Anna Djos

Emergence of New ALK Mutations at Relapse of Neuroblastoma

PURPOSE In neuroblastoma, the ALK receptor tyrosine kinase is activated by point mutations. We investigated the potential role of ALK mutations in neuroblastoma clonal evolution. METHODS We analyzed ALK mutations in 54 paired diagnosis-relapse neuroblastoma samples using Sanger sequencing. When an ALK mutation was observed in one paired sample, a minor mutated component in the other sample was searched for by more than 100,000× deep sequencing of the relevant hotspot, with a sensitivity of 0.17%. RESULTS All nine ALK-mutated cases at diagnosis demonstrated the same mutation at relapse, in one case in only one of several relapse nodules. In five additional cases, the mutation seemed to be relapse specific, four of which were investigated by deep sequencing. In two cases, no mutation evidence was observed at diagnosis. In one case, the mutation was present at a subclonal level (0.798%) at diagnosis, whereas in another case, two different mutations resulting in identical amino acid changes were detected, one only at diagnosis and the other only at relapse. Further evidence of clonal evolution of ALK-mutated cells was provided by establishment of a fully ALK-mutated cell line from a primary sample with an ALK-mutated cell population at subclonal level (6.6%). CONCLUSION In neuroblastoma, subclonal ALK mutations can be present at diagnosis with subsequent clonal expansion at relapse. Given the potential of ALK-targeted therapy, the significant spatiotemporal variation of ALK mutations is of utmost importance, highlighting the potential of deep sequencing for detection of subclonal mutations with a sensitivity 100-fold that of Sanger sequencing and the importance of serial samplings for therapeutic decisions.

Anaplastic Lymphoma Kinase (ALK) regulates initiation of transcription of MYCN in neuroblastoma cells

Neuroblastoma is a neural crest-derived embryonal tumour of the postganglionic sympathetic nervous system and a disease with several different chromosomal gains and losses, which include MYCN-amplified neuroblastoma on chromosome 2, deletions of parts of the chromosomes 1p and 11q, gain of parts of 17q and triploidy. Recently, activating mutations of the ALK (Anaplastic Lymphoma Kinase) RTK (Receptor Tyrosine Kinase) gene have been described in neuroblastoma. A meta-analysis of neuroblastoma cases revealed that ALK mutations (49 of 709 cases) in relation to genomic subtype were most frequently observed in MYCN amplified tumours (8.9%), correlating with a poor clinical outcome. MYCN proteins target proliferation and apoptotic pathways, and have an important role in the progression of neuroblastoma. Here, we show that both wild-type and gain-of-function mutants in ALK are able to stimulate transcription at the MYCN promoter and initiate mRNA transcription of the MYCN gene in both neuronal and neuroblastoma cell lines. Further, this stimulation of MYCN gene transcription and de novo MYCN protein expression is abrogated by specific ALK inhibitors, such as crizotinib (PF-2341066), NVP-TAE684, and by small interfering RNA to ALK resulting in a decrease in proliferation rate. Finally, co-transfection of ALK gain-of-function mutations together with MYCN leads to an increase in transformation potential. Taken together, our results indicate that ALK signalling regulates initiation of transcription of the MYCN gene providing a possible explanation for the poor clinical outcome observed when MYCN is amplified together with activated ALK.

The RASSF gene family members RASSF5, RASSF6 and RASSF7 show frequent DNA methylation in neuroblastoma

BackgroundHypermethylation of promotor CpG islands is a common mechanism that inactivates tumor suppressor genes in cancer. Genes belonging to the RASSF gene family have frequently been reported as epigenetically silenced by promotor methylation in human cancers. Two members of this gene family, RASSF1A and RASSF5A have been reported as methylated in neuroblastoma. Data from our previously performed genome-wide DNA methylation array analysis indicated that other members of the RASSF gene family are targeted by DNA methylation in neuroblastoma.ResultsIn the current study, we found that several of the RASSF family genes (RASSF2, RASSF4, RASSF5, RASSF6, RASSF7, and RASSF10) to various degrees were methylated in neuroblastoma cell lines and primary tumors. In addition, several of the RASSF family genes showed low or absent mRNA expression in neuroblastoma cell lines. RASSF5 and RASSF6 were to various degrees methylated in a large portion of neuroblastoma tumors and RASSF7 was heavily methylated in most tumors. Further, CpG methylation sites in the CpG islands of some RASSF family members could be used to significantly discriminate between biological subgroups of neuroblastoma tumors. For example, RASSF5 methylation highly correlated to MYCN amplification and INRG stage M. Furthermore, high methylation of RASSF6 was correlated to unfavorable outcome, 1p deletion and MYCN amplification in our tumor material.In conclusionThis study shows that several genes belonging to the RASSF gene family are methylated in neuroblastoma. The genes RASSF5, RASSF6 and RASSF7 stand out as the most promising candidate genes for further investigations in neuroblastoma.

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.

Genetic instability and intratumoral heterogeneity in neuroblastoma with MYCN amplification plus 11q deletion.

