Pediatric Erythroid Sarcoma Diagnostically Confirmed by Identification of a Recurrent NFIA::CBFA2T3 Fusion.

Erythroid sarcoma (ES) is exceedingly rare in the pediatric population with only a handful of reports of de novo cases, mostly occurring in the central nervous system (CNS) or orbit. It is clinically and pathologically challenging and can masquerade as a nonhematopoietic small round blue cell tumor. Clinical presentation of ES without bone marrow involvement makes diagnosis particularly difficult. We describe a 22-month-old female with ES who presented with a 2-cm mass involving the left parotid region and CNS. The presence of crush/fixation artifact from the initial biopsy made definitive classification of this highly proliferative and malignant neoplasm challenging despite an extensive immunohistochemical workup. Molecular studies including RNA-sequencing revealed a NFIA::CBFA2T3 fusion. This fusion has been identified in several cases of de novo acute erythroid leukemia (AEL) and gene expression analysis comparing this case to other AELs revealed a similar transcriptional profile. Given the diagnostically challenging nature of this tumor, clinical RNA-sequencing was essential for establishing a diagnosis.

Neonatal osteoblastic tumor with a novel PTBP1::FOSB fusion.

Congenital/neonatal bone neoplasms are extremely rare. We present the case of a patient with a neonatal bone tumor of the fibula that had osteoblastic differentiation and a novel PTBP1::FOSB fusion. FOSB fusions are described in several different tumor types, including osteoid osteoma and osteoblastoma; however, these tumors typically present in the second or third decade of life, with case reports as young as 4 months of age. Our case expands the spectrum of congenital/neonatal bone lesions. The initial radiologic, histologic, and molecular findings supported the decision for close clinical follow-up rather than more aggressive intervention. Since the time of diagnosis, this tumor has undergone radiologic regression without treatment.

EGFR internal tandem duplications in fusion-negative congenital and neonatal spindle cell tumors.

Next-generation sequencing (NGS) assays can sensitively detect somatic variation, and increasingly can enable the identification of complex structural rearrangements. A subset of infantile spindle cell sarcomas, particularly congenital mesoblastic nephromas with classic or mixed histology, have structural rearrangement in the form of internal tandem duplications (ITD) involving EGFR. We performed prospective analysis to identify EGFR ITD through clinical or research studies, as well as retrospective analysis to quantify the frequency of EGFR ITD in pediatric sarcomas. Within our institution, three tumors with EGFR ITD were prospectively identified, all occurring in patients less than 1 year of age at diagnosis, including two renal tumors and one mediastinal soft tissue tumor. These three cases exhibited both cellular and mixed cellular and classic histology. All patients had no evidence of disease progression off therapy, despite incomplete resection. To extend our analysis and quantify the frequency of EGFR ITD in pediatric sarcomas, we retrospectively analyzed a cohort of tumors (n = 90) that were previously negative for clinical RT-PCR-based fusion testing. We identified EGFR ITD in three analyzed cases, all in patients less than 1 year of age (n = 18; 3/18, 17%). Here we expand the spectrum of tumors with EGFR ITD to congenital soft tissue tumors and report an unusual example of an EGFR ITD in a tumor with cellular congenital mesoblastic nephroma histology. We also highlight the importance of appropriate test selection and bioinformatic analysis for identification of this genomic alteration that is unexpectedly common in congenital and infantile spindle cell tumors.

A developmentally prometastatic niche to hepatoblastoma in neonatal liver mediated by the Cxcl1/Cxcr2 axis.

