Metastatic rhabdomyosarcoma with exclusive distant lymph node involvement: A European Pediatric Soft tissue sarcoma Study Group (EpSSG) report.

BACKGROUND: The prognosis of patients with metastatic rhabdomyosarcoma (RMS) is not uniformly poor. Tumors with nodal involvement beyond the first lymph node station are currently considered to have distant metastases. The aim of this study is to evaluate the characteristics and outcome of RMS patients with distal nodal involvement as the only site of metastasis. METHODS: This study included all patients with a diagnosis of RMS and distant nodal involvement as the only metastatic site, enrolled in the European Pediatric Soft tissue sarcoma Study Group (EpSSG) protocols. Treatment comprised chemotherapy, surgery, and/or radiotherapy. The main outcome measures were event-free survival (EFS) and overall survival (OS). RESULTS: A total of 22 patients (median age 7.1 years, range 1.4-16.7) fit the inclusion criteria. The extremities were the most common primary tumor site (59%). Twenty-one patients had regional and distant nodal involvement, 12 were PAX3/7-FOXO1 positive. Twenty patients had radiotherapy including 16 to the nodal metastatic area. After a median follow-up of 53.9 months (range 22.8-110.5), 15 patients remain in complete remission, seven had progressive disease or relapse, and six of them died. The 3-year EFS and OS were 67.1% (95% confidence interval [CI]: 42.9-82.9) and 71.9% (95% CI: 47.7-86.3), respectively. Patients with fusion-negative tumors had better outcomes than those with fusion-positive tumors (3-year EFS 100% vs. 46.6%; p = .04). CONCLUSION: In our experience, patients with RMS and distant lymph node involvement as the only site of metastasis present an outcome superior than other metastatic patients and comparable to patients with locoregional nodal involvement. In particular, excellent outcomes were seen in the limited number of patients with fusion-negative tumors.

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.

Quantitative diffusion-weighted MRI response assessment in rhabdomyosarcoma: an international retrospective study on behalf of the European paediatric Soft tissue sarcoma Study Group Imaging Committee.

OBJECTIVE: To investigate the feasibility of diffusion-weighted magnetic resonance imaging (DW-MRI) as a predictive imaging marker after neoadjuvant chemotherapy in patients with rhabdomyosarcoma. MATERIAL AND METHODS: We performed a multicenter retrospective study including pediatric, adolescent and young adult patients with rhabdomyosarcoma, Intergroup Rhabdomyosarcoma Study group III/IV, treated according to the European paediatric Soft tissue sarcoma Study Group (EpSSG) RMS2005 or MTS2008 studies. DW-MRI was performed according to institutional protocols. We performed two-dimensional single-slice tumor delineation. Areas of necrosis or hemorrhage were delineated to be excluded in the primary analysis. Mean, median and 5th and 95th apparent diffusion coefficient (ADC) were extracted. RESULTS: Of 134 included patients, 82 had measurable tumor at diagnosis and response and DW-MRI scans of adequate quality and were included in the analysis. Technical heterogeneity in scan acquisition protocols and scanners was observed. Mean ADC at diagnosis was 1.1 (95% confidence interval [CI]: 1.1-1.2) (all ADC expressed in * 10-3 mm2/s), versus 1.6 (1.5-1.6) at response assessment. The 5th percentile ADC was 0.8 (0.7-0.9) at diagnosis and 1.1 (1.0-1.2) at response. Absolute change in mean ADC after neoadjuvant chemotherapy was 0.4 (0.3-0.5). Exploratory analyses for association between ADC and clinical parameters showed a significant difference in mean ADC at diagnosis for alveolar versus embryonal histology. Landmark analysis at nine weeks after the date of diagnosis showed no significant association (hazard ratio 1.3 [0.6-3.2]) between the mean ADC change and event-free survival. CONCLUSION: A significant change in the 5th percentile and the mean ADC after chemotherapy was observed. Strong heterogeneity was identified in DW-MRI acquisition protocols between centers and in individual patients.

Structural disruption of BAF chromatin remodeller impairs neuroblastoma metastasis by reverting an invasiveness epigenomic program.

