Disruption of the TP53 locus in osteosarcoma leads to TP53 promoter gene fusions and restoration of parts of the TP53 signalling pathway.

TP53 is the most frequently mutated gene in human cancer. This gene shows not only loss-of-function mutations but also recurrent missense mutations with gain-of-function activity. We have studied the primary bone malignancy osteosarcoma, which harbours one of the most rearranged genomes of all cancers. This is odd since it primarily affects children and adolescents who have not lived the long life thought necessary to accumulate massive numbers of mutations. In osteosarcoma, TP53 is often disrupted by structural variants. Here, we show through combined whole-genome and transcriptome analyses of 148 osteosarcomas that TP53 structural variants commonly result in loss of coding parts of the gene while simultaneously preserving and relocating the promoter region. The transferred TP53 promoter region is fused to genes previously implicated in cancer development. Paradoxically, these erroneously upregulated genes are significantly associated with the TP53 signalling pathway itself. This suggests that while the classical tumour suppressor activities of TP53 are lost, certain parts of the TP53 signalling pathway that are necessary for cancer cell survival and proliferation are retained. In line with this, our data suggest that transposition of the TP53 promoter is an early event that allows for a new normal state of genome-wide rearrangements in osteosarcoma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Osteosarcomas With Few Chromosomal Alterations or Adult Onset Are Genetically Heterogeneous.

Osteosarcoma is the most common primary bone malignancy, often detected in children and adolescents and commonly associated with TP53 alterations along with a high number of chromosomal rearrangements. However, osteosarcoma can affect patients of any age, and some tumors display less genetic complexity. Besides TP53 variants, data on key driving mutations are lacking for many osteosarcomas, particularly those affecting adults. To detect osteosarcoma-specific alterations, we screened transcriptomic and genomic sequencing and copy number data from 150 bone tumors originally diagnosed as osteosarcomas. To increase the precision in gene fusion detection, we developed a bioinformatic tool denoted as NAFuse, which extracts gene fusions that are verified at both the genomic and transcriptomic levels. Apart from the already reported genetic subgroups of osteosarcoma with TP53 structural variants, or MDM2 and/or CDK4 amplification, we did not identify any recurrent genetic driver that signifies the remaining cases. Among the plethora of mutations identified, we found genetic alterations characteristic of, or similar to, those of other bone and soft tissue tumors in 8 cases. These mutations were found in tumors with relatively few other genetic alterations or in adults. Due to the lack of clinical context and available tissue, we can question the diagnosis only on a genetic basis. However, our findings support the notion that osteosarcomas with few chromosomal alterations or adult onset seem genetically distinct from conventional osteosarcomas of children and adolescents.

Inactivation of RB1, CDKN2A, and TP53 have distinct effects on genomic stability at side-by-side comparison in karyotypically normal cells.

Chromosomal instability is a common feature in malignant tumors. Previous studies have indicated that inactivation of the classical tumor suppressor genes RB1, CDKN2A, and TP53 may contribute to chromosomal aberrations in cancer by disrupting different aspects of the cell cycle and DNA damage checkpoint machinery. We performed a side-by-side comparison of how inactivation of each of these genes affected chromosomal stability in vitro. Using CRISPR-Cas9 technology, RB1, CDKN2A, and TP53 were independently knocked out in karyotypically normal immortalized cells, after which these cells were followed over time. Bulk RNA sequencing revealed a distinct phenotype with upregulation of pathways related to cell cycle control and proliferation in all three knockouts. Surprisingly, the RB1 and CDKN2A knocked out cell lines did not harbor more copy number aberrations than wild-type cells, despite culturing for months. The TP53-knocked out cells, in contrast, showed a massive amount of copy number alterations and saltatory evolution through whole genome duplication. This side-by-side comparison indicated that the effects on chromosomal stability from inactivation of RB1 and CDKN2A are negligible compared to inactivation of TP53, under the same conditions in a nonstressful environment, even though partly overlapping regulatory pathways are affected. Our data suggest that loss of RB1 and CDKN2A alone is not enough to trigger surviving detectable aneuploid clones while inactivation of TP53 on its own caused massive CIN leading to saltatory clonal evolution in vitro and clonal selection.

