Rhabdomyosarcomas are oncogene addicted to the activation of AVIL.

Rhabdomyosarcoma (RMS) is one of the most common pediatric soft-tissue cancer. Previously, we discovered a gene fusion, MARS-AVIL formed by chromosomal inversion in RMS. Suspecting that forming a fusion with a housekeeping gene may be one of the mechanisms to dysregulate an oncogene, we investigated AVIL expression and its role in RMS. We first showed that MARS-AVIL translates into an in-frame fusion protein, which is critical for RMS cell tumorigenesis. Besides forming a gene fusion with the housekeeping gene, MARS, the AVIL locus is often amplified, and its RNA and protein expression are overexpressed in the majority of RMSs. Tumors with AVIL dysregulation exhibit evidence of oncogene addiction: Silencing MARS-AVIL in cells harboring the fusion, or silencing AVIL in cells with AVIL overexpression, nearly eradicated the cells in culture, as well as inhibited in vivo xenograft growth in mice. Conversely, gain-of-function manipulations of AVIL led to increased cell growth and migration, enhanced foci formation in mouse fibroblasts, and most importantly transformed mesenchymal stem cells in vitro and in vivo. Mechanistically, AVIL seems to serve as a converging node functioning upstream of two oncogenic pathways, PAX3-FOXO1 and RAS, thus connecting two types of RMS associated with these pathways. Interestingly, AVIL is overexpressed in other sarcoma cells as well, and its expression correlates with clinical outcomes, with higher levels of AVIL expression being associated with worse prognosis. AVIL is a bona fide oncogene in RMS, and RMS cells are addicted to its activity.

Late-onset Proteus syndrome with cerebriform connective tissue nevus and subsequent development of intraductal papilloma.

Proteus syndrome (PS) is a rare segmental overgrowth disorder caused by a mosaic activating variant in AKT1. The features of PS are often not present at birth but develop during the first few years of life. We describe a 55-year-old female, whose first symptom of overgrowth, a cerebriform connective tissue nevus, occurred at 19 years of age. We report the identification of the AKT1 c.49G > A p.(Glu17Lys) variant in this progressive lesion, the bony overgrowth, and recurrence after surgical intervention. In the sixth decade of life, this individual developed intraductal papillomas within her right breast which were confirmed to contain the same activating AKT1 variant as the connective tissue nevus. While similar neoplasms have been described in an individual with Proteus syndrome, none has been evaluated for the presence of the AKT1 variant. The tumor also contained two likely pathogenic variants in PIK3R1, c.1392_1403dupTAGATTATATGA p.(Asp464_Tyr467dup) and c.1728_1730delGAG p.(Arg577del). The finding of additional genetic variation putatively affecting the PI3K/AKT pathway in the neoplastic tissue may provide preliminary evidence of a molecular mechanism for tumorigenesis in PS. The late onset of symptoms and molecular characterization of the breast tumor expand the clinical spectrum of this rare disorder.

MYOD1 as a prognostic indicator in rhabdomyosarcoma.

BACKGROUND/OBJECTIVES: Rhabdomyosarcoma (RMS) is characterized by the expression of the myogenic regulatory protein MYOD1. Histologic types include alveolar, embryonal (ERMS), and spindle cell sclerosing RMS (SRMS). SRMS harbors MYOD1 mutations in a subset of adult cases in association with poor prognosis. DESIGN/METHODS: To study the level of MYOD1 protein expression and its clinical significance, we have analyzed variable numbers of pediatric (<18 years of age) and adult (age range ≥18 to 35 years) ERMS and SRMS cases for presence or absence of MYOD1 immunoreactivity in correlation with clinical outcome and MYOD1 L122R mutations. RESULTS: Lack of MYOD1 immunoreactivity, identified in 23.8% of nonalveolar RMS (non-ARMS) cases, was more prevalent in SRMS (44%) than ERMS (17.2%) and was significantly associated with low overall survival and unfavorable tumor sites (p < .05). Lack of MYOD1 immunoreactivity was not associated with MYOD1 L122R mutations, which were identified in 3/37 (8%) cases including only two of 31 (6.5%) pediatric cases, one of 11 or 9% pediatric SRMS, and one case of infant ERMS. CONCLUSION: These studies highlight the prognostic role of MYOD1 in non-ARMS. Lack of MYOD1 immunoreactivity is associated with poor prognosis in ERMS and SRMS. MYOD1 gene mutations are generally infrequent in pediatric RMS. Although mutations are predominant in SRMS, they may exceptionally occur in infantile ERMS.

Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma.

Our previous study of DNA methylation in the pediatric soft tissue tumor rhabdomyosarcoma (RMS) demonstrated that fusion-positive (FP) and fusion-negative (FN) RMS tumors exhibit distinct DNA methylation patterns. To further examine the significance of DNA methylation differences in RMS, we investigated genome-wide DNA methylation profiles in discovery and validation cohorts. Unsupervised analysis of DNA methylation data identified novel distinct subsets associated with the specific fusion subtype in FP RMS and with RAS mutation status in FN RMS. Furthermore, the methylation pattern in normal muscle is most similar to the FN subset with wild-type RAS mutation status. Several biologically relevant genes were identified with methylation and expression differences between the two fusion subtypes of FP RMS or between the RAS wild-type and mutant subsets of FN RMS. Genomic localization studies showed that promoter and intergenic regions were hypomethylated and the 3' untranslated regions were hypermethylated in FP compared to FN tumors. There was also a significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation. Moreover, genes with PAX3-FOXO1 binding sites and promoter hypomethylation exhibited the highest frequency of overexpression in FP tumors. Finally, a comparison of RMS model systems revealed that patient-derived xenografts most closely recapitulate the DNA methylation patterns found in human RMS tumors compared to cell lines and cell line-derived xenografts. In conclusion, these findings highlight the interaction of epigenetic changes with mutational alterations and transcriptional organization in RMS tumors, and contribute to improved molecular categorization of these tumors.

Fusion transcriptome profiling provides insights into alveolar rhabdomyosarcoma.

Gene fusions and fusion products were thought to be unique features of neoplasia. However, more and more studies have identified fusion RNAs in normal physiology. Through RNA sequencing of 27 human noncancer tissues, a large number of fusion RNAs were found. By analyzing fusion transcriptome, we observed close clusterings between samples of same or similar tissues, supporting the feasibility of using fusion RNA profiling to reveal connections between biological samples. To put the concept into use, we selected alveolar rhabdomyosarcoma (ARMS), a myogenic pediatric cancer whose exact cell of origin is not clear. PAX3-FOXO1 (paired box gene 3 fused with forkhead box O1) fusion RNA, which is considered a hallmark of ARMS, was recently found during normal muscle cell differentiation. We performed and analyzed RNA sequencing from various time points during myogenesis and uncovered many chimeric fusion RNAs. Interestingly, we found that the fusion RNA profile of RH30, an ARMS cell line, is most similar to the myogenesis time point when PAX3-FOXO1 is expressed. In contrast, full transcriptome clustering analysis failed to uncover this connection. Strikingly, all of the 18 chimeric RNAs in RH30 cells could be detected at the same myogenic time point(s). In addition, the seven chimeric RNAs that follow the exact transient expression pattern as PAX3-FOXO1 are specific to rhabdomyosarcoma cells. Further testing with clinical samples also confirmed their specificity to rhabdomyosarcoma. These results provide further support for the link between at least some ARMSs and the PAX3-FOXO1-expressing myogenic cells and demonstrate that fusion RNA profiling can be used to investigate the etiology of fusion-gene-associated cancers.

PAX3-FOXO1 is essential for tumour initiation and maintenance but not recurrence in a human myoblast model of rhabdomyosarcoma.

