Carmen de Torres

Paediatric high grade glioma and diffuse midline glioma (including DIPG) are comprised of multiple biological and clinical subgroups, the majority of which urgently require novel therapies. Patient-derived in vitro primary cell cultures represent potentially useful tools for mechanistic and preclinical investigation based upon their retention of key features of tumour subgroups under experimental conditions amenable to high-throughput approaches. We established 21 novel primary cultures derived from patients in London, Dublin and Rome, and together with cultures shared from Barcelona, Brisbane and Stanford we assembled a panel of 42 models under 2D (laminin matrix) and/or 3D (neurospheres) conditions, fully credentialed by phenotypic and molecular comparison to the original tumour sample (methylation BeadArray, panel/exome sequencing, RNAseq). Screening against a panel of ~400 approved chemotherapeutics and small molecules, we identified specific dependencies associated with tumour subgroups and/or specific molecular markers. Examples included cells with sensitizing (HSJD-GBM-001, PDGFRA_A385ins; HSJD-DIPG-008, PDGFRA_D846N) or resistance (HSJD-GBM-002, PDGFRA_D842Y) mutations to a range of PDGFRA inhibitors, and individual models showing profound sensitivity to multiple FGFR (QCTB-R006) or EGFR (HSJD-DIPG-012) inhibitors. H3.3G34R cells were differentially sensitive to agents targeting the proteasome, whilst H3.3 K27M cells were responsive to crizotinib. MAPK-dysregulated PXA-like cultures differentially responded to inhibitors of upstream signalling via PKC and CK2. In total, 85% cells were found to have at least one drug screening hit in short term assays linked to the underlying biology of the patient’s tumour, providing a rational approach for individualised clinical translation.

We have previously reported that the calcium‐sensing receptor (CaSR) is expressed in benign, differentiated neuroblastic tumors, and epigenetically silenced in undifferentiated, malignant cases. Furthermore, cinacalcet, an allosteric activator of the CaSR, reduces neuroblastoma tumor growth in preclinical models. However, to identify patients that might benefit from this treatment, a complete understanding of mechanisms governing CaSR expression in these tumors would be required. We have now analyzed two polymorphisms in the promoter region of theCASRgene (rs7652589 and rs1501899) by allelic discrimination in neuroblastoma patients and cell lines. Association of genotypes and haplotypes withCaSRmRNA levels andCASRpromoter P2 methylation status was determined. Data presented show that minor alleles rs7652589 and rs1501899, present either in homo‐ or heterozygosis, were correlated with reducedCaSRmRNA levels in matching primary tumors and this association was independent ofCASRpromoter P2 hypermethylation. Haplotype AA was independently associated with reducedCaSRexpression after adjusting by promoter P2 methylation status. These polymorphisms were identified in some ganglioneuromas in which CaSR expression is low despite exhibiting a high degree of differentiation. Furthermore, homozygous variants rs7652589 and rs1501899 were detected in SH‐SY5Y cells, which are devoid ofCaSRexpression in the absence of hypermethylation ofCASRpromoter P2. In summary, minor alleles rs7652589 and rs1501899 are associated with reduced CaSR expression in neuroblastic tumors and neuroblastoma cell lines in which theCASRgene promoter P2 is not hypermethylated. Therefore, they potentially represent an additional mechanism ofCASRtranscriptional regulation in this group of developmental malignancies.

