Comparative analysis of whole-genome sequencing of tumor and cfDNA in a neuroblastoma patient: a case report

High-risk neuroblastoma (NB) poses significant challenges in pediatric oncology due to its resistance to conventional therapies, leading to relapse and poor prognosis. Copy number variations (CNVs) are strong prognostic factors in NB, prompting exploration into alternative methods for CNV profiling. We conducted whole-genome sequencing (WGS) of the circulating cell-free DNA (cfDNA) from a patient with NB and compared the WGS of the primary and relapsed tumor tissue. Our analysis revealed concordance between the somatic single nucleotide variants (SNVs), insertions and deletions (indels), and CNVs identified in the cfDNA and tumor WGS. Notably, WGS detected numerical chromosome imbalances, large and focal structural aberrations including amplifications in MYCN, CDK4, and MDM2, using low-input cfDNA. Furthermore, additional variants unique to the cfDNA, such as the rare MET (p.R970C) variant, were identified, possibly representing sub-clonal populations or variants present at metastatic sites. In conclusion, WGS analysis of cfDNA offers a noninvasive, cost-effective, rapid, and sensitive alternative for CNV profiling in patients with NB. This approach holds promise for improving prognostication and for guiding personalized treatment strategies in NB

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

Thirteen families with hereditary VUR; relationship between 13 index cases and 28 affected relatives.

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Workflow overview and specific settings for bioinformatical handling of sequence data in CLC genomic workbench.

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Three possibly pathogenic variants identified in nephrogenesis-related genes in three families with hereditary VUR.

Three possibly pathogenic variants identified in nephrogenesis-related genes in three families with hereditary VUR.</p

Data selection process for the study.

Data selection process for the study.</p