The PAX3-FOXO1 fusion gene is generated by a 2;13 chromosomal translocation and is a characteristic feature of an aggressive subset of rhabdomyosarcoma (RMS). To dissect the mechanism of oncogene action during RMS tumourigenesis and progression, doxycycline-inducible PAX3-FOXO1 and constitutive MYCN expression constructs were introduced into immortalized human myoblasts. Although myoblasts expressing PAX3-FOXO1 or MYCN alone were not transformed in focus formation assays, combined PAX3-FOXO1 and MYCN expression resulted in transformation. Following intramuscular injection into immunodeficient mice, myoblasts expressing PAX3-FOXO1 and MYCN formed rapidly growing RMS tumours, whereas myoblasts expressing only PAX3-FOXO1 formed tumours after a longer latency period. Doxycycline withdrawal in myoblasts expressing inducible PAX3-FOXO1 and constitutive MYCN following tumour formation in vivo or focus formation in vitro resulted in tumour regression or smaller foci associated with myogenic differentiation and cell death. Following regression, most tumours recurred in the absence of doxycycline. Analysis of recurrent tumours revealed a subset without PAX3-FOXO1 expression, and cell lines derived from these recurrent tumours showed transformation in the absence of doxycycline. The doxycycline-independent oncogenicity in these recurrent tumour-derived lines persisted even after PAX3-FOXO1 was inactivated with a CRISPR/Cas9 editing strategy. Whereas cell lines derived from primary tumours were dependent on PAX3-FOXO1 and differentiated following doxycycline withdrawal, recurrent tumour-derived cells without PAX3-FOXO1 expression did not differentiate under these conditions. These findings indicate that PAX3-FOXO1 collaborates with MYCN during early RMS tumourigenesis to dysregulate proliferation and inhibit myogenic differentiation and cell death. Although most cells in the primary tumours are dependent on PAX3-FOXO1, recurrent tumours can develop by a PAX3-FOXO1-independent mechanism, in which rare cells are postulated to acquire secondary transforming events that were activated or selected by initial PAX3-FOXO1 expression. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Clonality and evolutionary history of rhabdomyosarcoma.

To infer the subclonality of rhabdomyosarcoma (RMS) and predict the temporal order of genetic events for the tumorigenic process, and to identify novel drivers, we applied a systematic method that takes into account germline and somatic alterations in 44 tumor-normal RMS pairs using deep whole-genome sequencing. Intriguingly, we find that loss of heterozygosity of 11p15.5 and mutations in RAS pathway genes occur early in the evolutionary history of the PAX-fusion-negative-RMS (PFN-RMS) subtype. We discover several early mutations in non-RAS mutated samples and predict them to be drivers in PFN-RMS including recurrent mutation of PKN1. In contrast, we find that PAX-fusion-positive (PFP) subtype tumors have undergone whole-genome duplication in the late stage of cancer evolutionary history and have acquired fewer mutations and subclones than PFN-RMS. Moreover we predict that the PAX3-FOXO1 fusion event occurs earlier than the whole genome duplication. Our findings provide information critical to the understanding of tumorigenesis of RMS.

Therapeutic Approaches Targeting PAX3-FOXO1 and Its Regulatory and Transcriptional Pathways in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a family of soft tissue cancers that are related to the skeletal muscle lineage and predominantly occur in children and young adults. A specific chromosomal translocation t(2;13)(q35;q14) that gives rise to the chimeric oncogenic transcription factor PAX3-FOXO1 has been identified as a hallmark of the aggressive alveolar subtype of RMS. PAX3-FOXO1 cooperates with additional molecular changes to promote oncogenic transformation and tumorigenesis in various human and murine models. Its expression is generally restricted to RMS tumor cells, thus providing a very specific target for therapeutic approaches for these RMS tumors. In this article, we review the recent understanding of PAX3-FOXO1 as a transcription factor in the pathogenesis of this cancer and discuss recent developments to target this oncoprotein for treatment of RMS.

Histology, fusion status, and outcome in metastatic rhabdomyosarcoma: A report from the Children's Oncology Group.

BACKGROUND: Distinguishing alveolar rhabdomyosarcoma (ARMS) from embryonal rhabdomyosarcoma (ERMS) has historically been of prognostic and therapeutic importance. However, classification has been complicated by shifting histologic criteria required for an ARMS diagnosis. Children's Oncology Group (COG) studies after IRS-IV, which included the height of this diagnostic shift, showed both an increased number of ARMS and an increase in the proportion of fusion-negative ARMS. Following diagnostic standardization and histologic re-review of ARMS cases enrolled during this era, analysis of low-risk (D9602) and intermediate-risk (D9803) rhabdomyosarcoma (RMS) studies showed that fusion status rather than histology best predicts prognosis for patients with RMS. This analysis remains to be completed for patients with high-risk RMS. PROCEDURE: We re-reviewed cases on high-risk COG studies D9802 and ARST0431 with an enrollment diagnosis of ARMS. We compared the event-free survival (EFS) and overall survival by histology, PAX-FOXO1 fusion, and clinical risk factors (Oberlin score) for patients with metastatic RMS using the log-rank test. RESULTS: Histology re-review resulted in reclassification as ERMS for 12% of D9802 cases and 5% of ARST0431 cases. Fusion-negative RMS had a superior EFS to fusion-positive RMS; however, poorer outcome for metastatic RMS was most related to clinical risk factors including age, primary site, and number of metastatic sites. CONCLUSIONS: In contrast to low- or intermediate-risk RMS, in metastatic RMS, clinical risk factors have the most impact on patient outcome. PAX-FOXO1 fusion is more common in patients with a high Oberlin score, but fusion status is not an independent biomarker of prognosis.