BACKGROUND AND AIMS: Hepatoblastoma (HB) is the most common pediatric liver cancer. Its predominant occurrence in very young children led us to investigate whether the neonatal liver provides a protumorigenic niche to HB development. APPROACH AND RESULTS: HB development was compared between orthotopic transplantation models established in postnatal day 5 (P5) and 60 (P60) mice (P5Tx and P60Tx models). Single-cell RNA-sequencing (sc-RNAseq) was performed using tumor and liver tissues from both models and the top candidate cell types and genes identified are investigated for their roles in HB cell growth, migration, and survival. CONCLUSIONS: We found that various HB cell lines including HepG2 cells were consistently and considerably more tumorigenic and metastatic in the P5Tx model than in the P60Tx models. Sc-RNAseq of the P5Tx and P60Tx HepG2 models revealed that the P5Tx tumor was more hypoxic and had a larger number of activated hepatic stellate cells (aHSCs) in the tumor-surrounding liver that express significantly higher levels of Cxcl1 than those from the P60Tx model. We found these differences were developmentally present in normal P5 and P60 liver. We showed that the Cxcl1/Cxcr2 axis mediated HB cell migration and was critical to HB cell survival under hypoxia. Treating HepG2 P60Tx model with recombinant CXCL1 protein induced intrahepatic and pulmonary metastasis and CXCR2 knockout (KO) in HepG2 cells abolished their metastatic potential in the P5Tx model. Lastly, we showed that in tumors from patients with metastatic HB, there was a similar larger population of aHSCs in the tumor-surrounding liver than in localized tumors, and tumor hypoxia was uniquely associated with prognosis of patients with HB among pediatric cancers. We demonstrated that the neonatal liver provides a prometastatic niche to HB development through the Cxcl1/Cxcr2 axis.

KRIT1-positive hyperkeratotic cutaneous capillary venous malformation.

Cerebral cavernous malformations (CCM) may present in sporadic or familial forms, with different cutaneous manifestations including deep blue nodules, capillary malformations, and hyperkeratotic cutaneous capillary venous malformations (HCCVM). We report the case of an infant with a KRIT1-positive HCCVM associated with familial CCM. Moreover, histopathology showed positive immunohistochemical stain with GLUT1, further expanding the differential diagnosis of GLUT1-positive vascular anomalies.

Novel morphologic findings in PLAG1-rearranged soft tissue tumors.

Oncogenesis in PLAG1-rearranged tumors often results from PLAG1 transcription factor overexpression driven by promoter-swapping between constitutively expressed fusion partners. PLAG1-rearranged tumors demonstrate diverse morphologies. This study adds to this morphologic heterogeneity by introducing two tumors with PLAG1 rearrangements that display distinct histologic features. The first arose in the inguinal region of a 3-year-old, appeared well-circumscribed with a multinodular pattern, and harbored two fusions: ZFHX4-PLAG1 and CHCHD7-PLAG1. The second arose in the pelvic cavity of a 15-year-old girl, was extensively infiltrative and vascularized with an adipocytic component, and demonstrated a COL3A1-PLAG1 fusion. Both showed low-grade cytomorphology, scarce mitoses, no necrosis, and expression of CD34 and desmin. The ZFHX4-/CHCHD7-PLAG1-rearranged tumor showed no evidence of recurrence after 5 months. By contrast, the COL3A1-PLAG1-rearranged tumor quickly recurred following primary excision with positive margins; subsequent re-excision with adjuvant chemotherapy resulted in no evidence of recurrence after 2 years. While both tumors show overlap with benign and malignant fibroblastic and fibrovascular neoplasms, they also display divergent features. These cases highlight the importance of appropriate characterization in soft tissue tumors with unusual clinical and histologic characteristics.

Gastroblastoma with a novel EWSR1-CTBP1 fusion presenting in adolescence.

Gastroblastomas are rare tumors with a biphasic epithelioid/spindle cell morphology that typically present in early adulthood and have recurrent MALAT1-GLI1 fusions. We describe an adolescent patient with Wiskott-Aldrich syndrome who presented with a large submucosal gastric tumor with biphasic morphology. Despite histologic features consistent with gastroblastoma, a MALAT1-GLI1 fusion was not found in this patient's tumor; instead, comprehensive molecular profiling identified a novel EWSR1-CTBP1 fusion and no other significant genetic alterations. The tumor also overexpressed NOTCH and FGFR by RNA profiling. The novel fusion and expression profile suggest a role for epithelial-mesenchymal transition in this tumor, with potential implications for the pathogenesis of biphasic gastric tumors such as gastroblastoma.

Discovery of clinically relevant fusions in pediatric cancer.