Insertion of the CXXC domain of KMT2A into YAP1: An unusual mechanism behind the formation of a chimeric oncogenic protein.

Most neoplasia-associated gene fusions are formed through the fusion of the 5'-part of one gene with the 3'-part of another. We here describe a unique mechanism, by which a part of the KMT2A gene through an insertion replaces part of the YAP1 gene. The resulting YAP1::KMT2A::YAP1 (YKY) fusion was verified by RT-PCR in three cases of sarcoma morphologically resembling sclerosing epithelioid fibrosarcoma (SEF-like sarcoma). In all cases, a portion (exons 4/5-6) encoding the CXXC domain of KMT2A was inserted between exon 4/5 and exon 8/9 of YAP1. The inserted sequence from KMT2A thus replaced exons 5/6-8 of YAP1, which encode an important regulatory sequence of YAP1. To evaluate the cellular impact of the YKY fusion, global gene expression profiles from fresh frozen and formalin-fixed YKY-expressing sarcomas were compared with control tumors. The effects of the YKY fusion, as well as YAP1::KMT2A and KMT2A::YAP1 fusion constructs, were further studied in immortalized fibroblasts. Analysis of differentially upregulated genes revealed significant overlap between tumors and cell lines expressing YKY, as well as with previously reported YAP1 fusions. Pathway analysis of upregulated genes in cells and tumors expressing YKY revealed an enrichment of genes included in key oncogenic signaling pathways, such as Wnt and Hedgehog. As these pathways are known to interact with YAP1, it seems likely that the pathogenesis of sarcomas with the YKY fusion is linked to distorted YAP1 signaling.

Amplification of ERBB2 (HER2) in embryonal rhabdomyosarcoma: A potential treatment target in rare cases?

The ERBB2 gene encodes a receptor tyrosine kinase also known as HER2. The gene is amplified and overexpressed in one-fifth of breast carcinomas; patients with such tumors benefit from targeted treatment with trastuzumab or other drugs blocking the receptor. In addition, ERBB2 has been shown to be amplified and/or overexpressed in a variety of other malignancies. Notably, both alveolar and embryonal rhabdomyosarcoma (RMS), especially in children, often show increased expression of ERBB2. Although high-level amplification of the gene has not been described in RMS, its frequent expression at the cell surface of RMS cells has been exploited for chimeric antigen receptor T-cell (CAR T)-based treatment strategies. We here describe two cases of pediatric, fusion-negative embryonal RMS with high-level amplification of the ERBB2 gene. One patient is currently treated with conventional chemotherapy for a recently detected standard risk RMS, whereas the other patient died from metastatic disease. Both tumors displayed focal amplicons (210 and 274 Kb, respectively) in chromosome band 17q12, with proximal and distal borders corresponding to those typically seen in breast cancer. In both tumors, the ERBB2 amplicon correlated with high expression at the RNA and protein levels. Thus, breast cancer-like ERBB2 amplification is a very rare, but recurrent feature of pediatric RMS, and should be exploited as an alternative treatment target.

The diagnostic utility of DNA copy number analysis of core needle biopsies from soft tissue and bone tumors.

Morphologic and immunohistochemical analysis of preoperative core needle biopsies (CNB) is important in the management of patients with soft tissue and bone tumors (STBTs). Most SBTB subtypes have more or less extensive DNA copy number aberrations (CNA), potentially providing useful diagnostic information. To evaluate the technical feasibility of single nucleotide polymorphism (SNP) array analysis and the diagnostic usefulness of the copy number profiles, we studied CNBs from 171 patients with suspected STBTs. SNP array analysis could be performed on 168 (98%) of the samples. The CNA profile was compatible with the CNB diagnosis in 87% of the cases. Discrepant cases were dominated by false-negative results due to nonrepresentative material or contamination with normal cells. 70 genomic profiles were indicative of specific histopathologic tumor entities and in agreement with the corresponding CNB diagnoses in 83%. In 96 of the cases with aberrant CNA profiles, the SNP profiles were of sufficient quality for segmentation, allowing clustering analysis on the basis of the Jaccard similarity index. The analysis of these segment files showed three major CNA clusters, based on the complexity levels and the predominance of gains versus losses. For 43 of these CNB samples, we had SNP array data also from their corresponding surgical samples. In 33 of these pairs, the two corresponding samples clustered next to each other, with Jaccard scores ranging from 0.61 to 0.99 (median 0.96). Also, for those tumor pairs that did not cluster together, the Jaccard scores were relatively high (median 0.9). 10 cases showed discrepant results, mainly due to varying degrees of normal cell contamination or technical issues. Thus, the copy number profile seen in a CNB is typically highly representative of the major cell population in the tumor.

