Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish.

Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc-/-, il2rga-/- zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.

PROX1 transcription factor controls rhabdomyosarcoma growth, stemness, myogenic properties and therapeutic targets.

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft-tissue cancer with features of skeletal muscle. Because of poor survival of RMS patients and severe long-term side effects of RMS therapies, alternative RMS therapies are urgently needed. Here we show that the prospero-related homeobox 1 (PROX1) transcription factor is highly expressed in RMS tumors regardless of their cell type of origin. We demonstrate that PROX1 is needed for RMS cell clonogenicity, growth and tumor formation. PROX1 gene silencing repressed several myogenic and tumorigenic transcripts and transformed the RD cell transcriptome to resemble that of benign mesenchymal stem cells. Importantly, we found that fibroblast growth factor receptors (FGFR) mediated the growth effects of PROX1 in RMS. Because of receptor cross-compensation, paralog-specific FGFR inhibition did not mimic the effects of PROX1 silencing, whereas a pan-FGFR inhibitor ablated RMS cell proliferation and induced apoptosis. Our findings uncover the critical role of PROX1 in RMS and offer insights into the mechanisms that regulate RMS development and growth. As FGFR inhibitors have already been tested in clinical phase I/II trials in other cancer types, our findings provide an alternative option for RMS treatment.

Diagnostic and Therapeutic Implications of a FUS::TFCP2 Fusion and ALK Activation in a Metastatic Rhabdomyosarcoma.

The identification of gene fusions in rare sarcoma subtypes can have diagnostic, prognostic, and therapeutic impacts for advanced cancer patients. Here, we present a case of a 31-year-old male with a lytic lesion of the left mandible initially diagnosed as an osteosarcoma but found to have a TFCP2 fusion and ALK alteration, redefining the diagnosis and providing rationale for a novel treatment strategy. Histologically, the tumor displayed hypercellular, spindled to epithelioid neoplasm and nuclear pleomorphism, while immunohistochemistry showed diffuse SATB2 and focal desmin staining. Whole genome and transcriptome analysis revealed a FUS::TFCP2 fusion, the defining alteration of a rare molecularly characterized subtype of soft tissue sarcoma termed intraosseous rhabdomyosarcoma. An internal ALK deletion and extremely high ALK RNA expression were also identified, suggesting potential benefit of an ALK inhibitor. This patient displayed a rapid and dramatic clinical and radiographic response to an ALK inhibitor, alectinib. Unfortunately, the response was short-lived, likely due to the advanced stage and aggressiveness of the disease. This report describes genome and transcriptome characterization of an intraosseous rhabdomyosarcoma, few of which exist in the literature, as well as providing evidence that inhibition of ALK may be a rational treatment strategy for patients with this exceedingly rare soft tissue sarcoma subtype characterized by TFCP2 fusions and ALK activation.

Addition of temsirolimus to chemotherapy in children, adolescents, and young adults with intermediate-risk rhabdomyosarcoma (ARST1431): a randomised, open-label, phase 3 trial from the Children's Oncology Group.

