11q deletion or ALK activity curbs DLG2 expression to maintain an undifferentiated state in neuroblastoma

High-risk neuroblastomas typically display an undifferentiated or poorly differentiated morphology. It is therefore vital to understand molecular mechanisms that block the differentiation process. We identify an important role for oncogenic ALK-ERK1/2-SP1 signaling in the maintenance of undifferentiated neural crest-derived progenitors through the repression of DLG2, a candidate tumor suppressor gene in neuroblastoma. DLG2 is expressed in the murine "bridge signature'' that represents the transcriptional transition state when neural crest cells or Schwann cell precursors differentiate to chromaffin cells of the adrenal gland. We show that the restoration of DLG2 expression spontaneously drives neuroblastoma cell differentiation, high-lighting the importance of DLG2 in this process. These findings are supported by genetic analyses of high-risk 11q deletion neuroblastomas, which identified genetic lesions in the DLG2 gene. Our data also suggest that further exploration of other bridge genes may help elucidate the mechanisms underlying the differentiation of NC-derived progenitors and their contribution to neuroblastomas

Genomic instability and genetic heterogeneity in neuroblastoma tumours

Javanmardi, N., 2017, Genomic instability and genetic heterogeneity in neuroblastoma Department of Pathology and Clinical Genetics, Institute of Biomedicine at Sahlgrenska Academy, University of Gothenburg, Sweden Neuroblastoma (NB), a tumour of the sympathetic nervous system and the most common malignant disease of early childhood, is responsible for 9% of paediatric cancer related deaths. Aggressive NB still constitutes a major clinical problem with survival rates of about 35%. It is therefore of great clinical interest to further study the biological parameters that can (i) better classify tumours so that the children may be given the right treatment (ii) identify new actionable targets. Aim - the objective of this thesis was to explore genes and chromosomal regions with potential involvement in the initiation/progression of NB that can be used for improved patient stratification. Results – In paper I and III we detected point mutations in the tyrosine kinase domain of the ALK oncogene. Minor population of cells with ALK mutations were detected with massive parallel deep DNA sequencing. It is likely that early detection of subclones with ALK mutation is critical in treatment of these tumours with recently derived small molecule ALK inhibitors. We propose increased serial sampling of tumour material from high-risk NB tumours and analysis with the new sequencing techniques. In paper II we observed that the distal part of chromosome arm 2p often is subjected to gain of an extra copy – i.e. 2p-gain. Interestingly, this region contains three genes, ALKAL2, MYCN and ALK, of strong importance for NB development. We suggest that the gain of this “cassette” of genes is beneficial to the NB tumor pathogenesis with potential to aid in therapeutical intervention. In the last study, paper IV, we analysed the high-risk 11q-deleted NB tumours. We show that 11q-deleted tumours with and without MYCN amplification present different 11q-deletion breakpoint patterns. The detailed analysis of these patterns enabled us to detect genes and chromosomal regions on 11q that may contain tumour suppressors in this severe child cancer subgroup. Furthermore, we propose DLG2 as a highly interesting 11q candidate NB gene. Conclusion - Our observation of a significant spatiotemporal variation of ALK mutations is of utmost importance in clinical practice. DLG2 stands out as a strong tumor suppressor candidate for the 11q-deleted NBs. It is important to note that the experiments we propose are expected to contribute to precision medicine

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

Tumor Development, Growth Characteristics and Spectrum of Genetic Aberrations in the TH-<em>MYCN</em> Mouse Model of Neuroblastoma

<div><h3>Background</h3><p>The TH-<em>MYCN</em> transgenic neuroblastoma model, with targeted MYCN expression to the developing neural crest, has been used to study neuroblastoma development and evaluate novel targeted tumor therapies.</p> <h3>Methods</h3><p>We followed tumor development in 395 TH-<em>MYCN</em> (129X1/SvJ) mice (125 negative, 206 hemizygous and 64 homozygous mice) by abdominal palpations up to 40 weeks of age. DNA sequencing of <em>MYCN</em> in the original plasmid construct and mouse genomic DNA was done to verify the accuracy. Copy number analysis with Affymetrix® Mouse Diversity Genotyping Arrays was used to characterize acquired genetic aberrations.</p> <h3>Results</h3><p>DNA sequencing confirmed presence of human <em>MYCN</em> cDNA in genomic TH-<em>MYCN</em> DNA corresponding to the original plasmid construct. Tumor incidence and growth correlated significantly to transgene status with event-free survival for hemizygous mice at 50%, and 0% for homozygous mice. Hemizygous mice developed tumors at 5.6–19 weeks (median 9.1) and homozygous mice at 4.0–6.9 weeks (5.4). The mean treatment window, time from palpable tumor to sacrifice, for hemizygous and homozygous mice was 15 and 5.2 days, respectively. Hemizygous mice developing tumors as early as homozygous mice had a longer treatment window. Age at tumor development did not influence treatment window for hemizygous mice, whereas treatment window in homozygous mice decreased significantly with increasing age. Seven out of 10 analysed tumors had a flat DNA profile with neither segmental nor numerical chromosomal aberrations. Only three tumors from hemizygous mice showed acquired genetic features with one or more numerical aberrations. Of these, one event corresponded to gain on the mouse equivalent of human chromosome 17.</p> <h3>Conclusion</h3><p>Hemizygous and homozygous TH-<em>MYCN</em> mice have significantly different neuroblastoma incidence, tumor growth characteristics and treatment windows but overlap in age at tumor development making correct early genotyping essential to evaluate therapeutic interventions. Contrasting previous studies, our data show that TH-<em>MYCN</em> tumors have few genetic aberrations.</p> </div

