Massive Bone Marrow Infiltration by Disseminated Alveolar Rhabdomyosarcoma Mimicking Acute Leukemia.

RMS is a malignant tumor of soft tissues affecting primarily children and adolescents. Around 6% to 23% RMS patients present bone marrow infiltration but leukemia-like involvement is very rare; in these patients cytomorphology on bone marrow smears can lead to misdiagnosis. Differential diagnosis with alveolar RMS should be kept in mind in every pediatric patient presenting with a marked bone marrow involvement in the absence of typical lymphoproliferative findings.

A rare adult case of primary uterine rhabdomyosarcoma with mixed pattern: a clinicopathological & immunohistochemical study with literature review.

BACKGROUND: Rhabdomyosarcomas are aggressive tumors that comprise a group of morphologically similar but biologically diverse lesions. Owing to its rarity, Mixed pattern RMS (ARMS and ERMS) constitutes a diagnostic and therapeutic dilemma. CASE: Herein is presented a very rare case of mixed alveolar & embryonal rhabdomyosarcoma in the uterus of a 68-year-old woman. The wall of the uterine corpus & cervix was replaced by multiple whitish-yellow, firm nodules, measuring up to 12 cm. Microscopically, the tumor was predominantly composed of round to polygonal cells arranged in nests with alveolar pattern intermingled with hypo- & hypercellular areas of more primitive cells with scattered multinucleated giant cells seen as well. Extensive sampling failed to show epithelial elements. Immunohistochemical staining showed positive staining for vimentin, desmin, myogenin, CD56 & WT-1. However, no staining was detected for CK, LCA, CD10, ER, SMA, CD99, S100, Cyclin-D1 & Olig-2. Metastatic deposits were found in the peritoneum. The patient received postoperative chemotherapy and radiotherapy but died of systemic metastases 3 months after surgery. CONCLUSION: The rarity of this histological tumor entity and its aggressive behavior and poor prognosis grab attention to improving recognition and treatment modalities in adults.

Developmental and therapeutic implications of IL4ra expression for rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a solid tumor whose metastatic progression can be accelerated through interleukin-4 receptor alpha (Il4ra) mediated interaction with normal muscle stem cells (satellite cells). To understand the function of Il4ra in this tumor initiation phase of RMS, we conditionally deleted Il4ra in genetically-engineered RMS mouse models. Nullizygosity of Il4ra altered the latency, site and/or stage distribution of RMS tumors compared to IL4RA intact models. Primary tumor cell cultures taken from the genetically-engineered models then used in orthotopic allografts further defined the interaction of satellite cells and RMS tumor cells in the context of tumor initiation: in alveolar rhabdomyosarcoma (ARMS), satellite cell co-injection was necessary for Il4ra null tumor cells engraftment, whereas in embryonal rhabdomyosarcoma (ERMS), satellite cell co-injection decreased latency of engraftment of Il4ra wildtype tumor cells but not Il4ra null tumor cells. When refocusing on Il4ra wildtype tumors by single cell sequencing and cytokine studies, we have uncovered a putative signaling interplay of Il4 from T-lymphocytes being received by Il4ra + rhabdomyosarcoma tumor cells, which in turn express Ccl2, the ligand for Ccr2 and Ccr5. Taken together, these results suggest that mutations imposed during tumor initiation have different effects than genetic or therapeutic intervention imposed once tumors are already formed. We also propose that CCL2 and its cognate receptors CCR2 and/or CCR5 are potential therapeutic targets in Il4ra mediated RMS progression.

FGFR1 fusions as a novel molecular driver in rhabdomyosarcoma.

