Massively Parallel Sequencing Reveals an Accumulation of De Novo Mutations and an Activating Mutation of LPAR1 in a Patient with Metastatic Neuroblastoma

Abstract

Neuroblastoma is one of the most genomically heterogeneous childhood malignances studied to date, and the molecular events that occur during the course of the disease are not fully understood. Genomic studies in neuroblastoma have showed only a few recurrent mutations and a low somatic mutation burden. However, none of these studies has examined the mutations arising during the course of disease, nor have they systemically examined the expression of mutant genes. Here we performed genomic analyses on tumors taken during a 3.5 years disease course from a neuroblastoma patient (bone marrow biopsy at diagnosis, adrenal primary tumor taken at surgical resection, and a liver metastasis at autopsy). Whole genome sequencing of the index liver metastasis identified 44 non-synonymous somatic mutations in 42 genes (0.85 mutation/MB) and a large hemizygous deletion in the ATRX gene which has been recently reported in neuroblastoma. Of these 45 somatic alterations, 15 were also detected in the primary tumor and bone marrow biopsy, while the other 30 were unique to the index tumor, indicating accumulation of de novo mutations during therapy. Furthermore, transcriptome sequencing on the 3 tumors demonstrated only 3 out of the 15 commonly mutated genes (LPAR1, GATA2, and NUFIP1) had high level of expression of the mutant alleles, suggesting potential oncogenic driver roles of these mutated genes. Among them, the druggable G-protein coupled receptor LPAR1 was highly expressed in all tumors. Cells expressing the LPAR1 R163W mutant demonstrated a significantly increased motility through elevated Rho signaling, but had no effect on growth. Therefore, this study highlights the need for multiple biopsies and sequencing during progression of a cancer and combinatorial DNA and RNA sequencing approach for systematic identification of expressed driver mutations.

MultiDimensional ClinOmics for Precision Therapy of Children and Adolescent Young Adults with Relapsed and Refractory Cancer: A Report from the Center for Cancer Research

“…1). We and others have reported the same phenomenon that approximately 50% of DNA somatic mutations are expressed in the RNA (22, 23). …”

“…Our WTS results showed that about one-half of all SNVs are not expressed in the transcriptome, and thus can be excluded as a driver mutation. We and others have reported the same phenomenon that approximately 50% of DNA mutations are expressed in the RNA (22, 23). The cause is usually that the RNA transcript, and hence, the gene, is not expressed at the RNA level.…”

MYCN controls an alternative RNA splicing program in high-risk metastatic neuroblastoma

“…Total RNA was extracted, RNA-seq libraries were made and sequenced as previously described [55, 56]. The integrity of total RNA was evaluated using an Agilent BioAnalyzer 2100 (Agilent, Palo Alto, CA), and only RNAs with RIN greater than 6.0 were used in this study.…”

Advances in the translational genomics of neuroblastoma: From improving risk stratification and revealing novel biology to identifying actionable genomic alterations

“…However, it is clear from early studies that clonal evolution is common in neuroblastoma and that relapsed neuroblastoma tumors acquire many additional segmental chromosomal alterations that may have prognostic implications in the relapsed setting . More recent focused studies using genomic deep‐sequencing efforts have suggested that clonal evolution also results in the acquisition of targetable somatic aberrations in known oncogenic pathways, and early evidence suggests that these converge on the mitogen‐activated protein (MAP) kinase (MAPK) pathway . Although the MAPK pathway is a key driver of oncogenicity in several human malignancies, until recently, this was not believed to be the case in neuroblastoma, because MAPK pathway mutations are rare in primary neuroblastoma tumors (although NRAS was discovered in neuroblastoma) .…”

“…68 More recent focused studies using genomic deep-sequencing efforts have suggested that clonal evolution also results in the acquisition of targetable somatic aberrations in known oncogenic pathways, and early evidence suggests that these converge on the mitogen-activated protein (MAP) kinase (MAPK) pathway. 88,89,106,107 Although the MAPK pathway is a key driver of oncogenicity in several human malignancies, until recently, this was not believed to be the case in neuroblastoma, because MAPK pathway mutations are rare in primary neuroblastoma tumors (although NRAS was discovered in neuroblastoma). 40,108 However, in a recent, comprehensive, deep-resequencing study of 23 relapsed/ refractory neuroblastomas, 78% of relapsed specimens ALK Mutation/focal amplification c 8%-14% (Sausen 2013, 39 Pugh 2013, 40 Molenaar 2012, 41 Cheung 2012, 42 Shukla 2012 87 ) 26%-43% (Schleiermacher 2014, 88 Eleveld 2015 89 ) c ALK mutation/amplification ALK inhibition (Barone 2013, 35 Carpenter & Mosse 2012 91 Gogolin 2013 92 ) CDK4/6 inhibition (Rader 2013, 91 Gogolin 2013 91 Gogolin 2013 92 ) CDK4/6 inhibition (Rader 2013, 91 Gogolin 2013 ) 13%-22% (Eleveld 2015, 89 Carr-Wilkinson 2010 94 ) MYCN amplification (Rader 2013, 91 Gogolin 2013 92 ) CDK4/6 inhibition (Rader 2013, 91 Gogolin 2013 96 Gamble 2012, 97 Van Maerken…”