Combination Therapies Targeting ALK-aberrant Neuroblastoma in Preclinical Models

PURPOSE ALK-activating mutations are identified in approximately 10% of newly diagnosed neuroblastomas and ALK amplifications in a further 1%-2% of cases. Lorlatinib, a third-generation anaplastic lymphoma kinase (ALK) inhibitor, will soon be given alongside induction chemotherapy for children with ALK-aberrant neuroblastoma. However, resistance to single-agent treatment has been reported and therapies that improve the response duration are urgently required. We studied the preclinical combination of lorlatinib with chemotherapy, or with the MDM2 inhibitor, idasanutlin, as recent data have suggested that ALK inhibitor resistance can be overcome through activation of the p53-MDM2 pathway. EXPERIMENTAL DESIGN We compared different ALK inhibitors in preclinical models prior to evaluating lorlatinib in combination with chemotherapy or idasanutlin. We developed a triple chemotherapy (CAV: cyclophosphamide, doxorubicin, and vincristine) in vivo dosing schedule and applied this to both neuroblastoma genetically engineered mouse models (GEMM) and patient-derived xenografts (PDX). RESULTS Lorlatinib in combination with chemotherapy was synergistic in immunocompetent neuroblastoma GEMM. Significant growth inhibition in response to lorlatinib was only observed in the ALK-amplified PDX model with high ALK expression. In this PDX, lorlatinib combined with idasanutlin resulted in complete tumor regression and significantly delayed tumor regrowth. CONCLUSIONS In our preclinical neuroblastoma models, high ALK expression was associated with lorlatinib response alone or in combination with either chemotherapy or idasanutlin. The synergy between MDM2 and ALK inhibition warrants further evaluation of this combination as a potential clinical approach for children with neuroblastoma

Cell-Free DNA as a Diagnostic and Prognostic Biomarker in Pediatric Rhabdomyosarcoma

PURPOSE: Total cell-free DNA (cfDNA) and tumor-derived cfDNA (ctDNA) can be used to study tumor-derived genetic aberrations. We analyzed the diagnostic and prognostic potential of cfDNA and ctDNA, obtained from pediatric patients with rhabdomyosarcoma. METHODS: cfDNA was isolated from diagnostic plasma samples from 57 patients enrolled in the EpSSG RMS2005 study. To study the diagnostic potential, shallow whole genome sequencing (shWGS) and cell-free reduced representation bisulphite sequencing (cfRRBS) were performed in a subset of samples and all samples were tested using droplet digital polymerase chain reaction to detect methylated RASSF1A ( RASSF1A-M). Correlation with outcome was studied by combining cfDNA RASSF1A-M detection with analysis of our rhabdomyosarcoma-specific RNA panel in paired cellular blood and bone marrow fractions and survival analysis in 56 patients. RESULTS: At diagnosis, ctDNA was detected in 16 of 30 and 24 of 26 patients using shallow whole genome sequencing and cfRRBS, respectively. Furthermore, 21 of 25 samples were correctly classified as embryonal by cfRRBS. RASSF1A-M was detected in 21 of 57 patients. The presence of RASSF1A-M was significantly correlated with poor outcome (the 5-year event-free survival [EFS] rate was 46.2% for 21 RASSF1A-M ‒positive patients, compared with 84.9% for 36 RASSF1A-M ‒negative patients [ P < .001]). RASSF1A-M positivity had the highest prognostic effect among patients with metastatic disease. Patients both negative for RASSF1A-M and the rhabdomyosarcoma-specific RNA panel (28 of 56 patients) had excellent outcome (5-year EFS 92.9%), while double-positive patients (11/56) had poor outcome (5-year EFS 13.6%, P < .001). CONCLUSION: Analyzing ctDNA at diagnosis using various techniques is feasible in pediatric rhabdomyosarcoma and has potential for clinical use. Measuring RASSF1A-M in plasma at initial diagnosis correlated significantly with outcome, particularly when combined with paired analysis of blood and bone marrow using a rhabdomyosarcoma-specific RNA panel

A SPRY1 domain cardiac ryanodine receptor variant associated with short-coupled torsade de pointes