BACKGROUND: Pediatric cancers typically have a distinct genomic landscape when compared to adult cancers and frequently carry somatic gene fusion events that alter gene expression and drive tumorigenesis. Sensitive and specific detection of gene fusions through the analysis of next-generation-based RNA sequencing (RNA-Seq) data is computationally challenging and may be confounded by low tumor cellularity or underlying genomic complexity. Furthermore, numerous computational tools are available to identify fusions from supporting RNA-Seq reads, yet each algorithm demonstrates unique variability in sensitivity and precision, and no clearly superior approach currently exists. To overcome these challenges, we have developed an ensemble fusion calling approach to increase the accuracy of identifying fusions. RESULTS: Our Ensemble Fusion (EnFusion) approach utilizes seven fusion calling algorithms: Arriba, CICERO, FusionMap, FusionCatcher, JAFFA, MapSplice, and STAR-Fusion, which are packaged as a fully automated pipeline using Docker and Amazon Web Services (AWS) serverless technology. This method uses paired end RNA-Seq sequence reads as input, and the output from each algorithm is examined to identify fusions detected by a consensus of at least three algorithms. These consensus fusion results are filtered by comparison to an internal database to remove likely artifactual fusions occurring at high frequencies in our internal cohort, while a "known fusion list" prevents failure to report known pathogenic events. We have employed the EnFusion pipeline on RNA-Seq data from 229 patients with pediatric cancer or blood disorders studied under an IRB-approved protocol. The samples consist of 138 central nervous system tumors, 73 solid tumors, and 18 hematologic malignancies or disorders. The combination of an ensemble fusion-calling pipeline and a knowledge-based filtering strategy identified 67 clinically relevant fusions among our cohort (diagnostic yield of 29.3%), including RBPMS-MET, BCAN-NTRK1, and TRIM22-BRAF fusions. Following clinical confirmation and reporting in the patient's medical record, both known and novel fusions provided medically meaningful information. CONCLUSIONS: The EnFusion pipeline offers a streamlined approach to discover fusions in cancer, at higher levels of sensitivity and accuracy than single algorithm methods. Furthermore, this method accurately identifies driver fusions in pediatric cancer, providing clinical impact by contributing evidence to diagnosis and, when appropriate, indicating targeted therapies.

Targeted Therapy in a Young Adult With a Novel Epithelioid Tumor Driven by a PRRC2B-ALK Fusion.

This case report describes an 18-year-old woman with an unusual epithelioid tumor of the omentum with a novel PRRC2B-ALK fusion. Although the atypical pathologic features raised significant diagnostic challenges, expression of CD30 on tumor cells and detection of an ALK rearrangement provided critical information for selecting targeted therapy in a patient not suitable for surgical resection. Despite an initially promising therapeutic response, the patient died. The efficacy of treatment was confirmed by the lack of viable tumor cells at autopsy. This case highlights the role of timely targeted therapy in patients with rare tumors and novel actionable molecular targets.

Synthesis of Oligoribonucleotides Containing a 2'-Amino-5'-S-phosphorothiolate Linkage.

Oligoribonucleotides containing a photocaged 2'-amino-5'-S-phophorothiolate linkage have potential applications as therapeutic agents and biological probes to investigate the RNA structure and function. We envisioned that oligoribonucleotides containing a 2'-amino-5'-S-phosphorothiolate linkage could provide an approach to identify the general base within catalytic RNAs by chemogenetic suppression. To enable preliminary tests of this idea, we developed synthetic approaches to a dinucleotide, trinucleotide, and oligoribonucleotide containing a photocaged 2'-amino-5'-S-phosphorothiolate linkage. We incorporated the photocaged 2'-amino-5'-S-phosphorothiolate linkage into an oligoribonucleotide substrate for the hepatitis delta virus (HDV) ribozyme and investigated the pH dependence of its cleavage following UV irradiation both in the presence and absence of the ribozyme. The substrate exhibited a pH-rate profile characteristic of the modified linkage but reacted slower when bound to the ribozyme. Cleavage inhibition by the HDV ribozyme could reflect a non-productive ground-state interaction with the modified substrate's nucleophilic 2'-NH2 or a poor fit of the modified transition state at the ribozyme's active site.

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.

Bilateral Lung Metastases From a Phalangeal Giant Cell Tumor of Bone.

We describe a rare pediatric case of a phalangeal giant cell tumor of bone with extensive bilateral lung metastases following curettage, wide resection, and amputation. Concurrent peripheral blood eosinophilia and pleural effusion with marked eosinophilia (47%) were present. To discover genetic changes driving tumor metastasis, genomic and transcriptome profiling of the metastatic lung mass as well as germline analysis were performed. Whole exome sequencing detected a histone H3F3A p.G35V missense mutation in tumor cells. RNA sequencing revealed overexpression of receptor activator of nuclear factor kappa-B ligand (RANKL). The patient is alive with no residual disease and uncompromised respiratory function 29 months after amputation of primary tumor and 19 months after surgical resection of his metastatic lung disease.

