Rhabdomyosarcoma Patients Research Articles

Adult Prostate Sarcoma: Demographics, Treatment Patterns, and Survival

BackgroundThis study aimed to examine clinicopathologic characteristics, treatment patterns, and survival rates in a contemporary population-based cohort of adult prostate sarcoma patients.MethodsIn the Surveillance, Epidemiology, and End Results database (2004–2020), adult patients with prostate sarcoma were identified. Descriptive statistics, Kaplan–Meier analyses, smoothed cumulative incidence plots, and Cox regression models were used.ResultsOf 125 patients, 45 (36%) harbored leiomyosarcoma, 17 (14%) had rhabdomyosarcoma, 15 (12%) had stromal sarcoma, 17 (14%) had sarcoma not otherwise specified (NOS), and 31 (25%) had other sarcoma subtypes. Metastatic stage was most common in the rhabdomyosarcoma patients (44%) and least common in the leiomyosarcoma (21%) and stromal sarcoma (20%) patients. Most of the rhabdomyosarcoma patients received the combination of systemic and radiation therapy with (24%) or without radical surgery (35%), whereas most of the leiomyosarcoma and stromal sarcoma patients underwent radical surgery with (22 and 13%) or without (22 and 47%) radiation. In the overall population, the median overall survival was 27 months. The 5-years overall versus cancer-specific versus other-cause mortality rates were respectively 71 versus 58 versus 13%. In the multivariable Cox regression models, the highest overall mortality was exhibited by the patients with metastatic disease (hazard ratio [HR] 2.87; 95% confidence interval [CI] 1.55–5.31; p < 0.001) or unknown disease stage (HR 2.94; 95% CI 2.20–7.21; p = 0.019). Conversely, of all the histologic subtypes, only stromal sarcoma distinguished itself by lower overall mortality (HR 0.41; 95% CI 0.18–0.96; p = 0.039).ConclusionsFour major histologic subtypes were identified. Among most adult sarcoma patients, treatment patterns vary according to histology, from multimodal therapy to radical prostatectomy alone. These treatment differences reflect equally important heterogeneity in survival patterns.

Improving Individualized Rhabdomyosarcoma Prognosis Predictions Using Somatic Molecular Biomarkers.

Molecular markers, such as FOXO1 fusion genes and TP53 and MYOD1 mutations, increasingly influence risk-stratified treatment selection for pediatric rhabdomyosarcoma (RMS). This study aims to integrate molecular and clinical data to produce individualized prognosis predictions that can further improve treatment selection. Clinical variables and somatic mutation data for 20 genes from 641 RMS patients in the United Kingdom and the United States were used to develop three Cox proportional hazard models for predicting event-free survival (EFS). The 'Baseline Clinical' (BC) model included treatment location, age, fusion status, and risk group. The 'Gene Enhanced 2' (GE2) model added TP53 and MYOD1 mutations to the BC predictors. The 'Gene Enhanced 6' (GE6) model further included NF1, MET, CDKN2A, and MYCN mutations, selected through LASSO regression. Model performance was assessed using likelihood ratio (LR) tests and optimism-adjusted, bootstrapped validation and calibration metrics. The GE6 model demonstrated superior predictive performance, offering 39% more predictive information than the BC model (LR p<0.001) and 15% more than the GE2 model (LR p<0.001). The GE6 model achieved the highest discrimination with a C-index of 0.7087, a Nagalkerke R2 of 0.205, and appropriate calibration. Mutations in TP53, MYOD1, CDKN2A, MET, and MYCN were associated with higher hazards, while NF1 mutation correlated with lower hazard. Individual prognosis predictions varied between models in ways that may suggest different treatments for the same patient. For example, the 5-year EFS for a 10-year-old patient with high-risk, fusion-negative, NF1-positive disease was 50.0% (95% confidence interval: 39-64%) from BC but 76% (64-90%) from GE6. Incorporating molecular markers into RMS prognosis models improves prognosis predictions. Individualized prognosis predictions may suggest alternative treatment regimens compared to traditional risk-classification schemas. Improved clinical variables and external validation are required prior to implementing these models into clinical practice.

Clinicopathological Analysis of Rhabdomyosarcoma Patients Undergone Radiotherapy in Two Referral Hospitals in West Java

Clinicopathological Analysis of Rhabdomyosarcoma Patients Undergone Radiotherapy in Two Referral Hospitals in West Java

PAX fusion proteins deregulate gene networks controlling mitochondrial translation in pediatric rhabdomyosarcoma.

