Impact of fusion gene status versus histology on riskâ stratification for rhabdomyosarcoma: Retrospective analyses of patients on UK trials

BackgroundLongâ term toxicities from current treatments are a major issue in paediatric cancer. Previous studies, including our own, have shown prognostic value for the presence of PAX3/7â FOXO1 fusion genes in rhabdomyosarcoma (RMS). It is proposed to introduce PAX3/7â FOXO1 positivity as a component of risk stratification, rather than alveolar histology, in future clinical trials.ProcedureTo assess the potential impact of this reclassification, we have determined the changes to risk category assignment of 210 histologically reviewed patients treated in the UK from previous malignant mesenchymal tumour clinical trials for nonâ metastatic RMS based on identification of PAX3/7â FOXO1 by fluorescence in situ hybridisation and/or reverse transcription PCR.ResultsUsing fusion gene positivity in the current risk stratification would reassign 7% of patients to different European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) risk groups. The next European trial would have 80% power to detect differences in eventâ free survival of 15% over 10 years and 20% over 5 years in reassigned patients. This would decrease treatment for over a quarter of patients with alveolar histology tumours that lack PAX3/7â FOXO1.ConclusionsFusion gene status used in stratification may result in significant numbers of patients benefitting from lower treatmentâ associated toxicity. Prospective testing to show this reassignment maintains current survival rates is now required and is shown to be feasible based on estimated recruitment to a future EpSSG trial. Together with developing novel therapeutic strategies for patients identified as higher risk, this may ultimately improve the outcome and quality of life for patients with RMS.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137481/1/pbc26386_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137481/2/pbc26386.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137481/3/pbc26386-sup-0002-FigureS2.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137481/4/pbc26386-sup-0001-FigureS1.pd

Delayed effects of a 20-min crushed ice application on knee joint position sense assessed by a functional task during a re-warming period

Delayed effects of a 20-minute crushed ice application on knee joint position sense assessed by a functional task during a re-warming period. Introduction The effect of cryotherapy on joint positioning presents conflicting debates as to whether individuals are at an increased risk of injury when returning to play following cryotherapy application at the lower limb. Objectives The aim of this study was to investigate whether a 20 minute application of crushed ice at the knee affects knee joint position sense immediately post and up to 20 mins post ice removal, during a small knee bend. Method 17 healthy male participants took part in the study performing a functional task. Using three-dimensional motion analysis (Qualisys Medical AB Gothenburg, Sweden), kinematics of the knee were measured during a weight bearing functional task pre and immediately post, 5, 10, 15 and 20 minutes post cryotherapy intervention. Skin surface temperature (Tsk) cooling was measured via infrared non-contact thermal imaging (Flir Systems, Danderyd, Sweden) over the anterior and medial aspect of the knee. Results Results demonstrated significant reductions in the ability to accurately replicate knee joint positioning. A significant increase (P ≧ 0.05) in rotational movement in the transverse plane occurred, 20 minutes post ice removal. Discussion A 20-minute application of crushed ice to the anterior aspect of the non-dominant knee has an adverse effect on knee joint repositioning and dynamic stability, 20 minutes after ice is removed. In consideration of returning a land-based athlete to dynamic functional activities, post cryotherapeutic intervention at the knee, clinicians should consider these findings due to the potential increase risk of injur

Genomic and Epigenetic Changes Drive Aberrant Skeletal Muscle Differentiation in Rhabdomyosarcoma

Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children and adolescents, represents an aberrant form of skeletal muscle differentiation. Both skeletal muscle development, as well as regeneration of adult skeletal muscle are governed by members of the myogenic family of regulatory transcription factors (MRFs), which are deployed in a highly controlled, multi-step, bidirectional process. Many aspects of this complex process are deregulated in RMS and contribute to tumorigenesis. Interconnected loops of super-enhancers, called core regulatory circuitries (CRCs), define aberrant muscle differentiation in RMS cells. The transcriptional regulation of MRF expression/activity takes a central role in the CRCs active in skeletal muscle and RMS. In PAX3::FOXO1 fusion-positive (PF+) RMS, CRCs maintain expression of the disease-driving fusion oncogene. Recent single-cell studies have revealed hierarchically organized subsets of cells within the RMS cell pool, which recapitulate developmental myogenesis and appear to drive malignancy. There is a large interest in exploiting the causes of aberrant muscle development in RMS to allow for terminal differentiation as a therapeutic strategy, for example, by interrupting MEK/ERK signaling or by interfering with the epigenetic machinery controlling CRCs. In this review, we provide an overview of the genetic and epigenetic framework of abnormal muscle differentiation in RMS, as it provides insights into fundamental mechanisms of RMS malignancy, its remarkable phenotypic diversity and, ultimately, opportunities for therapeutic intervention

Chemosensitivity profiling of osteosarcoma tumour cell lines identifies a model of BRCAness