BACKGROUND: The Children's Oncology Group defines intermediate-risk rhabdomyosarcoma as unresected FOXO1 fusion-negative disease arising at an unfavourable site or non-metastatic FOXO1 fusion-positive disease. Temsirolimus in combination with chemotherapy has shown promising activity in patients with relapsed or refractory rhabdomyosarcoma. We aimed to compare event-free survival in patients with intermediate-risk rhabdomyosarcoma treated with vincristine, actinomycin, and cyclophosphamide alternating with vincristine and irinotecan (VAC/VI) combined with temsirolimus followed by maintenance therapy versus VAC/VI alone with maintenance therapy. METHODS: ARST1431 was a randomised, open-label, phase 3 trial conducted across 210 institutions in Australia, Canada, New Zealand, and the USA. Eligible patients were those aged 40 years or younger with non-metastatic FOXO1-positive rhabdomyosarcoma or unresected FOXO1-negative rhabdomyosarcoma disease from unfavourable sites. Two other groups of patients were also eligible: those who had FOXO1-negative disease at a favourable site (excluding orbit) that was unresected; and those who were aged younger than 10 years with stage IV FOXO1-negative disease with distant metastases. Eligible patients had to have a Lansky performance status score of 50 or higher if 16 years or younger and a Karnofsky performance status score of 50 or higher if older than 16 years; all patients were previously untreated. Patients were randomised (1:1) in blocks of four and stratified by histology, stage, and group. Patients received intravenous VAC/VI chemotherapy with a cyclophosphamide dose of 1·2 g/m2 per dose per cycle with or without a reducing dose of intravenous weekly temsirolimus starting at 15 mg/m2 or 0·5 mg/kg per dose for those who weighed less than 10 kg. The total duration of therapy was 42 weeks followed by 6 months of maintenance therapy with oral cyclophosphamide plus intravenous vinorelbine for all patients. Temsirolimus was withheld during radiotherapy and for 2 weeks before any major surgical procedure. The primary endpoint was 3-year event-free survival. Data were analysed with a revised intention-to-treat approach. The study is registered with ClinicalTrials.gov (NCT02567435) and is complete. FINDINGS: Between May 23, 2016, and Jan 1, 2022, 325 patients were enrolled. In 297 evaluable patients (148 assigned to VAC/VI alone and 149 assigned to VAC/VI with temsirolimus), the median age was 6·3 years (IQR 3·0-11·3); 33 (11%) patients were aged 18 years or older; 179 (60%) of 297 were male. 113 (77%) of 148 patients were FOXO1 negative in the VAC/VI group, and 108 (73%) of 149 were FOXO1 negative in the VAC/VI with temsirolimus group. With a median follow-up of 3·6 years (IQR 2·8-4·5), 3-year event-free survival did not differ significantly between the two groups (64·8% [95% CI 55·5-74·1] in the VAC/VI group vs 66·8% [57·5-76·2] in the VAC/VI plus temsirolimus group (hazard ratio 0·86 [95% CI 0·58-1·26]; log-rank p=0·44). The most common grade 3-4 adverse events were anaemia (62 events in 60 [41%] of 148 patients in the VAC/VI group vs 89 events in 87 [58%] of 149 patients in the VAC/VI with temsirolimus group), lymphopenia (83 events in 65 [44%] vs 99 events in 71 [48%]), neutropenia (160 events in 99 [67%] vs 164 events in 105 [70%]), and leukopenia (121 events in 86 [58%] vs 132 events in 93 [62%]). There was one treatment-related death in the VAC/VI with temsirolimus group, categorised as not otherwise specified. INTERPRETATION: Addition of temsirolimus to VAC/VI did not improve event-free survival in patients with intermediate-risk rhabdomyosarcoma defined by their FOXO1 translocation status and clinical factors. Novel biology-based strategies are needed to improve outcomes in this population. FUNDING: The Children's Oncology Group (supported by the US National Cancer Institute, US National Institutes of Health).

A systematic review of early phase studies for children and young people with relapsed and refractory rhabdomyosarcoma: The REFoRMS-SR project.

Rhabdomyosarcoma is the commonest soft tissue sarcoma in children. Around one-third of children with rhabdomyosarcoma experience relapse or have refractory disease, which is associated with a poor prognosis. This systematic review of early phase studies in pediatric relapsed/refractory rhabdomyosarcoma was conducted to inform future research and provide accurate information to families and clinicians making difficult treatment choices. Nine databases and five trial registries were searched in June 2021. Early phase studies of interventions for disease control in patients under 18 years old with relapsed/refractory rhabdomyosarcoma were eligible. No language/geographic restrictions were applied. Studies conducted after 2000 were included. Survival outcomes, response rates, quality of life and adverse event data were extracted. Screening, data extraction and quality assessment (Downs and Black Checklist) were conducted by two researchers. Owing to heterogeneity in the included studies, narrative synthesis was conducted. Of 16,965 records screened, 129 published studies including over 1100 relapsed/refractory rhabdomyosarcoma patients were eligible. Most studies evaluated systemic therapies. Where reported, 70% of studies reported a median progression-free survival ≤6 months. Objective response rate was 21.6%. Adverse events were mostly hematological. One-hundred and seven trial registry records of 99 studies were also eligible, 63 of which report they are currently recruiting. Study quality was limited by poor and inconsistent reporting. Outcomes for children with relapsed/refractory rhabdomyosarcoma who enroll on early phase studies are poor. Improving reporting quality and consistency would facilitate the synthesis of early phase studies in relapsed/refractory rhabdomyosarcoma (PROSPERO registration: CRD42021266254).