Histology, Fusion Status, and Outcome in Alveolar Rhabdomyosarcoma With Low-Risk Clinical Features: A Report From the Children's Oncology Group.

BACKGROUND: Distinguishing alveolar rhabdomyosarcoma (ARMS) from embryonal rhabdomyosarcoma (ERMS) is of prognostic and therapeutic importance. Criteria for classifying these entities evolved significantly from 1995 to 2013. ARMS is associated with inferior outcome; therefore, patients with alveolar histology have generally been excluded from low-risk therapy. However, patients with ARMS and low-risk stage and group (Stage 1, Group I/II/orbit III; or Stage 2/3, Group I/II) were eligible for the Children's Oncology Group (COG) low-risk rhabdomyosarcoma (RMS) study D9602 from 1997 to 1999. The characteristics and outcomes of these patients have not been previously reported, and the histology of these cases has not been reviewed using current criteria. PROCEDURE: We re-reviewed cases that were classified as ARMS on D9602 using current histologic criteria, determined PAX3/PAX7-FOXO1 fusion status, and compared these data with outcome for this unique group of patients. RESULTS: Thirty-eight patients with ARMS were enrolled onto D9602. Only one-third of cases with slides available for re-review (11/33) remained classified as ARMS by current histologic criteria. Most cases were reclassified as ERMS (17/33, 51.5%). Cases that remained classified as ARMS were typically fusion-positive (8/11, 73%), therefore current classification results in a similar rate of fusion-positive ARMS for all clinical risk groups. In conjunction with data from COG intermediate-risk treatment protocol D9803, our data demonstrate excellent outcomes for fusion-negative ARMS with otherwise low-risk clinical features. CONCLUSIONS: Patients with fusion-positive RMS with low-risk clinical features should be classified and treated as intermediate risk, while patients with fusion-negative ARMS could be appropriately treated with reduced intensity therapy.

Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma.

Rhabdomyosarcoma comprises two major subtypes, fusion positive (PAX3-FOXO1 or PAX7-FOXO1) and fusion negative. To investigate the significance of DNA methylation in these subtypes, we analyzed methylation profiles of 37 rhabdomyosarcoma tumors and 10 rhabdomyosarcoma cell lines, as well as 8 normal tissues. Unsupervised clustering of DNA methylation clearly distinguished the fusion-positive and fusion-negative subsets. The fusion-positive tumors showed substantially lower overall levels of methylation compared with fusion-negative tumors. Comparison with the methylation pattern of normal skeletal muscle and bone marrow indicates that fusion-negative rhabdomyosarcoma is more similar to these normal tissues compared with fusion-positive rhabdomyosarcoma, and suggests that many of the methylation differences between these subtypes arise from 'aberrant' hyper- and hypomethylation events in fusion-positive rhabdomyosarcoma. Integrative methylation and gene expression analysis revealed that methylation differences between fusion-positive and fusion-negative tumors could either be positively or negatively associated with mRNA expression. There was no significant difference in the distribution of PAX3-FOXO1-binding sites between genes with and without differential methylation. However, the finding that PAX3-FOXO1-binding sites were enriched among genes that were both differentially methylated and differentially expressed suggests that the fusion protein interacts with DNA methylation to regulate target gene expression. An 11-gene DNA methylation signature, classifying the rhabdomyosarcoma tumors into fusion-positive and fusion-negative subsets, was established and validated by pyrosequencing assays. Notably, EMILIN1 (part of the 11-gene signature) showed higher methylation and lower mRNA expression in fusion-positive compared with fusion-negative tumors, and demonstrated demethylation and re-expression in multiple fusion-positive cell lines after treatment with 5-aza-2'-deoxycytidine. In conclusion, our study demonstrates that fusion-positive and fusion-negative rhabdomyosarcoma tumors possess characteristic methylation profiles that contribute to the expression differences between these fusion subtypes. These findings indicate an important relationship between fusion status and epigenetic changes in rhabdomyosarcoma, present a novel approach for ascertaining fusion status, and may identify new therapeutic targets in rhabdomyosarcoma.