Evidence That Nucleophile Deprotonation Exceeds Bond Formation in the HDV Ribozyme Transition State.

Steric constraints imposed by the active sites of protein and RNA enzymes pose major challenges to the investigation of structure-function relationships within these systems. As a strategy to circumvent such constraints in the HDV ribozyme, we have synthesized phosphoramidites from propanediol derivatives and incorporated them at the 5'-termini of RNA and DNA oligonucleotides to generate a series of novel substrates with nucleophiles perturbed electronically through geminal fluorination. In nonenzymatic, hydroxide-catalyzed intramolecular transphosphorylation of the DNA substrates, pH-rate profiles revealed that fluorine substitution reduces the maximal rate and the kinetic p Ka, consistent with the expected electron-withdrawing effect. In HDV ribozyme reactions, we observed that the RNA substrates undergo transphosphorylation relatively efficiently, suggesting that the conformational constraints imposed by a ribofuranose ring are not strictly required for ribozyme catalysis. In contrast to the nonenzymatic reactions, however, substrate fluorination modestly increases the ribozyme reaction rate, consistent with a mechanism in which (1) the 2'-hydroxyl nucleophile exists predominantly in its neutral, protonated form in the ground state and (2) the 2'-hydroxyl bears some negative charge in the rate-determining step, consistent with a transition state in which the extent of 2'-OH deprotonation exceeds the extent of P-O bond formation.

Transition State Features in the Hepatitis Delta Virus Ribozyme Reaction Revealed by Atomic Perturbations.

Endonucleolytic ribozymes constitute a class of non-coding RNAs that catalyze single-strand RNA scission. With crystal structures available for all of the known ribozymes, a major challenge involves relating functional data to the physically observed RNA architecture. In the case of the hepatitis delta virus (HDV) ribozyme, there are three high-resolution crystal structures, the product state of the reaction and two precursor variants, with distinct mechanistic implications. Here, we develop new strategies to probe the structure and catalytic mechanism of a ribozyme. First, we use double-mutant cycles to distinguish differences in functional group proximity implicated by the crystal structures. Second, we use a corrected form of the Brønsted equation to assess the functional significance of general acid catalysis in the system. Our results delineate the functional relevance of atomic interactions inferred from structure, and suggest that the HDV ribozyme transition state resembles the cleavage product in the degree of proton transfer to the leaving group.

Discovery of immunotherapy targets for pediatric solid and brain tumors by exon-level expression.

Immunotherapy with CAR T cells for pediatric solid and brain tumors is constrained by available targetable antigens. Cancer-specific exons (CSE) present a promising reservoir of targets; however, these have not been explored and validated systematically in a pan-cancer fashion. To identify CSE targets, we analyzed 1,532 RNA-seq datasets from 16 types of pediatric solid and brain tumors for comparison with normal tissues using a newly developed workflow. We found 2,933 exons in 157 genes encoding proteins of the surfaceome or matrisome with high cancer specificity either at the gene (n=148) or the alternatively spliced (AS) isoform (n=9) level. Expression of selected AS targets, including the EDB domain of FN1 (EDB), and gene targets, such as COL11A1, were validated in pediatric PDX tumors. We generated CAR T cells specific to EDB or COL11A1 and demonstrated that COL11A1-CAR T-cells have potent antitumor activity. The full target list, explorable via an interactive web portal (https://cseminer.stjude.org/), provides a rich resource for developing immunotherapy of pediatric solid and brain tumors using gene or AS targets with high expression specificity in cancer.

Discovery of immunotherapy targets for pediatric solid and brain tumors by exon-level expression.

Immunotherapy with chimeric antigen receptor T cells for pediatric solid and brain tumors is constrained by available targetable antigens. Cancer-specific exons present a promising reservoir of targets; however, these have not been explored and validated systematically in a pan-cancer fashion. To identify cancer specific exon targets, here we analyze 1532 RNA-seq datasets from 16 types of pediatric solid and brain tumors for comparison with normal tissues using a newly developed workflow. We find 2933 exons in 157 genes encoding proteins of the surfaceome or matrisome with high cancer specificity either at the gene (n = 148) or the alternatively spliced isoform (n = 9) level. Expression of selected alternatively spliced targets, including the EDB domain of fibronectin 1, and gene targets, such as COL11A1, are validated in pediatric patient derived xenograft tumors. We generate T cells expressing chimeric antigen receptors specific for the EDB domain or COL11A1 and demonstrate that these have antitumor activity. The full target list, explorable via an interactive web portal ( https://cseminer.stjude.org/ ), provides a rich resource for developing immunotherapy of pediatric solid and brain tumors using gene or AS targets with high expression specificity in cancer.