Mecapegfilgrastim for prophylaxis of febrile neutropenia in children and adolescents with rhabdomyosarcoma or Ewing sarcoma: a prospective, single-arm, pilot study.

BACKGROUND: The chemotherapy regimens recommended for both rhabdomyosarcoma (RMS) and Ewing sarcoma (ES) patients are myelosuppressive and can reduce the absolute neutrophil count (ANC) and subsequently increase the risk of febrile neutropenia (FN). However, only a few studies have focused on the efficacy and safety of granulocyte-colony stimulating factor (G-CSF) drugs in pediatric and adolescent patients with RMS and ES. Our objective was to investigate the efficacy and safety of mecapegfilgrastim, a biosimilar of pegfilgrastim, in prophylaxis of FN for pediatric and adolescent patients with RMS or ES. METHODS: In this single-arm, single-center, prospective study, pediatric and adolescent patients with RMS or ES were enrolled to receive either VAC (vincristine, cyclophosphamide, dactinomycin) regimen or VDC (vincristine, cyclophosphamide, doxorubicin) regimen in a 3-week cycle, followed by treatment with mecapegfilgrastim (100 µg/kg, maximum 6 mg) given at 24 h after completing chemotherapy. The primary endpoint was the incidence rate of FN. Secondary endpoints included the incidence rate of grade 4 neutropenia, duration of ANC ≤ 0.5 × 109/L, incidence rate of chemotherapy delay or reduction, use of antibiotics, and safety profile. RESULTS: In total, 2 of the 30 (6.7%, 95% CI: 0.82-22.07) patients experienced FN after the first cycle of chemotherapy. Eight (26.7%, 95% CI: 12.28-45.89) patients experienced grade 4 neutropenia after receiving prophylactic mecapegfilgrastim. Eight patients experienced ANC ≤ 0.5 × 109/L with a median duration of 4.5 days; among them, 6 patients reached the lowest point of their ANC level on day 7, and 5 of them recovered by day 10. No dose reductions, delays, or discontinuation of chemotherapy was reported. Twenty-one (70.0%) patients received antibiotics during the treatment period. No patient experienced FN in the 0-5 years and the 13-18 years groups, and 2 patients experienced FN in the 6-12 years group. Two patients, 6 patients, and no patient experienced grade 4 neutropenia in the 0-5 years, 6-12 years, and 13-18 years groups, respectively. CONCLUSION: Mecapegfilgrastim showed acceptable efficacy and safety profile in pediatric and adolescent patients with RMS or ES. Further randomized studies with large sample size are warranted. TRIAL REGISTRATION: This clinical trial was registered at Chictr.org.cn (No.ChiCTR1900022249). Registered on March 31, 2019.

Antitumour effects of SFX-01 molecule in combination with ionizing radiation in preclinical and in vivo models of rhabdomyosarcoma.

