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.     

P/CAF mediates PAX3–FOXO1‐dependent oncogenesis in alveolar rhabdomyosarcoma

Alveolar rhabdomyosarcoma (ARMS) is an aggressive paediatric cancer of skeletal muscle with poor prognosis. A PAX3–FOXO1 fusion protein acts as a driver of malignancy in ARMS by disrupting tightly coupled but mutually exclusive pathways of proliferation and differentiation. While PAX3–FOXO1 is an attractive therapeutic target, no current treatments are designed to block its oncogenic activity. The present work shows that the histone acetyltransferase P/CAF (KAT2B) is overexpressed in primary tumours from ARMS patients. Interestingly, in fusion‐positive ARMS cell lines, P/CAF acetylates and stabilizes PAX3–FOXO1 rather than MyoD, a master regulator of muscle differentiation. Silencing P/CAF, or pharmacological inhibition of its acetyltransferase activity, down‐regulates PAX3–FOXO1 levels concomitant with reduced proliferation and tumour burden in xenograft mouse models. Our studies identify a P/CAF–PAX3–FOXO1 signalling node that promotes oncogenesis and may contribute to MyoD dysfunction in ARMS. This work exemplifies the therapeutic potential of targeting chromatin‐modifying enzymes to inhibit fusion oncoproteins that are a frequent event in sarcomas. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Genetic heterogeneity in rhabdomyosarcoma revealed by SNP array analysis

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents. Alveolar (ARMS) and embryonal (ERMS) histologies predominate, but rare cases are classified as spindle cell/sclerosing (SRMS). For treatment stratification, RMS is further subclassified as fusion‐positive (FP‐RMS) or fusion‐negative (FN‐RMS), depending on whether a gene fusion involving PAX3 or PAX7 is present or not. We investigated 19 cases of pediatric RMS using high resolution single‐nucleotide polymorphism (SNP) array. FP‐ARMS displayed, on average, more structural rearrangements than ERMS; the single FN‐ARMS had a genomic profile similar to ERMS. Apart from previously known amplification (e.g., MYCN, CDK4, and MIR17HG) and deletion (e.g., NF1, CDKN2A, and CDKN2B) targets, amplification of ERBB2 and homozygous loss of ASCC3 or ODZ3 were seen. Combining SNP array with cytogenetic data revealed that most cases were polyploid, with at least one case having started as a near‐haploid tumor. Further bioinformatic analysis of the SNP array data disclosed genetic heterogeneity, in the form of subclonal chromosomal imbalances, in five tumors. The outcome was worse for patients with FP‐ARMS than ERMS or FN‐ARMS (6/8 vs. 1/9 dead of disease), and the only children with ERMS showing intratumor diversity or with MYOD1 mutation‐positive SRMS also died of disease. High resolution SNP array can be useful in evaluating genomic imbalances in pediatric RMS. © 2015 Wiley Periodicals, Inc.     

Low frequency of PAX8‐PPARγ rearrangement in follicular thyroid carcinomas in Japanese patients

Paired‐box gene 8 (PAX8)‐peroxisome proliferator‐activated receptor‐γ (PPARγ) gene fusion has been identified at significant frequency in follicular thyroid carcinomas (FTCs) with cytogenetically detectable translocation t(2;3)(q13;p25). This represents a possible specific molecular marker for follicular carcinoma. In this study, we examined PAX8‐PPARγ rearrangement in 24 FTC samples from Japanese patients by reverse transcribed‐polymerase chain reaction (RT‐PCR) using two upstream PAX8 primers located in exons 7 and 8 and a downstream primer in exon 1 of PPARγ. The fusion gene was detected in only one of 24 FTCs (4%). The FTC with PAX8‐PPARγ rearrangement from a 56‐year‐old man showed a product consistent with fusion between exon 8 of PAX8 and exon 1 of PPARγ. It was confirmed by direct sequencing. This FTC was histologically encapsulated, composed of trabeculae and small follicles and had complete penetration of the capsule by tumor tissues (minimally invasive type). The frequency of the fusion gene in this study was much lower than the 29–63% noted in reports from other countries suggesting that FTCs in Japanese patients may have a special genetic background, and that the high iodine intake from a typical Japanese diet might influence the frequency of the fusion gene in FTCs.     