The wide application of RNA sequencing in clinical practice has allowed the discovery of novel fusion genes, which have contributed to a refined molecular classification of rhabdomyosarcoma (RMS). Most fusions in RMS result in aberrant transcription factors, such as PAX3/7::FOXO1 in alveolar RMS (ARMS) and fusions involving VGLL2 or NCOA2 in infantile spindle cell RMS. However, recurrent fusions driving oncogenic kinase activation have not been reported in RMS. Triggered by an index case of an unclassified RMS (overlapping features between ARMS and sclerosing RMS) with a novel FGFR1::ANK1 fusion, we reviewed our molecular files for cases harboring FGFR1-related fusions. One additional case with an FGFR1::TACC1 fusion was identified in a tumor resembling embryonal RMS (ERMS) with anaplasia, but with no pathogenic variants in TP53 or DICER1 on germline testing. Both cases occurred in males, aged 7 and 24, and in the pelvis. The 2nd case also harbored additional alterations, including somatic TP53 and TET2 mutations. Two additional RMS cases (one unclassified, one ERMS) with FGFR1 overexpression but lacking FGFR1 fusions were identified by RNA sequencing. These two cases and the FGFR1::TACC1-positive case clustered together with the ERMS group by RNAseq. This is the first report of RMS harboring recurrent FGFR1 fusions. However, it remains unclear if FGFR1 fusions define a novel subset of RMS or alternatively, whether this alteration can sporadically drive the pathogenesis of known RMS subtypes, such as ERMS. Additional larger series with integrated genomic and epigenetic datasets are needed for better subclassification, as the resulting oncogenic kinase activation underscores the potential for targeted therapy.

Embryonal and alveolar rhabdomyosarcoma in adolescents/young adults, adults and older adults: a population-based cohort study.

BACKGROUND: The clinical characteristics, outcomes, and prognostic factors of adult embryonal rhabdomyosarcomas (ERMS) and alveolar rhabdomyosarcomas (ARMS), particularly the differences among adolescents/young adults (AYA), adults, and older adults, remain unclear. We assessed the clinicopathological features and survival outcomes of adult patients with ERMS and ARMS in Japan and to compare these features among AYA, adult, and older adult patients. METHODS: We retrospectively analyzed data from the Bone and Soft Tissue Tumor Registry of Japan and enrolled patients aged ≥15 years with ERMS and ARMS. Disease-specific overall survival (DOS) was estimated using the Kaplan-Meier method, and a Cox regression model was used to identify prognostic factors. RESULTS: Among 184 patients with ERMS and ARMS (median age, 27 years; interquartile range, 18-49 years), a high rate of distant and regional nodal metastases was initially observed in 65 (35%) and 66 (36%) cases, respectively. Older age and distant metastasis at first presentation were statistically poor prognostic factors, and histological subtype and site of tumor origin were not associated with DOS. In patients with localized ERMS and ARMS, older age and nodal metastasis were poor prognostic factors; the 5-year DOS rates of patients with and without nodal metastasis were 23% and 72%, respectively. CONCLUSIONS: Older patients with rhabdomyosarcoma had a dismal prognosis, and distant metastasis was a poor prognostic factor. The prognostic factors differed between adult and pediatric patients with rhabdomyosarcoma; biological analyses, such as genome analysis of adult rhabdomyosarcoma and clinical trials with pediatric oncologists, are needed to improve the prognosis of adult rhabdomyosarcoma.

Transforming Growth Factor Beta and Alveolar Rhabdomyosarcoma: A Challenge of Tumor Differentiation and Chemotherapy Response.

Alveolar rhabdomyosarcoma (ARMS), an invasive subtype of rhabdomyosarcoma (RMS), is associated with chromosomal translocation events resulting in one of two oncogenic fusion genes, PAX3-FOXO1 or PAX7-FOXO1. ARMS patients exhibit an overexpression of the pleiotropic cytokine transforming growth factor beta (TGF-ß). This overexpression of TGF-ß1 causes an increased expression of a downstream transcription factor called SNAIL, which promotes epithelial to mesenchymal transition (EMT). Overexpression of TGF-ß also inhibits myogenic differentiation, making ARMS patients highly resistant to chemotherapy. In this review, we first describe different types of RMS and then focus on ARMS and the impact of TGF-ß in this tumor type. We next highlight current chemotherapy strategies, including a combination of the FDA-approved drugs vincristine, actinomycin D, and cyclophosphamide (VAC); cabozantinib; bortezomib; vinorelbine; AZD 1775; and cisplatin. Lastly, we discuss chemotherapy agents that target the differentiation of tumor cells in ARMS, which include all-trans retinoic acid (ATRA) and 5-Azacytidine. Improving our understanding of the role of signaling pathways, such as TGF-ß1, in the development of ARMS tumor cells differentiation will help inform more tailored drug administration in the future.