Alveolar rhabdomyosarcoma (ARMS) patients harboring PAX3-FOXO1 and PAX7-FOXO1 fusion proteins exhibit a greater incidence of tumor relapse, metastasis, and poor survival outcome, thereby underscoring the urgent need to develop effective therapies to treat this subtype of childhood cancer. To uncover mechanisms that contribute to tumor initiation, we developed a novel muscle progenitor model and used epigenomic approaches to unravel genome re-wiring events mediated by PAX3/7 fusion proteins. Importantly, these regulatory mechanisms are conserved across established ARMS cell lines, primary tumors, and orthotopic-patient derived xenografts. Among the key targets of PAX3- and PAX7-fusion proteins, we identified a cohort of oncogenes, FGF receptors, and genes essential for mitochondrial metabolism and protein translation, which we successfully targeted in preclinical trials. Our data suggest an explanation for the relative paucity of recurring mutations in this tumor, provide a compelling list of actionable targets, and suggest promising new strategies to treat this tumor.

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

BackgroundDespite 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.MethodsRH30, 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.ResultsSFX-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.ConclusionsIn 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.

Correlation of Genetic Polymorphism of CYP3A5 to Cyclophosphamide Efficacy and Toxicity in Rhabdomyosarcoma Pediatric Egyptian Cancer Patients.

Rhabdomyosarcoma (RMS) accounts for 50% of soft tissue sarcomas and 7% of pediatric malignancies. Cyclophosphamide (CPA) is the cornerstone of therapy and is a prodrug that is activated by the highly polymorphic drug-metabolizing enzyme CYP3A5. We aim to examine the possible CYP3A5 polymorphism association with CPA efficacy, survival outcomes, and toxicity in Egyptian pediatric RMS patients. The three non-functional SNPs, CYP3A5*3 rs776746 (C_26201809_30), CYP3A5*6 rs10264272 (C_30203950_10), and CYP3A5*7 rs41303343 (C_32287188_10) were genotyped by real-time PCR. We conducted a cohort retrospective study of 150 pediatric RMS patients treated with CPA-based first-line treatment to analyze the association between these genotypes and CPA efficacy/toxicities in RMS patients. The frequency of having normal, intermediate, and poor metabolizers was 4.7%, 34%, and 61.3%, respectively. There was an association between these different phenotypes, genotypes, and CPA efficacy/toxicity. Hemorrhagic cystitis and pancytopenia were present in all patients, while nephrotoxicity incidence was 87.3%. There was a notable difference in the occurrence of hemorrhagic cystitis among CYP3A5 intermediate metabolizers *1/*3, *1/*6, and poor metabolizers *3/*3, *3/*6 with a significance level of p<0.05. Neither CYP3A5*7 polymorphism nor *6/*6 genotype was identified in our study. Our results demonstrate that CYP3A5*3 (rs776746) and CYP3A5*6 (rs10264272) have a great association with CPA efficacy and toxicity in RMS patients.

Targeted immunotherapy and nanomedicine for rhabdomyosarcoma: The way of the future.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. Histology separates two main subtypes: embryonal RMS (eRMS; 60%-70%) and alveolar RMS (aRMS; 20%-30%). The aggressive aRMS carry one of two characteristic chromosomal translocations that result in the expression of a PAX3::FOXO1 or PAX7::FOXO1 fusion transcription factor; therefore, aRMS are now classified as fusion-positive (FP) RMS. Embryonal RMS have a better prognosis and are clinically indistinguishable from fusion-negative (FN) RMS. Next to histology and molecular characteristics, RMS risk groupings are now available defining low risk tumors with excellent outcomes and advanced stage disease with poor prognosis, with an overall survival of about only 20% despite intensified multimodal treatment. Therefore, development of novel effective targeted strategies to increase survival and to decrease long-term side effects is urgently needed. Recently, immunotherapies and nanomedicine have been emerging for potent and effective tumor treatments with minimal side effects, raising hopes for effective and safe cures for RMS patients. This review aims to describe the most relevant preclinical and clinical studies in immunotherapy and targeted nanomedicine performed so far in RMS and to provide an insight in future developments.

The effectiveness of adjuvant therapy in adult patients with spindle cell/sclerosing rhabdomyosarcoma: A single-center retrospective study.

