Pediatric Soft Tissue Research Articles

Abstract 1093: Small molecule inhibitors of the PAX3::FOXO1 fusion protein in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. It is commonly divided into subtypes based on histopathological appearance. The oncogenic fusion proteins PAX3::FOXO1 or PAX7::FOXO1 are present in the alveolar subtype of RMS (ARMS). These chimeric proteins are formed by reciprocal chromosomal translocations and consist of an N-terminal portion of the PAX3 or PAX7 transcription factor fused to a C-terminal portion of the FOXO1 transcription factor. These fusion proteins have been shown to drive malignant transformation in ARMS, and patients with fusion-positive ARMS have a worse prognosis than patients with fusion-negative ARMS and embryonal RMS. We recently reported that piperacetazine, a phenothiazine derivative and first-generation antipsychotic, is capable of binding to and inhibiting the transcriptional activity of PAX3::FOXO1, reducing the expression of PAX3::FOXO1 target genes, and inhibiting the ability of fusion-positive RMS cells to grow in soft agar. We hypothesize that other phenothiazine derivatives may have stronger binding affinity for PAX3::FOXO1 and greater ability to reduce tumor growth in vivo. We employed a structure-activity relationship campaign to develop new phenothiazine derivatives that may be more potent inhibitors of PAX3::FOXO1 than piperacetazine. We synthesized and tested 10 new compounds, in addition to 9 existing FDA-approved phenothiazine derivatives, for their ability to bind to PAX3::FOXO1 and inhibit its transcriptional activity and target gene expression as well as inhibiting the growth of fusion-positive RMS cells in 3D culture. Seven derivatives inhibited PAX3::FOXO1 reporter activity better than piperacetazine, nine of them performed worse, and three of them showed comparable activity to piperacetazine. We demonstrated that piperacetazine does not alter PAX3::FOXO1 levels, subcellular localization, binding to a PAX3::FOXO1 target DNA sequence, or phosphorylation at Ser256 in the FOXO1 portion of the fusion protein. We therefore hypothesize that piperacetazine may be disrupting interactions between PAX3::FOXO1 and its partner proteins. We are employing a proximity-based biotinylation method to identify proteins that are competed away from PAX3::FOXO1 by piperacetazine. Our data suggest that it is possible to target PAX3::FOXO1 protein using small molecules that can directly bind to it and inhibit its activity in fusion-positive RMS cells. Citation Format: Taryn Shaw, Kay Nakazawa, Purushottam Tiwari, Eryn Nelson, Jeffrey Formen, Christian Wolf, Jeffrey Toretsky, Aykut Üren. Small molecule inhibitors of the PAX3::FOXO1 fusion protein in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1093.

Abstract 4739: Targeting resistance to BET inhibitors in Fusion-Positive Rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most frequent pediatric soft tissue sarcoma. Fusion-positive (FP-)RMS expressing the oncogenic chimeric transcription factor (TF) PAX3-FOXO1 (P3F) is at high risk of recurrence. FP-RMS cells show survival dependency on P3F. Despite P3F, as a TF, is considered undruggable, we and others demonstrated that P3F levels/functions can be epigenetically modulated. The BET protein and epigenetic reader BRD4 binds super-enhancers (SEs) to foster oncogenic transcription in cancer. We showed that P3F rewires the RMS enhancer landscape by recruiting BRD4 on oncogenic SEs. Accordingly, JQ1, a BET inhibitor (BETi) shuts down P3F functions halting tumor growth in vitro and in vivo. Therefore, BRD4 inhibition in FP-RMS results in a tumor subtype-specific vulnerability. Nonetheless, as for other targeted therapies, cancer cells can acquire resistance to BETi, thus, we investigated whether FP-RMS cells could develop BETi resistance. Therefore, we established resistant cells from two sensitive FP-RMS cell lines chronically exposed to escalating doses of JQ1, as a model of naturally acquired resistance. Our data show that the two established FP-RMS cell lines, acquired resistance by increasing JQ1 IC50 over the sensitive cells both in 2D and 3D settings. Compared to the sensitive ones, both the resistant cell lines show cross-resistance to other monovalent and bivalent BETi and degraders. Resistant cells also maintain the ability to form colonies in a clonogenic assay and to invade in a 3D Matrigel assay in the presence of JQ1. Moreover, conversely to sensitive cells, JQ1 treatment is unable to down-regulate P3F, MYCN and BCL2 (P3F target) levels in resistant cells. Furthermore, integration of transcriptomic and proteomic data reveals differentially modulated subset of genes/proteins among which MYCN and P3F targets, suggesting a restored activity of the TFs circuitry in resistant cells. Accordingly, MYCN silencing completely halts growth of resistant cells. In line, MYCN protein levels are reduced by JQ1 in a dose-dependent manner in sensitive but not in resistant cells and its half-life is 3 folds longer in resistant than in sensitive cells. Being MYCN protein stability affected by specific post-translational modifications among which the phosphorylation at Ser62 by ERKs, we evaluated the expression of phosphorylated MYCN and the activated form of ERK (pERK) and found both of them increased in resistant cells. In agreement, resistant cells are more sensitive to MEK-ERKs pathway inhibition, showing a lower IC50 of Trametinib (MEKi) compared to sensitive cells. Moreover, Trametinib and JQ1 co-treatment downregulates MYCN protein levels in resistant but not in sensitive cells, suggesting that ERKs overactivation could participate to BETi acquired resistance by stabilizing MYCN, thus representing an acquired vulnerability. The study has received funding from Ministero della Salute to SP. Citation Format: Matteo Cassandri, Erika Ferraro, Lucrezia D'Archivio, Francesca Aiello, Hsien-Chao Chou, Young K. Song, Berkley E. Gryder, Jun S. Wei, Simone Sidoli, Robert G. Hawley, Rossella Rota, Franco Locatelli, Javed Khan, Silvia Pomella. Targeting resistance to BET inhibitors in Fusion-Positive Rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4739.