Primary alveolar rhabdomyosarcoma of the brain: a case report.

BACKGROUND: Primary brain rhabdomyosarcoma is a rare primary brain malignancy with few case reports. The vast majority of cases of primary brain rhabdomyosarcoma occur in pediatric patients, and immunohistochemistry can distinguish it from embryonal subtypes; however, few cases of primary brain rhabdomyosarcoma in adults have been reported in the literature. CASE PRESENTATION: We report the case of a 26-year-old White male patient who was found to have primary brain alveolar rhabdomyosarcoma after developing headaches for several months. A brain MRI revealed a mixed cystic and solid tumor along the vermis of the cerebellum. The patient underwent a gross total surgical resection, which confirmed the diagnosis of alveolar rhabdomyosarcoma. Further staging workup for another primary focus or disseminated disease yielded negative results, confirming the diagnosis of primary alveolar rhabdomyosarcoma of the brain. CONCLUSION: The standard of care for managing this rare type of brain tumor involves surgery with adjuvant chemoradiotherapy. Further studies should be conducted for a better diagnostic and therapeutic understanding.

KDM3B inhibitors disrupt the oncogenic activity of PAX3-FOXO1 in fusion-positive rhabdomyosarcoma.

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.

Neopetrotaurines A-C, Isoquinoline Alkaloids with an Unprecedented Taurine Bridge from the Sponge Neopetrosia sp.

Neopetrotaurines A-C (1-3), unusual alkaloids possessing two isoquinoline-derived moieties that are linked via a unique taurine bridge, were isolated from a Neopetrosia sp. marine sponge. These new compounds have proton-deficient structural scaffolds that are difficult to unambiguously assign using only conventional 2- and 3-bond 1H-13C and 1H-15N heteronuclear correlation data. Thus, the application of LR-HSQMBC and HMBC NMR experiments optimized to detect 4- and 5-bond long-range 1H-13C heteronuclear correlations facilitated the structure elucidation of these unusual taurine-bridged marine metabolites. Neopetrotaurines A-C (1-3) showed significant inhibition of transcription driven by the oncogenic fusion protein PAX3-FOXO1, which is associated with alveolar rhabdomyosarcoma, and cytotoxic activity against PAX3-FOXO1-positive cell lines.

Receptor tyrosine kinase gene expression profiling of orbital rhabdomyosarcoma unveils MET as a potential biomarker and therapeutic target.

Receptor tyrosine kinases (RTKs) serve as molecular targets for the development of novel personalized therapies in many malignancies. In the present study, expression pattern of receptor tyrosine kinases and its clinical significance in orbital RMS has been explored. Eighteen patients with histopathologically confirmed orbital RMS formed part of this study. Comprehensive q-PCR gene expression profiles of 19 RTKs were generated in the cases and controls. The patients were followed up for 59.53 ± 20.93 years. Clustering and statistical analysis tools were applied to identify the significant combination of RTKs associated with orbital rhabdomyosarcoma patients. mRNA overexpression of RTKs which included MET, AXL, EGFR was seen in 60-80% of cases; EGFR3, IGFR2, FGFR1, RET, PDGFR1, VEGFR2, PDGFR2 in 30-60% of cases; and EGFR4, FGFR3,VEGFR3 and ROS,IGFR1, EGFR1, FGFR2, VEGFR1 in 10-30% of cases. Immunoexpression of MET was seen in 89% of cases. A significant association was seen between MET mRNA and its protein expression. In all the cases MET gene expression was associated with worst overall survival (P = 0.03).There was a significant correlation of MET mRNA expression with RET, ROS, AXL, FGFR1, FGFR3, PDGFR1, IGFR1, VEGFR2, and EGFR3 genes. Association between MET gene and collective expression of RTKs was further evaluated by semi-supervised gene cluster analysis and Principal component analysis, which showed well-separated tumor clusters. MET gene overexpression could be a useful biomarker for identifying high risk orbital rhabdomyosarcoma patients. Well-separated tumor clusters confirmed the association between MET gene and collective expression of RTK genes. Therefore, the therapeutic potential of multi-kinase inhibitors targeting MET and the 9 other significant RTKs needs to be explored.