An integrated single-cell RNA-seq map of human neuroblastoma tumors and preclinical models uncovers divergent mesenchymal-like gene expression programs.

BACKGROUND: Neuroblastoma is a common pediatric cancer, where preclinical studies suggest that a mesenchymal-like gene expression program contributes to chemotherapy resistance. However, clinical outcomes remain poor, implying we need a better understanding of the relationship between patient tumor heterogeneity and preclinical models. RESULTS: Here, we generate single-cell RNA-seq maps of neuroblastoma cell lines, patient-derived xenograft models (PDX), and a genetically engineered mouse model (GEMM). We develop an unsupervised machine learning approach ("automatic consensus nonnegative matrix factorization" (acNMF)) to compare the gene expression programs found in preclinical models to a large cohort of patient tumors. We confirm a weakly expressed, mesenchymal-like program in otherwise adrenergic cancer cells in some pre-treated high-risk patient tumors, but this appears distinct from the presumptive drug-resistance mesenchymal programs evident in cell lines. Surprisingly, however, this weak-mesenchymal-like program is maintained in PDX and could be chemotherapy-induced in our GEMM after only 24 h, suggesting an uncharacterized therapy-escape mechanism. CONCLUSIONS: Collectively, our findings improve the understanding of how neuroblastoma patient tumor heterogeneity is reflected in preclinical models, provides a comprehensive integrated resource, and a generalizable set of computational methodologies for the joint analysis of clinical and pre-clinical single-cell RNA-seq datasets.

An integrated single-cell RNA-seq map of human neuroblastoma tumors and preclinical models uncovers divergent mesenchymal-like gene expression programs.

Neuroblastoma is a common pediatric cancer, where preclinical studies suggest that a mesenchymal-like gene expression program contributes to chemotherapy resistance. However, clinical outcomes remain poor, implying we need a better understanding of the relationship between patient tumor heterogeneity and preclinical models. Here, we generated single-cell RNA-seq maps of neuroblastoma cell lines, patient-derived xenograft models (PDX), and a genetically engineered mouse model (GEMM). We developed an unsupervised machine learning approach ('automatic consensus nonnegative matrix factorization' (acNMF)) to compare the gene expression programs found in preclinical models to a large cohort of patient tumors. We confirmed a weakly expressed, mesenchymal-like program in otherwise adrenergic cancer cells in some pre-treated high-risk patient tumors, but this appears distinct from the presumptive drug-resistance mesenchymal programs evident in cell lines. Surprisingly however, this weak-mesenchymal-like program was maintained in PDX and could be chemotherapy-induced in our GEMM after only 24 hours, suggesting an uncharacterized therapy-escape mechanism. Collectively, our findings improve the understanding of how neuroblastoma patient tumor heterogeneity is reflected in preclinical models, provides a comprehensive integrated resource, and a generalizable set of computational methodologies for the joint analysis of clinical and pre-clinical single-cell RNA-seq datasets.

Bone morphogenetic protein (BMP) signaling determines neuroblastoma cell fate and sensitivity to retinoic acid.

Retinoic acid (RA) is a standard-of-care neuroblastoma drug thought to be effective by inducing differentiation. Curiously, RA has little effect on primary human tumors during upfront treatment but can eliminate neuroblastoma cells from the bone marrow during post-chemo consolidation therapy-a discrepancy that has never been explained. To investigate this, we treated a large cohort of neuroblastoma cell lines with RA and observed that the most RA-sensitive cells predominantly undergo apoptosis or senescence, rather than differentiation. We conducted genome-wide CRISPR knockout screens under RA treatment, which identified BMP signaling as controlling the apoptosis/senescence vs differentiation cell fate decision and determining RA's overall potency. We then discovered that BMP signaling activity is markedly higher in neuroblastoma patient samples at bone marrow metastatic sites, providing a plausible explanation for RA's ability to clear neuroblastoma cells specifically from the bone marrow, seemingly mimicking interactions between BMP and RA during normal development.