BACKGROUND: Epigenetic programming during development is essential for determining cell lineages, and alterations in this programming contribute to the initiation of embryonal tumour development. In neuroblastoma, neural crest progenitors block their course of natural differentiation into sympathoadrenergic cells, leading to the development of aggressive and metastatic paediatric cancer. Research of the epigenetic regulators responsible for oncogenic epigenomic networks is crucial for developing new epigenetic-based therapies against these tumours. Mammalian switch/sucrose non-fermenting (mSWI/SNF) ATP-dependent chromatin remodelling complexes act genome-wide translating epigenetic signals into open chromatin states. The present study aimed to understand the contribution of mSWI/SNF to the oncogenic epigenomes of neuroblastoma and its potential as a therapeutic target. METHODS: Functional characterisation of the mSWI/SNF complexes was performed in neuroblastoma cells using proteomic approaches, loss-of-function experiments, transcriptome and chromatin accessibility analyses, and in vitro and in vivo assays. RESULTS: Neuroblastoma cells contain three main mSWI/SNF subtypes, but only BRG1-associated factor (BAF) complex disruption through silencing of its key structural subunits, ARID1A and ARID1B, impairs cell proliferation by promoting cell cycle blockade. Genome-wide chromatin remodelling and transcriptomic analyses revealed that BAF disruption results in the epigenetic repression of an extensive invasiveness-related expression program involving integrins, cadherins, and key mesenchymal regulators, thereby reducing adhesion to the extracellular matrix and the subsequent invasion in vitro and drastically inhibiting the initiation and growth of neuroblastoma metastasis in vivo. CONCLUSIONS: We report a novel ATPase-independent role for the BAF complex in maintaining an epigenomic program that allows neuroblastoma invasiveness and metastasis, urging for the development of new BAF pharmacological structural disruptors for therapeutic exploitation in metastatic neuroblastoma.

Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics.

MicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (<150 nm) and composition. These nanovesicles are colloidal stable (>24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.

Distinct molecular profile of IRF4-rearranged large B-cell lymphoma.

Pediatric large B-cell lymphomas (LBCLs) share morphological and phenotypic features with adult types but have better prognosis. The higher frequency of some subtypes such as LBCL with IRF4 rearrangement (LBCL-IRF4) in children suggests that some age-related biological differences may exist. To characterize the genetic and molecular heterogeneity of these tumors, we studied 31 diffuse LBCLs (DLBCLs), not otherwise specified (NOS); 20 LBCL-IRF4 cases; and 12 cases of high-grade B-cell lymphoma (HGBCL), NOS in patients ≤25 years using an integrated approach, including targeted gene sequencing, copy-number arrays, and gene expression profiling. Each subgroup displayed different molecular profiles. LBCL-IRF4 had frequent mutations in IRF4 and NF-κB pathway genes (CARD11, CD79B, and MYD88), losses of 17p13 and gains of chromosome 7, 11q12.3-q25, whereas DLBCL, NOS was predominantly of germinal center B-cell (GCB) subtype and carried gene mutations similar to the adult counterpart (eg, SOCS1 and KMT2D), gains of 2p16/REL, and losses of 19p13/CD70. A subset of HGBCL, NOS displayed recurrent alterations of Burkitt lymphoma-related genes such as MYC, ID3, and DDX3X and homozygous deletions of 9p21/CDKN2A, whereas other cases were genetically closer to GCB DLBCL. Factors related to unfavorable outcome were age >18 years; activated B-cell (ABC) DLBCL profile, HGBCL, NOS, high genetic complexity, 1q21-q44 gains, 2p16/REL gains/amplifications, 19p13/CD70 homozygous deletions, and TP53 and MYC mutations. In conclusion, these findings further unravel the molecular heterogeneity of pediatric and young adult LBCL, improve the classification of this group of tumors, and provide new parameters for risk stratification.

Congenital rhabdomyosarcoma: A report from the European paediatric Soft tissue sarcoma Study Group.