Prognostic Factors in Pediatric Alveolar Rhabdomyosarcoma: SEER Analysis of 277 Cases.

Alveolar rhabdomyosarcoma (ARMS) is a rare but highly aggressive cancer predominantly affecting children and adolescents. This study explores prognostic factors for pediatric and adolescent ARMS, using the Surveillance, Epidemiology, and End Results (SEER) database. Leveraging SEER data (2000-2019), we analyzed 277 cases. Employing Kaplan-Meier survival analysis and Cox proportional hazards models, we identified significant prognostic factors. Gender distribution was nearly equal (56.0% boys, 44.0% girls), with the majority (70.8%) from the white ethnic group. Primary tumors were predominantly in extremities (37.2%). Distant metastases significantly increased mortality risk (hazard ratio [HR], 3.13; 95% CI: 2.14-4.58) and regional lymph node involvement raised mortality risk (HR, 1.36; 95% CI: 0.96-1.92). Chemotherapy-only treatment had higher mortality risk than chemoradiotherapy (HR, 1.16; 95% CI: 0.97-2.67). Conclusively, our study identifies distant metastases, regional lymph node involvement, and treatment modality as crucial predictors of overall survival in pediatric ARMS.

PAX3-FOXO1 uses its activation domain to recruit CBP/P300 and shape RNA Pol2 cluster distribution.

Activation of oncogenic gene expression from long-range enhancers is initiated by the assembly of DNA-binding transcription factors (TF), leading to recruitment of co-activators such as CBP/p300 to modify the local genomic context and facilitate RNA-Polymerase 2 (Pol2) binding. Yet, most TF-to-coactivator recruitment relationships remain unmapped. Here, studying the oncogenic fusion TF PAX3-FOXO1 (P3F) from alveolar rhabdomyosarcoma (aRMS), we show that a single cysteine in the activation domain (AD) of P3F is important for a small alpha helical coil that recruits CBP/p300 to chromatin. P3F driven transcription requires both this single cysteine and CBP/p300. Mutants of the cysteine reduce aRMS cell proliferation and induce cellular differentiation. Furthermore, we discover a profound dependence on CBP/p300 for clustering of Pol2 loops that connect P3F to its target genes. In the absence of CBP/p300, Pol2 long range enhancer loops collapse, Pol2 accumulates in CpG islands and fails to exit the gene body. These results reveal a potential novel axis for therapeutic interference with P3F in aRMS and clarify the molecular relationship of P3F and CBP/p300 in sustaining active Pol2 clusters essential for oncogenic transcription.

PAX3-FOXO1 dictates myogenic reprogramming and rhabdomyosarcoma identity in endothelial progenitors.