BACKGROUND: Despite a multimodal approach including surgery, chemo- and radiotherapy, the 5-year event-free survival rate for rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in childhood, remains very poor for metastatic patients, mainly due to the selection and proliferation of tumour cells driving resistance mechanisms. Personalised medicine-based protocols using new drugs or targeted therapies in combination with conventional treatments have the potential to enhance the therapeutic effects, while minimizing damage to healthy tissues in a wide range of human malignancies, with several clinical trials being started. In this study, we analysed, for the first time, the antitumour activity of SFX-01, a complex of synthetic d, l-sulforaphane stabilised in alpha-cyclodextrin (Evgen Pharma plc, UK), used as single agent and in combination with irradiation, in four preclinical models of alveolar and embryonal RMS. Indeed, SFX-01 has shown promise in preclinical studies for its ability to modulate cellular pathways involved in inflammation and oxidative stress that are essential to be controlled in cancer treatment. METHODS: RH30, RH4 (alveolar RMS), RD and JR1 (embryonal RMS) cell lines as well as mouse xenograft models of RMS were used to evaluate the biological and molecular effects induced by SFX-01 treatment. Flow cytometry and the modulation of key markers analysed by q-PCR and Western blot were used to assess cell proliferation, apoptosis, autophagy and production of intracellular reactive oxygen species (ROS) in RMS cells exposed to SFX-01. The ability to migrate and invade was also investigated with specific assays. The possible synergistic effects between SFX-01 and ionising radiation (IR) was studied in both the in vitro and in vivo studies. Student's t-test or two-way ANOVA were used to test the statistical significance of two or more comparisons, respectively. RESULTS: SFX-01 treatment exhibited cytostatic and cytotoxic effects, mediated by G2 cell cycle arrest, apoptosis induction and suppression of autophagy. Moreover, SFX-01 was able to inhibit the formation and the proliferation of 3D tumorspheres as monotherapy and in combination with IR. Finally, SFX-01, when orally administered as single agent, displayed a pattern of efficacy at reducing the growth of tumour masses in RMS xenograft mouse models; when combined with a radiotherapy regime, it was observed to act synergistically, resulting in a more positive outcome than would be expected by adding each exposure alone. CONCLUSIONS: In summary, our results provide evidence for the antitumour properties of SFX-01 in preclinical models of RMS tumours, both as a standalone treatment and in combination with irradiation. These forthcoming findings are crucial for deeper investigations of SFX-01 molecular mechanisms against RMS and for setting up clinical trials in RMS patients in order to use the SFX-01/IR co-treatment as a promising therapeutic approach, particularly in the clinical management of aggressive RMS disease.

Local Treatment of Children Suffering From Parameningeal Rhabdomyosarcoma: A Retrospective Single-Center Study From China.

BACKGROUND: Treatment for parameningeal rhabdomyosarcoma (PM-RMS) has been a challenge since local control is difficult. The goal of this study was to analyse the impact of different local treatment approaches on childhood PM-RMS patients and help dispel the doubt that whether secondary radical surgery (SRS) should be encouraged in the management of PM-RMS. METHODS: A total of 17 children with PM-RMS who received unified systemic chemotherapy and individualized local therapy such as radiotherapy (RT) and/or SRS were included in this retrospective study. The overall survival (OS) and event free survival (EFS) were compared between groups adopting different local strategies. RESULTS: The 3-year OS and EFS of our PM-RMS patients was 75.5% and 56.5% respectively. The OS and EFS of patients who received SRS were both significantly lower than that of the non-SRS group (3-year OS: 50.0% vs 90.0%, P = .031; 3-year EFS: 33.3% vs 60.6%, P = .020). The OS and EFS of the patients who received RT was higher than that of the patients of the non-RT group (3-year OS: 85.6% vs 0%, P = .001; 3-year EFS: 64.0% vs 0%, P = .011). CONCLUSION: This study illustrates that SRS was associated with poor prognosis of PM-RMS and should not be routinely performed. Optimized RT strategies along with more intensive chemotherapy may be alternative options to improve the survival of patients with PM-RMS. Multi-center, large sample and prospective studies are needed to further validate these findings.

Event-free survival in relapsed and refractory rhabdomyosarcoma treated on cooperative group phase II trials: A report from the Children's Oncology Group.

BACKGROUND: Novel therapies are needed for relapsed and refractory rhabdomyosarcoma (RRMS). Phase II clinical trials in RRMS have typically utilized radiologic response as the primary activity endpoint, an approach that poses several limitations in RRMS. In this analysis, we aimed to estimate an event-free survival (EFS) endpoint for RRMS that could be used as a benchmark for future studies. PROCEDURE: We performed a retrospective study of patients with RRMS enrolling on 13 single-agent phase II Children's Oncology Group and legacy group trials from 1997 to 2016. All included trials used radiographic response as their primary activity endpoint. Six-month EFS was estimated from time of trial enrollment with 95% confidence intervals. Clinical characteristics, including trial of enrollment, sex, age, race, histology, number of prior chemotherapies, and radiographic response were evaluated for their impact on 6-month EFS. RESULTS: We identified 175 patients across 13 trials. The 6-month EFS was 16.8% (11.6%-22.8%). No differences were seen in 6-month EFS based on age, sex, race, or histology. There were nonsignificant trends toward improved 6-month EFS for patients with less than or equal to two prior lines of therapy versus higher than two, for patients enrolled on trials that achieved their primary radiographic response endpoint versus trials that did not, and for patients who achieved complete or partial response compared to those achieving stable disease. CONCLUSIONS: The prognosis of RRMS enrolled on single-agent phase II trials is poor. This pooled 6-month EFS of RRMS on single-agent trials may be used as a RRMS-specific benchmark for future single-agent phase II trials.