Copy number alterations determined by single nucleotide polymorphism array testing in the clinical laboratory are indicative of gene fusions in pediatric cancer patients

Gene fusions resulting from structural rearrangements are an established mechanism of tumorigenesis in pediatric cancer. In this clinical cohort, 1,350 single nucleotide polymorphism (SNP)‐based chromosomal microarrays from 1,211 pediatric cancer patients were evaluated for copy number alterations (CNAs) associated with gene fusions. Karyotype or fluorescence in situ hybridization studies were performed in 42% of the patients. Ten percent of the bone marrow or solid tumor specimens had SNP array‐associated CNAs suggestive of a gene fusion. Alterations involving ETV6, ABL1‐NUP214, EBF1‐PDGFRB, KMT2A(MLL), LMO2‐RAG, MYH11‐CBFB, NSD1‐NUP98, PBX1, STIL‐TAL1, ZNF384‐TCF3, P2RY8‐CRLF2, and RUNX1T1‐RUNX1 fusions were detected in the bone marrow samples. The most common alteration among the low‐grade gliomas was a 7q34 tandem duplication resulting in a KIAA1549‐BRAF fusion. Additional fusions identified in the pediatric brain tumors included FAM131B‐BRAF and RAF1‐QKI. COL1A1‐PDGFB, CRTC1‐MAML2, EWSR1, HEY1, PAX3‐ and PAX7‐FOXO1, and PLAG1 fusions were determined in a variety of solid tumors and a novel potential gene fusion, FGFR1‐USP6, was detected in an aneurysmal bone cyst. The identification of these gene fusions was instrumental in tumor diagnosis. In contrast to hematologic and solid tumors in adults that are predominantly driven by mutations, the majority of hematologic and solid tumors in children are characterized by CNAs and gene fusions. Chromosomal microarray analysis is therefore a robust platform to identify diagnostic and prognostic markers in the clinical setting.     

Genomic and clinical analyses of 2p24 and 12q13‐q14 amplification in alveolar rhabdomyosarcoma: A report from the Children's Oncology Group

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer that is related to the skeletal muscle lineage and characterized by recurrent chromosomal translocations. Within the ARMS category, there is clinical and genetic heterogeneity, consistent with the premise that “primary” genetic events collaborate with “secondary” events to give rise to subsets with varying clinical features. Previous studies demonstrated that genomic amplification occurs frequently in ARMS. In the current study, we used oligonucleotide arrays to localize two common amplicons to the 2p24 and 12q13‐q14 chromosomal regions. Based on the copy number array data, we sublocalized the minimum common regions of 2p24 and 12q13‐q14 amplification to a 0.83 Mb region containing the DDX1 and MYCN genes, and a 0.55 Mb region containing 27 genes, respectively. Using fluorescent in situ hybridization assays to measure copy number of the 2p24 and 12q13‐q14 regions in over 100 cases, we detected these amplicons in 13% and 12% of cases, respectively. Comparison with fusion status revealed that 2p24 amplification occurred preferentially in cases positive for PAX3‐FOXO1 or PAX7‐FOXO1 while 12q13‐q14 amplification occurred preferentially in PAX3‐FOXO1‐positive cases. Expression studies demonstrated that MYCN was usually overexpressed in cases with 2p24 amplification while multiple genes were overexpressed in cases with 12q13‐q14 amplification. Finally, although 2p24 amplification did not have a significant association with clinical outcome, 12q13‐q14 amplification was associated with significantly worse failure‐free and overall survival that was independent of gene fusion status. © 2009 Wiley‐Liss, Inc.     

Ph‐like acute lymphoblastic leukemia with a novel PAX5‐KIDINS220 fusion transcript

Although “paired box 5” (PAX5)‐related fusion genes are well documented in childhood B‐cell precursor acute lymphoblastic leukemia (ALL), these types of fusion with the exception of PAX5‐JAK2 are rarely seen in patients with gene expression profiles similar to those of BCR‐ABL1 (Philadelphia)‐positive ALL (Ph‐like ALL). We report a novel fusion of the genes PAX5 and “kinase D‐interacting substrate of 220 kDa” (KIDINS220, also known as ARMS) in a Ph‐like ALL. As PAX5 is a master regulator of B‐lymphocyte differentiation, PAX5 rearrangements induce a differentiation block in B lymphocytes. KIDINS220 is a mediator of multiple receptor signaling pathways, interacts with both T‐ and B‐cell receptors, and is necessary for sustained extracellular signal‐regulated kinase (ERK) signaling. Although functional studies are needed, the PAX5‐KIDINS220 fusion protein might not only inhibit wild‐type PAX5 function, but also promote sustained activation of the ERK signaling pathway through upregulation of KIDINS220. © 2016 Wiley Periodicals, Inc.     