Abstract 136: Determining the genomic basis of PAX3-FOXO1 mediated transformation in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Despite rigorous scientific advances and clinical trials, the survival rate for high-risk RMS has not increased above 20% in the last three decades. A deeper understanding of the basic biology driving RMS tumorigenesis is needed to discover novel therapeutic targets. RMS can be divided into two major histologic subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS). ARMS accounts for 20% of pediatric cases and has poorer survival. The majority of ARMS tumors harbor t(2;13)(q35;q14) or t(1;13)(p36;q14) chromosomal translocations resulting in the PAX3-FOXO1 (P3F) and PAX7-FOXO1 (P7F) fusion oncoproteins, respectively. ARMS tumors that lack oncofusions more closely resemble ERMS. Classifying on fusion status more accurately describes the disease and highlights the significance of P3/7F. ARMS tumor genomes only contain an average of 6.4 somatic mutations suggesting the oncofusion protein is the key oncogenic driver and a potential candidate for therapeutic intervention. RMS is commonly thought to arise from skeletal muscle progenitor cells that fail to terminally differentiate. However, RMS occurs throughout the body in sites devoid of skeletal muscle, underscoring the potential for alternative cells of origin. Our lab established that endothelial progenitors are indeed a cell of origin for ARMS by generating a genetically engineered mouse model with endothelial cell specific P3F expression coupled with Cdkn2a loss. We further generated a human model system derived from TP53-null human induced pluripotent stem cells (iPSCs), in which we direct differentiation to endothelial cell fate and then force P3F expression. P3F expression during endothelial cell directed differentiation blocked endothelial maturation and instead reprogrammed cells shifting cell fate to skeletal muscle-like cells that form ARMS tumors in mice. Generation of iPSC-derived ARMS (iARMS) occurs over 15 days and provides a unique model to determine the kinetics and specific genomic contributions facilitated by P3F during RMS transformation. To investigate the genomic changes occurring during iARMS transformation I utilized CUT&RUN for P3F and H3K27ac to assess P3F occupancy and enhancer landscape, ATAC-seq to assess chromatin accessibility, and RNA-seq to assess gene expression changes at timepoints throughout the transformation event. These results define the fundamental genomic changes needed for transformation into FP-RMS by P3F and uncovers the key determinants of ARMS cell identity. The iARMS system provides a unique platform to uncover how P3F establishes and maintains RMS cell state and to dissect the specific dependencies required to maintain the ARMS cell state. Understanding the fundamental mechanism of P3F-driven tumorigenesis could provide greater resolution into the key disease determinants to focus pre-clinical efforts on pivotal targets. Citation Format: Bradley T. Stevens, Yang Zhang, Randolph K. Larsen, Grace E. Adkins, Jack D. Hopkins, Darden W. Kimbrough, Matthew R. Garcia, Brian J. Abraham, Mark E. Hatley. Determining the genomic basis of PAX3-FOXO1 mediated transformation in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 136.

