Use of Optical Genome Mapping to Detect Structural Variants in Neuroblastoma

\ua9 2023 by the authors.Background: Neuroblastoma is the most common extracranial solid tumour in children, accounting for 15% of paediatric cancer deaths. Multiple genetic abnormalities have been identified as prognostically significant in neuroblastoma patients. Optical genome mapping (OGM) is a novel cytogenetic technique used to detect structural variants, which has not previously been tested in neuroblastoma. We used OGM to identify copy number and structural variants (SVs) in neuroblastoma which may have been missed by standard cytogenetic techniques. Methods: Five neuroblastoma cell lines (SH-SY5Y, NBLW, GI-ME-N, NB1691 and SK-N-BE2(C)) and two neuroblastoma tumours were analysed using OGM with the Bionano Saphyr\uae instrument. The results were analysed using Bionano Access software and compared to previous genetic analyses including G-band karyotyping, FISH (fluorescent in situ hybridisation), single-nucleotide polymorphism (SNP) array and RNA fusion panels for cell lines, and SNP arrays and whole genome sequencing (WGS) for tumours. Results: OGM detected copy number abnormalities found using previous methods and provided estimates for absolute copy numbers of amplified genes. OGM identified novel SVs, including fusion genes in two cell lines of potential clinical significance. Conclusions: OGM can reliably detect clinically significant structural and copy number variations in a single test. OGM may prove to be more time- and cost-effective than current standard cytogenetic techniques for neuroblastoma

Characterisation of the p53 pathway in cell lines established from TH-MYCN transgenic mouse tumours

Cell lines established from the TH-MYCN transgenic murine model of neuroblastoma are a valuable preclinical, immunocompetent, syngeneic model of neuroblastoma, for which knowledge of their p53 pathway status is important. In this study, the Trp53 status and functional response to Nutlin-3 and ionising radiation (IR) were determined in 6 adherent TH-MYCN transgenic cell lines using Sanger sequencing, western blot analysis and flow cytometry. Sensitivity to structurally diverse MDM2 inhibitors (Nutlin-3, MI-63, RG7388 and NDD0005) was determined using XTT proliferation assays. In total, 2/6 cell lines were Trp53 homozygous mutant (NHO2A and 844MYCN+/+) and 1/6 (282MYCN+/-) was Trp53 heterozygous mutant. For 1/6 cell lines (NHO2A), DNA from the corresponding primary tumour was found to be Trp53 wt. In all cases, the presence of a mutation was consistent with aberrant p53 signalling in response to Nutlin-3 and IR. In comparison to TP53 wt human neuroblastoma cells, Trp53 wt murine control and TH-MYCN cell lines were significantly less sensitive to growth inhibition mediated by MI-63 and RG7388. These murine Trp53 wt and mutant TH-MYCN cell lines are useful syngeneic, immunocompetent neuroblastoma models, the former to test p53-dependent therapies in combination with immunotherapies, such as anti-GD2, and the latter as models of chemoresistant relapsed neuroblastoma when aberrations in the p53 pathway are more common. The spontaneous development of Trp53 mutations in 3 cell lines from TH-MYCN mice may have arisen from MYCN oncogenic driven and/or ex vivo selection. The identified species-dependent selectivity of MI-63 and RG7388 should be considered when interpreting in vivo toxicity studies of MDM2 inhibitors

Schedule-dependent response of neuroblastoma cell lines to combinations of etoposide and cisplatin

The growth inhibitory effects of cisplatin and etoposide on neuroblastoma cell lines were investigated in several scheduled combinations. Results were analyzed using median effect and combination index analyses. In all schedules in which cisplatin was administered prior to etoposide a synergistic effect was observed. Conversely, an antagonistic effect was seen in all schedules where etoposide was administered before cisplatin

Acceleration of generalized hypergeometric functions through precise remainder asymptotics

We express the asymptotics of the remainders of the partial sums {s_n} of the generalized hypergeometric function q+1_F_q through an inverse power series z^n n^l \sum_k c_k/n^k, where the exponent l and the asymptotic coefficients {c_k} may be recursively computed to any desired order from the hypergeometric parameters and argument. From this we derive a new series acceleration technique that can be applied to any such function, even with complex parameters and at the branch point z=1. For moderate parameters (up to approximately ten) a C implementation at fixed precision is very effective at computing these functions; for larger parameters an implementation in higher than machine precision would be needed. Even for larger parameters, however, our C implementation is able to correctly determine whether or not it has converged; and when it converges, its estimate of its error is accurate.Comment: 36 pages, 6 figures, LaTeX2e. Fixed sign error in Eq. (2.28), added several references, added comparison to other methods, and added discussion of recursion stabilit