Overlapping morphological, immunohistochemical and genetic features of superficial CD34-positive fibroblastic tumor and PRDM10-rearranged soft tissue tumor.

Superficial CD34-positive fibroblastic tumor (SCD34FT) is a recently recognized soft tissue tumor that is considered to be of borderline malignancy. The pathogenesis of this tumor remains incompletely understood, but it has been suggested that SCD34FT overlaps with tumors showing fusions involving the PRDM10 gene. Previous analyses of PRDM10-rearranged tumors have demonstrated that they have a distinct gene expression profile, resulting in high expression of CADM3 (also known as SynCam3), which can be detected immunohistochemically. Here, we investigated a series (n = 43) of SCD34FT or PRDM10-rearranged tumors and potential mimics (n = 226) with regard to morphological, genetic, and immunohistochemical features. The results show that SCD34FT and PRDM10-rearranged tumor are morphologically indistinguishable; 41 of 43 tumors of both entities are CADM3-positive. Hence, we suggest that they constitute a single entity, preferably referred to as SCD34FT. Expression of CADM3 was only rarely seen in other soft tissue tumors, except in tumors with Schwann cell differentiation. Thus, IHC for CADM3, in combination with the characteristic morphological features, is a valuable adjunct in the diagnosis of SCD34FT.

Deep sequencing of myxoinflammatory fibroblastic sarcoma.

Myxoinflammatory fibroblastic sarcoma (MIFS) has recurrent genetic features in the form of a translocation t(1;10)(p22-31;q24-25), BRAF gene fusions, and/or an amplicon in 3p11-12 including the VGLL3 gene. The breakpoints on chromosomes 1 and 10 in the t(1;10) cluster in or near the TGFBR3 and OGA genes, respectively. We here used a combination of deep sequencing of the genome (WGS), captured sequences (Cap-seq), and transcriptome (RNA-seq) and genomic arrays to investigate the molecular outcome of the t(1;10) and the VGLL3 amplicon, as well as to assess the spectrum of other recurrent genomic features in MIFS. Apart from a ROBO1-BRAF chimera in a t(1;10)-negative MIFS-like tumor, no fusion gene was found at RNA-seq. This was in line with WGS and Cap-seq results, revealing variable breakpoints in chromosomes 1 and 10 and genomic breakpoints that should not yield functional fusion transcripts. The most common genomic rearrangements were breakpoints in or around the OGA, NPM3, and FGF8 genes in chromosome band 10q24, and loss of 1p11-p21 and 10q26-qter (all simultaneously present in 6/7 MIFS); a breakpoint in or near TGFBR3 in chromosome 1 was found in four of these tumors. Amplification and overexpression of VGLL3 was a consistent feature in MIFS and MIFS-like tumors with amplicons in 3p11-12. The significant molecular genetic outcome of the recurrent t(1;10) could be loss of genetic material from 1p and 10q. Other recurrent genomic imbalances in MIFS, such as homozygous loss of CDKN2A and 3p- and 13q-deletions, are shared with other sarcomas, suggesting overlapping pathogenetic pathways.

PHF1 fusions cause distinct gene expression and chromatin accessibility profiles in ossifying fibromyxoid tumors and mesenchymal cells.

