Neomorphic DNA-binding enables tumor-specific therapeutic gene expression in fusion-addicted childhood sarcoma

Chimeric fusion transcription factors are oncogenic hallmarks of several devastating cancer types including pediatric sarcomas, such as Ewing sarcoma (EwS) and alveolar rhabdomyosarcoma (ARMS). Despite their exquisite specificity, these driver oncogenes have been considered largely undruggable due to their lack of enzymatic activity. Here, we show in the EwS model that - capitalizing on neomorphic DNA-binding preferences - the addiction to the respective fusion transcription factor EWSR1-FLI1 can be leveraged to express therapeutic genes. We genetically engineered a de novo enhancer-based, synthetic and highly potent expression cassette that can elicit EWSR1-FLI1-dependent expression of a therapeutic payload as evidenced by episomal and CRISPR-edited genomic reporter assays. Combining in silico screens and immunohistochemistry, we identified GPR64 as a highly specific cell surface antigen for targeted transduction strategies in EwS. Functional experiments demonstrated that anti-GPR64-pseudotyped lentivirus harboring our expression cassette can specifically transduce EwS cells to promote the expression of viral thymidine kinase sensitizing EwS for treatment to the otherwise relatively non-toxic (Val)ganciclovir and leading to strong anti-tumorigenic, but no adverse effects in vivo. Further, we prove that similar vector designs can be applied in PAX3-FOXO1-driven ARMS, and to express immunomodulatory cytokines, such as IL-15 and XCL1, in tumor types typically considered to be immunologically cold. Collectively, these results generated in pediatric sarcomas indicate that exploiting, rather than suppressing, the neomorphic functions of chimeric transcription factors may open inroads to innovative and personalized therapies, and that our highly versatile approach may be translatable to other cancers addicted to oncogenic transcription factors with unique DNA-binding properties.

Cytoplasmic P53 Immunostaining With N-Terminus P53 Antibody And Absence Of Staining With C-Terminus P53 Antibody: A Report Of Two Pediatric Sarcomas With Distal Truncating TP53 Mutations Affecting Nuclear Localization Domain

P53 immunohistochemical staining with antibodies targeted to epitopes at or near the N-terminus are commonly used in diagnostic pathology practice as a surrogate for TP53 mutations. The abnormal staining patterns indicating TP53 mutations include nuclear overexpression, null, and the recently described cytoplasmic staining. The latter staining pattern occurs with the less common TP53 mutations affecting its nuclear localization and/or tetramerization domains that are located toward the C-terminus. Here we describe the first two cases of pediatric sarcomas with cytoplasmic staining with P53 antibody against N-terminus epitope and the absence of staining with P53 antibody against C-terminus epitope. We propose that a more precise description of P53 immunohistochemical staining patterns should include the nature of the antibody used.

Prognostic Value of Cell-Surface Vimentin-Positive CTCs in Pediatric Sarcomas

BackgroundDespite advances in care, the 5 year overall survival for patients with relapsed and or metastatic sarcoma remains as low as < 35%. Currently, there are no biomarkers available to assess disease status in patients with sarcomas and as such, disease surveillance remains reliant on serial imaging which increases the risk of secondary malignancies and heightens patient anxiety.MethodsHere, for the first time reported in the literature, we have enumerated the cell surface vimentin (CSV+) CTCs in the blood of 92 sarcoma pediatric and adolescent and young adult (AYA) patients as a possible marker of disease.ResultsWe constructed a ROC with an AUC of 0.831 resulting in a sensitivity of 85.3% and a specificity of 75%. Additionally, patients who were deemed to be CSV+ CTC positive were found to have a worse overall survival compared to those who were CSV+ CTC negative. We additionally found the use of available molecular testing increased the accuracy of our diagnostic and prognostic tests.ConclusionsOur findings indicate that CSV+ CTCs have prognostic value and can possibly serve as a measure of disease burden.

Fusion Oncoproteins in Childhood Cancers: Potential Role in Targeted Therapy

Cancer remains the leading cause of death from disease in children. Historically, in contrast to their adult counterparts, the causes of pediatric malignancies have remained largely unknown, with most pediatric cancers displaying low mutational burdens. Research related to molecular genetics in pediatric cancers is advancing our understanding of potential drivers of tumorigenesis and opening new opportunities for targeted therapies. One such area is fusion oncoproteins, which are a product of chromosomal rearrangements resulting in the fusion of different genes. They have been identified as oncogenic drivers in several sarcomas and leukemias. Continued advancement in the understanding of the biology of fusion oncoproteins will contribute to the discovery and development of new therapies for childhood cancers. Here we review the current scientific knowledge on fusion oncoproteins, focusing on pediatric sarcomas and hematologic cancers, and highlight the challenges and current efforts in developing drugs to target fusion oncoproteins.