e23550 Background: In recent years, the WHO classification of soft tissue tumours has classified spindle cell/sclerosing rhabdomyosarcoma (RMS) as a novel subtype of RMS. However, the prognosis and standard treatment modalities for operable spindle cell/sclerosing RMS patients remain unclear. This study aims to investigate the effectiveness and influencing factors of postoperative adjuvant therapy in adult patients with spindle cell/sclerosing RMS. Materials and Methods: This single-center retrospective study enrolled adult patients with spindle cell/sclerosing RMS who underwent surgery and subsequent adjuvant therapy at West China Hospital, Sichuan University, from August 2014 to March 2023. The included patients initially underwent surgical excision, followed by one of the adjuvant treatments: radiotherapy, chemotherapy, or a combination of radiotherapy and chemotherapy. Disease-Free Survival (DFS) and Overall Survival (OS) were calculated for the included patients in this study. Univariate analysis and multivariate COX regression were conducted to determine prognostic factors. Results: This study included a total of 17 patients diagnosed with spindle cell/sclerosing RMS. Among them, 10 (58.8%) patients received postoperative adjuvant chemoradiotherapy, while 7 (41.2%) patients underwent postoperative chemotherapy alone. 12 (70.6%) patients received chemotherapy following the VAC regimen, and 5 (29.4%) patients received non-VAC chemotherapy. The prognosis analysis indicates that the median DFS was not reached, with a 1-year DFS rate of approximately 87.8% and a 2-year DFS rate of approximately 52.4%. The median OS was not reached, with a 1-year OS rate of 100% and a 2-year OS rate of approximately 92.9%. Univariate analysis shows that compared to undergoing non-R0 resection, undergoing R0 surgical resection significantly improves patient DFS (P = 0.02). Compared to no postoperative radiotherapy, receiving postoperative prophylactic radiotherapy significantly improved both DFS (P = 0.01) and OS (P = 0.02). In contrast, using VAC as adjuvant chemotherapy did not significantly increase DFS and OS compared to non-VAC regimens (P = 0.83 and 0.57, respectively). Multivariate Cox regression analysis demonstrated that the receipt of postoperative prophylactic radiotherapy independently predicted DFS (P = 0.03). Conclusions: In adult patients with spindle cell/sclerosing RMS, R0 resection, when compared with non-R0 resection, delays disease recurrence but does not enhance overall survival. Long-term outcomes may be improved in patients who receive postoperative adjuvant radiotherapy. However, choosing a VAC regimen as adjuvant chemotherapy may not contribute to an improved long-term prognosis when compared with a non-VAC regimen. Keywords: adjuvant therapy, spindle cell, sclerosing, rhabdomyosarcoma, effectiveness

Outcome of children and young adults with localized extremity rhabdomyosarcoma treated on Children's Oncology Group trials.

10006 Background: Extremity rhabdomyosarcoma (RMS) accounts for 15-20% of patients with localized RMS. The long-term survival and associated prognostic factors of extremity RMS patients treated on the contemporary Children’s Oncology Group (COG) trials have not been reported. Methods: Patients with localized extremity RMS enrolled on the D9602, D9803, ARST0331 and ARST0531 COG trials were analyzed. The Kaplan-Meyer method estimated event-free (EFS) and overall survival (OS). The Log-rank test and Cox regression were used to estimate the prognostic significance of age at diagnosis, sex, race, tumor site &amp; size, IRS group, stage, histology, FOXO1 fusion status, anaplasia, lymph node involvement, delayed primary excision (DPE) and radiation on survival. Results: We identified 159 patients with localized extremity RMS; 108 (68%) were &lt; 10 years at diagnosis, 84(53%) had primary tumor &gt; 5 cm, and 35 (22%) and 98 (62%) had IRS group II and III disease. Histology was alveolar in 106 (67%), and 43 (27%) had regional lymph node involvement on imaging or pathology. Seventeen were treated on low-risk (LR) studies D9602 (N = 10) and ARST0331 (N = 7), whereas 142 were treated on intermediate-risk (IR) trials D9803 (N = 85) and ARST0531 (N = 57). Eighty patients (50%) underwent primary surgical excision and 121 (76%) received radiotherapy. DPE was attempted in 60 patients (38%), resulting in an R0 (microscopically negative) margin in 48 (80%). Relapse/progression occurred in 52 patients: metastatic 26 (50%), local 22 (42%), and combined 3(6%). The 5-year EFS and OS of the whole cohort were 62.6% (95% CI: 54%-71.2%) and 78.7%% (95%CI: 71.4%-85.9%). LR patients treated on D9602 and ARST0331 trials had comparable 5-year EFS (70% vs. 71.4%, p-0.90) and OS (90% vs. 85.7%, p-0.54), respectively, while IR patients had significantly better 5-year EFS (68.9% vs. 51%, p-0.023) and OS (86.2% vs. 64.4%, p-0.007) on D9803 trial than ARST0531 trial. The 5-year EFS and OS of IRS group III patients with DPE with R0 margins were comparable to IRS group II patients (70.4% vs. 66.6%) &amp; (86.5% vs. 84.6%), respectively. On univariable analysis, age at diagnosis and sex were associated with EFS, while age at diagnosis, sex, tumor size, IRS group, stage and fusion status were associated with OS (p &lt; 0.05). None of the analyzed factors demonstrated independent statistical association with EFS, whereas female sex was the only factor independently associated with OS [Hazard ratio (HR): 0.4, 95% CI 0.20-0.85)]. Conclusions: IR localized extremity RMS patients treated on ARST0531 had suboptimal outcome. Survival of IRS group III patients with R0 DPE was comparable to IRS group II patients. Females with localized extremity RMS had better OS than males. Clinical trial information: NCT00354835 ; NCT00003958 ; NCT00075582 ; NCT00002995 .