11002 Background: This is the first Spanish trial of Ewing sarcoma (ES) with the aim to reproduce the mP6 results and test the efficacy of the Gemcitabine/Docetaxel (G/D) regimen. Methods: Prospective, multicentric, non-randomized study of ES pts ≤40 years with tumor rearrangements of EWSR1. Centralized bone marrow screening for micrometastatic spread and radiological review was performed. All pts received 5 mP6 courses of chemotherapy; surgical resection after course 3; and radiotherapy after course 5. High-risk (HR) pts received 2 window cycles of G/D, Gemcitabine 1000 mg/m2 IV on days 1 and 8, and Docetaxel 100 mg/m2 on day 8 of a 21-day cycle. HR pts with an objective response (OR) to G/D received 12 monthly cycles of G/D after mP6. Results: 43 (22 SR, 21 HR) pts were enrolled, median age 17y (range, 3 - 40). 10/21 HR pts evaluable for G/D window therapy response showed 1 complete (CR) and 3 partial (PR) responses, 3 stable disease (SD). 30/43 pts were evaluable at the end of mP6. 24 (80%) CR; 3 (10%) PR; 1 SD (3%); and 2 (7%) progressed. 11 HR entered maintenance and 1 year later 5 remain CR. Grade 4 adverse events during mP6 occurred in 28/39 (72%) and did not correlate with age. Statistically significant differences were detected for risk groups: SR 4-y OS 74% (CI=51,100) and 4-y EFS 67% (CI=47,95); HR 4-y OS 42% (CI=25,73) p=0.011 and EFS 27% (CI=13,56) p=0.0028; and age: 18y (CI= 15,69) p18y, p= 0.0070; HR and >18y vs SR and < 18y, p 18y vs SR and <18y, p<0.001; 18y vs same risk group, p= 0.0021, superior to same age vs risk group, p=0.014. Conclusions: The G/D regimen resulted in objective responses to 70% HR-ES. Age is stronger predictive marker than risk group. Monthly G/D benefited 50% of HR-ES providing a backbone regimen for managing minimal residual disease. Clinical trial information: EudraCT 2009-016027-62.

During normal development of the nervous system (NS), neural progenitor cells (NPCs) produce specialized populations of neurons and glial cells upon cell fate restriction and terminal differentiation. These sequential processes require the dynamic regulation of thousands of genes. The calcium-sensing receptor (CaSR) is temporally and spatially regulated in both neurons and glial cells during development of the NS. In particular, CaSR expression and function have been shown to play a significant role during differentiation of NPCs towards the oligodendrocyte lineage and also in maturation of cerebellar granule cell precursors. Moreover, CaSR regulates axonal and dendritic growth in both central and peripheral nervous systems, a process necessary for proper construction of mature neuronal networks. On the other hand, several lines of evidence support a role for CaSR in promotion of cell differentiation and inhibition of proliferation in neuroblastoma, a tumor arising from precursor cells of developing peripheral nervous system. Thus, among the variety of NS functions in which the CaSR participates, this mini-review focuses on its role in differentiation of normal and tumoral cells. Current knowledge of the mechanisms responsible for CaSR regulation and function in these contexts is also discussed, together with the therapeutic opportunities provided by CaSR allosteric modulators.

Cinacalcet inhibits neuroblastoma tumor growth and upregulates cancer-testis antigens

Neuroblastic tumors include the neuroblastomas, ganglioneuroblastomas, and ganglioneuromas. Clinical behavior of these developmental malignancies varies from regression to aggressive growth with metastatic dissemination. Several clinical, histological, genetic, and biological features are associated with this diversity of clinical presentations. The calcium-sensing receptor (CaSR) is a G-protein coupled receptor with a key role in calcium homeostasis. We have previously reported that it is expressed in benign, differentiated neuroblastic tumors, but silenced by genetic and epigenetic events in unfavorable neuroblastomas. We have now analyzed three functionally relevant polymorphisms clustered at the signal transduction region of the CaSR (rs1801725, rs1042636 and rs1801726) to assess if genetic variants producing a less active receptor are associated with more aggressive disease course. Polymorphisms were analyzed in DNA samples from 65 patients using specific Taqman Genotyping Assays. Mildly inactivating variant rs1801725 was associated with clinical stage 4 (P = 0.002) and the histological subgroup of undifferentiated neuroblastomas (P = 0.046). Patients harboring this polymorphism had significantly lower overall (P = 0.022) and event-free survival (P = 0.01) rates than those who were homozygous for the most common allele among Caucasians. However, this single locus genotype was not independently associated with outcome in multivariate analyses. Conversely, the tri-locus haplotype TAC was independently associated with an increased risk of death in the entire cohort (Hazard Ratio = 2.45; 95% Confidence Interval [1.14–5.29]; P = 0.022) and also in patients diagnosed with neuroblastomas (Hazard Ratio = 2.74; 95% Confidence Interval [1.20–6.25]; P = 0.016). The TAC haplotype includes the moderately inactivating variant rs1801725 and absence of the gain-of-function rs1042636 polymorphism. Thus, its association with metastatic disease and poor outcome would add to our previous data and further support that inactivation of the CaSR gene is a mechanism associated with neuroblastoma malignant behavior.