Integrated diagnosis based on transcriptome analysis in suspected pediatric sarcomas

Pediatric solid tumors are a heterogeneous group of neoplasms with over 100 subtypes. Clinical and histopathological diagnosis remains challenging due to the overlapping morphological and immunohistochemical findings and the presence of atypical cases. To evaluate the potential utility of including RNA-sequencing (RNA-seq) in the diagnostic process, we performed RNA-seq in 47 patients with suspected pediatric sarcomas. Histopathologists specialized in pediatric cancer re-evaluated pathological specimens to reach a consensus diagnosis; 42 patients were diagnosed with known subtypes of solid tumors whereas 5 patients were diagnosed with undifferentiated sarcoma. RNA-seq analysis confirmed and refined consensus diagnoses and further identified diagnostic genetic variants in four of the five patients with undifferentiated sarcoma. Genetic lesions were detected in 23 patients, including the novel SMARCA4-THOP1 fusion gene and 22 conventional or recently reported genetic events. Unsupervised clustering analysis of the RNA-seq data identified a distinct cluster defined by the overexpression of rhabdomyosarcoma-associated genes including MYOG and CHRNG. These findings suggest that RNA-seq-based genetic analysis may aid in the diagnosis of suspected pediatric sarcomas, which would be useful for the development of stratified treatment strategies.

Malignant Mesothelioma With EWSR1-ATF1 Fusion in Two Adolescent Male Patients

Malignant mesothelioma is a neoplasm of serosal surfaces, most commonly affecting the pleura. The peritoneum, pericardium, and tunica vaginalis are less frequently involved. Malignant mesothelioma with EWSR1-ATF1 fusion in young adults was recently reported in the literature. Here, we present two pediatric cases of EWSR1-ATF1 translocation-associated malignant mesothelioma in the peritoneum and pericardium respectively. Both cases lacked a known exposure history. Microscopy in both cases showed predominantly epithelioid morphology with ample eosinophilic cytoplasm, and immunohistochemistry was positive for pan-keratin, calretinin, and WT1. Both cases showed EWSR1-ATF1 gene rearrangement by RNA sequencing, which was instrumental in confirming the diagnosis of malignant mesothelioma and to exclude more common pediatric sarcomas, especially in the context of limited sampling.

Phenotypic Similarities Within The Morphologic Spectrum Of DICER1-Associated Sarcomas and Pleuropulmonary Blastoma -- Histopathologic Features Suggesting Genetic Testing and Counselling

Context - DICER1-associated sarcomas are rare. These are currently described under a wide variety of appellations; morphologic characterizations in reported cases and sites of occurrence have also been disparate. Design – We aimed to review pediatric sarcomas associated with DICER1-mutation reported at our center, along with literature review, to identify histologic hallmarks for diagnosis. Results - A 12 year old girl with intracranial sarcoma mimicking mesenchymal chondrosarcoma, a 16 year old girl with broad ligament sarcoma mimicking fibrosarcoma and a 5 month old girl with vaginal sarcoma mimicking embryonal rhabdomyosarcoma showed DICER1-mutation. All three tumors though seemingly diverse, had an uncanny resemblance, comprising of a primitive mesenchyme-like spindle cell component with rhabdomyoblastic differentiation on immunohistochemistry. Primitive blastema, chondroid differentiation and foci of anaplasia mimicking pleuropulmonary blastoma histology were variably present. One case showed primitive neuroblastic differentiation. Though the constellation of features reported in literature is quite varied, rhabdomyoblastic differentiation has been ubiquitously reported in tumours across sites. Molecular testing showed gain of chromosome 8 in 2 cases. All 3 cases responded to alternating Vincristine, doxorubicin and cyclophosphamide / Cisplatin, etoposide and ifosfamide (VDC/PEI) backbone followed by maintenance chemotherapy. Conclusion - We highlight this morphologic hallmark of rhabdomyoblastic differentiation with or without chondroid differentiation, in primitive appearing pediatric sarcomas, especially of female genital tract and brain, which should raise a flag to test for DICER1 pathogenic variation. This is crucial in low / middle income countries where sequencing is not done routinely. Timely diagnosis can ensure appropriate treatment and implementation of surveillance protocols for those with germline mutation.

Pediatric sarcoma survivorship: A call for a developmental cascades approach

Survivors of pediatric sarcomas often experience greater psychological and psychosocial difficulties than their non-afflicted peers. We consider findings related to poorer outcomes from a developmental cascade perspective. Specifically, we discuss how physical, neurocognitive, psychological, and psychosocial costs associated with pediatric sarcomas and their treatment function transactionally to degrade well-being in long-term pediatric sarcoma survivors. We situate the sarcoma experience as a broad developmental threat – one stemming from both the presence and treatment of a life-imperiling disease, and the absence of typical childhood experiences. Ways in which degradation in one developmental domain spills over and effects other domains are highlighted. We argue that the aggregate effect of these cascades is two-fold: first, it adds to the typical stress involved in meeting developmental milestones and navigating developmental transitions; and second, it deprives survivors of crucial coping strategies that mitigate these stressors. This position suggests specific moments of intervention and raises specific hypotheses for investigators to explore.