Bone marrow disease in rhabdomyosarcoma visualized by 2-[18F]fluorodeoxyglucose positron emission tomography/computed tomography

Bone marrow metastases—noted in 6% of patients with rhabdomyosarcoma—have been linked to very poor outcomes. Bilateral bone marrow sampling from iliac crests has been the gold standard for bone marrow examination in rhabdomyosarcoma, but sampling errors due to patchy bone marrow involvement may limit its sensitivity. Here, we report the case of a 6-year-old boy with embryonal rhabdomyosarcoma of the skull base and multiple 2-[18F]fluoro-2-deoxy-D-glucose (2-[18F]FDG)-avid bone marrow metastases visualized by positron emission tomography and computed tomography (2-[18F]FDG PET/CT). His bone marrow aspirates were tumor-free. This case illustrates the diagnostic value of 2-[18F]FDG PET/CT in the detection of bone marrow metastases in rhabdomyosarcoma patients, which may re-shape the definition of bone marrow disease and, ultimately, alter disease staging and risk stratification.

FOXO3 polymorphisms influence the risk and prognosis of rhabdomyosarcoma in children.

Rhabdomyosarcoma(RMS) is the most common soft tissue sarcoma in children and single nucleotide polymorphisms(SNPs) in certain genes influence risk of RMS. Although FOXO3 had been reported in multiple cancers including RMS, the role of FOXO3 polymorphisms in RMS remains unclear. In this case-control study, we evaluated the association of FOXO3 SNPs with RMS risk and prognosis in children. Four FOXO3 SNPs(rs17069665 A>G, rs4946936 T>C, rs4945816 C>T and rs9400241 C>A) were genotyped in 110 RMS cases and 359 controls. The associations between FOXO3 polymorphisms and RMS risk were determined by odds ratios(ORs) with 95% confidence intervals(CIs). The associations of rs17069665 and rs4946936 with overall survival in RMS children were estimated using the Kaplan-Meier method and log-rank test. Functional analysis in silico was performed to estimate the probability that rs17069665 and rs4946936 might influence the regulation of FOXO3. We found that rs17069665 (GG vs. AA+AG, adjusted OR=2.96; 95%CI [1.10-3.32]; P=0.010) and rs4946936 (TC+CC vs. TT, adjusted OR=0.48; 95%CI [0.25-0.90]; P=0.023) were related to the increased and decreased RMS risk, respectively. Besides, rs17069665(P<0.001) and rs4946936(P<0.001) were associated with decreased and increased overall survival in RMS patients, respectively. Functional analysis showed that rs17069665 and rs4946936 might influence the transcription and expression of FOXO3 via altering the bindings to MYC, CTCF, and/or RELA. This study revealed that FOXO3 polymorphisms influence the RMS susceptibility and prognosis in children, and might altered the expression of FOXO3. FOXO3 polymorphism was suggested as a biomarker for RMS susceptibility and prognosis.

Infiltration of Round Blue Cell Tumors in Bone Marrow

Objective: To identify different types of round blue cell tumors on bone marrow biopsy and their pattern of marrow infiltration. Methodology: This cross-sectional descriptive study was conducted at the Department of Hematology, Armed Forces Institute of Pathology (AFIP), Rawalpindi, from July 2021 to December 2021. A total of 60 diagnosed cases of round blue cell tumors were included. Patients with acute lymphoblastic leukemia (ALL) and those receiving treatment for round blue cell tumors were excluded. Bone marrow aspiration and biopsy were performed. Peripheral film results, clinical observations, and patterns of marrow infiltration of round blue cell malignancies were analyzed using Statistical Package for the Social Sciences (SPSS) version 23. Results: The study revealed a male to female ratio of 2:1, with 40 (66.6%) males and 20 (33.3%) females. The overall average age was 5.69 ± 5.0 years. Ewing sarcoma and rhabdomyosarcoma patients did not exhibit infiltration, while three instances of retinoblastoma (9.37%) and two cases of neuroblastoma (10.52%) did. Immunohistochemistry (IHC) results for synaptophysin and chromogranin were positive for retinoblastoma and neuroblastoma, while S100 results were positive for neuroblastoma infiltration and negative for retinoblastoma. Both retinoblastoma and neuroblastoma showed diffuse, focal, and interstitial patterns of infiltration on trephine biopsy. Conclusion: Small round blue cell tumors were noted in bone marrow infiltration in 5 (8.33%) cases. Early detection of malignant cancers necessitates appropriate medical care, and in more severe cases, the need for neoadjuvant chemotherapy.