Recurrent NCOA2 gene rearrangements in congenital/infantile spindle cell rhabdomyosarcoma

Spindle cell rhabdomyosarcoma (RMS) is a rare form of RMS with different clinical characteristics between children and adult patients. Its genetic hallmark remains unknown and it remains debatable if there is pathogenetic relationship between the spindle cell and the so‐called sclerosing RMS. We studied two pediatric and one adult spindle cell RMS by next generation RNA sequencing and FusionSeq data analysis to detect novel fusions. An SRF‐NCOA2 fusion was detected in a spindle cell RMS from the posterior neck in a 7‐month‐old child. The fusion matched the tumor karyotype and was confirmed by FISH and RT‐PCR, which showed fusion of SRF exon 6 to NCOA2 exon 12. Additional 14 spindle cell (from 8 children and 6 adults) and 4 sclerosing (from 2 children and 2 adults) RMS were tested by FISH for the presence of abnormalities in NCOA2, SRF, as well as for PAX3 and NCOA1. NCOA2 rearrangements were found in two additional spindle cell RMS from a 3‐month‐old and a 4‐week‐old child. In the latter tumor, TEAD1 was identified by rapid amplification of cDNA ends (RACE) to be the NCOA2 gene fusion partner. None of the adult tumors were positive for NCOA2 rearrangement. Despite similar histomorphology in adults and young children, these results suggest that spindle cell RMS is a heterogeneous disease genetically as well as clinically. Our findings also support a relationship between NCOA2‐rearranged spindle cell RMS occurring in young childhood and the so‐called congenital RMS, which often displays rearrangements at 8q13 locus (NCOA2). © 2013 Wiley Periodicals, Inc.     

Preoperative diagnosis of clear cell sarcoma of the kidney by detection of BCOR internal tandem duplication in circulating tumor DNA

Clear cell sarcoma of the kidney (CCSK) is the second most common renal malignancy in children. The prognosis is poorer in CCSK than in Wilms’ tumor, and multimodal treatment including surgery, intensive chemotherapy, and radiation is required to improve the outcome for children with CCSK. Histological evaluation is required for the diagnosis. However, biopsies of tumors to obtain diagnostic specimens are not routinely performed because of the risk of spreading tumor cells during the procedure. Recently, internal tandem duplication (ITD) of BCOR has been recognized as a genetic hallmark of CCSK. We herein established a novel BCOR‐ITD‐specific polymerase chain reaction method with well‐designed primers, and then performed a liquid biopsy for cell‐free DNA (cfDNA) obtained from plasma of three children with nonmetastatic renal tumors (stage II) and from one control. BCOR‐ITD was positively detected in the cfDNA of two cases, both of which were later diagnosed as CCSK based on histological feature of the resected tumor specimen, while it was not detected for a normal control and a patient diagnosed with Wilms’ tumor. Our study is the first one of preoperative circulating tumor DNA assay in pediatric renal tumors. The liquid biopsy method enables less invasive, preoperative diagnosis of CCSK with no risk of tumor spillage, which can avoid iatrogenic upstaging.     

Detection of HEY1‐NCOA2 fusion by fluorescence in‐situ hybridization in formalin‐fixed paraffin‐embedded tissues as a possible diagnostic tool for mesenchymal chondrosarcoma

Mesenchymal chondrosarcoma (MC) is an extremely rare subtype of chondrosarcoma. A tumor specific fusion gene, HEY1‐NCOA2 fusion, was recently identified in this tumor. The finding raises the possibility that the diagnosis of MC can be improved by examining the fusion gene. In the present study, we aimed to evaluate the efficacy of fluorescence in situ hybridization (FISH) in detecting HEY1‐NCOA2 fusion for the diagnosis of MC. Specimens from 10 patients diagnosed with MC were used for the study. Dual‐color FISH was performed using two different probes that specifically hybridize to HEY1 and NCOA2, respectively. Fusion signals were identified in all but two specimens, in which no signal was detected, presumably because of inadequate sample preparation. In accordance with results of a previous study, FISH analysis was highly sensitive in detecting HEY1‐NCOA2 fusion in adequately prepared MC samples. The current study adds further support for the use of HEY1‐NCOA2 fusion as a valid diagnostic marker for MC.     

Spindle‐cell rhabdomyosarcoma of the thumb: Rare site,rare tumor in a child

Spindle‐cell rhabdomyosarcoma (RMS) is a relatively recently recognized favorable variant of embryonal RMS occurring mainly in the paratesticular and head and neck regions. Cytological reports of spindle‐cell RMS have been sparse in the literature and the awareness of its cytological features is not very wide among cytopathologists. A 2‐year‐old girl was brought to clinical attention for a progressively enlarging swelling of right thumb. Imaging studies showed it to be a soft‐tissue tumor in the subcutaneous region. Fine‐needle aspiration yielded moderately cellular smears composed of small, round cells and few spindle cells with tapered cytoplasm. A cytological impression of RMS was rendered, which was later confirmed as spindle‐cell RMS on excision biopsy. Spindle‐cell RMS, a favorable prognostic variant of embryonal RMS, should be considered in the cytologic diagnosis of soft‐tissue tumors with round cell and spindle cell morphology. This is especially true for tumors occurring in hitherto unreported sites. Diagn. Cytopathol. 2016;44:1094–1097. © 2016 Wiley Periodicals, Inc.