Structure-activity relationship of dihydropyridines for rhabdomyosarcoma.

Childhood muscle-related cancer rhabdomyosarcoma is a rare disease with a 50-year unmet clinical need for the patients presented with advanced disease. The rarity of ∼350 cases per year in North America generally diminishes the viability of large-scale, pharmaceutical industry driven drug development efforts for rhabdomyosarcoma. In this study, we performed a large-scale screen of 640,000 compounds to identify the dihydropyridine (DHP) class of anti-hypertensives as a priority compound hit. A structure-activity relationship was uncovered with increasing cell growth inhibition as side chain length increases at the ortho and para positions of the parent DHP molecule. Growth inhibition was consistent across n = 21 rhabdomyosarcoma cell line models. Anti-tumor activity in vitro was paralleled by studies in vivo. The unexpected finding was that the action of DHPs appears to be other than on the DHP receptor (i.e., L-type voltage-gated calcium channel). These findings provide the basis of a medicinal chemistry program to develop dihydropyridine derivatives that retain anti-rhabdomyosarcoma activity without anti-hypertensive effects.

Advances in the clinical management of high-risk Wilms tumors.

Outcomes are excellent for the majority of patients with Wilms tumors (WT). However, there remain WT subgroups for which the survival rate is approximately 50% or lower. Acknowledging that the composition of this high-risk group has changed over time reflecting improvements in therapy, we introduce the authors' view of the historical and current approach to the classification and treatment of high-risk WT. For this review, we consider high-risk WT to include patients with newly diagnosed metastatic blastemal-type or diffuse anaplastic histology, those who relapse after having been initially treated with three or more different chemotherapeutics, or those who relapse more than once. In certain low- or low middle-income settings, socio-economic factors expand the definition of what constitutes a high-risk WT. As conventional therapies are inadequate to cure the majority of high-risk WT patients, advancement of laboratory and early-phase clinical investigations to identify active agents is urgently needed.

Functional genomics of human clear cell sarcoma: genomic, transcriptomic and chemical biology landscape for clear cell sarcoma.

BACKGROUND: Systemic therapy for metastatic clear cell sarcoma (CCS) bearing EWSR1-CREB1/ATF1 fusions remains an unmet clinical need in children, adolescents, and young adults. METHODS: To identify key signaling pathway vulnerabilities in CCS, a multi-pronged approach was taken: (i) genomic and transcriptomic landscape analysis, (ii) integrated chemical biology interrogations, (iii) development of CREB1/ATF1 inhibitors, and (iv) antibody-drug conjugate testing (ADC). The first approach encompassed DNA exome and RNA deep sequencing of the largest human CCS cohort yet reported consisting of 47 patient tumor samples and 8 cell lines. RESULTS: Sequencing revealed recurrent mutations in cell cycle checkpoint, DNA double-strand break repair or DNA mismatch repair genes, with a correspondingly low to intermediate tumor mutational burden. DNA multi-copy gains with corresponding high RNA expression were observed in CCS tumor subsets. CCS cell lines responded to the HER3 ADC patritumab deruxtecan in a dose-dependent manner in vitro, with impaired long term cell viability. CONCLUSION: These studies of the genomic, transcriptomic and chemical biology landscape represent a resource 'atlas' for the field of CCS investigation and drug development. CHK inhibitors are identified as having potential relevance, CREB1 inhibitors non-dependence of CCS on CREB1 activity was established, and the potential utility of HER3 ADC being used in CCS is found.

Advances in the clinical management of high-risk Wilms tumors.

Outcomes are excellent for the majority of patients with Wilms tumors (WT). However, there remain WT subgroups for which the survival rate is approximately 50% or lower. Acknowledging that the composition of this high-risk group has changed over time reflecting improvements in therapy, we introduce the authors' view of the historical and current approach to the classification and treatment of high-risk WT. For this review, we consider high-risk WT to include patients with newly diagnosed metastatic blastemal-type or diffuse anaplastic histology, those who relapse after having been initially treated with three or more different chemotherapeutics, or those who relapse more than once. In certain low- or low middle-income settings, socio-economic factors expand the definition of what constitutes a high-risk WT. As conventional therapies are inadequate to cure the majority of high-risk WT patients, advancement of laboratory and early-phase clinical investigations to identify active agents is urgently needed.

Functional genomic analysis of epithelioid sarcoma reveals distinct proximal and distal subtype biology.