Summary of genomic aberrations in ten different TH-<i>MYCN</i> tumors.

<p>Abbreviations:</p>a<p>homozygous;</p>b<p>hemizygous;</p>c<p>whole chromosome gain;</p>d<p>whole chromosome loss.</p

Time from a palpable tumor to sacrifice – the treatment window.

<p><b>A.</b> Treatment window for homozygous and hemizygous mice. Mean number of treatment days for homozygous mice was 5.2 days (median 5, n = 30) and for hemizygous mice 15 days (median 11, n = 54). Statistical analysis was performed using two-sided <i>t</i> test on log-transformed data; p<0.01. <b>B.</b> Treatment window for hemizygous mice as a function of age at tumor palpation. Linear regression analysis yielded R² =  0.012 and the correlation was assessed using two-sided Spearman test; p = 0.83. <b>C.</b> Treatment window for homozygous mice as a function of age at tumor palpation. Linear regression analysis yielded R² =  0.30 and the correlation was assessed using two-sided Spearman test; p<0.05. <b>D.</b> Treatment window for homozygous mice versus hemizygous mice with an early tumor development (5.6–7.0 weeks of age). Mean number of treatment days for homozygous mice was 5.2 days (median 5, n = 30) and for hemizygous mice with an early tumor development 15 days (median 18, n = 5). Statistical analysis was performed using two-sided t test on log-transformed data; p<0.01. Box plots show median, inter quartile range (25–75%), min and max, and + represent the mean.</p

Whole genomic profile of two representative cases of the ten analyzed TH-<i>MYCN</i> tumors.

<p>TG1–TG7 showed flat profiles with no segmental or numerical aberrations (TG7 is shown here). TG8, TG9 and TG10 showed a few numerical aberrations. TG8 shown here display whole chromosome gain of mouse chromosome 3 and 11 (indicated by arrows).</p

Tumor development of TH-<i>MYCN</i> mice on the 129X1/SvJ background.

<p><b>A.</b> Event-free survival. The y-axis shows the proportion of animals that remain free of an event, here defined as palpable tumor development, spontaneous death by unknown cause including missing animals, and euthanization due to general signs of discomfort. The x-axis shows weeks from birth. Negative n = 125, hemizygous n = 206 and homozygous n = 64. <b>B.</b> Frequency distribution diagram showing the age at palpable tumor. Homozygous mice developed palpable tumors between 4.0–6.9 weeks of age, mean of 5.6 and median of 5.4 weeks (n = 45). The hemizygous mice that developed tumors were palpated with a tumor between 5.6–19 weeks of age, mean 9.9 and median 9.1 weeks (n = 88). The two-sided unpaired t test was used for comparison between the two groups p<0.01.</p

Amplification of CDK4 and MDM2 : a detailed study of a high-risk neuroblastoma subgroup

In neuroblastoma, MYCN amplification and 11q-deletion are important, although incomplete, markers of high-risk disease. It is therefore relevant to characterize additional alterations that can function as prognostic and/or predictive markers. Using SNP-microarrays, a group of neuroblastoma patients showing amplification of one or multiple 12q loci was identified. Two loci containing CDK4 and MDM2 were commonly co-amplified, although amplification of either locus in the absence of the other was observed. Pharmacological inhibition of CDK4/6 with ribociclib or abemaciclib decreased proliferation in a broad set of neuroblastoma cell lines, including CDK4/MDM2-amplified, whereas MDM2 inhibition by Nutlin-3a was only effective in p53(wild-type) cells. Combined CDK4/MDM2 targeting had an additive effect in p53(wild-type) cell lines, while no or negative additive effect was observed in p53(mutated) cells. Most 12q-amplified primary tumors were of abdominal origin, including those of intrarenal origin initially suspected of being Wilms' tumor. An atypical metastatic pattern was also observed with low degree of bone marrow involvement, favoring other sites such as the lungs. Here we present detailed biological data of an aggressive neuroblastoma subgroup hallmarked by 12q amplification and atypical clinical presentation for which our in vitro studies indicate that CDK4 and/or MDM2 inhibition also could be beneficial