Multifocal Neuroblastoma and Central Hypoventilation in An Infant with Germline ALK F1174I Mutation

Funding Information: This work has been supported by grants from the Swedish Cancer Society (TM 15-794; TM 20-1213; PK 19-566), the Swedish Childhood Cancer Foundation (TM 16-147; TM 17-166; TM 19-139; PK 17-122; SF 15-61, 18-99; DT 12-002, NC12-0026), the Swedish Research Council (TM 521-2014-3031), the Swedish state under the LUA/ALF agreement (TM ALFGBG-447171) and the Swedish Foundation for Strategic Research (TM/PK RB13-0204, www.nnbcr.se). SF was the recipient of a Research Assistant Fellowship (14-64), by the Swedish Childhood Cancer Foundation. Publisher Copyright: © 2022 by the authors.A preterm infant with central hypoventilation was diagnosed with multifocal neuroblastoma. Congenital anomalies of the autonomic nervous system in association with neuroblastoma are commonly associated with germline mutations in PHOX2B. Further, the ALK gene is frequently mutated in both familial and sporadic neuroblastoma. Sanger sequencing of ALK and PHOX2B, SNP microarray of three tumor samples and whole genome sequencing of tumor and blood were performed. Genetic testing revealed a germline ALK F1174I mutation that was present in all tumor samples as well as in normal tissue samples from the patient. Neither of the patient’s parents presented the ALK variant. Array profiling of the three tumor samples showed that two of them had only numerical aberrations, whereas one sample displayed segmental alterations, including a gain at chromosome 2p, resulting in two copies of the ALK-mutated allele. Whole genome sequencing confirmed the presence of the ALK variant and did not detect any aberrations in the coding or promotor region of PHOX2B. This study is to our knowledge the first to report a de novo ALK F1174I germline mutation. This may not only predispose to congenital multifocal neuroblastoma but may also contribute to the respiratory dysfunction seen in this patient.Peer reviewe

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

Identification of epigenetically regulated genes that predict patient outcome in neuroblastoma

<p>Abstract</p> <p>Background</p> <p>Epigenetic mechanisms such as DNA methylation and histone modifications are important regulators of gene expression and are frequently involved in silencing tumor suppressor genes.</p> <p>Methods</p> <p>In order to identify genes that are epigenetically regulated in neuroblastoma tumors, we treated four neuroblastoma cell lines with the demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC) either separately or in conjunction with the histone deacetylase inhibitor trichostatin A (TSA). Expression was analyzed using whole-genome expression arrays to identify genes activated by the treatment. These data were then combined with data from genome-wide DNA methylation arrays to identify candidate genes silenced in neuroblastoma due to DNA methylation.</p> <p>Results</p> <p>We present eight genes (<it>KRT19</it>, <it>PRKCDBP</it>, <it>SCNN1A</it>, <it>POU2F2</it>, <it>TGFBI</it>, <it>COL1A2</it>, <it>DHRS3 </it>and <it>DUSP23</it>) that are methylated in neuroblastoma, most of them not previously reported as such, some of which also distinguish between biological subsets of neuroblastoma tumors. Differential methylation was observed for the genes <it>SCNN1A </it>(p < 0.001), <it>PRKCDBP </it>(p < 0.001) and <it>KRT19 </it>(p < 0.01). Among these, the mRNA expression of <it>KRT19 </it>and <it>PRKCDBP </it>was significantly lower in patients that have died from the disease compared with patients with no evidence of disease (fold change -8.3, p = 0.01 for <it>KRT19 </it>and fold change -2.4, p = 0.04 for <it>PRKCDBP</it>).</p> <p>Conclusions</p> <p>In our study, a low methylation frequency of <it>SCNN1A</it>, <it>PRKCDBP </it>and <it>KRT19 </it>is significantly associated with favorable outcome in neuroblastoma. It is likely that analysis of specific DNA methylation will be one of several methods in future patient therapy stratification protocols for treatment of childhood neuroblastomas.</p

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

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

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

Genetic Instability and Intratumoral Heterogeneity in Neuroblastoma with <em>MYCN</em> Amplification Plus 11q Deletion

<div><h3>Background/Aim</h3><p>Genetic analysis in neuroblastoma has identified the profound influence of <em>MYCN</em> 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.</p> <h3>Methods</h3><p>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.</p> <h3>Results and Conclusion</h3><p>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.</p> </div

FISH results in the heterogeneous cases (hetMNA and het11q-del).

<p>Disomic cells (ratio 2∶2) and numeric alterations (ratios 3∶3; 4∶4…), balanced ratio between the signal numbers of chromosomal region of interest and the reference signals on the opposite arm of the chromosome; Cells with gain (ie ratios 2∶4; 3∶4…), signal numbers of the chromosomal region of interest exceed up to 4-fold the number of reference signals; Cells with imbalance (ie ratios 3∶2; 4∶3…), imbalance ratio between the signal numbers of chromosomal region of interest and the reference signals with more than 1 signals of chromosomal region of interest; Cells with deletion (ie. ratios 2∶1; 3∶1 4∶1…), unbalanced ratio between the signal numbers of the chromosomal region of interest and the reference signals with only 1 signal of the chromosomal region of interest; hetMNA, occurrence of clusters or as single cells with amplification (at least five cells per slide) surrounded by non-amplified tumor cells.</p><p>ND, not done.</p><p>The cases have been listed according to chromosome 2 aberrations using pangenomic techniques.</p

MLPA data.

<p>INRG Biology Committee definitions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053740#pone.0053740-Schleiermacher2" target="_blank">[11]</a>. <i>MYCN</i> amplification (MNA), up to 4-fold excess of signal numbers of the chromosomal region of interest compared with the reference signals; Gain (+), unbalanced ratio (high signal excess) between the signals of a gene and all other probes located on the same chromosome; Loss (−), unbalanced ratio (low signal excess) between the signals of the chromosomal region of interest (at least two adjacent probes) and the reference signals (at least two) of the chromosomal region of interest.</p><p>The cases have been listed according to chromosome 2 aberrations using pangenomic techniques.</p