BACKGROUND: Metastatic epithelioid sarcoma (EPS) remains a largely unmet clinical need in children, adolescents and young adults despite the advent of EZH2 inhibitor tazemetostat. METHODS: In order to realise consistently effective drug therapies, a functional genomics approach was used to identify key signalling pathway vulnerabilities in a spectrum of EPS patient samples. EPS biopsies/surgical resections and cell lines were studied by next-generation DNA exome and RNA deep sequencing, then EPS cell cultures were tested against a panel of chemical probes to discover signalling pathway targets with the most significant contributions to EPS tumour cell maintenance. RESULTS: Other biologically inspired functional interrogations of EPS cultures using gene knockdown or chemical probes demonstrated only limited to modest efficacy in vitro. However, our molecular studies uncovered distinguishing features (including retained dysfunctional SMARCB1 expression and elevated GLI3, FYN and CXCL12 expression) of distal, paediatric/young adult-associated EPS versus proximal, adult-associated EPS. CONCLUSIONS: Overall results highlight the complexity of the disease and a limited chemical space for therapeutic advancement. However, subtle differences between the two EPS subtypes highlight the biological disparities between younger and older EPS patients and emphasise the need to approach the two subtypes as molecularly and clinically distinct diseases.

Machine learning for rhabdomyosarcoma histopathology.

Correctly diagnosing a rare childhood cancer such as sarcoma can be critical to assigning the correct treatment regimen. With a finite number of pathologists worldwide specializing in pediatric/young adult sarcoma histopathology, access to expert differential diagnosis early in case assessment is limited for many global regions. The lack of highly-trained sarcoma pathologists is especially pronounced in low to middle-income countries, where pathology expertise may be limited despite a similar rate of sarcoma incidence. To address this issue in part, we developed a deep learning convolutional neural network (CNN)-based differential diagnosis system to act as a pre-pathologist screening tool that quantifies diagnosis likelihood amongst trained soft-tissue sarcoma subtypes based on whole histopathology tissue slides. The CNN model is trained on a cohort of 424 centrally-reviewed histopathology tissue slides of alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma and clear-cell sarcoma tumors, all initially diagnosed at the originating institution and subsequently validated by central review. This CNN model was able to accurately classify the withheld testing cohort with resulting receiver operating characteristic (ROC) area under curve (AUC) values above 0.889 for all tested sarcoma subtypes. We subsequently used the CNN model to classify an externally-sourced cohort of human alveolar and embryonal rhabdomyosarcoma samples and a cohort of 318 histopathology tissue sections from genetically engineered mouse models of rhabdomyosarcoma. Finally, we investigated the overall robustness of the trained CNN model with respect to histopathological variations such as anaplasia, and classification outcomes on histopathology slides from untrained disease models. Overall positive results from our validation studies coupled with the limited worldwide availability of sarcoma pathology expertise suggests the potential of machine learning to assist local pathologists in quickly narrowing the differential diagnosis of sarcoma subtype in children, adolescents, and young adults.

White paper: Oncofertility in pediatric patients with Wilms tumor.

The survival of childhood Wilms tumor is currently around 90%, with many survivors reaching reproductive age. Chemotherapy and radiotherapy are established risk factors for gonadal damage and are used in both COG and SIOP Wilms tumor treatment protocols. The risk of infertility in Wilms tumor patients is low but increases with intensification of treatment including the use of alkylating agents, whole abdominal radiation or radiotherapy to the pelvis. Both COG and SIOP protocols aim to limit the use of gonadotoxic treatment, but unfortunately this cannot be avoided in all patients. Infertility is considered one of the most important late effects of childhood cancer treatment by patients and their families. Thus, timely discussion of gonadal damage risk and fertility preservation options is important. Additionally, irrespective of the choice for preservation, consultation with a fertility preservation (FP) team is associated with decreased patient and family regret and better quality of life. Current guidelines recommend early discussion of the impact of therapy on potential fertility. Since most patients with Wilms tumors are prepubertal, potential FP methods for this group are still considered experimental. There are no proven methods for FP for prepubertal males (testicular biopsy for cryopreservation is experimental), and there is just a single option for prepubertal females (ovarian tissue cryopreservation), posing both technical and ethical challenges. Identification of genetic markers of susceptibility to gonadotoxic therapy may help to stratify patient risk of gonadal damage and identify patients most likely to benefit from FP methods.

Bromodomain 4 inhibition leads to MYCN downregulation in Wilms tumor.

BACKGROUND: Wilms tumor is the most common childhood kidney cancer. Two distinct histological subtypes of Wilms tumor have been described: tumors lacking anaplasia (the favorable subtype) and tumors displaying anaplastic features (the unfavorable subtype). Children with favorable disease generally have a very good prognosis, whereas those with anaplasia are oftentimes refractory to standard treatments and suffer poor outcomes, leading to an unmet clinical need. MYCN dysregulation has been associated with a number of pediatric cancers including Wilms tumor. PROCEDURES: In this context, we undertook a functional genomics approach to uncover novel therapeutic strategies for those patients with anaplastic Wilms tumor. Genomic analysis and in vitro experimentation demonstrate that cell growth can be reduced by modulating MYCN overexpression via bromodomain 4 (BRD4) inhibition in both anaplastic and nonanaplastic Wilms tumor models. RESULTS: We observed a time-dependent reduction of MYCN and MYCC protein levels upon BRD4 inhibition in Wilms tumor cell lines, which led to cell death and proliferation suppression. BRD4 inhibition significantly reduced tumor volumes in Wilms tumor patient-derived xenograft (PDX) mouse models. CONCLUSIONS: We suggest that AZD5153, a novel dual-BRD4 inhibitor, can reduce MYCN levels in both anaplastic and nonanaplastic Wilms tumor cell lines, reduces tumor volume in Wilms tumor PDXs, and should be further explored for its therapeutic potential.

