Josep Sánchez de Toledo

Notch Pathway Inhibition Significantly Reduces Rhabdomyosarcoma Invasiveness and Mobility In Vitro

Purpose: Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children and can be divided into two main subtypes: embryonal and alveolar RMS. Patients with metastatic disease continue to have very poor prognosis although aggressive therapies and recurrences are common in advanced localized disease. The oncogenic potential of the Notch pathway has been established in some cancers of the adult and in some pediatric malignancies. Experimental Design: A real-time PCR assay was used to ascertain the expression of several Notch pathway components in a wide panel of RMS and cell lines. Four γ-secretase inhibitors (GSIs) were tested for pathway inhibition and the degree of inhibition was assessed by analysis of Hes1 and Hey1 expression. The putative effects of Notch pathway inhibition were evaluated by wound-healing, matrigel/transwell invasion, cell-cycle, and apoptosis assays. Results: The Notch pathway was widely expressed and activated in RMS and underwent substantial inhibition when treated with GSIs or transfected with a dominant negative form of MAML1. RMS cells showed a significant decrease in its mobility and invasiveness when the Notch pathway was properly inhibited; conversely, its inhibition had no noticeable effect on cell cycle or apoptosis. Conclusion: Pharmacological or genetic blockage of the pathway significantly reduced invasiveness of RMS cell lines, thereby suggesting a possible role of the Notch pathway in the regulation of the metastatic process in RMS. Clin Cancer Res; 17(3); 505–13. ©2010 AACR.

Notch, Wnt, and Hedgehog Pathways in Rhabdomyosarcoma: From Single Pathways to an Integrated Network

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. Regarding histopathological criteria, RMS can be divided into 2 main subtypes: embryonal and alveolar. These subtypes differ considerably in their clinical phenotype and molecular features. Abnormal regulation or mutation of signalling pathways that regulate normal embryonic development such as Notch, Hedgehog, and Wnt is a recurrent feature in tumorigenesis. Herein, the general features of each of the three pathways, their implication in cancer and particularly in RMS are reviewed. Finally, the cross-talking among these three pathways and the possibility of better understanding of the horizontal communication among them, leading to the development of more potent therapeutic approaches, are discussed.

Detection of bone marrow micrometastasis and microcirculating disease in rhabdomyosarcoma by a real-time RT-PCR assay

Purpose: To assess if molecular detection of minimal disseminated disease by real-time reverse transcription and polymerase chain reaction (RT-PCR) could contribute to a better treatment stratification in patients with rhabdomyosarcoma (RMS). Methods: Relative quantification of the tumor-mRNA present in serial samples of bone marrow (BM) and peripheral blood (PB) from 16 patients with RMS (7 alveolar and 9 embryonal) was performed by a real-time RT-PCR assay. Expression of MyoD1 and acetylcholine receptor (AChR) was analyzed in all samples, along with PAX3/7-FKHR in samples from alveolar tumors. Results: A good correlation was found between the expression of PAX3/7-FKHR and AChR, while MyoD1 was more sensitive but less specific. In this study, patients with positive PB at the end of treatment showed a poorer prognosis than patients with negative PB. Moreover, in this patient cohort, metastatic relapses were preceded by the detection of microcirculating disease in all cases. Conclusion: The detection of minimal circulating and micrometastatic disease by real-time RT-PCR, based on the expression of multiple genes, yields highly reproducible results. Patients with positive PB after treatment show poorer survival than patients without microcirculating disease.

PET/CT in paediatrics: it is time to increase its use!