Local treatment in initially unresected non-rhabdomyosarcoma soft-tissue sarcomas of children and adolescents: A retrospective single-center experience.

BACKGROUND: Pediatric non-rhabdomyosarcoma soft-tissue sarcomas (NRSTS) are a heterogeneous group of aggressive tumors. Patients with locally advanced/initially unresected disease represent a subset of patients with unsatisfactory outcome: limited data are available on the best treatment approach, in particular regarding local therapy. METHODS: This retrospective analysis concerned 71 patients < 21 years old with nonmetastatic, initially unresected adult-type NRSTS, treated at a referral center for pediatric sarcomas from 1990 to 2021. Patients were treated using a multimodal approach, based on the protocols adopted at the time of their diagnosis. RESULTS: The series included a selected group of patients with unfavorable clinical characteristics, i.e., most cases had high-grade and large tumors, arising from axial sites in 61% of cases. All patients received neoadjuvant chemotherapy, 58 (82%) had delayed surgery (R0 in 45 cases), and 50 (70%) had radiotherapy. Partial response to chemotherapy was observed in 46% of cases. With a median follow-up of 152 months (range, 18-233), 5-year event-free survival (EFS) and overall survival (OS) were 39.9% and 56.5%, respectively. Survival was significantly better for patients who responded to chemotherapy, and those who had a delayed R0 resection. Local relapse at 5 years was 7.7% for patients who did not undergo delayed surgery. CONCLUSIONS: Our series underscores the unsatisfactory outcome of initially unresected NRSTS patients. Improving the outcome of this patient category requires therapeutic strategies able to combine novel effective systemic therapies with a better-defined local treatment approach to offer patients the best chances to have R0 surgery.

Acetylated galactopyranosyl N-heterocyclic monocarbene complexes of Silver(I) as novel anti-proliferative agents in a rhabdomyosarcoma cell line.

Herein, four silver(I) complexes bearing acetylated d-galactopyranoside-based N-heterocyclic carbene ligands were synthesized and fully characterized by elemental analysis, NMR, and X-ray photoelectron spectroscopy. All complexes were obtained with an anomeric ß-configuration and as monocarbene species. In this study, we investigated the biological effects of the silver(I) complexes 2a-d on the human rhabdomyosarcoma cell line, RD. Our results show concentration-dependent effects on cell density, growth inhibition, and activation of key signaling pathways such as Akt 1/2, ERK 1/2, and p38-MAPK, indicating their potential as anticancer agents. Notably, at 35.5 µM, the complexes induced mitochondrial network disruption, as observed with 2b and 2c, whereas with 2a, this disruption was accompanied by nuclear content release. These results provide insight into the utility of carbohydrate incorporated NHC complexes of silver(I) as new agents in cancer therapy.

What are the Optimal Systemic Treatment Options for Rhabdomyosarcoma?

OPINION STATEMENT: Rhabdomyosarcoma, a soft tissue sarcoma commonly observed in childhood, requires multidisciplinary treatment, including surgical tumor resection, chemotherapy, and radiation therapy. Although long-term survival can be expected in patients with localized rhabdomyosarcoma, the clinical outcomes in patients with metastatic or unresectable rhabdomyosarcoma remain unsatisfactory. To improve the outcomes of rhabdomyosarcoma, it is important to explore effective systemic treatments for metastatic rhabdomyosarcoma. Currently, multiagent chemotherapy comprising vincristine, actinomycin D, and ifosfamide/cyclophosphamide remains standard systemic treatment for rhabdomyosarcoma. On the other hand, new treatment, such as immune checkpoint inhibitors and molecular targeted drugs, have demonstrated superior clinical outcomes compared to those of standard treatments in various type of malignancies. Therefore, it is necessary to assess the efficacies of these treatments in patients with rhabdomyosarcoma. Recent clinical studies have shown efficacies and safeties of temozolomide combined with vincristine/irinotecan, olaratumab combined with doxorubicin or vincristine/irinotecan, and long-term maintenance therapy. Furthermore, basic researches demonstrated new therapeutic targets. Future studies using these approaches are required to assess their clinical significances.

