The majority of neuroblastoma patients have tumors that initially respond to chemotherapy, but a large proportion of patients will experience therapy-resistant relapses. The molecular basis of this aggressive phenotype is unknown. Whole genome sequencing of 23 paired diagnostic and relapsed neuroblastomas showed clonal evolution from the diagnostic tumor with a median of 29 somatic mutations unique to the relapse sample. Eighteen of the 23 relapse tumors (78%) showed mutations predicted to activate the RAS-MAPK signaling pathway. Seven events were detected only in the relapse tumor while the others showed clonal enrichment. In neuroblastoma cell lines we also detected a high frequency of activating mutations in the RAS-MAPK pathway (11/18, 61%) and these lesions predicted for sensitivity to MEK inhibition in vitro and in vivo. Our findings provide the rationale for genetic characterization of relapse neuroblastoma and show that RAS-MAPK pathway mutations may function as a biomarker for new therapeutic approaches to refractory disease.

Mutations affecting the RAS-MAPK pathway frequently occur in relapsed neuroblastoma tumors, which suggests that activation of this pathway is associated with a more aggressive phenotype. To explore this hypothesis, we generated several model systems to define a neuroblastoma RAS-MAPK pathway signature. Activation of this pathway in primary tumors indeed correlated with poor survival and was associated with known activating mutations in ALK and other RAS-MAPK pathway genes. Integrative analysis showed that mutations in PHOX2B, CIC, and DMD were also associated with an activated RAS-MAPK pathway. Mutation of PHOX2B and deletion of CIC in neuroblastoma cell lines induced activation of the RAS-MAPK pathway. This activation was independent of phosphorylated ERK in CIC knockout systems. Furthermore, deletion of CIC caused a significant increase in tumor growth These results show that the RAS-MAPK pathway is involved in tumor progression and establish CIC as a powerful tumor suppressor that functions downstream of this pathway in neuroblastoma. This work identifies CIC as a powerful tumor suppressor affecting the RAS-MAPK pathway in neuroblastoma and reinforces the importance of mutation-driven activation of this pathway in cancer. .

Background: Children with cancer are in urgent need of new therapies, as approximately 25% of patients experience a relapse and 20% succumb to their disease. Moreover, the majority of survivors suffer from clinically relevant health problems. Repurposing of targeted agents developed for adult indications could provide novel therapeutic options for paediatric cancer patients. To prioritise targeted drugs for paediatric clinical development, we applied a systematic review methodology to develop a Target Actionability Review (TAR) strategy. These TARs assess the strength and completeness of published preclinical proof-of-concept (PoC) data by structured critical appraisal of and summarising the available scientific literature for a specific target (pathway) and the associated drugs in paediatric tumours. Methods: A sensitive literature search in PubMed was performed and relevant papers were identified. For each paper, the individual experimental findings were extracted, marked for

Large-scale chromosomal deletions are a prevalent and defining feature of cancer. A high degree of tumor-type and subtype specific recurrencies suggest a selective oncogenic advantage. However, due to their large size it has been difficult to pinpoint the oncogenic drivers that confer this advantage. Suitable functional genomics approaches to study the oncogenic driving capacity of large-scale deletions are limited. Here, we present an effective technique to engineer large-scale deletions by CRISPR-Cas9 and create isogenic cell line models. We simultaneously induce double-strand breaks (DSBs) at two ends of a chromosomal arm and select the cells that have lost the intermittent region. Using this technique, we induced large-scale deletions on chromosome 11q (65 Mb) and chromosome 6q (53 Mb) in neuroblastoma cell lines. A high frequency of successful deletions (up to 30% of selected clones) and increased colony forming capacity in the 11q deleted lines suggest an oncogenic advantage of these deletions. Such isogenic models enable further research on the role of large-scale deletions in tumor development and growth, and their possible therapeutic potential.

Germ cell tumors (GCTs) are considered to be highly curable; however, there are major differences in the outcomes related to histology and anatomical localization. GCTs originating from the testis are, overall, sensitive to platinum-based chemotherapy, whereas GCTs originating from the mediastinum show a worse response, which remains largely unexplained. Here, we address the differences among GCTs from two different anatomical locations (testicular versus mediastinal/extragonadal), with a specific focus on the role of the P53 pathway. It was recently shown that GCTs with TP53 mutations most often localize to the mediastinum. To elucidate the underlying mechanism, TP53 knock-out lines were generated in cisplatin-sensitive and -resistant clones of the representative 2102Ep cell line (wild-type TP53 testicular GCT) and NCCIT cell line (hemizygously mutated TP53, mutant TP53 mediastinal GCT). The full knock-out of TP53 in 2102Ep and resistant NCCIT resulted in an increase in cisplatin resistance, suggesting a contributing role for P53, even in NCCIT, in which P53 had been reported to be non-functional. In conclusion, these results suggest that TP53 mutations contribute to the cisplatin-resistant phenotype of mediastinal GCTs and, therefore, are a potential candidate for targeted treatment. This knowledge provides a novel model system to elucidate the underlying mechanism of clinical behavior and possible alternative treatment of the TP53 mutant and mediastinal GCTs.