Efficacy and Toxicity of Vincristine and CYP3A5 Genetic Polymorphism in Rhabdomyosarcoma Pediatric Egyptian Patients.

Rhabdomyosarcoma (RMS) is a rare cancer that develops in soft tissue, particularly skeletal muscle tissue and occasionally hollow organs like the bladder or uterus. Vincristine (VCR) is the main therapy used in treatment of RMS, it is an alkaloid produced from vinca and it is one of the most commonly prescribed drugs in pediatric oncology for the treatment of a number of tumors. The CYP3A5 enzyme is responsible for vincristine metabolism. The effect of CYP3A5 genetic polymorphism on the efficacy and toxicity of VCR on RMS patients still needs further research. Genotyping for CYP3A5 SNPs rs776746, rs10264272 and rs41303343 was performed using Taqman Real-Time PCR assays in a retrospective cohort study of 150 RMS pediatric patients treated with vincristine. The relationship between these genotypes and RMS survival was then examined. We found that patients with CYP3A5*3/*3 had the highest incidence of vincristine-induced neuropathy reaching 61.3%. Patients with CYP3A5*1/*3, CYP3A5*3/*6 and the normal metabolizers with CYP3A5*1/*1 had frequencies of 22%, 10.7%, and 4.7%. patients with the lowest frequency of 1.3% were those with the CYP3A5*1/*6 genotype. There was no correlation between the genotypes of CYP3A5*3, CYP3A5*6, CYP3A5*7, and RMS survival. Initial risk, metastasis, response, convulsions, unsteady gait and hepatotoxicity grade had a significant effect on overall survival with p<0.05. CYP3A5*1/*1 have less severe vincristine-induced neuropathy than CYP3A5 *1/*3, CYP3A5 *1/*6 and CYP3A5 *3/*3, CYP3A5 *3/*6. There is a significant influence of CYP3A5 mutation on neuropathy grade and assist of ADL as a part of neurotoxicity.

Nomogram to predict prognosis of head and neck rhabdomyosarcoma patients in children and adolescents.

This study aims to explore the prognostic factors of head and neck rhabdomyosarcoma (HNRMS) in children and adolescents and construct a simple but reliable nomogram model for estimating overall survival (OS) of patients. Data of all HNRMS patients during 2004-2018 were identified from the Surveillance, Epidemiology, and End Result database. Kaplan-Meier method was performed to calculate OS stratified by subgroups and comparison between subgroups was completed by log-rank test. Univariate and multivariate Cox regressions analysis were employed for identifying independent predictors, which subsequently were used for a predictive model by R software, and the efficacy of the model was evaluated by applying receiver operating curve (ROC), calibration and decision curve analysis (DCA). A total of 446 patients were included in the study. The 1-, 3-, and 5-year OS rate of the whole cohort was 90.6%, 80.0%, and 75.5%, respectively. The results of univariate and multivariate Cox regression analysis indicated that the primary site in parameningeal region, alveolar RMS histology, M1 stage, IRS stage 4, surgery, and chemotherapy were significant prognostic factors (all P<0.05). The performance of nomogram model was validated by discrimination and calibration, with AUC values of 1, 3, and 5 years OS of 0.843, 0.851, and 0.890, respectively. We constructed a prognostic nomogram model for predicting the OS in HNRMS patients in children and adolescents and this model presented practical and applicable clinical value to predict survival when choosing treatment strategies.

Abstract 5610: Investigating the role of BRCA2 in the development of pediatric rhabdomyosarcoma