Ossifying fibromyxoid tumor (OFMT) is a soft tissue tumor frequently displaying gene fusions, most of which affect the PHF1 gene. PHF1 encodes plant homeodomain finger protein 1, which is involved in various processes regulating gene transcription, including those orchestrated by the polycomb repressor complex 2. Here, a series of 37 OFMTs, including 18 typical, 9 atypical, and 10 malignant variants, was analyzed with regard to transcriptomic features, gene fusion and copy number status, and/or single-nucleotide variants. The effects on gene expression and chromatin accessibility of three detected fusions (EP400-PHF1, MEAF6-PHF1, and PHF1-TFE3) were further evaluated in fibroblasts. Genomic imbalances showed a progression-related pattern, with more extensive copy number changes among atypical/malignant lesions than among typical OFMTs; loss of the RB1 gene was restricted to atypical/malignant OFMTs, occurring in one-third of the cases. RNA sequencing identified fusion transcripts in >80% of the cases analyzed, including a novel CSMD1-MEAF6. The gene-expression profile of OFMT was distinct from that of other soft tissue tumors, with extensive transcriptional upregulation of genes in OFMT. These findings were largely recapitulated in gene fusion-expressing fibroblast lines, suggesting that genes involved in, e.g., Wnt signaling and/or being regulated through trimethylation of lysine 27 in histone 3 (H3K27me3) are pivotal for OFMT development. The genes showing differentially higher expression in fusion-expressing cells paralleled increased chromatin accessibility, as revealed by ATAC sequencing. Thus, the present study suggests that OFMT develops through gene fusions that have extensive epigenetic consequences.

Undifferentiated pleomorphic sarcomas with PRDM10 fusions have a distinct gene expression profile.

Undifferentiated pleomorphic sarcoma (UPS) is a highly aggressive soft tissue tumor. A subset of UPS is characterized by a CITED2-PRDM10 or a MED12-PRDM10 gene fusion. Preliminary data suggest that these so-called PRDM10-rearranged tumors (PRT) are clinically more indolent than classical high-grade UPS, and hence important to recognize. Here, we assessed the spectrum of accompanying mutations and the gene expression profile in PRT using genomic arrays and sequencing of the genome (WGS) and transcriptome (RNA-seq). The fusion protein's function was further investigated by conditional expression of the CITED2-PRDM10 fusion in a fibroblast cell line, followed by RNA-seq and an assay for transposase-accessible chromatin (ATAC-seq). The CADM3 gene was found to be differentially up-regulated in PRT and cell lines and was also evaluated for expression at the protein level using immunohistochemistry (IHC). The genomic analyses identified few and nonrecurrent mutations in addition to the structural variants giving rise to the gene fusions, strongly indicating that the PRDM10-fusions represent the critical driver mutations. RNA-seq of tumors showed a distinct gene expression profile, separating PRT from high-grade UPS and other soft tissue tumors. CADM3 was among the genes that was consistently and highly expressed in both PRT and fibroblasts expressing CITED2-PRDM10, suggesting that it is a direct target of the PRDM10 transcription factor. This conclusion is in line with sequencing data from ATAC-seq, showing enrichment of PRDM10 binding sites, suggesting that the amino-terminal fusion partner contributes by making the DNA more accessible to PRDM10 binding. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Aberrant receptor tyrosine kinase signaling in lipofibromatosis: a clinicopathological and molecular genetic study of 20 cases.

Lipofibromatosis is a rare pediatric soft tissue tumor with predilection for the hands and feet. Previously considered to represent "infantile fibromatosis", lipofibromatosis has distinctive morphological features, with mature adipose tissue, short fascicles of bland fibroblastic cells, and lipoblast-like cells. Very little is known about the genetic underpinnings of lipofibromatosis. Prompted by our finding of the FN1-EGF gene fusion, previously shown to be a characteristic feature of calcifying aponeurotic fibroma (CAF), in a morphologically typical case of lipofibromatosis that recurred showing features of CAF, we studied a cohort of 20 cases of lipofibromatosis for this and other genetic events. The cohort was composed of 14 males and 6 females (median age 3 years; range 1 month-14 years). All primary tumors showed classical lipofibromatosis morphology. Follow-up disclosed three local recurrences, two of which contained calcifying aponeurotic fibroma-like nodular calcifications in addition to areas of classic lipofibromatosis, and no metastases. By FISH and RNA sequencing, four cases were positive for FN1-EGF and one case each showed an EGR1-GRIA1, TPR-ROS1, SPARC-PDGFRB, FN1-TGFA, EGFR-BRAF, VCL-RET, or HBEGF-RBM27 fusion. FN1-EGF was the only recurrent fusion, suggesting that some cases of "lipofibromatosis" may represent calcifying aponeurotic fibroma lacking hallmark calcifications. Several of the genes involved in fusions (BRAF, EGFR, PDGFRB, RET, and ROS1) encode receptor tyrosine kinases (RTK), or ligands to the RTK EGFR (EGF, HBEGF, TGFA), suggesting a shared deregulation of the PI3K-AKT-mTOR pathway in a large subset of lipofibromatosis cases.