Renal toxicity of ifosfamide in children with cancer: an exploratory study integrating aldehyde dehydrogenase enzymatic activity data and a wide-array urinary metabolomics approach.

BACKGROUND: Ifosfamide is a major anti-cancer drug in children with well-known renal toxicity. Understanding the mechanisms underlying this toxicity could help identify children at increased risk of toxicity. METHODS: The IFOS01 study included children undergoing ifosfamide-based chemotherapy for Ewing sarcoma or rhabdomyosarcoma. A fully evaluation of renal function was performed during and after chemotherapy. Proton nuclear magnetic resonance (NMR) and conventional biochemistry were used to detect early signs of ifosfamide-induced tubulopathy. The enzymatic activity of aldehyde dehydrogenase (ALDH) was measured in the peripheral blood lymphocytes as a marker of ifosfamide-derived chloroacetaldehyde detoxification capacity. Plasma and urine concentrations of ifosfamide and dechloroethylated metabolites were quantified. RESULTS: The 15 participants received a median total ifosfamide dose of 59 g/m2 (range: 24-102), given over a median of 7 cycles (range: 4-14). All children had acute proximal tubular toxicity during chemotherapy that was reversible post-cycle, seen with both conventional assays and NMR. After a median follow-up of 31 months, 8/13 children presented overall chronic toxicity among which 7 had decreased glomerular filtration rate. ALDH enzymatic activity showed high inter- and intra-individual variations across cycles, though overall activity looked lower in children who subsequently developed chronic nephrotoxicity. Concentrations of ifosfamide and metabolites were similar in all children. CONCLUSIONS: Acute renal toxicity was frequent during chemotherapy and did not allow identification of children at risk for long-term toxicity. A role of ALDH in late renal dysfunction is possible so further exploration of its enzymatic activity and polymorphism should be encouraged to improve the understanding of ifosfamide-induced nephrotoxicity.

Survival of patients with orbital and eyelid rhabdomyosarcoma treated on Children's Oncology Group studies from 1997 to 2013: A report from the Children's Oncology Group.

BACKGROUND: Orbital rhabdomyosarcoma (ORMS) commonly presents as low-risk disease (stage 1, group I-III, embryonal RMS) with excellent outcome. Long-term follow-up of patients with low-risk ORMS and outcomes of less common subgroups of ORMS treated on recent Children's Oncology Group (COG) trials have not been reported. METHODS: Patients with ORMS enrolled on COG trials from 1997 to 2013 were identified. Demographic information and disease characteristics were collected. Outcomes were determined for the following subgroups: 1) low-risk ORMS, 2) resected (group I/II) low-risk ORMS, 3) non-low-risk ORMS, and 4) recurrent ORMS. Event-free survival (EFS) and overall survival (OS) were estimated using the Kaplan-Meier method. ResultsThe authors identified 218 patients with ORMS. Most tumors were embryonal/botryoid (n = 169; 77.5%), <5 cm (n = 213; 97.7%), group III (n = 170; 78.0%), and without lymph node involvement (N0; n = 215; 98.6%). For 192 patients with low-risk ORMS, the 10-year EFS and OS rates were 85.5% (95% confidence interval [CI], 77.0%-94.0%) and 95.6% (95% CI, 90.8%-100.0%), respectively. Those with group I/II low-risk ORMS (n = 5 in group I; n = 39 in group IIA) had 10-year EFS and OS rates of 88.0% (95% CI, 72.6%-100.0%) and 97.6% (95% CI, 90.0%-100.0%), respectively. Twenty-six patients with non-low-risk ORMS had 5-year EFS and OS rates of 88.5% (95% CI, 75.6%-100.0%) and 95.8% (95% CI, 87.7%-100.0%), respectively. For patients with recurrent ORMS, the 10-year OS rate from the time of recurrence was 69.4% (95% CI, 50.0%-88.8%). CONCLUSIONS: Patients with ORMS had favorable long-term survival outcomes on COG studies from 1997 to 2013, including those who had both low-risk and non-low-risk disease. A significant proportion of patients with recurrent ORMS may achieve long-term survival.