Abstract Introduction: In a large-scale exome sequencing study of pediatric rhabdomyosarcoma (RMS) patients, we identified a statistical enrichment of patients with germline variants in BRCA2 (Li, et al. JNCI, 2020), which prompted this study to assess the role of heterozygous BRCA2 variants in the development of pediatric RMS in both precursor cell and mouse models. Methods: We engineered BRCA2 loss of function (LoF) variants in lines of immortalized mesenchymal stem cells, a potential precursor cell to RMS, using CRISPR and Prime Editing (PE). CRISPR was used to induce frameshift alleles in Exons 10 and 11 and with PE, we created two RMS patient specific LOF variants in Exons 5 and 17. We generated a genetically engineered mouse model (GEMM) with heterozygous excision of Exon 11 of Brca2. This allele is now being crossed into two different previously established GEMMs of RMS: a myoblast-specific (MyoD1-Cre) conditional loss of p53, and a gain-of-function mutant p53, LSL-p53R172H (Nakahata, et al. Dis. Model. Mech., 2022), to assess changes in the pace of tumorigenesis and metastatic potential. Through an X01 agreement with the Gabriella Miller Kids First Program, we have submitted paired tumor-normal samples for additional whole-genome sequencing and RNA sequencing (tumor only) on &amp;gt;500 patients with RMS, including those with germline variants in BRCA2. Results: Using CRISPR, we generated cell lines heterozygous for the frameshift variants c.1345dup, c.5680del, and c.5680dup. With Prime Editing, we created heterozygous lines mimicking two specific variants detected in the RMS cohort, c.462_463del and c.7857G&amp;gt;A. Generation of a C57BL6 GEMM with a heterozygous removal of Exon 11 in Brca2 has been successful. No homozygous mice were obtained following 7 heterozygous crosses (50 pups), a departure from Mendelian ratios (p=0.0001) consistent with embryonic lethality. Heterozygous Brca2 mice derived from crosses into two RMS models are being monitored for tumor development. We have completed tumor-normal whole-genome and tumor RNA sequencing for 609 RMS patients, including 228 subjects not previously analyzed. We will directly assess whether there is evidence for mutation or loss of expression of the remaining BRCA2 allele in tumors and whether these tumor genomes exhibit signatures of DNA repair deficiency. Conclusions: The goal of this project is to determine the molecular basis for the enrichment in germline BRCA2 LOF variants in pediatric malignancies, including RMS. Ongoing experiments using the engineered heterozygous BRCA2 in vitro cell line models will assess genomic stability, homologous recombination proficiency, and DNA replication fork stability. Assessment of the effect of heterozygous Brca2 variants in GEMMs of RMS, and subsequent analysis of additional sequencing data, will further elucidate what role this gene may play in increasing risk or progression of pediatric cancers. Citation Format: P. Adam Weinstein, Ryan Zabriskie, Cem Ozdemir, Kyle M. Miller, Olga Medina-Martinez, Jason D. Heaney, Aniko Sabo, Philip J. Lupo, Sharon E. Plon. Investigating the role of BRCA2 in the development of pediatric 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 5610.

Abstract 2862: Evolution of clinically relevant variants in advanced pediatric sarcomas

Abstract Objective Few pediatric sarcoma studies have investigated how gene alterations detected by targeted sequencing change in the same patient over time. We leveraged targeted somatic DNA next-generation sequencing (NGS) data from a large cohort of young patients with osteosarcoma (OS), Ewing sarcoma (EWS), or rhabdomyosarcoma (RMS) to identify changes in known oncogenic variants with the aim of addressing the clinical utility of resequencing at recurrence. Methods The iCat2/GAIN Consortium Study is a cohort study involving 13 US institutions evaluating outcomes after receipt of matched targeted therapy (MTT) in young patients with advanced extra-cranial solid tumors. Tumors are sequenced using OncoPanel, a targeted DNA NGS test performed in a CLIA/CAP certified laboratory assessing single nucleotide variants (SNV), copy number variants (CNV) and structural variants (SV) in up to 447 cancer genes and rearrangements in up to 60 cancer genes. In 2 cases, similar targeted NGS tests were performed. For OS, EWS, or RMS patients with at least 2 OncoPanels from different timepoints (initial diagnosis or relapse) and/or tumor sites (metastatic or primary), we assessed oncogenic variants in genes previously known to be recurrently altered in the respective diagnosis. For each variant, we assigned patterns of change as emergence, disappearance, or persistence. Results Of 784 iCat2/GAIN patients, there were 157, 107, and 100 patients with OS, EWS, and RMS respectively. The analytic cohort consisted of 34, 20, and 15 OS, EWS, and RMS patients respectively with at least 2 tumor sequenced samples. The median number of sequenced samples per patient was 2 (range 2 to 6). In EWS, 9 (45%) patients had an oncogenic variant (other than an expected fusion) in at least one sample. In 4 patients, the predominant pattern of change was emergence. Specifically, a new pathogenic SNV in STAG2 (N=1) or TP53 (N=3) were observed. In OS, all 34 patients had an oncogenic variant in at least one sample. In RMS, 12 (80%) patients had an oncogenic variant in at least one sample. There was no predominant pattern of change in OS and RMS. In OS, 41% percent of variants persisted in all samples; 32% of variants disappeared in subsequent samples; 22% of variants emerged in subsequent samples; 5% of variants were variably present over time. Interestingly, while TP53 was most often persistent over time, there were also 7 cases with emergence of TP53 variants and 8 cases with disappearance of TP53 variants. In RMS, 26% percent of variants persisted in all samples; 31% of variants disappeared in subsequent samples; 43% of variants emerged in subsequent samples. Interestingly, in three cases there was emergence of a new variant activating the RAF/MAPK or AKT pathway. Conclusion Pediatric sarcomas display a range of genomic changes over time. Given the observed changes in oncogenic alterations, there may be utility to repeat sequencing of relapsed tumor biopsies to understand these changes. Citation Format: Hannah Y. Comeau, Lorena Lazo De La Vega, Alanna J. Church, Luke D. Maese, Navin Pinto, Margaret E. Macy, Mark A. Applebaum, Amit J. Sabnis, AeRang Kim, Susan I. Colace, Filemon Dela Cruz, Steven G. DuBois, Natalie B. Collins, Katherine A. Janeway. Evolution of clinically relevant variants in advanced pediatric sarcomas [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 2862.