Loss of the tumour suppressor gene AIP mediates the browning of human brown fat tumours.

Human brown fat tumours (hibernomas) show concomitant loss of the tumour suppressor genes MEN1 and AIP. We hypothesized that the brown fat phenotype is attributable to these mutations. Accordingly, in this study, we demonstrate that silencing of AIP in human brown preadipocytic and white fat cell lines results in the induction of the brown fat marker UCP1. In human adipocytic tumours, loss of MEN1 was found both in white (one of 51 lipomas) and in brown fat tumours. In contrast, concurrent loss of AIP was always accompanied by a brown fat morphology. We conclude that this white-to-brown phenotype switch in brown fat tumours is mediated by the loss of AIP. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Frequent low-level mutations of protein kinase D2 in angiolipoma.

Tumours displaying differentiation towards normal fat constitute the most common subgroup of soft tissue neoplasms. A series of such tumours was investigated by whole-exome sequencing followed by targeted ultra-deep sequencing. Eighty per cent of angiolipomas, but not any other tumour type, displayed mutations in the protein kinase D2 (PRKD2) gene, typically in the part encoding the catalytic domain. The absence of other aberrations at the chromosome or RNA level suggests that PRKD2 mutations are critical for angiolipoma development. Consistently, the mutated PRKD2 alleles were present at low (3-15%) frequencies, indicating that only a subset of the tumour cells is affected. Indeed, by sequencing mature fat cells and other cells separately, the former typically showed the highest mutation frequencies. Thus, we hypothesize that altered PRKD2 signalling in the adipocytic cells drives tumourigenesis and, in agreement with its pivotal role in angiogenesis, induces the vessel formation that is characteristic for angiolipoma. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

FN1-EGF gene fusions are recurrent in calcifying aponeurotic fibroma.

Calcifying aponeurotic fibroma (CAF) is a soft tissue neoplasm with a predilection for the hands and feet in children and adolescents. Its molecular basis is unknown. We used chromosome banding analysis, fluorescence in situ hybridization (FISH), mRNA sequencing (RNA-seq), RT-PCR, and immunohistochemistry to characterize a series of CAFs. An insertion ins(2;4)(q35;q25q?) was identified in the index case. Fusion of the FN1 and EGF genes, mapping to the breakpoint regions on chromosomes 2 and 4, respectively, was detected by RNA-seq and confirmed by RT-PCR in the index case and two additional cases. FISH on five additional tumours identified FN1-EGF fusions in all cases. CAFs analysed by RT-PCR showed that FN1 exon 23, 27 or 42 was fused to EGF exon 17 or 19. High-level expression of the entire FN1 gene in CAF suggests that strong FN1 promoter activity drives inappropriate expression of the biologically active portion of EGF, which was detected immunohistochemically in 8/9 cases. The FN1-EGF fusion, which has not been observed in any other neoplasm, appears to be the main driver mutation in CAF. Although further functional studies are required to understand the exact pathogenesis of CAF, the composition of the chimera suggests an autocrine/paracrine mechanism of transformation. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Gene fusion detection in formalin-fixed paraffin-embedded benign fibrous histiocytomas using fluorescence in situ hybridization and RNA sequencing.