F-FDG PET and PET/CT have proved to beuseful tools in oncological patients [1]. For example, it hasbeen reported that PET can modify the initial staging inabout 40% of patients with lymphoma, leading tomodification of therapy in 18% of them [2]. Similarresults in terms of clinical management, i.e. changes instaging and treatment, have been described in large seriesof patients with other malignancies. PET and PET/CThave also been shown to exert a high impact ononcological management in the paediatric population [3–5]. However, the low incidence of the paediatric malig-nancies has made it difficult to elucidate the precise role ofthese examinations and to attain evidence-based indica-tions for them. Nevertheless, more and more papers havebeen published demonstrating that FDG PET/CT is alsouseful in children. Results obtained in paediatric lympho-ma patients have shown high rates (up to 23%) of therapychanges owing to FDG PET results [6], and several recentpapers have described the usefulness of this technique inmost other paediatric malignancies, such as neuroblastomaand sarcomas.What is the situation regarding correlative image? Inboth children and adults it is clear that imaging diagnosis ismore accurate when anatomical and metabolic images areinterpreted together. Hybrid equipment and fusion imaging(and PET/CT is probably the reference) provide betterinformation than is acquired by separate anatomical andmetabolic examinations or even by comparison of them.This fact is encouraging imaging specialists (in CT, MRI,nuclear medicine, etc.) to collaborate more closely, therebyincreasing communication, improving reports and increas-ing the clinical impact of imaging modalities [1, 2].Several papers, even in paediatric oncology, havereported that fused PET/CT improves diagnostic accuracycompared with PET alone or side by side comparison ofPET and CT images [6, 7]. An excellent example of thisbenefit is given in a recently published paper from theGerman Society of Paediatric Oncology and Hematologyon Hodgkin’s disease. This prospective, multicentre studycompares the accuracy of conventional imaging methodsand FDG PET in the initial staging of paediatric patientsdiagnosed with Hodgkin’s lymphoma. Twenty-one nodaland six extranodal regions were evaluated by conventionalimaging methods and FDG PET independently, and laterthe images were also compared side by side and fused.Results were statistically superior when anatomical regionswere evaluated using both methods in combination. Nodifferences were observed according to the way in whichthe methods were combined, i.e. side by side viewing orimage fusion. However, the confidence of analysts in theresults was clearly superior when the fusion method wasused [8].In this issue, Zvi Bar-Sever et al. describe the results of aprospective study on 46 consecutive paediatric patients whounderwent

Improving Survival of Medulloblastoma: Results in Two Groups of Patients

This retrospective study compares the results obtained in 56 children with medulloblastoma treated with two different protocols at our center between 1975 and 1990. Since 1985 we have been enrolled in the medulloblastoma SIOP II protocol in which we have entered 27 patients. These patients from the SIOP group (SG) and the 29 children treated before 1985, the historical group (HG). When the two groups were compared for age and sex distribution, no differences were found; however, prognostic factors were worse in the HG than in SG: 21 versus 15 T3-T4 and 13 versus 8 high-risk, respectively. The relapse rate was 63% for the HG and 39% for the SG. Five-year disease-free survival was 31% for the HG and 56% for the SG (p = 0.037). Five-year survival was 52 and 70%, respectively (p = 0.055). When SG and HG were compared by stratifying for tumor size, surgical resection and the risk variable, better disease-free survival was obtained in SG than in HG. In a multivariate analysis, the HG and the high-risk proved to be variable independent predictors of poor survival. We observe an increase in survival in our patients with medulloblastoma.

Characterisation of novel splicing variants of the tyrosine hydroxylase C-terminal domain in human neuroblastic tumours.