Background/Aim Genetic analysis in neuroblastoma has identified the profound influence of MYCN amplification and 11q deletion in patients’ prognosis. These two features of high-risk neuroblastoma usually occur as mutually exclusive genetic markers, although in rare cases both are present in the same tumor. The purpose of this study was to characterize the genetic profile of these uncommon neuroblastomas harboring both these high-risk features. Methods We selected 18 neuroblastomas with MNA plus 11q loss detected by FISH. Chromosomal aberrations were analyzed using Multiplex Ligation-dependent Probe Amplification and Single Nucleotide Polymorphism array techniques. Results and Conclusion This group of tumors has approximately the same high frequency of aberrations as found earlier for 11q deleted tumors. In some cases, DNA instability generates genetic heterogeneity, and must be taken into account in routine genetic diagnosis.

Comparative genetic study of intratumoral heterogenous MYCN amplified neuroblastoma versus aggressive genetic profile neuroblastic tumors.

Intratumoral heterogeneous MYCN amplification (hetMNA) is an unusual event in neuroblastoma with unascertained biological and clinical implications. Diagnosis is based on the detection of MYCN amplification surrounded by non-amplified tumor cells by fluorescence in situ hybridization (FISH). To better define the genetic features of hetMNA tumors, we studied the Spanish cohort of neuroblastic tumors by FISH and single nucleotide polymorphism arrays. We compared hetMNA tumors with homogeneous MNA (homMNA) and nonMNA tumors with 11q deletion (nonMNA w11q-). Of 1091 primary tumors, 28 were hetMNA by FISH. Intratumoral heterogeneity of 1p, 2p, 11q and 17q was closely associated with hetMNA tumors when analyzing different pieces for each case. For chromosome 2, 16 cases showed 2p intact, 4 focal gain at 2p24.3 and 8 MNA. The lengths of the smallest regions of overlap (SROs) for 2p gains and 1p deletions were between the SRO lengths observed in homMNA and nonMNA w11q- tumors. Co-occurrence of 11q- and +17q was frequently found with the largest SROs for both aberrations. The evidence for and frequency of different genetic subpopulations representing a hallmark of the hetMNA subgroup of NB indicates, on one hand, the presence of a considerable genetic instability with different SRO of either gains and losses compared with those of the other NB groups and highlights and, on the other hand, the need for multiple sampling from distant and macroscopically and microscopically distinct tumor areas. Narrowing down the different SRO for both deletions and gains in NB groups would be crucial to pinpointing the candidate gene(s) and the critical gene dosage with prognostic and therapeutic significance. This complexity of segmental chromosomal aberration patterns reinforces the necessity for a larger cohort study using FISH and pangenomic techniques to develop a suitable therapeutic strategy for these patients.

Estimation of copy number aberrations: Comparison of exome sequencing data with SNP microarrays identifies homozygous deletions of 19q13.2 and CIC in neuroblastoma

In the pediatric cancer neuroblastoma, analysis of recurrent chromosomal aberrations such as loss of chromosome 1p, 11q, gain of 17q and MYCN amplification are used for patient stratification and subsequent therapy decision making. Different analysis techniques have been used for detection of segmental abnormalities, including fluorescence in situ hybridization (FISH), comparative genomic hybridization (CGH)-microarrays and multiplex ligation-dependent probe amplification (MLPA). However, as next-generation sequencing becomes available for clinical use, this technique could also be used for assessment of copy number alterations simultaneously with mutational analysis. In this study we compare genomic profiles generated through exome sequencing data with profiles generated from high resolution Affymetrix single nucleotide polymorphism (SNP) microarrays on 30 neuroblastoma tumors of different stages. Normalized coverage reads for tumors were calculated using Control-FREEC software and visualized through a web based Shiny application, prior to comparison with corresponding SNP-microarray data. The two methods show high-level agreement for breakpoints and copy number of larger segmental aberrations and numerical aneuploidies. However, several smaller gene containing deletions that could not readily be detected through the SNP-microarray analyses were identified through exome profiling, most likely due to difference between spatial distribution of microarray probes and targeted regions of the exome capture. These smaller aberrations included focal ATRX deletion in two tumors and three cases of novel deletions in chromosomal region 19q13.2 causing homozygous loss of multiple genes including the CIC (Capicua) gene. In conclusion, genomic profiles generated from normalized coverage of exome sequencing show concordance with SNP microarray generated genomic profiles. Exome sequencing is therefore a useful diagnostic tool for copy number variant (CNV) detection in neuroblastoma tumors, especially considering the combination with mutational screening. This enables detection of theranostic targets such as ALK and ATRX together with detection of significant segmental aneuploidies, such as 2p-gain, 17q-gain, 11q-deletion as well as MYCN amplification.

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

BackgroundChromosomal 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.MethodAs 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 conclusionAs no STAG2 mutation was detected in this study, we conclude that inactivating mutation of STAG2 is not likely causative to neuroblastoma aneuploidy.

Abstract 4867: Exome- and whole genome sequencing for clinical evaluation and precision medicine in neuroblastoma

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

Abstract 2430: Neuroblastoma: Telomere elongation is responsible for aggressive behavior

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 Sjoberg, 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.