Benign fibrous histiocytomas (FH) can be subdivided into several morphological and clinical subgroups. Recently, gene fusions involving either one of two protein kinase C genes (PRKCB and PRKCD) or the ALK gene were described in FH. We here wanted to evaluate the frequency of PRKCB and PRKCD gene fusions in FH. Using interphase fluorescence in situ hybridization on sections from formalin-fixed paraffin-embedded (FFPE) tumors, 36 cases could be analyzed. PRKCB or PRKCD rearrangements were seen in five tumors: 1/7 regular, 0/3 aneurysmal, 0/6 cellular, 2/7 epithelioid, 0/1 atypical, 2/10 deep, and 0/2 metastatic lesions. We also evaluated the status of the ALK gene in selected cases, finding rearrangements in 3/7 epithelioid and 0/1 atypical lesions. To assess the gene fusion status of FH further, deep sequencing of RNA (RNA-Seq) was performed on FFPE tissue from eight cases with unknown gene fusion status, as well as on two FH and six soft tissue sarcomas with known gene fusions; of the latter eight positive controls, the expected fusion transcript was found in all but one, while 2/8 FH with unknown genetic status showed fusion transcripts, including a novel KIRREL/PRKCA chimera. Thus, also a third member of the PRKC family is involved in FH tumorigenesis. We conclude that gene fusions involving PRKC genes occur in several morphological (regular, cellular, aneurysmal, epithelioid) and clinical (cutaneous, deep) subsets of FH, but they seem to account for only a minority of the cases. In epithelioid lesions, however, rearrangements of PRKC or ALK were seen, as mutually exclusive events, in the majority (5/7) of cases. Finally, the study also shows that RNA-Seq is a promising tool for identifying gene fusions in FFPE tissues.

RNA sequencing of sarcomas with simple karyotypes: identification and enrichment of fusion transcripts.

Gene fusions are neoplasia-associated mutations arising from structural chromosomal rearrangements. They have a strong impact on tumor development and constitute important diagnostic markers. Malignant soft tissue tumors (sarcomas) constitute a heterogeneous group of neoplasms with >50 distinct subtypes, each of which is rare. In addition, there is considerable morphologic overlap between sarcomas and benign lesions. Several subtypes display distinct gene fusions, serving as excellent biomarkers. The development of methods for deep sequencing of the complete transcriptome (RNA-Seq) has substantially improved the possibilities for detecting gene fusions. With the aim of identifying new gene fusions of biological and clinical relevance, eight sarcomas with simple karyotypes, ie, only one or a few structural rearrangements, were subjected to massively parallel paired-end sequencing of mRNA. Three different algorithms were used to identify fusion transcripts from RNA-Seq data. Three novel (KIAA2026-NUDT11, CCBL1-ARL1, and AFF3-PHF1) and two previously known fusions (FUS-CREB3L2 and HAS2-PLAG1) were found and could be verified by other methods. These findings show that RNA-Seq is a powerful tool for detecting gene fusions in sarcomas but also suggest that it is advisable to use more than one algorithm to analyze the output data as only two of the confirmed fusions were reported by more than one of the gene fusion detection software programs. For all of the confirmed gene fusions, at least one of the genes mapped to a chromosome band implicated by the karyotype, suggesting that sarcomas with simple karyotypes constitute an excellent resource for identifying novel gene fusions.

A novel SERPINE1-FOSB fusion gene results in transcriptional up-regulation of FOSB in pseudomyogenic haemangioendothelioma.

Pseudomyogenic haemangioendothelioma (PHE) is an intermediate malignant vascular soft tissue tumour primarily affecting children and young adults. The molecular basis of this neoplasm is unknown. We here used chromosome banding analysis, fluorescence in situ hybridization (FISH), mRNA sequencing, RT-PCR and quantitative real-time PCR on a series of morphologically well-characterized PHEs to show that a balanced translocation, t(7;19)(q22;q13), detected as the sole cytogenetic aberration in two cases, results in fusion of the SERPINE1 and FOSB genes. This translocation has not been observed in any other bone or soft tissue tumour. Interphase FISH on sections from eight additional PHEs identified the same SERPINE1-FOSB fusion in all cases. The role of SERPINE1, which is highly expressed in vascular cells, in this gene fusion is probably to provide a strong promoter for FOSB, which was found to be expressed at higher levels in PHEs than in other soft tissue tumours. FOSB encodes a transcription factor belonging to the FOS family of proteins, which, together with members of the JUN family of transcription factors, are major components of the activating protein 1 (AP-1) complex. Further studies are needed to understand the cellular impact of the aberrant expression of the FOSB gene, but as the t(7;19) resulting in the SERPINE1-FOSB fusion seems to be pathognomonic for PHE, FISH or RT-PCR could be useful for differential diagnostic purposes.