Abstract 4015: Improvement of CAR T cell potency against low-density targets like L1CAM and FGFR4 in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Overall survival rates are low for metastatic RMS, emphasizing the urgent need of improved therapies. Chimeric Antigen Receptor (CAR) Ts have shown remarkable results against hematological malignancies, but CAR T cell therapy remains challenging in solid tumors, mainly due to a lack of ideal tumor specific targets, and of the immunosuppressive tumor microenvironment (TME). To maximize antitumoral activity against RMS and to address optimal CAR T persistence in vivo, novel CAR constructs were investigated against upregulated RMS targets. By surfaceome profiling, we recently identified L1CAM as promising candidate for RMS CAR T cell therapy, and confirmed CD276 and FGFR4 expression. CD276- (CD276.CD28HD.28TM.28CSD.3z) and Dual-CAR Ts targeting both CD276 and FGFR4 (F8-FR4.CD28HD.28TM.28CSD.3z) showed tumor eradication in an orthotopic RMS model, whereas FGFR4-CAR Ts exhibited only a partial response in vivo. Moreover, CD276-CAR Ts showed a limited activity against RMS tumors expressing lower amounts of CD276, eradicating the tumor in 1/5 mice, underlying the need of improved CAR T cell potency against low-density target-expressing tumors. First, to improve FGFR4-CAR Ts activity, we optimized the recognition of FGFR4 epitope by modifying the CAR moiety “hinge”. We replaced the CD28-derived hinge with various hinges of different length: no hinge, IgG4 (12aa), IgG4CH3 (119aa), and IgG4CH2CH3 (229aa). Luciferase assays showed effective in vitro killing capacity of FGFR4-CAR Ts expressing an IgG4-derived hinge, with a tumor cell survival less than 10% at Effector to Target (E:T) ratio of 5:1. They also released the double amount of IFN-γ (50pg/ml) compared to the other constructs, when co-incubated with Rh4 cells. Next, to verify the potential of L1CAM as CAR T target for RMS, we generated a panel of L1CAM-CARs expressing either a long (IgG4CH2CH3, 229aa) or a short (CD28, 35aa) Hinge, and a CD28- or 41BB-derived costimulatory domain (CSD). L1CAM.IgG4CH2CH3Hinge.28TM.28CSD.3z CAR Ts outperformed the other constructs, killing ~60% of Rh30 at the E:T ratio of 1:1 and more than 90% at the E:T ratio of 5:1. They also exhibited a 3-fold higher IFN-γ release (120 pg/ml) compared to the other constructs. However, although our examination of patient-derived TMA revealed a strong immunoreactivity for L1CAM, more expressed in aggressive RMS subtypes, the selected L1CAM-CAR Ts against Rh30 cells, exhibiting ~10’000 L1CAM copies, showed limited activity in an in vivo pilot study. Based on these results and on the RMS TME investigation, which revealed consistent collagen (COL4A1, COL4A2 and COL18A1) deposition in RMS-derived mice tumor xenografts, we hypothesize that co-expressing a collagenase, able to partially degrade the dense extracellular matrix surrounding tumors, will lead to an improved CAR T cell infiltration and activity in vivo. Citation Format: Caroline Piccand, Andrea Timpanaro, Stenija Anton-Joseph, Michele Bernasconi, Jochen Karl Rössler. Improvement of CAR T cell potency against low-density targets like L1CAM and FGFR4 in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4015.

Abstract 722: A cancer-associated fibroblast model to discover mechanisms of growth, survival, and treatment sensitivity in PAX3/7-FKHR fusion-positive alveolar rhabdomyosarcoma