Metastatic adult-type non-rhabdomyosarcoma soft tissue sarcomas in children and adolescents: A cohort study from the European paediatric Soft tissue sarcoma Study Group.

BACKGROUND: Limited data exist on the clinical behavior of pediatric non-rhabdomyosarcoma soft tissue sarcomas (NRSTS) with distant metastases at onset, and a clear standard of care has not yet been defined. METHODS: This cohort study reports on pediatric adult-type metastatic NRSTS enrolled in two concurrent prospective European studies, i.e., the randomized BERNIE study and the single-arm MTS 2008 study developed by the European paediatric Soft tissue sarcoma Study Group. Treatment programs were originally designed for patients with metastatic rhabdomyosarcoma, i.e., nine courses of multidrug chemotherapy (with or without bevacizumab in the BERNIE study), followed by 12 cycles of maintenance therapy, whereas radiotherapy and/or surgery (on primary tumor and/or metastases) were delayed until after seven courses of chemotherapy had been administered. RESULTS: The study included 61 patients <21 years old treated from July 2008 to December 2016. The lung was the site of metastases in 75% of the cases. All patients received multi-agent chemotherapy, 44% had local therapy to primary tumor, and 18% had treatment of metastases. Median time to progression/relapse was 6 months. A high rate of tumor progression was observed during the initial part of the chemotherapy program. With a median follow-up of 41.5 months (range, 2-111 months), 3-year event-free survival and overall survival were 15.4% (95% confidence interval [CI], 7.6-25.7) and 34.9% (95% CI, 22.7-47.5), respectively. There were no statistically significant differences in outcome depending on the type of treatment administered. CONCLUSIONS: The study confirmed the overall poor outcome for patients with metastatic NRSTS, whose treatment remains a challenge. PLAIN LANGUAGE SUMMARY: Pediatric non-rhabdomyosarcoma soft tissue sarcomas form a heterogeneous group of rare tumors. Although recent international studies have defined the standard of care for patients with localized disease, limited data are available on the clinical behavior of patients with distant metastases. This study on 61 metastatic cases treated on two prospective European protocols confirms that the chances of survival of such patients are often dismal and a standard treatment is still lacking.

Pediatric phase 2 trial of a WEE1 inhibitor, adavosertib (AZD1775), and irinotecan for relapsed neuroblastoma, medulloblastoma, and rhabdomyosarcoma.

BACKGROUND: Inhibition of the WEE1 kinase by adavosertib (AZD1775) potentiates replicative stress from genomic instability or chemotherapy. This study reports the pediatric solid tumor phase 2 results of the ADVL1312 trial combining irinotecan and adavosertib. METHODS: Pediatric patients with recurrent neuroblastoma (part B), medulloblastoma/central nervous system embryonal tumors (part C), or rhabdomyosarcoma (part D) were treated with irinotecan and adavosertib orally for 5 days every 21 days. The combination was considered effective if there were at least three of 20 responses in parts B and D or six of 19 responses in part C. Tumor tissue was analyzed for alternative lengthening of telomeres and ATRX. Patient's prior tumor genomic analyses were provided. RESULTS: The 20 patients with neuroblastoma (part B) had a median of three prior regimens and 95% had a history of prior irinotecan. There were three objective responses (9, 11, and 18 cycles) meeting the protocol defined efficacy end point. Two of the three patients with objective responses had tumors with alternative lengthening of telomeres. One patient with pineoblastoma had a partial response (11 cycles), but parts C and D did not meet the protocol defined efficacy end point. The combination was well tolerated and there were no dose limiting toxicities at cycle 1 or beyond in any parts of ADVL1312 at the recommended phase 2 dose. CONCLUSION: This is first phase 2 clinical trial of adavosertib in pediatrics and the first with irinotecan. The combination may be of sufficient activity to consider further study of adavosertib in neuroblastoma.