PROCEDURE: Congenital rhabdomyosarcoma (RMS) represents a challenging disease due to its characteristics and the difficulties in delivering treatment in this immature population. METHODS: We analyzed treatment and outcome of patients with congenital RMS, defined as tumor diagnosed in the first 2 months of life, enrolled in the European paediatric Soft tissue sarcoma Study Group protocols. RESULTS: Twenty-four patients with congenital RMS were registered. All, except one patient (PAX3-FOXO1-positive metastatic RMS), had favorable histology and localized disease. Three patients had VGLL2-CITED2/NCOA2 fusion. Complete tumor resection was achieved in 10 patients. No radiotherapy was given. Chemotherapy doses were adjusted to age and weight. Only two patients required further dose reduction for toxicity. The 5-year event-free survival (EFS) and overall survival (OS) were 75.0% (95% confidence interval [CI] 52.6-87.9) and 87.3% (95% CI 65.6-95.7), respectively. Progressive disease was the main cause of treatment failure. CONCLUSION: Patients with congenital RMS presented with a favorable disease, allowing weight- and age-adjusted doses of chemotherapy and avoidance of irradiation, without compromising the outcome.

Alveolar rhabdomyosarcoma with regional nodal involvement: Results of a combined analysis from two cooperative groups.

BACKGROUND: Treatment of children and adolescents with alveolar rhabdomyosarcoma (ARMS) and regional nodal involvement (N1) have been approached differently by North American and European cooperative groups. In order to define a better therapeutic strategy, we analyzed two studies conducted between 2005 and 2016 by the European paediatric Soft tissue sarcoma Study Group (EpSSG) and Children's Oncology Group (COG). METHODS: We retrospectively identified patients with ARMS N1 enrolled in either EpSSG RMS2005 or in COG ARST0531. Chemotherapy in RMS2005 comprised ifosfamide + vincristine + dactinomycin + doxorubicin (IVADo), IVA and maintenance (vinorelbine, cyclophosphamide); in ARST0531, it consisted of either vincristine + dactinomycin + cyclophosphamide (VAC) or VAC alternating with vincristine + irinotecan (VI). Local treatment was similar in both protocols. RESULTS: The analysis of the clinical characteristics of 239 patients showed some differences between study groups: in RMS2005, advanced Intergroup Rhabdomyosarcoma Study Group (IRS) and large tumors predominated. There were no differences in outcomes between the two groups: 5-year event-free survival (EFS), 49% (95% confidence interval [CI]: 39-59) and 44% (95% CI: 30-58), and overall survival (OS), 51% (95% CI: 41-61) and 53.6% (95% CI: 40-68) in RMS2005 and ARST0531, respectively. In RMS2005, EFS of patients with FOXO1-positive tumors was significantly inferior to those with FOXO1-negative (49.3% vs 73%, P = .034). In contrast, in ARST0531, EFS of patients with FOXO1-positive tumors was 45% compared with 43.8% for those with FOXO1-negative. CONCLUSIONS: The outcome of patients with ARMS N1 was similar in both protocols. However, patients with FOXO1 fusion-negative tumors enrolled in RMS2005 showed a significantly better outcome, suggesting that different strategies of chemotherapy may have an impact in the outcome of this subgroup of patients.

Dickkopf-1 Inhibition Reactivates Wnt/ß-Catenin Signaling in Rhabdomyosarcoma, Induces Myogenic Markers In Vitro and Impairs Tumor Cell Survival In Vivo.

The Wnt/ß-catenin signaling pathway plays a pivotal role during embryogenesis and its deregulation is a key mechanism in the origin and progression of several tumors. Wnt antagonists have been described as key modulators of Wnt/ß-catenin signaling in cancer, with Dickkopf-1 (DKK-1) being the most studied member of the DKK family. Although the therapeutic potential of DKK-1 inhibition has been evaluated in several diseases and malignancies, little is known in pediatric tumors. Only a few works have studied the genetic inhibition and function of DKK-1 in rhabdomyosarcoma. Here, for the first time, we report the analysis of the therapeutic potential of DKK-1 pharmaceutical inhibition in rhabdomyosarcoma, the most common soft tissue sarcoma in children. We performed DKK-1 inhibition via shRNA technology and via the chemical inhibitor WAY-2626211. Its inhibition led to ß-catenin activation and the modulation of focal adhesion kinase (FAK), with positive effects on in vitro expression of myogenic markers and a reduction in proliferation and invasion. In addition, WAY-262611 was able to impair survival of tumor cells in vivo. Therefore, DKK-1 could constitute a molecular target, which could lead to novel therapeutic strategies in RMS, especially in those patients with high DKK-1 expression.