Abstract Introduction: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Among various subtypes, alveolar RMS (ARMS) is generally more aggressive and usually carries the fusion transcription factor, PAX3/7-FKHR. High-risk ARMS has an extremely poor prognosis, with ~25% five-year survival. Hence, novel therapeutic approaches are urgently needed. However, potential therapies are often hindered by the immunosuppressive tumor microenvironment (TME) associated with sarcomas. The TME is highly dynamic and includes fibroblasts, immune cells, and extracellular matrix. Cancer-associated fibroblasts (CAFs) may promote tumor growth and survival by secreting distinct factors to regulate proliferation, metastasis, and resistance to therapy. In this study, we investigated mechanisms involved in this phenomenon and identified a novel therapeutic strategy targeting the TME of ARMS. Methods: Cells were isolated from a PAX3-FKHR fusion-positive ARMS tumor specimen, following ethics approval and informed consent. The PAX3-FKHR fusion status and mesenchymal biomarkers were analyzed by immunoblotting. The patient’s CAFs (designated KCCF14), RMS lines (RD, RH30, RH41), and control fibroblasts (BJ, HS68, WI-38) were cultured in vitro and their respective supernatant media was harvested for subsequent experiments. Cell proliferation and viability were determined by hemocytometer and alamar blue assay, respectively. The scratch wound healing assay was used to assess cell migration. Cytokine analysis was performed with a 96-plex array. For drug screening, cells were treated with a panel of CXCR4 antagonists to generate dose-response curves. Results: KCCF14 CAFs isolated from an ARMS tumor exhibit elongated spindle morphology and are positive for mesenchymal biomarkers, including PDGFR-β and smooth muscle α-actin. As expected, these cells lack characteristics of the patient’s tumor, notably the expression of PAX3-FKHR fusion and N-myc. KCCF14-conditioned media significantly enhances ARMS cell proliferation and migration in a concentration-dependent manner. Analysis of the conditioned media revealed upregulation of key growth factors and cytokines, including PDGF-AA, VEGF-A, MCP-1, and IL-11, that promote tumor aggressiveness through proliferative signaling, angiogenesis, activation of migration, and immune evasion. Because SDF-1 (CXCR4 ligand) was also increased, we tested targeted inhibition of CXCR4, which induced cytotoxicity with increased sensitivity (IC50 = 0.5 µM) in KCCF14 cells compared to control fibroblasts. Conclusions: This study provides, for the first time, findings from a continuously growing CAF model system for RMS. Our data describes key tumor growth and survival mechanisms supported by the TME and establishes a method to identify treatments with novel therapeutic strategies. Citation Format: Patrick Sipila, Son Tran, Chunfen Zhang, Anne–Marie Langevin, Aru Narendran. A cancer-associated fibroblast model to discover mechanisms of growth, survival, and treatment sensitivity in PAX3/7-FKHR fusion-positive alveolar rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 722.

Frontline and Relapsed Rhabdomyosarcoma (FaR-RMS) Clinical Trial: A Report from the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG).

The Frontline and Relapsed Rhabdomyosarcoma (FaR-RMS) clinical trial is an overarching, multinational study for children and adults with rhabdomyosarcoma (RMS). The trial, developed by the European Soft Tissue Sarcoma Study Group (EpSSG), incorporates multiple different research questions within a multistage design with a focus on (i) novel regimens for poor prognostic subgroups, (ii) optimal duration of maintenance chemotherapy, and (iii) optimal use of radiotherapy for local control and widespread metastatic disease. Additional sub-studies focusing on biological risk stratification, use of imaging modalities, including [18F]FDG PET-CT and diffusion-weighted MRI imaging (DWI) as prognostic markers, and impact of therapy on quality of life are described. This paper forms part of a Special Issue on rhabdomyosarcoma and outlines the study background, rationale for randomisations and sub-studies, design, and plans for utilisation and dissemination of results.

Pediatric Hemangiomas in the Female Genital Tract: A Literature Review.

Hemangiomas are aberrant proliferations of blood vessels and the most frequent benign pediatric soft tissue tumors. Although they are common, genital localization is rare. This study aimed to assist doctors in the diagnosis, management, and treatment of pediatric vulvovaginal hemangiomas by conducting a review of the literature. We conducted a literature review including papers published between August 2009 and May 2023. While most hemangiomas are usually indolent and resolve with time, in some cases, especially cervicovaginal and uterine ones, they might present with severe symptoms like heavy bleeding and need further instrumental investigation for diagnosis, like CT or MRI. As for the treatment, many options are available, with medical therapy or expectant management being the first choice. Vulvovaginal pediatric hemangiomas are rare and require more research on how to detect and manage these lesions, especially the symptomatic and the psychologically impacting ones. For the time being, treatment should be personalized based on the patient's situation and clinician's expertise.

Reappraisal of prognostic factors used in the European Pediatric Soft Tissue Sarcoma Study Group RMS 2005 study for localized rhabdomyosarcoma to optimize risk stratification and generate a prognostic nomogram.