Myoepithelioma of bone with a novel FUS-POU5F1 fusion gene.

AIMS: Myoepithelial tumours of soft tissue are rare lesions with a broad morphological and clinical spectrum. Previous studies have found EWSR1 rearrangements in approximately half of all cases and PBX1, ZNF44 and POU5F1 have been identified as recurrent fusion partners. In bone, only a small number of myoepithelial tumours have been described. We investigated an intraosseous myoepithelioma of the sacrum in a 54-year-old man without EWSR1 rearrangement for the presence of other fusion genes. METHODS AND RESULTS: G-banding analysis, SNP-array and fluorescence in situ hybridisation suggested rearrangement of the FUS and POU5F1 genes. RT-PCR confirmed a chimeric in-frame transcript fusing FUS exon 5 to POU5F1 exon 2. The clinical course after en bloc resection was without recurrence or metastasis over a period of 87 months. CONCLUSION: We report a novel FUS-POU5F1 fusion gene in an intraosseous myoepithelioma of the sacrum. This case highlights that FUS can replace EWSR1 as the N-terminal transactivator in oncogenic fusion genes in myoepithelial tumours, similar to that which has previously been demonstrated in other tumour entities. Thus, in addition to EWSR1, also FUS needs to be considered as a potential fusion partner in the molecular work up of myoepithelial tumours.

Comprehensive genetic analysis identifies a pathognomonic NAB2/STAT6 fusion gene, nonrandom secondary genomic imbalances, and a characteristic gene expression profile in solitary fibrous tumor.

Solitary fibrous tumor (SFT) is a mesenchymal neoplasm displaying variable morphologic and clinical features. To identify pathogenetically important genetic rearrangements, 44 SFTs were analyzed using a variety of techniques. Chromosome banding and fluorescence in situ hybridization (FISH) showed recurrent breakpoints in 12q13, clustering near the NAB2 and STAT6 genes, and single nucleotide polymorphism array analysis disclosed frequent deletions affecting STAT6. Quantitative real-time PCR revealed high expression levels of the 5'-end of NAB2 and the 3'-end of STAT6, which at deep sequencing of enriched DNA corresponded to NAB2/STAT6 fusions. Subsequent reverse-transcriptase PCR (RT-PCR) analysis identified a NAB2/STAT6 fusion in 37/41 cases, confirming that this fusion gene underlies the pathogenesis of SFT. The hypothesis that the NAB2/STAT6 fusions will result in altered properties of the transcriptional co-repressor NAB2--a key regulator of the early growth response 1 (EGR1) transcription factor - was corroborated by global gene expression analysis; SFTs showed deregulated expression of EGR1 target genes, as well as of other, developmentally important genes. We also identified several nonrandom secondary changes, notably loss of material from 13q and 14q. As neither chromosome banding nor FISH analysis identify more than a minor fraction of the fusion-positive cases, and because multiple primer combinations are required to identify all possible fusion transcripts by RT-PCR, alternative diagnostic markers might instead be found among deregulated genes identified at global gene expression analysis. Indeed, using immunohistochemistry on tissue microarrays, the top up-regulated gene, GRIA2, was found to be differentially expressed also at the protein level.

MDM2 amplification in rod-shaped chromosomes provides clues to early stages of circularized gene amplification in liposarcoma.

Well-differentiated liposarcoma (WDLS) displays amplification of genes on chromosome 12 (Chr12) in supernumerary ring or giant marker chromosomes. These structures have been suggested to develop through chromothripsis, followed by circularization and breakage-fusion-bridge (BFB) cycles. To test this hypothesis, we compared WDLSs with Chr12 amplification in rod-shaped chromosomes with WDLSs with rings. Both types of amplicons share the same spectrum of structural variants (SVs), show higher SV frequencies in Chr12 than in co-amplified segments, have SVs that fuse the telomeric ends of co-amplified chromosomes, and lack interspersed deletions. Combined with the finding of cells with transient rod-shaped structures in tumors with ring chromosomes, this suggests a stepwise process starting with the gain of Chr12 material that, after remodeling which does not fit with classical chromothripsis, forms a dicentric structure with other chromosomes. Depending on if and when telomeres from other chromosomes are captured, circularized or linear gain of 12q sequences will predominate.