Abstract Alternative splicing of human tyrosine hydroxylase (hTH) transcripts appears to occur mainly in the N-terminal domain, giving rise to at least eight different isoforms. We recently reported the existence of hTH transcript variants resulting from splicing of exons 8 and 9, within a region previously thought to be constant. The mRNA distribution of these novel hTH isoforms in neuroblastic tumours and in foetal adrenal glands was analysed by conventional and real-time RT-PCR. The presence of the target protein was determined by Western blotting, immunoprecipitation and protein analysis. Transcripts lacking exons 8 and 9 were widely distributed in the tissues analysed. Characterisation of full-length mRNA revealed that splicing of exons 8 and 9 was always associated with splicing of exons 2 (hTH-Δ2,8,9) or 1b and 2 (hTH-Δ1b,2,8,9). In addition, one variant detected on Western blots in several tumours fits the predicted size (58 kDa) of the isoforms lacking exons 8 and 9. In conclusion, the two novel isoforms reported here (hTH-Δ2,8,9 and hTH-Δ1b,2,8,9) represent the first full-length isoforms with alternative splicing in the hTH C-terminal domain. These results demonstrate for the first time the existence of hTH isoforms Δ2,8,9 and Δ1b,2,8,9. Their general distribution in neuroblastoma and adrenal glands and translation into protein suggest a significant functional role for these novel hTH isoforms, which merit further study.

Nuevas estrategias terapéuticas para el neuroblastoma basadas en el uso de microRNAs

Neuroblastoma (NB) is the most common solid tumour in children and adolescents, and accounts for up to 15% of all cancer deaths in this group. It originates in the sympathetic nervous system, and its behaviour can be very aggressive and become resistant to current treatments. A review is presented, summarising the new alternative therapies based on epigenetics, i.e., modulators of gene expression, such as microRNAs and their potential application in the clinical practice of NB treatment.

Optimization of rhabdomyosarcoma disseminated disease assessment by flow cytometry

Rhabdomyosarcoma (RMS) is the most common type of soft tissue sarcoma in children. Circulating tumor cells in peripheral blood or disseminated to bone marrow, a concept commonly referred to as minimal residual disease (MRD), are thought to be key to the prediction of metastasis and treatment efficacy. To date, two MRD markers, MYOD and MYOGENIN, have been tested; however, MRD detection continues to be challenging mainly owing to the closeness of the detection limit and the discordance of both markers in some samples. Therefore, the addition of a third marker could be useful for more accurate MRD assessment. The PAX3 gene is expressed during embryo development in all myogenic precursor cells in the dermomyotome. As RMS cells are thought to originate from these muscle precursor cells, they are expected to be positive for PAX3. In this study, PAX3 expression was characterized in cancer cell lines and tumors, showing wide expression in RMS. Detection sensitivities by quantitative polymerase chain reaction (qPCR) of the previously proposed markers, MYOD and MYOGENIN, were similar to that of PAX3, thereby indicating the feasibility of its detection. Interestingly, the flow cytometry experiments supported the usefulness of this technique in the quantification of MRD in RMS using PAX3 as a marker. These results indicate that flow cytometry, albeit in some cases slightly less sensitive, can be considered a good approach for MRD assessment in RMS and more consistent than qPCR, especially owing to its greater specificity. Furthermore, fluorescence‐activated cell sorting permits the recovery of cells, thereby providing material for further characterization of circulating or disseminated cancer cells.