PURPOSE Cisplatin is the main systemic treatment modality for male type II germ cell tumors (GCTs). Although generally very effective, 5%-10% of patients suffer from cisplatin-resistant disease. Identification of the driving mechanisms of resistance will enable improved risk stratification and development of alternative treatments. METHODS We developed and characterized cisplatin-resistant GCT cell line models and compared their molecular characteristics with patient samples with cisplatin resistance and/or a poor clinical outcome. Subsequently, the association between the overlapping genetic features and clinical data was assessed. Finally, we used Cox regression to determine the prognostic relevance of these features within the currently used risk classification. RESULTS Gain of chromosome 3p25.3 was detected in all cisplatin-resistant cell lines, and copy number of this region correlated with the level of resistance (R = 0.96, P = 1.5e-04). Gain of this region was detected at low frequencies in primary tumors and at higher frequencies in relapsed and/or cisplatin-resistant tumors. Chromosome 3p25.3 gain was associated with shorter progression-free survival and overall survival, with the strongest association observed in nonseminomas excluding pure teratomas. 3p25.3 gain was more frequently observed in tumors with yolk sac tumor histology and predicted adverse outcome independent of the International Germ Cell Cancer Collaborative Group risk classification and the presence of TP53/ MDM2 alterations. CONCLUSION On the basis of both in vitro analyses and clinical data, we found 3p25.3 to be strongly associated with cisplatin resistance and poor clinical outcome in male type II GCTs. Using genomic profiling, 3p25.3 status could help to improve risk stratification in male patients with type II GCT. Further characterization of this locus and underlying mechanisms of resistance is warranted to guide development of novel treatment approaches for cisplatin-resistant disease.

We recently identified that mutations affecting the RAS-MAPK pathway occur frequently in relapsed neuroblastoma, which suggests that activation of this pathway may play an important role in the aggressive phenotype of these tumors. Furthermore, it was shown that the presence of these mutations may serve as a biomarker for treatment with MEK inhibitors both in vitro and in vivo. These results suggest that MEK inhibitors may be a viable treatment option for aggressive neuroblastoma tumors and warrant further investigation into the role of the RAS-MAPK pathway in neuroblastoma. To identify if, and to what extent, the RAS-MAPK pathway is also activated in primary neuroblastoma tumors we have developed a RAS-MAPK mRNA signature. Genes were included that were I) regulated by mutant ALK, which is a known activator of the RAS-MAPK pathway in neuroblastoma, and II) were conversely regulated by treatment with the MEK inhibitor U0126. We ended up with a core signature consisting of 8 genes, which was tested in a panel of 17 neuroblastoma cell lines. The expression of our gene signature in this panel correlates with mutations in the RAS-MAPK pathway, with the expression of phospho-MEK and phospho-ERK on Western Blot and with sensitivity to the MEK inhibitor Trametinib. Clustering analyses in our set of 122 primary neuroblastoma tumors shows that high expression of this signature is associated with significantly poorer survival and similar analyses in other publicly available datasets corroborate these results, even if applied to only stage 4 tumors . In our data set all tumors containing mutations in known RAS-MAPK pathway genes, such as ALK and NF1, show high signature scores, which makes it likely that this signature can indeed detect the activation state of the pathway through mRNA profiling. However, there are also tumors with high signature scores that do not show mutations that are known to be associated with RAS-MAPK signaling (as detectable by Whole Genome Sequencing). Therefore we looked in these tumors if there were other mutations that could explain activation of this pathway. We detected mutations in known RAS-MAPK genes, such as SOS1, but also recurrent aberrations in genes not previously known to be related to RAS-MAPK signaling, such as PHOX2B, DMD and ERF. We are currently characterizing the effect of these mutations on the RAS-MAPK signaling pathway in neuroblastoma cell lines. Our findings show that activation of the RAS-MAPK pathway, as determined by our mRNA gene signature, is associated with poor prognosis in neuroblastoma. In some tumors this activation is caused by known activating mutations in this pathway, but in most cases the causative events remain unknown. We present several other candidate genes, which when mutated may also lead to pathway activation. MEK inhibitors were already shown to be effective against neuroblastoma xenografts with RAS or NF1 mutations, and our results indicate that a broader selection of neuroblastoma tumors might respond to these compounds. Citation Format: T.F. Eleveld, L Schild, M.E. Ebus, P.G. van Sluis, E.M. Westerhout, H.N. Caron, J.J.B. Koster, R. Versteeg, J.J. Molenaar. Activation of the RAS-MAPK pathway in primary neuroblastoma tumors is associated with poor prognosis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A31.

Background and case: An adolescent male presented with a second mediastinal tumor 1.5 years after treatment of a proven malignant germ-cell tumor in that location. The differential diagnosis included a recurrent germ-cell tumor or a non-germ cell malignancy. Serum tumor markers alpha-fetoprotein (AFP) and human chorionic gonadotrophin (HCG) were negative. The first biopsy was not informative, and the second biopsy gave a broad differential diagnosis including secondary non-germ cell malignancy using histology and immunohistochemistry. DNA methylation profiling, RNA sequencing, and targeted microRNA371a-3p profiling was subsequently performed, without a supportive result. After resection of the tumor the definitive diagnosis yielded two secondary non-germ cell malignancies in the form of a leiomyosarcoma and a solitary neuro endocrine carcinoma (NEC). In spite of the differences between the molecular profiles of the initial germ-cell tumor, the leiomyosarcoma and large-cell NEC are clonally related, as determined by the presence of identical chromosomal breakpoints. The copy number profiles suggest an initial polyploidization step, followed by various independent chromosomal gains and losses. This case demonstrates that germ-cell tumors must be evaluated carefully, including molecularly, in which the non-germ cell malignancy is negative for miR-371a-3p, both in tissue as well as in serum, in contrast to the primary tumor. We conclude that the patient presented with a primary type II mediastinal GCT and, a year and a half later, followed by a leiomyosarcoma and a large-cell NEC presenting as two secondary somatic-type malignancies clonally related to the original GCT. Conclusions: Malignant germ-cell tumors are known to recur as a somatic-type malignancy in very rare cases. This case report illustrates the challenges faced in defining the nature and clonality of the secondary somatic-type malignancies.