The objective of this study was to investigate the role of clinical factors together with FOXO1 fusion status in patients with nonmetastatic rhabdomyosarcoma (RMS) to develop a predictive model for event-free survival and provide a rationale for risk stratification in future trials. The authors used data from patients enrolled in the European Pediatric Soft Tissue Sarcoma Study Group (EpSSG) RMS 2005 study (EpSSG RMS 2005; EudraCT number 2005-000217-35). The following baseline variables were considered for the multivariable model: age at diagnosis, sex, histology, primary tumor site, Intergroup Rhabdomyosarcoma Studies group, tumor size, nodal status, and FOXO1 fusion status. Main effects and significant second-order interactions of candidate predictors were included in a multiple Cox proportional hazards regression model. A nomogram was generated for predicting 5-year event-free survival (EFS) probabilities. The EFS and overall survival rates at 5years were 70.9% (95% confidence interval, 68.6%-73.1%) and 81.0% (95% confidence interval, 78.9%-82.8%), respectively. The multivariable model retained five prognostic factors, including ageat diagnosis interacting with tumor size, tumor primary site, Intergroup Rhabdomyosarcoma Studies clinical group, and FOXO1 fusion status. Based on each patient's total score in the nomogram, patients were stratified into four groups. The 5-year EFS rates were 94.1%, 78.4%, 65.2%, and 52.1% in the low-risk, intermediate-risk, high-risk, and very-high-risk groups, respectively, and the corresponding 5-year overall survival rates were 97.2%, 91.5%, 74.3%, and 60.8%, respectively. The results presented here provide the rationale to modify the EpSSG stratification, with the most significant change represented by the replacement of histology with fusion status. This classification was adopted in the new international trial launched by the EpSSG.

Statin-Sensitive Akt1/Src/Caveolin-1 Signaling Enhances Oxidative Stress Resistance in Rhabdomyosarcoma.

Identifying the molecular mechanisms underlying radioresistance is a priority for the treatment of RMS, a myogenic tumor accounting for approximately 50% of all pediatric soft tissue sarcomas. We found that irradiation (IR) transiently increased phosphorylation of Akt1, Src, and Cav1 in human RD and RH30 lines. Synthetic inhibition of Akt1 and Src phosphorylation increased ROS levels in all RMS lines, promoting cellular radiosensitization. Accordingly, the elevated activation of the Akt1/Src/Cav1 pathway, as detected in two RD lines characterized by overexpression of a myristoylated Akt1 form (myrAkt1) or Cav1 (RDCav1), was correlated with reduced levels of ROS, higher expression of catalase, and increased radioresistance. We found that treatment with cholesterol-lowering drugs such as lovastatin and simvastatin promoted cell apoptosis in all RMS lines by reducing Akt1 and Cav1 levels and increasing intracellular ROS levels. Combining statins with IR significantly increased DNA damage and cell apoptosis as assessed by γ histone 2AX (γH2AX) staining and FACS analysis. Furthermore, in combination with the chemotherapeutic agent actinomycin D, statins were effective in reducing cell survival through increased apoptosis. Taken together, our findings suggest that the molecularly linked signature formed by Akt1, Src, Cav1, and catalase may represent a prognostic determinant for identifying subgroups of RMS patients with higher probability of recurrence after radiotherapy. Furthermore, statin-induced oxidative stress could represent a treatment option to improve the success of radiotherapy.

Evaluation of clinical and imaging features for differentiating rhabdomyosarcoma from neuroblastoma in pediatric soft tissue.

In this study, we developed a nomogram predictive model based on clinical, CT, and MRI parameters to differentiate soft tissue rhabdomyosarcoma (RMS) from neuroblastoma (NB) in children preoperatively. A total of 103 children with RMS (n=37) and NB (n=66) were enrolled in the study from December 2012 to July 2023. The clinical and imaging data (assessed by two experienced radiologists) were analyzed using univariate analysis, and significant factors were further analyzed by multivariable logistic regression using the forward LR method to develop the clinical model, radiological model, and integrated nomogram model, respectively. The diagnostic performances, goodness of fit, and clinical utility of the integrated nomogram model were assessed using the area under the curve (AUC) of the receiver operator characteristics curve (ROC) with a 95% confidence interval (95% CI), calibration curve, and decision curve analysis (DCA) curves, respectively. Diagnostic efficacy between the model and radiologists' interpretations was examined. The median age at diagnosis in the RMS group was significantly older than the NB group (36.0 months vs. 14.5 months; P=0.003); the fever rates in RMS patients were significantly lower than in patients with NB (0.0% vs.16.7%; P=0.022), and the incidence of palpable mass was higher in patients with RMS compared with the NB patients (89.2% vs. 34.8%; P<0.001). Compare NB on image features: RMS occurred more frequently in the head and neck and displayed homogeneous density on non-enhanced CT than NB (48.6% vs. 9.1%; 35.3% vs. 13.8%, respectively; all P<0.05), and the occurrence of characteristics such as calcification, encasing vessels, and intraspinal tumor extension was significantly less frequent in RMS children compared to children with NB (18.9% vs. 84.8%; 13.5% vs. 34.8%; 2.7% vs. 50.0%, respectively; all P <0.05). Two, three, and four features were identified as independent parameters by multivariate logistic regression analysis to develop the clinical, radiological, and integrated nomogram models, respectively. The AUC value (0.962), calibration curve, and DCA showed that the integrated nomogram model may provide better diagnostic performance, good agreement, and greater clinical net benefits than the clinical model, radiological model, and radiologists' subjective diagnosis. The clinical and imaging features-based nomogram has potential for helping radiologists distinguish between pediatric soft tissue RMS and NB patients preoperatively, and reduce unnecessary interventions.