Abstract A27: Ligand-dependent Hedgehog pathway activation in rhabdomyosarcoma

The Hedgehog (HH) pathway is known to develop an oncogenic role in some cancers, including rhabdomyosarcoma (RMS). However, the molecular mechanism that drives activation of the pathway in RMS is not well understood. Currently, a consistent activation of the pathway is well established and generally accepted in RMS. However, despite controversy regarding the importance of mutation-driven constitutive activation of the pathway in RMS patients, the activator mutations reported to date can only account---in the best scenario---for the activation of the pathway in small subsets of patients; therefore, the mechanism of activation remains elusive for the majority of cases. Purposes of the Study: The main purpose of this study is to demonstrate the importance of the ligand-dependent activation of Hedgehog pathway in this neoplasia. A secondary purpose is the description and characterization of a deleterious effect of the SMO inhibitor vismodegib, which promotes cell invasiveness throughout activation of Unfolded Protein Response (UPR). Experimental Procedures: The expression of Hedgehog ligands was studied in RMS samples by quantitative PCR, Western blot, and immunohistochemistry. Functional assays (cell cycle, cell proliferation, cell invasion, and apoptosis) and animal model studies were carried out with cells transduced with shRNAs (against the 3 Hedgehog ligands and Gli1) or treated with Hedgehog-specific inhibitors (vismodegib and MEDI-5304). Finally, the molecular characterization of an off-target effect of vismodegib was also made by cDNA microarrays, Transwell assay, Western blot, and shRNA-mediated TRIB3 inhibition. Summary of New Data: Results showed a prominent expression of HH ligands (especially Indian and Desert Hedgehog) in RMS cells and tumors, thereby supporting the existence of autocrine ligand-dependent activation of the pathway in this neoplasia. Moreover, although the shRNA-mediated downregulation of the ligands was not complete and performed separately for each ligand, the effects observed on tumor growth were remarkable. Thus, the downregulation of each single HH ligand clearly impaired tumor growth, suggesting that the participation of all ligands is crucial for RMS pathogenesis. Interestingly, a first description of pernicious off-target effect of vismodegib is also reported. Unexpectedly, vismodegib enhanced the invasive properties of all 3 cell lines by 2-fold. The mechanistic characterization of this biologic response suggested an off-target effect of vismodegib on cell invasiveness and chemoresistance via UPR activation. Given that the cell lines studied covered all the main subtypes of RMS, the results strongly suggest that UPR activation may be a general mechanism of cell invasiveness in RMS under vismodegib treatment and not a negligible peculiarity of a single cell line. Conclusions: The clarification of the HH pathway activation mechanism in RMS opens a door for targeted therapies against HH ligands as a possible alternative in the future development of better treatment protocols for RMS and other ligand-dependent cancers. Moreover, the description of a pernicious off-target effect of vismodegib may mechanistically explain its previously reported inefficiency in several ligand-dependent cancers or even the premature halting of some of them owing to survival worsening. Therefore, the findings herein described may have significant translational implication not only for RMS but also for other HH ligand-dependent cancers. Citation Format: Josep Roma, Ana Almazan-Moga, Patricia Zarzosa, Pablo Velasco, Carla Molist, Irina Giralt, Isaac Vidal, Natalia Navarro, Miguel Segura, Aroa Soriano, Josep Sanchez de Toledo, Soledad Gallego. Ligand-dependent Hedgehog pathway activation in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A27.

Targeting of epigenetic regulators in neuroblastoma

Approximately 15,000 new cases of pediatric cancer are diagnosed yearly in Europe, with 8–10% corresponding to neuroblastoma, a rare disease with an incidence of 8–9 cases per million children <15 years of age. Although the survival rate for low-risk and intermediate-risk patients is excellent, half of children with high-risk, refractory, or relapsed tumors will be cured, and two-thirds of the other half will suffer major side effects and life-long disabilities. Epigenetic therapies aimed at reversing the oncogenic alterations in chromatin structure and function are an emerging alternative against aggressive tumors that are or will become resistant to conventional treatments. This approach proposes targeting epigenetic regulators, which are proteins that are involved in the creation, detection, and interpretation of epigenetic signals, such as methylation or histone post-translational modifications. In this review, we focused on the most promising epigenetic regulators for targeting and current drugs that have already reached clinical trials.Cancer therapy: Exploring an epigenetic approachTreatments that target chromatin, the combination of DNA and proteins, are emerging as alternative ways to treat aggressive neuroblastomas, cancers of neural tissue. Altering the structure and function of chromatin is a form of “epigenetic therapy”, treatment that affects inheritable molecular signals controlling the activity of genes, rather than targeting the genes directly. Researchers in Spain led by Miguel Segura at the Vall d’Hebron Research Institute in Barcelona review progress in developing epigenetic therapies for neuroblastomas. A growing body of fundamental research and evidence from clinical trials suggest this approach could open promising new avenues to treating aggressive and drug-resistant cancers. The authors recommend an increased effort to identify and explore the activities of small molecules that could form the basis of effective epigenetic therapies for various cancers.