The PAX Genes: Roles in Development, Cancer, and Other Diseases.

Since their 1986 discovery in Drosophila, Paired box (PAX) genes have been shown to play major roles in the early development of the eye, muscle, skeleton, kidney, and other organs. Consistent with their roles as master regulators of tissue formation, the PAX family members are evolutionarily conserved, regulate large transcriptional networks, and in turn can be regulated by a variety of mechanisms. Losses or mutations in these genes can result in developmental disorders or cancers. The precise mechanisms by which PAX genes control disease pathogenesis are well understood in some cases, but much remains to be explored. A deeper understanding of the biology of these genes, therefore, has the potential to aid in the improvement of disease diagnosis and the development of new treatments.

Classification of rhabdomyosarcoma and its molecular basis.

Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, has traditionally been classified into embryonal rhabdomyosarcoma (ERMS) and alveolar rhabdomyosarcoma (ARMS) for pediatric oncology practice. This review outlines the historical development of classification of childhood RMS and the challenges that have been associated with it, particularly problems with the diagnosis of "solid variant" ARMS and its distinction from ERMS. In addition to differences in clinical presentation and outcome, a number of genetic features underpin separation of ERMS from ARMS. Genetic differences associated with RMS subclassification include the presence of reciprocal translocations and their associated fusions in ARMS, amplification of genes in ARMS and its fusion subsets, chromosomal losses and gains that mostly occur in ERMS, and allelic losses and mutations usually associated with ERMS. Chimeric proteins encoded in most ARMS from the fusion of PAX3 or PAX7 with FOXO1 are expressed, result in a distinct pattern of downstream protein expression, and appear to be the proximate cause of the bad outcome associated with this subtype. A sizeable minority of ARMS lacks these fusions and shares the clinical and biological features of ERMS. A battery of immunohistochemical tests may prove useful in separating ERMS from ARMS and fusion-positive ARMS from fusion-negative ARMS. Because of limitation of predicting outcome solely based on histologic classification, treatment protocols will begin to utilize fusion testing for stratification of affected patients into low-risk, intermediate-risk, and high-risk groups.

PAX-FOXO1 fusion status drives unfavorable outcome for children with rhabdomyosarcoma: a children's oncology group report.

BACKGROUND: Rhabdomyosarcoma (RMS) is divided into two major histological subtypes: alveolar (ARMS) and embryonal (ERMS), with most ARMS expressing one of two oncogenic genes fusing PAX3 or PAX7 with FOXO1 (P3F and P7F, respectively). The Children's Oncology Group (COG) carried out a multi-institutional clinical trial to evaluate the prognostic value of PAX-FOXO1 fusion status. METHODS: Study participants were treated on COG protocol D9803 for intermediate risk ARMS or ERMS using multi-agent chemotherapy, radiotherapy, and surgery. Central diagnostic pathology review and molecular testing for fusion genes were carried out on prospectively collected specimens. Event-free (EFS) and overall survival (OS) at 5 years were correlated with histological subtype and PAX-FOXO1 status. RESULTS: Of 616 eligible D9803 enrollees, 434 cases had adequate clinical, molecular, and pathology data for definitive classification as ERMS, ARMS P3F+ or P7F+, or ARMSn (without detectable fusion). EFS was worse for those with ARMS P3F+ (54%) and P7F+ (65%) than those with ERMS (77%; P < 0.001). EFS for ARMSn and ERMS were not statistically different (90% vs. 77%, P = 0.15). ARMS P3F+ had poorer OS (64%) than ARMS P7F+ (87%), ARMSn (89%), and ERMS (82%; P = 0.006). CONCLUSIONS: ARMSn has an outcome similar to ERMS and superior EFS compared to ARMS with either P3F or P7F, when given therapy designed for children with intermediate risk RMS. This prospective analysis supports incorporation of PAX-FOXO1 fusion status into risk stratification and treatment allocation.