Clinical Characteristics and Treatment Outcomes of Pediatric Bacterial Skin and Soft Tissue Infections in Central Vietnam: A Prospective Study.

Objective. This study examined bacterial skin and soft tissue infections (SSTIs) in pediatric patients at a hospital in Vietnam. Methods. An April 2022-June 2023 prospective obsevational study at a Tertiary Paediatric Centre in Vietnam. Results. From We analyzed 87 cases and found that the greatest occurrence rates were reported during the summer season. Common symptoms were fever, edema, and lower leg involvement. Curiously, although blood cultures seldom verified the offender, Staphylococcus aureus was the most common cause. Additionally, the study found that children with infections including pus were administered a more extended course of antibiotics. Approximately one-third of the patients underwent surgical procedures, and most of them were administered intravenous antibiotics. Fortunately, all the youngsters made a full recovery. Conclusions. In summary, the results indicate that children with purulent SSTIs, primarily attributed to Staphylococcus aureus, may need longer courses of antibiotics. Intravenous antibiotic administration emerged as the predominant therapeutic modality.

Solitary myofibroma in children: a report of two cases

Myofibromas are rare benign tumors of myofibroblasts, seen more commonly in children. These tumors typically involve soft tissues with a predilection for the head and neck. Malignant neoplasia is often suspected for these rapidly growing tumors in early childhood. Clinical and radiological findings are not typical, and histopathological examination makes the definitive diagnosis. This pathology requires the intervention of a multidisciplinary team and regular follow-up. We report our experience with two children with myofibromas, one in the right arm of a 5-year-old girl and the other in the right axilla of a 9-year-old boy. Tumors were totally resected in both children, who are under follow-up with no adjuvant treatment free of disease after surgery. Myofibroma should be considered in the differential diagnosis of pediatric soft tissue tumors. Surgical resection is sufficient for treatment and patients should be followed regularly for possible recurrences.

Abstract A096: RAS mutation status predicts activity of the SHP2 inhibitor TNO155 in RAS-pathway driven fusion-negative rhabdomyosarcoma

Abstract Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue sarcoma. Fusion-negative RMS (FN-RMS, the subset lacking FOXO1 translocations), are characterized by oncogenic point mutations in genes that regulate activity of the RAS/ERK signaling pathway. Clinically available agents that directly target oncogenic RAS in cancer have only recently become FDA approved, and their activity is limited to cancers with G12C mutations in KRAS. Alternate molecular strategies to target RAS activation in cancer include inhibitors of RAS regulatory proteins, including the nonreceptor tyrosine phosphatase SHP2. SHP2 regulates RAS signaling by dephosphorylating receptor tyrosine kinases (RTKs), preventing effective RAS GTPase-activating protein (GAP) recruitment. Additionally, SHP2 recruits SOS1, a RAS guanine nucleotide exchange factor (GEF), promoting RAS activation. Novel small molecule inhibitors of SHP2 are now in clinical trials in several cancer types. We set out to determine whether SHP2 inhibitors demonstrate preclinical activity in models of RAS-pathway driven FN-RMS. We hypothesize that specific oncogenic mutations in HRAS, NRAS and KRAS, affecting codons 12, 13 and 61, will determine SHP2 inhibitor sensitivity in models of FN-RMS, and that sensitivity will be determined based on the interactions between RAS and RAS-GAPs/RAS-GEFs. We tested the effects of SHP2 inhibition on ERK pathway signaling, using the small molecule inhibitor TNO155, in a panel of FN-RMS cell lines with varied RAS mutation status. In FN-RMS cells with Q61-mutant HRAS or NRAS, phospho-MEK and phospho-ERK were unchanged, while in cells with RTK-amplification and wild-type RAS, or those with G12 or G13-mutant HRAS we observed reductions in p-MEK and p-ERK following treatment with TNO155. Reduced 2-D and 3-D cell growth were also observed in WT and G12/G13-mutant HRAS cell lines, but not those with Q61 mutations, using both high throughput proliferation assays and soft-agar colony formation assays, respectively. Responses in sensitive cells were modest and cytostatic at best, suggesting single agent SHP2i may have insufficient activity in these tumors. RNAseq analysis of TNO155-sensitive RAS-WT RMS cells reveals a set of differentially expressed genes that may inform the selection of agents for combination therapy. In addition, a subset of FN-RMS cells also harbor activating mutations in FGFR and PI3K-associated genes, and we therefore tested whether TNO155 might synergize with FGFR or PI3K small-molecule inhibitors and found additive effects between TNO155, the PI3Ki alpelisib, and the pan-FGFRi erdafitinib. In conclusion, FN-RMS cells driven by RTK-amplification, or by RAS G12 or G13 mutations, are sensitive to TNO155 while cells driven by RAS Q61 mutations are not. Additionally, FN-RMS cells treated with TNO155 may rely on the PI3K pathway for adaptive resistance, and combination treatment involving TNO155 and FGFR or PI3K inhibitors may reduce tumor growth with better efficacy than either compound alone. Citation Format: Andrew Baker, Patience Odeniyide, Alyza Skaist, Alla Lisok, Elana Fertig, Jiawan Wang, Christine Pratilas. RAS mutation status predicts activity of the SHP2 inhibitor TNO155 in RAS-pathway driven fusion-negative rhabdomyosarcoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A096.