Structure-activity relationship of dihydropyridines for rhabdomyosarcoma.

Childhood muscle-related cancer rhabdomyosarcoma is a rare disease with a 50-year unmet clinical need for the patients presented with advanced disease. The rarity of ∼350 cases per year in North America generally diminishes the viability of large-scale, pharmaceutical industry driven drug development efforts for rhabdomyosarcoma. In this study, we performed a large-scale screen of 640,000 compounds to identify the dihydropyridine (DHP) class of anti-hypertensives as a priority compound hit. A structure-activity relationship was uncovered with increasing cell growth inhibition as side chain length increases at the ortho and para positions of the parent DHP molecule. Growth inhibition was consistent across n = 21 rhabdomyosarcoma cell line models. Anti-tumor activity in vitro was paralleled by studies in vivo. The unexpected finding was that the action of DHPs appears to be other than on the DHP receptor (i.e., L-type voltage-gated calcium channel). These findings provide the basis of a medicinal chemistry program to develop dihydropyridine derivatives that retain anti-rhabdomyosarcoma activity without anti-hypertensive effects.

Rhabdomyosarcoma xenotransplants in zebrafish embryos.

Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue sarcomas. High-risk and metastatic disease continues to be associated with very poor prognosis. RMS model systems that faithfully recapitulate the human disease and provide rapid, cost-efficient estimates of antitumor efficacy of candidate drugs are needed to facilitate drug development and personalized medicine approaches. Here, we present a new zebrafish-based xenotransplant model allowing for rapid and easily accessible drug screening using low numbers of viable tumor cells and relatively small amounts of water-soluble chemicals. Under optimized temperature conditions, embryonal RMS xenografts were established in zebrafish embryos at 3 h postfertilization (hpf). In proof-of-principle experiments, chemotherapy drugs with established clinical anti-RMS efficacy (vincristine, dactinomycin) and the mitogen-activated protein kinase kinase inhibitor trametinib were shown to significantly reduce the cross-sectional area of the tumors by 120 hpf. RMS xenograft models in zebrafish embryos henceforth could serve as a valuable addition to cell culture and mammalian models of RMS and represent a rapid and cost-effective solution for preclinical candidate drug testing.

Orthotopic patient-derived xenografts of paediatric solid tumours.

Paediatric solid tumours arise from endodermal, ectodermal, or mesodermal lineages. Although the overall survival of children with solid tumours is 75%, that of children with recurrent disease is below 30%. To capture the complexity and diversity of paediatric solid tumours and establish new models of recurrent disease, here we develop a protocol to produce orthotopic patient-derived xenografts at diagnosis, recurrence, and autopsy. Tumour specimens were received from 168 patients, and 67 orthotopic patient-derived xenografts were established for 12 types of cancer. The origins of the patient-derived xenograft tumours were reflected in their gene-expression profiles and epigenomes. Genomic profiling of the tumours, including detailed clonal analysis, was performed to determine whether the clonal population in the xenograft recapitulated the patient's tumour. We identified several drug vulnerabilities and showed that the combination of a WEE1 inhibitor (AZD1775), irinotecan, and vincristine can lead to complete response in multiple rhabdomyosarcoma orthotopic patient-derived xenografts tumours in vivo.

Integrin alpha9 emerges as a key therapeutic target to reduce metastasis in rhabdomyosarcoma and neuroblastoma.

The majority of current cancer therapies are aimed at reducing tumour growth, but there is lack of viable pharmacological options to reduce the formation of metastasis. This is a paradox, since more than 90% of cancer deaths are attributable to metastatic progression. Integrin alpha9 (ITGA9) has been previously described as playing an essential role in metastasis; however, little is known about the mechanism that links this protein to this process, being one of the less studied integrins. We have now deciphered the importance of ITGA9 in metastasis and provide evidence demonstrating its essentiality for metastatic dissemination in rhabdomyosarcoma and neuroblastoma. However, the most translational advance of this study is to reveal, for the first time, the possibility of reducing metastasis by pharmacological inhibition of ITGA9 with a synthetic peptide simulating a key interaction domain of ADAM proteins, in experimental metastasis models, not only in childhood cancers but also in a breast cancer model.