Osteosarcoma (OS) is an aggressive sarcoma, where novel treatment approaches are required. Genomic studies suggest that a subset of OS, including OS tumour cell lines (TCLs), exhibit genomic loss of heterozygosity (LOH) patterns reminiscent of BRCA1 or BRCA2 mutant tumours. This raises the possibility that PARP inhibitors (PARPi), used to treat BRCA1/2 mutant cancers, could be used to target OS. Using high-throughput drug sensitivity screening we generated chemosensitivity profiles for 79 small molecule inhibitors, including three clinical PARPi. Drug screening was performed in 88 tumour cell lines, including 18 OS TCLs. This identified known sensitivity effects in OS TCLs, such as sensitivity to FGFR inhibitors. When compared to BRCA1/2 mutant TCLs, OS TCLs, with the exception of LM7, were PARPi resistant, including those with previously determined BRCAness LoH profiles. Post-screen validation experiments confirmed PARPi sensitivity in LM7 cells as well as a defect in the ability to form nuclear RAD51 foci in response to DNA damage. LM7 provides one OS model for the study of PARPi sensitivity through a potential defect in RAD51-mediated DNA repair. The drug sensitivity dataset we generated in 88 TCLs could also serve as a resource for the study of drug sensitivity effects in OS

The long non-coding RNA MYCNOS-01 regulates MYCN protein levels and affects growth of MYCN-amplified rhabdomyosarcoma and neuroblastoma cells

BACKGROUND: MYCN is amplified in small cell lung cancers and several pediatric tumors, including alveolar rhabdomyosarcomas and neuroblastomas. MYCN protein is known to play a key oncogenic role in both alveolar rhabdomyosarcomas and neuroblastomas. MYCN opposite strand (MYCNOS) is a gene located on the antisense strand to MYCN that encodes alternatively spliced transcripts, two of which (MYCNOS-01 and MYCNOS-02) are known to be expressed in neuroblastoma and small cell lung cancer with reciprocal regulation between MYCNOS-02 and MYCN reported for neuroblastomas. We sought to determine a functional role for MYCNOS-01 in alveolar rhabdomyosarcoma and neuroblastoma cells and identify any associated regulatory effects between MYCN and MYCNOS-01.METHODS: MYCNOS-01, MYCNOS-02 and MYCN expression levels were assessed in alveolar rhabdomyosarcoma and neuroblastoma cell lines and tumor samples from patients using Affymetrix microarray data and quantitative RT-PCR. Following MYCNOS-01 or MYCN siRNA knockdown and MYCNOS-01 overexpression, transcript levels were assayed by quantitative RT-PCR and MYCN protein expression assessed by Western blot and immunofluorescence. Additionally, effects on cell growth, apoptosis and cell cycle profiles were determined by a metabolic assay, caspase activity and flow cytometry, respectively.RESULTS: MYCNOS-01 transcript levels were generally higher in NB and RMS tumor samples and cell lines with MYCN genomic amplification. RNA interference of MYCNOS-01 expression did not alter MYCN transcript levels but decreased MYCN protein levels. Conversely, MYCN reduction increased MYCNOS-01 transcript levels, creating a negative feedback loop on MYCN protein levels. Reduction of MYCNOS-01 or MYCN expression decreased cell growth in MYCN-amplified alveolar rhabdomyosarcoma and neuroblastoma cell lines. This is consistent with MYCNOS-01-mediated regulation of MYCN contributing to the phenotype observed.CONCLUSIONS: An alternative transcript of MYCNOS, MYCNOS-01, post-transcriptionally regulates MYCN levels and affects growth in MYCN-amplified rhabdomyosarcoma and neuroblastoma cells.</p

Additional file 5: of The long non-coding RNA MYCNOS-01 regulates MYCN protein levels and affects growth of MYCN-amplified rhabdomyosarcoma and neuroblastoma cells

Figure S5. Effect of MYCNOS-01 knockdown on MYCN protein stability. (A) RMS-01 cells treated with three MYCNOS-01 siRNAs for 48 h including 4 h treatment with DMSO or MG132. Densitometry values shown above each blot normalised to GAPDH and relative to NT control for each condition. Blots representative of experiments run in triplicate. (B) MYCNOS-01 transcript expression after MYCNOS-01 knockdown for 24 h measured by qRT-PCR. NT = non-targeting control. (PDF 584 kb

Additional file 8: of The long non-coding RNA MYCNOS-01 regulates MYCN protein levels and affects growth of MYCN-amplified rhabdomyosarcoma and neuroblastoma cells

Figure S8. Knockdown of MYCNOS-02 inhibits cell viability. (Ai) MTS assay showing cell viability of RMS-01 over 96 h after transfection with three MYCNOS-02 siRNAs relative to NT siRNA. (Bi) KELLY as in (Ai). Corresponding qRT-PCR of MYCNOS-02 (ii) at 24 h shown below line graph. Data representative of two repeats. Statistical analysis relative to non-targeting control. NT = non-targeting. (PDF 82 kb

Additional file 6: of The long non-coding RNA MYCNOS-01 regulates MYCN protein levels and affects growth of MYCN-amplified rhabdomyosarcoma and neuroblastoma cells

Figure S6. Effect of MYCNOS-02 knockdown on MYCN transcript and protein expression in RMS and NB. qRT-PCR detecting MYCNOS-02 transcript level after MYCNOS-02 knockdown with three siRNAs in (A) RMS-01 and (D) KELLY. Corresponding MYCN transcript level shown in (B) RMS-01 and (E) KELLY. Expression relative to NT control. Western blots showing MYCN protein levels after MYCNOS-02 knockdown with three siRNAs shown in (C) RMS-01 and (F) KELLY. GAPDH used as loading control. Densitometry values shown above each blot normalised to GAPDH and relative to NT control. Data representative of 3 repeats. NT = non-targeting control. Relative expression of transcripts in these cell lines are indicated in Additional file 2: Figure S2E. (PDF 3709 kb