Dermatofibrosarcoma protuberans in children and adolescents: The European Paediatric Soft Tissue Sarcoma Study Group prospective trial (EpSSG NRSTS 2005).

BACKGROUND: As dermatofibrosarcoma protuberans (DFSP) are rare with no prospective series within paediatric sarcoma trials, the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) examined the clinical data and outcomes of DFSP enrolled in a multinational study of non-rhabdomyosarcoma soft tissue sarcomas (NRSTS). PATIENTS AND METHODS: Forty-six patients with confirmed DFSP were enrolled into the EpSSG NRSTS 2005 study. All had surgical resection and none had any further therapy at diagnosis. RESULTS: The median age at diagnosis was 6.9 years (range 0.4-17.5). All patients had localised disease, and the majority had small <5 cm tumours (93%), and 76% had Intergroup Rhabdomyosarcoma Study (IRS) I tumours. All patients had up front surgery, 32 requiring two operations. There were 11 patients with IRS II tumours, of which only two went on to have a local recurrence. After a median follow up of 49.0 months (range 4.2-130.9), all patients were alive at the time of this report, with 5-year event-free survival of 92.6% (CI 78.8-97.6) with a 100% overall survival. CONCLUSION: This report demonstrates the ability to run prospective paediatric studies in NRSTS in multiple European countries, with reasonable numbers of DFSP patients, with few events and no deaths, and hence excellent outcomes.

Functional high-throughput screening reveals miR-323a-5p and miR-342-5p as new tumor-suppressive microRNA for neuroblastoma.

Current therapies for most non-infectious diseases are directed at or affect functionality of the human translated genome, barely 2% of all genetic information. By contrast, the therapeutic potential of targeting the transcriptome, ~ 70% of the genome, remains largely unexplored. RNA therapeutics is an emerging field that widens the range of druggable targets and includes elements such as microRNA. Here, we sought to screen for microRNA with tumor-suppressive functions in neuroblastoma, an aggressive pediatric tumor of the sympathetic nervous system that requires the development of new therapies. We found miR-323a-5p and miR-342-5p to be capable of reducing cell proliferation in multiple neuroblastoma cell lines in vitro and in vivo, thereby providing a proof of concept for miRNA-based therapies for neuroblastoma. Furthermore, the combined inhibition of the direct identified targets such as CCND1, CHAF1A, INCENP and BCL-XL could reveal new vulnerabilities of high-risk neuroblastoma.

Fusion status in patients with lymph node-positive (N1) alveolar rhabdomyosarcoma is a powerful predictor of prognosis: Experience of the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG).

BACKGROUND: Alveolar rhabdomyosarcoma (aRMS) with lymph node involvement (N1 classification) accounts for up to 10% of all cases of RMS. The prognosis is poor, and is comparable to that of distant metastatic disease. In the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) RMS2005 protocol, patients with a histologic diagnosis of aRMS/N1 received intensified chemotherapy with systematic locoregional treatment. METHODS: Patients with aRMS/N1 were enrolled prospectively after primary surgery/biopsy and fusion status was assessed in tumor samples. All patients received 9 cycles of induction chemotherapy and 6 months of maintenance therapy. Local treatment included radiotherapy to the primary site and lymph nodes with or without secondary surgical resection. RESULTS: A total of 103 patients were enrolled. The clinical characteristics of the patients were predominantly unfavorable: 90% had macroscopic residual disease after initial surgery/biopsy, 63% had locally invasive tumors, 77% had a tumor measuring >5 cm, and 81% had disease at unfavorable sites. Fusion genes involving forkhead box protein O1 (FOXO1) were detected in 56 of 84 patients. Events occurred in 52 patients: 43 developed disease recurrence, 7 had disease that was refractory to treatment, and 2 patients developed second neoplasms. On univariate analysis, unfavorable disease site, tumor invasiveness, Intergroup Rhabdomyosarcoma Study group III, and fusion-positive status correlated with worse prognosis. The 5-year event-free survival rate of patients with fusion-positive tumors was 43% compared with 74% in patients with fusion-negative tumors (P = .01). On multivariate analysis, fusion positivity and tumor invasiveness proved to be unfavorable prognostic markers. CONCLUSIONS: Fusion status and tumor invasiveness appear to have a strong impact on prognosis in patients with aRMS/N1. Fusion status will be used to stratify these patients in the next EpSSG RMS study, and treatment will be intensified in patients with fusion-positive tumors. Cancer 2018. © 2018 American Cancer Society.