Abstract 73: Spatial profiling of pediatric rhabdomyosarcoma to elucidate their immunosuppressive tumor microenvironment

Abstract Background: Pediatric rhabdomyosarcoma (RMS) is a mesenchymal soft tissue malignancy. Despite intensive chemotherapy regimens, there is currently a gap in improving treatment outcomes for RMS. Chemotherapy can modulate the tumor microenvironment (TME) by altering the immune cell composition or their functions. However, the impact of chemotherapy in pediatric RMS TME is unclear. RMS tumors are also considered as “cold” tumors due to low immune infiltration, which could be a determinant of why immunotherapy is unlikely to work. Additional investigations are required to determine the reasons of why RMS is “cold” and whether a combined immunotherapy and chemotherapy approach should be administered for this hard-to-cure cancer. Methods We conducted for the first time spatial GeoMx whole transcriptome atlas and 51-plex Akoya CODEX profiling for a tissue microarray (TMA) from a cohort of pediatric RMS patients (n = 12) with pre-chemotherapy (n = 5) and post-chemotherapy (n = 5) samples. Regions of interest (ROIs) were selected based on tumor areas with immune cell infiltration, with the lymphocytes, tumor cells, and nuclei visualized using fluorescent morphological markers CD45, Desmin, and DAPI. ROIs were segmented into tumor stroma compartments and immune cell compartments. Computational analyses were conducted on the transcriptomics data using R package standR including quality control, normalization, differential expression analysis, gene set enrichment analysis, and cell type deconvolution. For CODEX data, we preformed cell segmentation, followed by further quality control, cell phenotyping, cellular neighborhood and cellular interaction analysis. Results: From differential expression analysis of the immune compartment, we have identified type I interferon-related genes (DTX3L, ADAP2, RNF135) and reactive oxygen species (ROS)-induced gene (NCF1) are upregulated, and an antioxidant enzyme gene (PRDX2) is downregulated in post-chemotherapy samples compared to pre-chemotherapy samples. Gene set enrichment analysis of the immune compartment revealed an enriched antigen presentation pathway in post-chemotherapy samples. In the tumor stroma compartment, we identified increased TGF-β binding pathway in post-chemotherapy samples. Cellular deconvolution revealed a higher proportion of endothelial cells in post-chemotherapy samples, which is validated via CODEX imaging analysis. Cellular neighborhoods and cell-cell interaction analysis for CODEX data also reveal how chemotherapy affect functional units and cellular interactions in RMS TME with spatial context preserved. Conclusion: By applying spatial transcriptomic and multiplexed imaging to RMS samples, we have revealed key genes, pathways and cell types involved in chemotherapy-induced alterations in the TME. This will provide a new perspective for developing novel chemoimmunotherapy for RMS patients. Citation Format: Cui Tu, Chin Wee Tan, Ning Liu, Arutha Kulasinghe, Fernando Souza-Fonseca-Guimaraes. Spatial profiling of pediatric rhabdomyosarcoma to elucidate their immunosuppressive tumor microenvironment [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 73.