Fusion-positive rhabdomyosarcoma (FP-RMS) driven by the expression of the PAX3-FOXO1 (P3F) fusion oncoprotein is an aggressive subtype of pediatric rhabdomyosarcoma. FP-RMS histologically resembles developing muscle yet occurs throughout the body in areas devoid of skeletal muscle highlighting that FP-RMS is not derived from an exclusively myogenic cell of origin. Here we demonstrate that P3F reprograms mouse and human endothelial progenitors to FP-RMS. We show that P3F expression in aP2-Cre expressing cells reprograms endothelial progenitors to functional myogenic stem cells capable of regenerating injured muscle fibers. Further, we describe a FP-RMS mouse model driven by P3F expression and Cdkn2a loss in endothelial cells. Additionally, we show that P3F expression in TP53-null human iPSCs blocks endothelial-directed differentiation and guides cells to become myogenic cells that form FP-RMS tumors in immunocompromised mice. Together these findings demonstrate that FP-RMS can originate from aberrant development of non-myogenic cells driven by P3F.

ErbB2 (HER2)-CAR-NK-92 cells for enhanced immunotherapy of metastatic fusion-driven alveolar rhabdomyosarcoma.

Introduction: Metastatic rhabdomyosarcoma (RMS) is a challenging tumor entity that evades conventional treatments and endogenous antitumor immune responses, highlighting the need for novel therapeutic strategies. Applying chimeric antigen receptor (CAR) technology to natural killer (NK) cells may offer safe, effective, and affordable therapies that enhance cancer immune surveillance. Methods: Here, we assess the efficacy of clinically usable CAR-engineered NK cell line NK-92/5.28.z against ErbB2-positive RMS in vitro and in a metastatic xenograft mouse model. Results: Our results show that NK-92/5.28.z cells effectively kill RMS cells in vitro and significantly prolong survival and inhibit tumor progression in mice. The persistence of NK-92/5.28.z cells at tumor sites demonstrates efficient antitumor response, which could help overcome current obstacles in the treatment of solid tumors. Discussion: These findings encourage further development of NK-92/5.28.z cells as off-the-shelf immunotherapy for the treatment of metastatic RMS.

Piperacetazine Directly Binds to the PAX3::FOXO1 Fusion Protein and Inhibits Its Transcriptional Activity.

The tumor-specific chromosomal translocation product, PAX3::FOXO1, is an aberrant fusion protein that plays a key role for oncogenesis in the alveolar subtype of rhabdomyosarcoma (RMS). PAX3::FOXO1 represents a validated molecular target for alveolar RMS and successful inhibition of its oncogenic activity is likely to have significant clinical applications. Even though several PAX3::FOXO1 function-based screening studies have been successfully completed, a directly binding small-molecule inhibitor of PAX3::FOXO1 has not been reported. Therefore, we screened small-molecule libraries to identify compounds that were capable of directly binding to PAX3::FOXO1 protein using surface plasmon resonance technology. Compounds that directly bound to PAX3::FOXO1 were further evaluated in secondary transcriptional activation assays. We discovered that piperacetazine can directly bind to PAX3::FOXO1 protein and inhibit fusion protein-derived transcription in multiple alveolar RMS cell lines. Piperacetazine inhibited anchorage-independent growth of fusion-positive alveolar RMS cells but not embryonal RMS cells. On the basis of our findings, piperacetazine is a molecular scaffold upon which derivatives could be developed as specific inhibitors of PAX3::FOXO1. These novel inhibitors could potentially be evaluated in future clinical trials for recurrent or metastatic alveolar RMS as novel targeted therapy options. SIGNIFICANCE: RMS is a malignant soft-tissue tumor mainly affecting the pediatric population. A subgroup of RMS with worse prognosis harbors a unique chromosomal translocation creating an oncogenic fusion protein, PAX3::FOXO1. We identified piperacetazine as a direct inhibitor of PAX3::FOXO1, which may provide a scaffold for designing RMS-specific targeted therapy.

Changes on chromosome 11p15.5 as specific marker for embryonal rhabdomyosarcoma?