Serotonin [5-hydroxytryptamine (5-HT)] is involved in modulating an array of complex behaviors including learning, depression, and circadian rhythms. Additionally, HTR2 receptors on layer V pyramidal neurons are thought to mediate the actions of psychedelic drugs; the native function of these receptors at this site, however, remains unknown. Previously, we found that activation of HTR2 receptors in the zebra finch forebrain song premotor structure the robust nucleus of the arcopallium (RA) led to increased excitation, and that endogenous 5-HT could roughly double spontaneous firing rate. Here, using in vivo single-unit recordings, we found that direct application of 5-HT to these same RA projection neurons, which are analogous to layer V cortical pyramidal neurons, caused a significant increase in the number of action potentials per song-related burst, and a dramatic decrease in signal-to-noise ratio. Injection of the serotonergic neurotoxin 5,7-dihydroxytryptamine into the third ventricle greatly reduced telencephalic 5-HT and resulted in decreased fundamental frequency of harmonic syllables as well as increased goodness of pitch. Both of these results can be explained by the observed actions of 5-HT on RA projection neurons, and both effects recovered to baseline within 2 weeks following the toxin injection. These results show that 5-HT is involved in modulating spectral properties of song, likely via effects on RA projection neurons, but that adult zebra finches can partially compensate for this deficit within 7 d.

Neuroblastic tumors (NTs) include the neuroblastomas, ganglioneuroblastomas and ganglioneuromas. We have reported previously that the calcium-sensing receptor is expressed in differentiated, favorable NTs but almost undetectable in unfavorable neuroblastomas. We have now detected hypermethylation of a particular region within the CpG island encompassing the CaSR gene promoter 2 in neuroblastoma cell lines and 25% primary neuroblastomas. Hypermethylation of this region was associated with reduced CaSR messenger RNA expression and several predictors of poor outcome in neuroblastomas, including MYCN amplification. Treatment with 5′aza-2-deoxycitidine and/or trichostatin A restored CaSR expression in MYCN-amplified cell lines. Following 5′aza-2-deoxycitidine exposure, decreased percentages of methylated CpG sites were observed at the above-mentioned region. By interphase fluorescence in situ hybridization, variable percentages of nuclei with monosomy of chromosome 3, where the human CaSR gene resides, were observed in more than 90% of primary NTs of all subgroups. Nuclei harboring this alteration were heterogeneously distributed among tumor cells. Ectopic overexpression of the calcium-sensing receptor in two MYCN-amplified neuroblastoma cell lines in which this gene is silenced by promoter hypermethylation significantly reduced their in vitro proliferation rates and almost abolished their capacity to generate xenografts in immunocompromised mice. Finally, upon acute exposure to calcium, the primary activator of this receptor, calcium-sensing receptor-overexpressing neuroblastoma cells underwent apoptosis, a process dependent on sustained activation of ERK1/2. These data would support the hypothesis that epigenetic silencing of the CaSR gene is neither an in vitro artefact in neuroblastoma cell lines nor an irrelevant, secondary event in primary NTs, but a significant mechanism for neuroblastoma survival.

The chromodomain, helicase DNA-binding protein 5 (CHD5) is a potential tumor suppressor gene located on chromosome 1p36, a region recurrently deleted in high risk neuroblastoma (NB). Previous data have shown that CHD5 mRNA is present in normal neural tissues and in low risk NB, nevertheless, the distribution of CHD5 protein has not been explored. The aim of this study was to investigate CHD5 protein expression as an immunohistochemical marker of outcome in NB. With this purpose, CHD5 protein expression was analyzed in normal neural tissues and neuroblastic tumors (NTs). CHD5 gene and protein expression was reexamined after induction chemotherapy in a subset of high risk tumors to identify potential changes reflecting tumor response.