Abstract 138: UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma

Abstract PAX3-FOXO1 positive rhabdomyosarcoma (RMS) is an aggressive pediatric sarcoma with a terrible prognosis. The resultant fusion acts as a chimeric transcription factor, in part remodeling chromatin to inhibit terminal differentiation and maintain cancer cell proliferation. As this oncoprotein is not directly druggable, we have used genetic screens to identify dependencies downstream of PAX3-FOXO1 to improve our understanding of pathogenesis and develop new therapeutic targets. We performed pooled CRISPRi screening in an isogenic system of a patient-derived RMS cell line with or without knockdown of PAX3-FOXO1. Loss of the ubiquitin hydroxylase UCHL5 led to selective depletion of PAX3-FOXO1 positive Rh30 cells compared to PAX3-FOXO1 knockdown cells, suggesting that the fusion creates UCHL5 dependence. We validated this finding in competition growth assays in additional fusion positive (FP) and fusion negative (FN) RMS cell lines, and observed UCHL5 dependency only in the former. By contrast, UCHL5 was dispensable for cell growth and survival in FN RMS cell lines. These data were recapitulated in the large DepMap dataset, which also identified the top genetic co-dependencies of UCHL5 as INO80-complex (INO80c) members. We therefore investigated whether the fusion selective effects of UCHL5 were mediated by the INO80c, of which UCHL5 is an N-terminal member. We observed that knockdown of another N-terminal component of INO80c, NFRKB, phenocopies the fusion-selective effects of UCHL5 loss. Conversely, the catalytic ATPase INO80 was pan-essential, suggesting that only N-terminal subunits of INO80c regulate PAX3-FOXO1 specific functions. INO80c plays an essential role in mitigating DNA replication stress. Indeed, loss of UCHL5 led to DNA damage as measured by γH2AX and slowing of replication forks as measured by fiber assays in FP, but not FN RMS. Unexpectedly, acute degradation of PAX3-FOXO1 also slowed DNA replication fork speed, suggesting a genetic model in which UCHL5 and PAX3-FOXO1 cooperate to resolve replication stress in FP RMS. Finally, in vivo experiments using cell line xenografts confirms that loss of UCHL5 slows the growth of FP RMS, but not FN RMS. These findings suggest that loss of UCHL5 induces replication stress in a fusion-specific manner, and offers both an opportunity to study the mechanisms by which the PAX3-FOXO1 oncoprotein mitigates replication stress, and to develop UCHL5 inhibitors as precision therapies for FP RMS patients. Citation Format: Amit J. Sabnis, Pushpendra K. Sahu, Yue Pan. UCHL5 and PAX3-FOXO1 resolve replication stress 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 138.

The Capacity of Drug-Metabolising Enzymes in Modulating the Therapeutic Efficacy of Drugs to Treat Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a rare soft tissue sarcoma (STS) that predominantly affects children and teenagers. It is the most common STS in children (40%) and accounts for 5-8% of total childhood malignancies. Apart from surgery and radiotherapy in eligible patients, standard chemotherapy is the only therapeutic option clinically available for RMS patients. While survival rates for this childhood cancer have considerably improved over the last few decades for low-risk and intermediate-risk cases, the mortality rate remains exceptionally high in high-risk RMS patients with recurrent and/or metastatic disease. The intensification of chemotherapeutic protocols in advanced-stage RMS has historically induced aggravated toxicity with only very modest therapeutic gain. In this review, we critically analyse what has been achieved so far in RMS therapy and provide insight into how a diverse group of drug-metabolising enzymes (DMEs) possess the capacity to modify the clinical efficacy of chemotherapy. We provide suggestions for new therapeutic strategies that exploit the presence of DMEs for prodrug activation, targeted chemotherapy that does not rely on DMEs, and RMS-molecular-subtype-targeted therapies that have the potential to enter clinical evaluation.

Breast metastasis from primary hand rhabdomyosarcoma

BackgroundRhabdomyosarcoma can occur almost anywhere in the body; however, the most common primary sites include the head and neck (50%), genitourinary tract (~ 25%), and extremities (~ 15%). Approximately 20% of rhabdomyosarcoma patients present with metastases at diagnosis, most commonly to the lungs, bone marrow, and lymph nodes. Rhabdomyosarcoma metastasizing to distant sites such as the breast highlights its aggressive behavior.Case presentationA 20-year-old girl presented with left-hand swelling and pain which was unfortunately excised without imaging evaluation. Postoperative histopathology revealed a malignant spindle cell tumor. After two months, the patient complained of rapidly growing bilateral breast masses. As regards the patient’s age diagnostic ultrasound was done that revealed bilateral breast masses with bilateral axillary lymphadenopathy (Fig. 1), and then, magnetic resonance imaging was done that revealed bilateral breast masses of malignant criteria and pathological left axillary lymph nodes (Fig. 2). An ultrasound-guided biopsy was taken from both breast masses which revealed a metastatic malignant spindle cell tumor. After immunophenotyping, the diagnosis of rhabdomyosarcoma is established. Unfortunately, a metastatic work-up was done that revealed a visceral and nodal metastasis as well as a myocardial deposit (Fig. 3).ConclusionsMetastasis of rhabdomyosarcoma to the breast is rare and occurs in young women as in our case with mainly primary site in limbs. It is important to be clinically aware of the risk of breast metastasis in female patients with primary extra-mammary rhabdomyosarcoma.