Rapid liposomal formulation for nucleolin targeting to rhabdomyosarcoma cells

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. More effective and less toxic therapies are urgently needed for high-risk patients. Peptide-guided targeted drug delivery can increase the therapeutic index of encapsulated drugs and improve patients’ well-being. To apply this strategy to RMS, we identified the peptide F3 in a screening for peptides binding to RMS cells surface. F3 binds to nucleolin, which is present on the surface of RMS cells and is abundantly expressed at the mRNA level in RMS patients’ biopsies compared to healthy tissues. We developed a rapid microfluidic formulation of F3-decorated PEGylated liposomes and remote loading of the chemotherapeutic drug vincristine. Size, surface charge, drug loading and retention of targeted and control liposomes were studied. Enhanced cellular binding and uptake were observed in three different nucleolin-positive RMS cell lines. Importantly, F3-functionalized liposomes loaded with vincristine were up to 11 times more cytotoxic than non-targeted liposomes for RMS cell lines. These results demonstrate that F3-functionalized liposomes are promising for targeted drug delivery to RMS and warrant further in vivo investigations.

Pediatric Microsurgery and Free-Tissue Transfer.

Advancements in microsurgery, along with increased microsurgical experience in pediatric patients, have made free-tissue transfer a reliable modality for pediatric bone and soft tissue reconstruction today. Free-tissue transfer is most commonly used in children for the coverage of large or complex defects resulting from traumatic, oncologic, or congenital etiologies. While flap success and complication rates between pediatric and adult populations are similar, special considerations must be taken into account within the pediatric population. In this article, we will describe common indications, technical nuances, and clinical considerations for the management of the pediatric free-tissue transfer patient.

Management of Soft Tissue Injuries in Children-A Comprehensive Review.

Airway injury, Ocular injury and neurovascular tissue damage, burns is all a spectrum of pediatric soft tissue injury complex. Soft tissue injuries to the head and neck area in children are challenging to manage, because these injuries significantly affect the child's overall health and development. Management of such injuries requires a multidisciplinary approach involving surgical and nonsurgical interventions and close collaboration among health care professionals, parents, and caregivers. This article reviews the various causes of injuries, specific considerations for each region of the head and neck, and approaches to the surgical management of soft tissue injuries in pediatric patients, including surgical and adjuvant therapies. Specific anatomic regions reviewed include the scalp/forehead, periorbital region, nose, cheeks, lips, ears, and neck/airway.Laceration repair in the growing pediatric populations may require revisions in the future. Facial soft tissue injuries are prone to poor cosmesis as in many occasions as may be constrained by available surgical specialists, thus proper multispecialty team approach along with surgical alignment and symmetry should be considered comprehensively.

Indeterminate pulmonary nodules in non-rhabdomyosarcoma soft tissue sarcoma: A study of the European paediatric Soft Tissue Sarcoma Study Group.