Genomic and clinical analysis of fusion gene amplification in rhabdomyosarcoma: a report from the Children's Oncology Group.

Alveolar rhabdomyosarcoma (RMS) is an aggressive pediatric cancer of the myogenic lineage with frequent chromosomal translocations involving the PAX3 or PAX7 and FOXO1 genes. Based on previous studies indicating that the fusion genes are amplified in a subset of these cancers, we conducted a comprehensive molecular and clinical investigation of these amplification events. Using oligonucleotide arrays to localize amplicons, we found that the minimal 1p36 amplicon measured 0.13 Mb and only contained PAX7 whereas the minimal 13q14 amplicon measured 0.53 Mb and contained FOXO1 and the poorly characterized LOC646982 gene. Application of a fluorescence in situ hybridization assay to over 100 fusion-positive cases revealed that the fusion gene is amplified in 93% of PAX7-FOXO1-positive and 9% of PAX3-FOXO1-positive cases. While most cells in amplified PAX7-FOXO1-positive cases contained the amplicon, only a fraction of cells in the amplified PAX3-FOXO1-positive cases contained the amplicon. Expression studies demonstrated that the fusion transcripts were generally expressed at higher levels in amplified cases, and that the PAX7-FOXO1 fusion transcript was expressed at higher levels than the PAX3-FOXO1 fusion transcript. Finally, fusion gene amplification and PAX7-FOXO1 fusion status were each associated with significantly improved outcome; a multivariate analysis demonstrated that this predictive value was independent of other standard prognostic parameters. These findings therefore provide further evidence for a novel good prognosis subset of fusion-positive 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.

The Combination of Trametinib and Ganitumab is Effective in RAS-Mutated PAX-Fusion Negative Rhabdomyosarcoma Models.

PURPOSE: PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS. EXPERIMENTAL DESIGN: Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. RESULTS: Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis. CONCLUSIONS: We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.

NanoString Digital Molecular Profiling of Protein and microRNA in Rhabdomyosarcoma.

PURPOSE: Rhabdomyosarcoma (RMS) exhibits a complex prognostic algorithm based on histologic, biologic and clinical parameters. The embryonal (ERMS) and spindle cell-sclerosing RMS (SRMS) histologic subtypes warrant further studies due to their heterogenous genetic background and variable clinical behavior. NanoString digital profiling methods have been previously highlighted as robust novel methods to detect protein and microRNA expression in several cancers but not in RMS. METHODS/PATIENTS: To identify prognostic biomarkers, we categorized 12 ERMS and SRMS tumor cases into adverse (n = 5) or favorable (n = 7) prognosis groups and analyzed their signaling pathways and microRNA profiles. The digital spatial profiling of protein and microRNA analysis was performed on formalin-fixed, paraffin-embedded (FFPE) tumor tissue using NanoString technology. RESULTS: The detectable expression of several component members of the PI3K/AKT, MAPK and apoptosis signaling pathways was highlighted in RMS, including INPP4B, Pan-AKT, MET, Pan-RAS, EGFR, phospho-p90 RSK, p44/42 ERK1/2, BAD, BCL-XL, cleaved caspase-9, NF1, PARP and p53. Compared to cases with favorable prognosis, the adverse-prognosis tumor samples had significantly increased expression of INPP4B, which was confirmed with traditional immunohistochemistry. The analysis of microRNA profiles revealed that, out of 798 microRNAs assessed, 228 were overexpressed and 134 downregulated in the adverse prognosis group. Significant over-expression of oncogenic/tumor suppressor miR-3144-3p, miR-612, miR-302d-3p, miR-421, miR-548ar-5p and miR-548y (p < 0.05) was noted in the adverse prognosis group. CONCLUSION: This study highlights the utility of NanoString digital profiling methods in RMS, where it can detect distinct molecular signatures with the expression of signaling pathways and microRNAs from FFPE tumor tissue that may help identify prognostic biomarkers of interest. The overexpression of INPP4B and miR-3144-3p, miR-612, miR-302d-3p, miR-421, miR-548y and miR-548ar-5p may be associated with worse overall survival in ERMS and SRMS.

Novel GLCCI1-BRAF fusion drives kinase signaling in a case of pheochromocytomatosis.