Methyl-CpG-binding domain sequencing reveals a prognostic methylation signature in neuroblastoma

Accurate assessment of neuroblastoma outcome prediction remains challenging. Therefore, this study aims at establishing novel prognostic tumor DNA methylation biomarkers. In total, 396 low- and high-risk primary tumors were analyzed, of which 87 were profiled using methyl-CpG-binding domain (MBD) sequencing for differential methylation analysis between prognostic patient groups. Subsequently, methylationspecific PCR (MSP) assays were developed for 78 top-ranking differentially methylated regions and tested on two independent cohorts of 132 and 177 samples, respectively. Further, a new statistical framework was used to identify a robust set of MSP assays of which the methylation score (i.e. the percentage of methylated assays) allows accurate outcome prediction. Survival analyses were performed on the individual target level, as well as on the combined multimarker signature. As a result of the differential DNA methylation assessment by MBD sequencing, 58 of the 78 MSP assays were designed in regions previously unexplored in neuroblastoma, and 36 are located in non-promoter or non-coding regions. In total, 5 individual MSP assays (located in CCDC177, NXPH1, lnc-MRPL3-2, lnc-TREX1-1 and one on a region from chromosome 8 with no further annotation) predict event-free survival and 4 additional assays (located in SPRED3, TNFAIP2, NPM2 and CYYR1) also predict overall survival. Furthermore, a robust 58-marker methylation signature predicting overall and eventfree survival was established. In conclusion, this study encompasses the largest DNA methylation biomarker study in neuroblastoma so far. We identified and independently validated several novel prognostic biomarkers, as well as a prognostic 58-marker methylation signature

A protein kinase Cβ inhibitor attenuates multidrug resistance of neuroblastoma cells

BACKGROUND: The acquisition of drug resistance is a major reason for poor outcome of neuroblastoma. Protein kinase C (PKC) has been suggested to influence drug resistance in cancer cells. The aim of this study was to elucidate whether inhibition of PKCβ isoforms influences drug-resistance of neuroblastoma cells. METHODS: The effect of the PKCβ inhibitor LY379196 on the growth-suppressing effects of different chemotherapeutics on neuroblastoma cells was analyzed with MTT assays. The effect of LY379196 on the accumulation of [(3)H]vincristine was also investigated RESULTS: The PKCβ inhibitor LY379196 suppressed the growth of three neuroblastoma cell lines. LY379196 also augmented the growth-suppressive effect of doxorubicin, etoposide, paclitaxel, and vincristine, but not of carboplatin. The effect was most marked for vincristine and for the cell-line (SK-N-BE(2)) that was least sensitive to vincristine. No effect was observed on the non-resistant IMR-32 cells. Two other PKC inhibitors, Gö6976 and GF109203X, also enhanced the vincristine effect. The PKC inhibitors caused an increased accumulation of [(3)H]vincristine in SK-N-BE(2) cells. CONCLUSIONS: This indicates that inhibition of PKCβ could attenuate multidrug resistance in neuroblastoma cells by augmenting the levels of natural product anticancer drugs in resistant cells

Antagomir-17-5p Abolishes the Growth of Therapy-Resistant Neuroblastoma through p21 and BIM

We identified a key oncogenic pathway underlying neuroblastoma progression: specifically, MYCN, expressed at elevated level, transactivates the miRNA 17-5p-92 cluster, which inhibits p21 and BIM translation by interaction with their mRNA 3′ UTRs. Overexpression of miRNA 17-5p-92 cluster in MYCN-not-amplified neuroblastoma cells strongly augments their in vitro and in vivo tumorigenesis. In vitro or in vivo treatment with antagomir-17-5p abolishes the growth of MYCN-amplified and therapy-resistant neuroblastoma through p21 and BIM upmodulation, leading to cell cycling blockade and activation of apoptosis, respectively. In primary neuroblastoma, the majority of cases show a rise of miR-17-5p level leading to p21 downmodulation, which is particularly severe in patients with MYCN amplification and poor prognosis. Altogether, our studies demonstrate for the first time that antagomir treatment can abolish tumor growth in vivo, specifically in therapy-resistant neuroblastoma