The aim of this study was to assess the clinical impact of indeterminate pulmonary nodules (no more than four pulmonary nodules of less than 5mm or one nodule measuring between 5 and less than 10mm by computed tomography [CT]) in children and adolescents with adult-type non-rhabdomyosarcoma soft tissue sarcoma (NRSTS) at diagnosis. Patients with NRSTS treated in 11 centers as part of the European paediatric Soft Tissue Sarcoma Study Group (EpSSG) were retrospectively assessed. Local radiologists, blinded to clinical information except for patients' age and tumor histotype, reviewed the chest CT at diagnosis and filled out a case report form. Because patients with or without indeterminate nodules in the EpSSG NRSTS 2005 study received the same type of treatment, event-free survival (EFS) and overall survival (OS) between groups by log-rank test were compared. Overall, 206 patients were examined: 109 (52.9%) were without any nodules, 78 (38%) had at least one indeterminate nodule, and 19 (9.2%) had nodules meeting the definition of metastases, which were then considered to be misclassified and were excluded from further analyses. Five-year EFS was 78.5% (95% CI, 69.4%-85.1%) for patients without nodules and 69.6% (95% CI, 57.9%-78.7%) for patients with indeterminate nodules (p=.135); 5-year OS was 87.4% (95% CI, 79.3%-92.5%) and 79.0% (95% CI, 67.5%-86.8%), respectively (p=.086). This study suggests that survival does not differ in otherwise nonmetastatic patients with indeterminate pulmonary nodules compared to nonmetastatic patients without pulmonary nodules. Radiologists should be aware of the classification of indeterminate pulmonary nodules in non-rhabdomyosarcoma soft tissue sarcomas and use it in their reports. More than a third of patients with non-rhabdomyosarcoma soft tissue sarcoma can be affected by indeterminate pulmonary nodules. Indeterminate pulmonary nodules do not significantly affect the overall survival of pediatric patients with non-rhabdomyosarcoma soft tissue sarcoma.

Impact of Consolidative Radiation on Overall and Progression-Free Survival in Pediatric, Adolescent, and Young Adult Metastatic Bone and Soft Tissue Sarcoma

To determine the association between consolidative radiation (RT) and survival in children, adolescents, and young adults with metastatic sarcoma. Eligibility criteria included patients aged ≤39 years with newly diagnosed metastatic bone or soft tissue sarcoma who completed local control of the primary tumor without disease progression. Consolidative RT was defined as RT to all known sites of metastatic disease. The Kaplan-Meier method was used to estimate overall survival (OS) and progression-free survival (PFS). The least absolute shrinkage and selection operator Cox provided adjusted estimates. To account for immortal time bias, consolidative RT was used as a time-varying covariate in a time dependent Cox model. Distant failure was estimated using the Fine-Gray model. Patients (n=85) had a median age at diagnosis of 14.8 years. Most common histology was Ewing Sarcoma (45.9%) followed by rhabdomyosarcoma (40.0%). Receipt of consolidative RT was associated with Ewing Sarcoma (P < .001) and local control modality as those who underwent local control with surgery and RT compared with surgery alone were more likely to be treated with consolidative RT (P=.034). Consolidative RT was independently associated with improved OS (hazard ratio [HR], 0.41; 95% CI, 0.17-0.98; P=.045) and improved PFS (HR, 0.37; 95% CI, 0.16-0.88; P=.024) after adjusting for confounding variables and immortal time bias. Patients treated with consolidative RT also experienced a lower risk of distant failure (HR, 0.33; 95% CI, 0.17-0.64; P=.001). In an independent data set of patients with metachronous progression (n=36), consolidative RT remained independently associated with improved OS. Consolidative RT was independently associated with improved OS and PFS and decreased risk of distant failure in child, adolescent, and young adult patients with metastatic sarcoma. Future work should evaluate biomarkers to optimize patient selection, timing, and dose for consolidative RT.

The Incidence of Multiple Fusions in a Series of Pediatric Soft Tissue and Bone Tumors.

Next generation sequencing (NGS) has increased the detection of fusion genes in cancer. NGS has found multiple fusions in single tumor samples; however, the incidence of this in pediatric soft tissue and bone tumors (PSTBTs) is not well documented. The aim of this study is to catalogue the incidence of multiple fusions in a series of PSTBTs, and apply a modified gene fusion classification system to determine clinical relevance. RNA from 78 bone and soft tissue tumors and 7 external quality assessment samples were sequenced and analyzed using recently-described Metafusion (MF) software and classified using a modification of previously-published schema for fusion classification into 3 tiers: 1, strong clinical significance; 2, potential clinical significance; and 3, unknown clinical significance. One-hundred forty-five fusions were detected in 85 samples. Fifty-five samples (65%) had a single fusion and 30 (35%) had more than 1 fusion. No samples contained more than 1 tier 1 fusion. There were 40 tier 1 (28%), 36 tier 2 (24%), and 69 (48%) tier 3 fusions. A significant percentage of PSTBTs harbor more than 1 fusion, and by applying a modified fusion classification scheme, the potential clinical relevance of such fusions can be determined.

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

ObjectiveTo 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 methodsWe 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.ResultsOf 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.ConclusionA 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.Graphical