Introduction: Recurrent and metastatic pheochromocytoma (PCC) are rare advanced endocrine neoplasms with limited treatment options. Insight into the pathogenic molecular alterations in patients with advanced PCC can provide therapeutic options for precisely targeting dysregulated pathways. Objective: We report the discovery and characterization of a novel BRAF-containing fusion transcript and its downstream molecular alterations in a patient with recurrent PCC with peritoneal seeding (pheochromocytomatosis). Methods: We reviewed the medical record of a patient with pheochromocytomatosis. A comprehensive pan-cancer molecular profiling using next-generation sequencing (NGS) as well as confirmatory real-time-quantitative PCR were performed on surgical specimens. BRAF rearrangement and downstream molecular changes were assayed using fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC), respectively. Western blot was used to assess the in vitro activation of the mitogen-activated protein kinase (MAPK) signaling pathway and the EMT markers in transfected HEK-293 cells. Results: The NGS analysis of a specimen from a 72-year-old female patient with pheochromocytomatosis showed an in-frame fusion of exon 3 of Glucocorticoid Induced 1 (GLCCI1) to exon 9 of BRAF. The upstream auto-inhibitory domain of BRAF was excluded from the GLCCI1-BRAF fusion; however, the downstream BRAF kinase domain was intact. A BRAF rearrangement was confirmed via a BRAF-specific break-apart FISH assay. Four separate tumor foci harbored GLCCI1-BRAF fusion. IHC demonstrated increased phosphorylated MEK. HEK-293 cells transfected with the GLCCI1-BRAF fusion demonstrated increased phosphorylated MEK as well as higher expression of EMT markers SNAI1 and ZEB1 in vitro. Conclusion: We demonstrate a novel pathogenic gene fusion of GLCCI1 with the oncogenic kinase domain of BRAF, resulting in an activation of the MAPK signaling pathway and EMT markers. Thus, this patient may benefit from clinically available MEK and/or BRAF inhibitors when systemic therapy is indicated. Summary Statement: This report is the first of GLCCI1 fused to BRAF in a human neoplasm and only the second BRAF-containing fusion transcript in PCC. Detailed molecular characterization of PCC can be a valuable tool in managing patients with recurrent PCC and pheochromocytomatosis that represents a significant clinical challenge.

Congenital spindle cell rhabdomyosarcoma: An international cooperative analysis.

BACKGROUND: Spindle cell rhabdomyosarcoma (RMS) is a rare variant of RMS accounting for up to 10% of cases in infants. In older children and adults, spindle cell RMS is associated with MYOD1 mutations and a poor prognosis. In infants, it is associated with recurring fusions involving NCOA2 and VGLL2. Reports in the literature suggest a favorable prognosis for this subset, however, little is known about treatment and outcome data of infants with spindle cell RMS. METHODS: Characteristics, treatment, and outcome of an international cohort of 40 patients aged ≤ 12 months with spindle cell RMS treated from 1997 to 2018 were evaluated. RESULTS: Localized disease (LD) was diagnosed in 39 patients. The median age at diagnosis was 2.5 months (range 0-12 months). Expert pathologic review confirmed the diagnosis of spindle cell RMS in all patients. Among 26 tumors that had molecular evaluation, 13 had rearrangements of NCOA and/or VGLL. Multimodal treatment of infants with LD included conventional (age adjusted) chemotherapy (n = 37), resection (n = 31) and radiotherapy (RT) (n = 5, brachytherapy in 3). Complete remission was achieved in 37/39 patients. Progressive disease occurred in two infants, relapsed disease in three. Microscopically complete surgical resection was associated with five-year event-free survival (EFS) and overall survival (OS) of 100%. Two patients with tumors ≤ 5 cm were treated with microscopically complete resection only and were alive 1 and 4.2 years after diagnosis. The 5-year EFS and OS for infants with LD were 86% (±11; CI 95%) and 91% (±9; CI 95%), respectively. One patient had metastatic disease (NCOA fusion positive) with primary tumor in head and neck and brain metastases. This patient died despite chemotherapy and delayed resection of the primary tumor due to respiratory failure secondary to cytomegalovirus infection 1.2 years after diagnosis. CONCLUSION: Infants with spindle cell RMS have an excellent prognosis. Multimodal treatment including microscopically complete resection of the tumor is strongly recommended.