Prognostic relevance of early radiologic response to induction chemotherapy in pediatric rhabdomyosarcoma: A report from the International Society of Pediatric Oncology Malignant Mesenchymal Tumor 95 study.

BACKGROUND: Early response to induction chemotherapy is used in current European guidelines to evaluate the efficacy of chemotherapy and subsequently to adapt treatment in pediatric patients with rhabdomyosarcoma (RMS). However, existing literature on the prognostic value of early radiologic response on survival is contradictory; here the prognostic value is analyzed with data from the International Society of Pediatric Oncology (SIOP) Malignant Mesenchymal Tumor 95 (MMT-95) study. METHODS: This study examined 432 Intergroup Rhabdomyosarcoma Study Grouping III (macroscopic residue) patients enrolled in the SIOP MMT-95 study with a response assessment after 3 courses of chemotherapy (a 2-dimensional assessment). Patients with progressive disease (PD) after 3 courses of chemotherapy were excluded (n = 7). Failure-free survival (FFS) and overall survival (OS), calculated with the Kaplan-Meier method, were compared for 3 groups (complete response [CR]/partial response [PR], objective response [OR], and no response [NR]). The prognostic impact of early response was assessed through the calculation of Cox proportional hazards. RESULTS: After 3 courses of chemotherapy, 85.2% of the patients had CR/PR, 8.6% had OR, and 6.3% had NR. For all patients, the 5-year FFS and OS rates were 60% (95% confidence interval [CI], 56%-65%) and 74% (95% CI, 70%-78%), respectively. However, a Cox proportional hazards regression analysis revealed no significant difference in FFS or OS between the response groups. The adjusted hazard ratios for an OR and NR were 1.09 (95% CI, 0.63-1.88) and 0.81 (95% CI, 0.39-1.67), respectively, for FFS and 0.91 (95% CI, 0.47-1.76) and 1.27 (95% CI, 0.61-2.64), respectively, for OS. CONCLUSIONS: No evidence was found for the idea that early radiologic response to chemotherapy is prognostic for survival for patients with RMS. Treatment adaptation based on early response (except for patients with PD) should, therefore, no longer be incorporated into future studies. Cancer 2018;124:1016-24. © 2017 American Cancer Society.

Alveolar soft part sarcoma in children and adolescents: The European Paediatric Soft Tissue Sarcoma study group prospective trial (EpSSG NRSTS 2005).

BACKGROUND: As alveolar soft part sarcomas (ASPS) are rare with no prospective series within pediatric sarcoma trials, the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) examined the clinical data and outcomes of ASPS enrolled in a multinational study of nonrhabdomyosarcoma soft tissue sarcomas (NRSTS). PATIENTS AND METHODS: Twenty-two patients with ASPS were enrolled into the EpSSG NRSTS 2005 study. After surgical resection, subsequent treatment depended on the stratification of patients for completeness of resection and Intergroup Rhabdomyosarcoma Study (IRS) stage, size, and French Federation of Cancer Centres Sarcoma Group (FNCLCC) grade. Chemotherapy using ifosfamide and doxorubicin was performed in IRS group III. Radiotherapy was performed in IRS groups II and III, and FNCLCC grades 2 and 3 tumors. RESULTS: The median age at diagnosis was 11.5 years (range 2.7-17.5 years). The majority in the series had localized disease (20), with small IRS I tumors (12), and in total 19 had surgical resection upfront. Of the four patients who received conventional chemotherapy, there were no responses. Three of 20 patients with localized tumors and all metastatic patients developed metastases. The median follow up of patients with localized disease is 61.7 months (range 25.7-135.5 months) from diagnosis. The 5-year event-free survival is 94.7% (95% confidence interval: 68.1-99.2), and therefore the overall survival (OS) is 100%. CONCLUSION: This report demonstrates the ability to run prospective pediatric studies in NRSTS in multiple European countries, despite the small numbers of ASPS patients. We can conclude that for the majority with small resected tumors, there were few events and no deaths.

