structural rearrangements in a series of 275 NBs of all stages using high density SNP microarrays. Exome sequencing was also performed on 15 tumor/constitutional DNA pairs and 25 additional tumors, mainly high-risk NBs, using paired-end sequencing on Illumina instrument. Normalized coverage data were used to generate array comparative genomic hybridization (CGH)-like profiles. Structural rearrangements leading to gain of TERT gene were observed in 15 of 275 neuroblastomas as judged by SNP microarray. 12 out of these 15 tumors with TERT rearrangement belong to 11q-deleted subgroup of NB patients that marks tumors with a poor prognosis. These rearrangements occurred only in high-risk NBs in an almost mutually exclusive fashion with MYCN amplifications, which is a known genetic event in this tumor type. Furthermore, in the small series of NBs analyzed by exome sequencing, we detect extensive structural rearrangements of chromosome 2, 5 and 7 leading to amplification of MYCN, TERT and CDK6 respectively in one tumor. The exome sequencing approach detected at average 14 (range 2-77) somatic protein-changing alterations per tumor. Combining structural and mutational data from the exome sequencing show recurrent alterations in ATRX gene. In our NB cohort we see alterations of ATRX in four non MYCN-amplified tumors: two focal deletions, one nonsense mutation and one deleterious missense mutation. ATRX inactivation results in alternative lengthening of telomeres (ALT). This means that high-risk neuroblastomas cancer cells can preserve their telomeres by ALT or by activation of telomerase reverse transcriptase (encoded by TERT). Alterations in TERT and ATRX were mutually exclusive, which is in agreement with the independent activation by these genes of telomere lengthening. Moreover, three cases with chromothripsis of chromosome 5 with rearrangements affecting the TERT were identified through SNP microarrays. These results identify TERT as at least one of the target genes activated by chromothripsis of chromosome 5. This remodeling of the genomic context likely abrogates transcriptional silencing of TERT in high-risk neuroblastoma and places telomerase activation in the centre of transformation in a large fraction of these tumors. This study identifies recurrent TERT and ATRX rearrangements and telomere lengthening as an important mechanism characterizing high-risk tumors and it supports the pharmacological inhibition of these targets to improve the outcome for this patient group. Citation Format: Niloufar Javanmardi, Susanne Fransson, Rose-Marie Sjöberg, Anna Djos, Per Kogner, Tommy Martinsson. Neuroblastoma: Telomere elongation is responsible for aggressive behavior. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2430.

Neuroblastoma tumors (NBs) show complex patterns of genetic abnormalities, which may include amplification or oncogenic mutations of ALK kinase in sporadic and familial cases. In analyzing genomic deviations it is sometimes highly efficient to focus on deep sequencing of specific parts of the genome with high clinical relevance to NB prognosis. This can be achieved by studying specific regions using targeted or amplicon sequencing, and thereby reducing the complexity of the analysis. The main benefits of this approach are reduced costs and shorter analysis time, due to the sequencing of fewer bases and the associated reduction in the volume of data generated. Additional benefit comes from achieving higher coverage that provides means for detection tumor specific variants present at subclonal level. The HaloPlex method, the custom target enrichment kit from Agilent, uses a different approach among enrichment kits, namely the fragmentation of DNA by restriction enzymes in conjunction with biotinylated probes designed to hybridize to both ends of a targeted DNA restriction fragment. This will form circular DNA strands that will be then closed by ligation (http://www.genomics.agilent.com) and it requires only 250 ng of input material. We targeted 35kb of genomic sequence including exons of related genes, as well as 50 bp of flanking exon regions. Samples were sequenced in a single MiSeq run (Illumina) using 2×150 paired-end sequencing. We compiled a gene panel for Next Generation Sequencing (NGS) comprising of three genes, ALK, ATRX and PPM1D, where the two first are associated with different subgroups of NBs. The Haloplex panel was evaluated on a pilot cohort of 40 patients including four controls with known and defined mutations (missense and indels), which produced a total of 4 Gb of data. Data were analyzed using an “in-house” bioinformatics pipeline based on mapping reads against hg19 using bwa, with realignment and recalibration using GATK haplotype caller, and variant annotation using ANNOVAR. For each position, the frequency for each base was calculated. The analysis revealed that we were able to generate sufficient sequencing data with very low background variability for mutation as well as deletion analysis using our diagnostic panels. We identified all expected mutations in our control samples. Sanger sequencing were used to validate the results and for analyzing sensitivity and specificity of the detected variants. Overall, the panel fulfills diagnostic criteria and will offer a fast and reliable procedure that will be useful in clarifying molecular genetic defects underlying NBs. Citation Format: Niloufar Javanmardi, Susanne Fransson, Malin Ostensson, Rose-marie Sjoberg, Per Kogner, Tommy Martinsson. Analysis of genetic variations in neuroblastoma using deep targeted sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4831. doi:10.1158/1538-7445.AM2015-4831