International audienceIdiopathic ventricular fibrillation (IVF) causes sudden death in young adult patients without structural or ischemic heart disease. Most IVF cases are sporadic and some patients present with short-coupled torsade de pointes, the genetics of which are poorly understood. A man who had a first syncope at the age of 35 presented with frequent short-coupled premature ventricular beats with bursts of polymorphic ventricular tachycardia and then died suddenly. By exome sequencing, we identified three rare variants: p.I784F in the SPRY1 of the ryanodine receptor 2 (RyR2), p.A96S in connexin 40 (Cx40), reported to affect electrical coupling and cardiac conduction, and a nonsense p.R244X in the cardiac-specific troponin I-interacting kinase (TNNI3K). We assessed intracellular Ca2+ handling in WT and mutant human RYR2 transfected HEK293 cells by fluorescent microscopy and an enhanced store overload-induced Ca2+ release in response to cytosolic Ca2+ was observed in RyR2-I784F cells. In addition, crystal structures and thermal melting temperatures revealed a conformational change in the I784F-SPRY1 domain compared to the WT-domain. The novel RyR2-I784F variant in SPRY1 domain causes a leaky channel under non-stress conditions. The presence of several variants affecting Ca2+ handling and cardiac conduction suggests a possible oligogenic origin for the ectopies originating from Purkinje fibres

The European MAPPYACTS Trial: Precision Medicine Program in Pediatric and Adolescent Patients with Recurrent Malignancies

International audienceMAPPYACTS (NCT02613962) is an international prospective precision medicine trial aiming to define tumor molecular profiles in pediatric patients with recurrent/refractory malignancies in order to suggest the most adapted salvage treatment. From February 2016 to July 2020, 787 patients were included in France, Italy, Ireland, and Spain. At least one genetic alteration leading to a targeted treatment suggestion was identified in 436 patients (69%) with successful sequencing; 10% of these alterations were considered ``ready for routine use.'' Of 356 patients with follow-up beyond 12 months, 107 (30%) received one or more matched targeted therapies-56% of them within early clinical trials-mainly in the AcSe-ESMART platform trial (NCT02813135). Overall, matched treatment resulted in a 17% objective response rate, and of those patients with ready for routine use alterations, it was 38%. In patients with extracerebral tumors, 76% of actionable alterations detected in tumor tissue were also identified in circulating cell-free DNA (cfDNA)

The feasibility of using liquid biopsies as a complementary assay for copy number aberration profiling in routinely collected paediatric cancer patient samples

Background: Paediatric tumours are often characterised by the presence of recurrent DNA copy number alterations (CNAs). These DNA copy number profiles, obtained from a tissue biopsy, can aid in the correct prognostic classification and therapeutic stratification of several paediatric cancer entities (e.g. MYCN amplification in neuroblastoma) and are part of the routine diagnostic practice. Liquid biopsies (LQBs) offer a potentially safer alternative for such invasive tumour tissue biopsies and can provide deeper insight into tumour heterogeneity. Procedure: The robustness and reliability of LQB CNA analyses was evaluated. We performed retrospective CNA profiling using shallow whole-genome sequencing (sWGS) on paired plasma circulating cell-free DNA (cfDNA) and tissue DNA samples from routinely collected samples from paediatric patients (n = 128) representing different tumour entities, including osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, Wilms tumour, brain tumours and neuroblastoma. Results: Overall, we observed a good concordance between CNAs in tissue DNA and cfDNA. The main cause of CNA discordance was found to be low cfDNA sample quality (i.e. the ratio of cfDNA (700 bp)). Furthermore, CNAs were observed that were present in cfDNA and not in tissue DNA, or vice-versa. In neuroblastoma samples, no false-positives or false-negatives were identified for the detection of the prognostic marker MYCN amplification. Conclusion: In future prospective studies, CNA analysis on LQBs that are of sufficient quality can serve as a complementary assay for CNA analysis on tissue biopsies, as either cfDNA or tissue DNA can contain CNAs that cannot be identified in the other biomaterial

Molecular Characterization of Circulating Tumor DNA in Pediatric Rhabdomyosarcoma: A Feasibility Study

Rhabdomyosarcomas (RMS) are rare neoplasms affecting children and young adults. Efforts to improve patient survival have been undermined by a lack of suitable disease markers. Plasma circulating tumor DNA (ctDNA) has shown promise as a potential minimally invasive biomarker and monitoring tool in other cancers; however, it remains underexplored in RMS. We aimed to determine the feasibility of identifying and quantifying ctDNA in plasma as a marker of disease burden and/or treatment response using blood samples from RMS mouse models and patients