Rhabdomyosarcomas (RMS) constitute a heterogeneous spectrum of tumors with respect to clinical behavior and tumor morphology. The paternal uniparental disomy (pUPD) of 11p15.5 is a molecular change described mainly in embryonal RMS. In addition to LOH, UPD, the MLPA technique (ME030kit) also determines copy number variants and methylation of H19 and KCNQ1OT1 genes, which have not been systematically investigated in RMS. All 127 RMS tumors were divided by histology and PAX status into four groups, pleomorphic histology (n = 2); alveolar RMS PAX fusion-positive (PAX+; n = 39); embryonal RMS (n = 70) and fusion-negative RMS with alveolar pattern (PAX-RMS-AP; n = 16). The following changes were detected; negative (n = 21), pUPD (n = 75), gain of paternal allele (n = 9), loss of maternal allele (n = 9), hypermethylation of H19 (n = 6), hypomethylation of KCNQ1OT1 (n = 6), and deletion of CDKN1C (n = 1). We have shown no difference in the frequency of pUPD 11p15.5 in all groups. Thus, we have proven that changes in the 11p15.5 are not only specific to the embryonal RMS (ERMS), but are often also present in alveolar RMS (ARMS). We have found changes that have not yet been described in RMS. We also demonstrated new potential diagnostic markers for ERMS (paternal duplication and UPD of whole chromosome 11) and for ARMS PAX+ (hypomethylation KCNQ1OT1).

A systematic review of the prevalence of pathogenic or likely pathogenic germline variants in individuals with FOXO1 fusion-positive rhabdomyosarcoma.

Several cancer predisposition syndromes (CPS) are reported to predispose to rhabdomyosarcoma, most frequently in children with embryonal rhabdomyosarcoma. There are lingering questions over the role of CPS in individuals with alveolar rhabdomyosarcoma (ARMS), which are frequently driven by FOXO1 fusion oncoproteins. We conducted a systematic review to identify patients with FOXO1 fusion-positive ARMS (FP-ARMS) who underwent germline DNA sequencing. We estimated the prevalence of pathogenic/likely pathogenic (P/LP) variants in cancer predisposing genes (CPGs) and of CPSs. We included 19 publications reporting on 191 patients with FP-ARMS. P/LP variants in CPGs were identified in 26/191 (13.6%) patients, nine (4.9%) of which were associated with a CPS diagnosis. Evidence for causal associations between CPSs and FP-ARMS could not be assessed with available data from this review. Only one patient was affected with a CPS known to predispose to rhabdomyosarcoma, Li-Fraumeni syndrome. Typical CPS associations with rhabdomyosarcoma are rare, but not nonexistent, in patients with FP-ARMS. FOXO1 fusion status, alone, is insufficient for clinicians to rely on to distinguish between patients with/without CPS.

A perfusion-based three-dimensional cell culture system to model alveolar rhabdomyosarcoma pathological features.

Although a rare disease, rhabdomyosarcoma (RMS) is one of the most common cancers in children the more aggressive and metastatic subtype is the alveolar RMS (ARMS). Survival outcomes with metastatic disease remain dismal and the need for new models that recapitulate key pathological features, including cell-extracellular matrix (ECM) interactions, is warranted. Here, we report an organotypic model that captures cellular and molecular determinants of invasive ARMS. We cultured the ARMS cell line RH30 on a collagen sponge in a perfusion-based bioreactor (U-CUP), obtaining after 7 days a 3D construct with homogeneous cell distribution. Compared to static culture, perfusion flow induced higher cell proliferation rates (20% vs. 5%), enhanced secretion of active MMP-2, and upregulation of the Rho pathway, associated with cancer cell dissemination. Consistently, the ECM genes LAMA1 and LAMA2, the antiapoptotic gene HSP90, identified in patient databases as hallmarks of invasive ARMS, were higher under perfusion flow at mRNA and protein level. Our advanced ARMS organotypic model mimics (1) the interactions cells-ECM, (2) the cell growth maintenance, and (3) the expression of proteins that characterize tumor expansion and aggressiveness. In the future, the perfusion-based model could be used with primary patient-derived cell subtypes to create a personalized ARMS chemotherapy screening system.