Localized vaginal/uterine rhabdomyosarcoma-results of a pooled analysis from four international cooperative groups.

BACKGROUND: Vaginal/uterine rhabdomyosarcoma (VU RMS) is one of the most favorable RMS sites. To determine the optimal therapy, the experience of four cooperative groups (Children's Oncology Group [COG], International Society of Pediatric Oncology (SIOP) Malignant Mesenchymal Tumor Group [MMT], Italian Cooperative Soft Tissue Sarcoma Group [ICG], and European pediatric Soft tissue sarcoma Study Group [EpSSG]) was analyzed. PROCEDURE: From 1981 to 2009, 237 patients were identified. Median age (years) at diagnosis differed by tumor location; it was 1.9 for vagina (n = 160), 2.7 for uterus corpus (n = 26), and 13.5 for uterus cervix (n = 51). Twenty-eight percent of patients received radiation therapy (RT) as part of primary therapy (23% COG, 27% MMT, 46% ICG, and 42% EpSSG), with significant differences in the use of brachytherapy between the cooperative groups (23% COG, 76% MMT, 64% ICG, and 88% EpSSG). RESULTS: Ten-year event-free (EFS) and overall survival (OS) were 74% (95% CI, 67-79%) and 92% (95% CI, 88-96%), respectively. In univariate analysis, OS was inferior for patients with uterine RMS and for those with regional lymph node involvement. Although EFS was slightly lower in patients without initial RT (71% without RT vs. 81% with RT; P = 0.08), there was no difference in OS (94% without RT vs. 89% with RT; P = 0.18). Local control using brachytherapy was excellent (93%). Fifty-one (51.5%) of the 99 survivors with known primary therapy and treatment for relapse were cured with chemotherapy with or without conservative surgery. CONCLUSIONS: About half of all patients with VU RMS can be cured without systematic RT or radical surgery. When RT is indicated, modalities that limit sequelae should be considered, such as brachytherapy.

Paratesticular rhabdomyosarcoma in children and adolescents-Outcome and patterns of relapse when utilizing a nonsurgical strategy for lymph node staging: Report from the International Society of Paediatric Oncology (SIOP) Malignant Mesenchymal Tumour 89 and 95 studies.

PURPOSE: To report the results from International Society of Pediatric Oncology (SIOP) Malignant Mesenchymal Tumors studies (MMT 89 and 95) of males with nonmetastatic paratesticular rhabdomyosarcoma. METHODS: From 1989 to 2003, 159 patients were included. Radical inguinal orchidectomy was recommended, but retroperitoneal lymph node (LN) assessment was based on imaging alone. The treatment was stratified by stage (SIOP tumor-node-metastasis staging system) and histology. RESULTS: Median age at presentation was 5.6 years (range 0.3-17.6) and 120 patients were of <10 years (75%). Patients ≥10 years had tumors of >5 cm more frequently compared to patients of <10 years (54% vs. 22%, P = 0.0004). The 5- year overall and progression-free survivals were 94% and 83%, respectively. Seventy-eight percent of relapses occurred in the retroperitoneal LN. Thirty-one percent of stage N0 patients of age ≥10 years developed node relapse, compared with 8% of N0 patients aged <10 years (P = 0.0005). CONCLUSIONS: Older patients with paratesticular rhabdomyosarcoma have a significant risk of LN relapse. These results support a surgical approach to LN staging in this subgroup of patients.

Access to clinical trials for adolescents with soft tissue sarcomas: Enrollment in European pediatric Soft tissue sarcoma Study Group (EpSSG) protocols.