Metastatic pediatric sclerosing epithelioid fibrosarcoma.

Sclerosing epithelioid fibrosarcoma (SEF) is a rare and aggressive soft-tissue sarcoma thought to originate in fibroblasts of the tissues comprising tendons, ligaments, and muscles. Minimally responsive to conventional cytotoxic chemotherapies, >50% of SEF patients experience local recurrence and/or metastatic disease. SEF is most commonly discovered in middle-aged and elderly adults, but also rarely in children. A common gene fusion occurring between the EWSR1 and CREB3L1 genes has been observed in 80%-90% of SEF cases. We describe here the youngest SEF patient reported to date (a 3-yr-old Caucasian male) who presented with numerous bony and lung metastases. Additionally, we perform a comprehensive literature review of all SEF-related articles published since the disease was first characterized. Finally, we describe the generation of an SEF primary cell line, the first such culture to be reported. The patient described here experienced persistent disease progression despite aggressive treatment including multiple resections, radiotherapy, and numerous chemotherapies and targeted therapeutics. Untreated and locally recurrent tumor and metastatic tissue were sequenced by whole-genome, whole-exome, and deep-transcriptome next-generation sequencing with comparison to a patient-matched normal blood sample. Consistent across all sequencing analyses was the disease-defining EWSR1-CREB3L1 fusion as a single feature consensus. We provide an analysis of our genomic findings and discuss potential therapeutic strategies for SEF.

Refractory alveolar rhabdomyosarcoma in an 11-year-old male.

Rhabdomyosarcoma (RMS) is a mesenchymal malignancy phenocopying muscle and is among the leading causes of death from childhood cancer. Metastatic alveolar rhabdomyosarcoma is the most aggressive subtype with an 8% 5-yr disease-free survival rate when a chromosomal fusion is present and a 29% 5-yr disease-free survival rate when negative for a fusion event. The underlying biology of PAX-fusion-negative alveolar rhabdomyosarcoma remains largely unexplored and is exceedingly rare in Li-Fraumeni syndrome patients. Here, we present the case of an 11-yr-old male with fusion-negative alveolar rhabdomyosarcoma studied at end of life with a comprehensive functional genomics characterization, resulting in identification of potential therapeutic targets for broader investigation.

Comprehensive Biology and Genetics Compendium of Wilms Tumor Cell Lines with Different WT1 Mutations.

PURPOSE: WT1 mutant Wilms tumors represent a distinct subgroup, frequently associated with CTNNB1 mutations. The genetic basis for the development of this subtype is currently not fully understood. METHODS: Live WT1 mutant Wilms tumors were collected during surgery of patients and cell cultures established in mesenchymal stem cell medium. They were studied for mutations in WT1 and CTNNB1, their differentiation capacity and protein activation status. Four cell lines were immortalized with a triple mutant ts SV40 largeT antigen and Telomerase. RESULTS: 11 cell lines were established from Wilms tumors of nine patients, including a left and right tumor from the same patient and a primary and second tumor from another patient. Six patients had germ line and three were tumor specific mutations. All cell lines harbored only mutant or deleted WT1 genes. CTNNB1 was wild type in three, all others carried mutations affecting amino acid S45. They had variable and limited capacities for mesenchymal differentiation, a high migratory capacity and a low invasive potential. All cells showed an activation of multiple receptor tyrosine kinases and downstream signaling pathways. CONCLUSIONS: These cell lines represent an important new tool to study WT1 mutant Wilms tumors, potentially leading to new treatment approaches.

Undifferentiated small round cell sarcoma in a young male: a case report.

CIC-rearranged sarcomas (CRSs) have recently been characterized as a distinct sarcoma subgroup with a less favorable prognosis compared to other small round cell sarcomas. CRSs share morphologic features with Ewing's sarcoma and prior to 2013 were grouped under undifferentiated sarcomas with round cell phenotype by the WHO classification. In this report, whole-genome sequencing and RNA sequencing were performed for an adolescent male patient with CRS who was diagnosed with undifferentiated pleomorphic sarcoma (UPS) by three contemporary institutions. Somatic mutation analysis identified mutations in IQGAP1, CCNC, and ATXN1L in pre- and post-treatment tissue samples, as well as a CIC-DUX4 fusion that was confirmed by qPCR and DUX4 immunohistochemistry. Of particular interest was the overexpression of the translation factor eEF1A1, which has oncogenic properties and has recently been identified as a target of the investigational agent plitidepsin. This case may provide a valuable waypoint in the understanding and classification of CRSs and may provide a rationale for targeting eEF1A1 in similar soft tissue sarcoma cases.