BACKGROUND: Adolescents with cancer are enrolled in clinical trials at far lower rates than children. This report compares the number of adolescents (15-19-year-olds) and children (0-14-year-olds) enrolled in the protocols of the European pediatric Soft tissue sarcoma Study Group (EpSSG) with the number of cases expected to occur. METHODS: The observed-to-expected (O/E) ratio was detected in the EpSSG countries contributing most of the cases, that is, Italy, France, Spain, the Netherlands, United Kingdom, and Ireland. The observed cases included patients enrolled in any of the EpSSG protocols from October 2008 to October 2015, when all EpSSG protocols were open in these countries. The number of expected cases was calculated from the incidence rates estimated throughout the RARECAREnet database in the countries' population-based cancer registries. RESULTS: In the countries considered, 2,118 cases aged 0-19 years were enrolled in the EpSSG trials from 2008 to 2015: 82.8% were children and 17.2% were adolescents. The O/E ratio was 0.30 among patients 15-19 years old, as opposed to 0.64 for those 0-14 years old. The O/E ratio differed for the different subtypes: in adolescents, it was 0.64 and 0.18 for rhabdomyosarcoma (RMS) and non-rhabdomyosarcoma soft tissue sarcomas (NRSTS), respectively; in children, it was 0.77 and 0.50, respectively. The O/E ratios differed across the countries considered. CONCLUSIONS: Adolescents were less well represented than children on the EpSSG protocols, with better enrolment for RMS than for NRSTS for all age groups.

Modulation of chemotherapeutic drug resistance in neuroblastoma SK-N-AS cells by the neural apoptosis inhibitory protein and miR-520f.

The acquisition of multidrug resistance is a major impediment to the successful treatment of neuroblastoma, a clinically heterogeneous cancer accounting for ∼15% of all pediatric cancer deaths. The MYCN transcription factor, whose gene is amplified in ∼30% of high-risk neuroblastoma cases, influences drug resistance by regulating a cadre of genes, including those involved with drug efflux, however, other high-risk subtypes of neuroblastoma lacking MYCN amplification, such as those with chromosome 11q deletions, also acquire multidrug resistance. To elucidate additional mechanisms involved with drug resistance in non-MYCN amplified tumour cells, an SK-N-AS subline (SK-N-AsCis24) that is significantly resistant to cisplatin and cross resistant to etoposide was developed through a pulse-selection process. High resolution aCGH analysis of SK-N-AsCis24 revealed a focal gain on chromosome 5 containing the coding sequence for the neural apoptosis inhibitory protein (NAIP). Significant overexpression of NAIP mRNA and protein was documented, while experimental modulation of NAIP levels in both SK-N-AsCis24 and in parental SK-N-AS cells confirmed that NAIP was responsible for the drug resistant phenotype by apoptosis inhibition. Furthermore, a decrease in the NAIP targeting microRNA, miR-520f, was also demonstrated to be partially responsible for increased NAIP levels in SK-N-AsCis24. Interestingly, miR-520f levels were determined to be significantly lower in postchemotherapy treatment tumours relative to matched prechemotherapy samples, consistent with a role for this miRNA in the acquisition of drug resistance in vivo, potentially through decreased NAIP targeting. Our findings provide biological novel insight into neuroblastoma drug-resistance and have implications for future therapeutic research.

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.

Conservative approach in localised rhabdomyosarcoma of the bladder and prostate: results from International Society of Paediatric Oncology (SIOP) studies: malignant mesenchymal tumour (MMT) 84, 89 and 95.

BACKGROUND: The three sequential SIOP MMT studies provide the largest dataset available to date, to define the patient and tumour characteristics, treatment modalities and event-free and overall survival for children with non metastatic rhabdomyosarcoma (RMS) of the bladder and/or prostate (BP). PROCEDURE: The combined dataset of 172 patients with BP RMS treated on the SIOP MMT 84, 89 and 95 studies was reviewed to determine tumour characteristics, details of treatment and outcome. RESULTS: Median age at diagnosis was 2.5 years (range 2 months-17.8 years) and 138 (79%) were males. Median follow-up was 11.4 years (range 3 months-22 years). The 5-year overall survival of the combined cohort was 77% (CI 70-83%). The 5-year event-free survival was 63% and included 7 patients (4%) who did not achieve complete remission (CR), and 57 (33%) who relapsed. Age ≥ 10 years (RR 3.7) and alveolar pathology (RR 3.3) were identified as independent prognostic factors on multivariate analysis. Fifty-nine (50%) of the 119 survivors were cured without significant local therapy, improving from 31% in MMT84 study to 61% in MMT95 study. CONCLUSION: The clinical strategy of the MMT studies aims to minimise the burden of therapy whilst maintaining survival rates. Overall survival is comparable to that of other international groups, despite the lower use of radiotherapy and or radical surgery, although number of events experienced is higher. Further assessment of the late effects of therapy is required to confirm whether this approach results in lower morbidity in the long-term.