Elsa Arbajian

Recurrent EWSR1-CREB3L1 gene fusions in sclerosing epithelioid fibrosarcoma.

Sclerosing epithelioid fibrosarcoma (SEF) and low-grade fibromyxoid sarcoma (LGFMS) are 2 distinct types of sarcoma, with a subset of cases showing overlapping morphologic and immunohistochemical features. LGFMS is characterized by expression of the MUC4 protein, and about 90% of cases display a distinctive FUS-CREB3L2 gene fusion. In addition, SEF is often MUC4 positive, but is genetically less well studied. Fluorescence in situ hybridization (FISH) studies have shown involvement of the FUS gene in the majority of so-called hybrid LGFMS/SEF and in 10% to 25% of sarcomas with pure SEF morphology. In this study, we investigated a series of 10 primary tumors showing pure SEF morphology, 4 cases of LGFMS that at local or distant relapse showed predominant SEF morphology, and 1 primary hybrid LGFMS/SEF. All but 1 case showed diffuse expression for MUC4. Using FISH, reverse transcription polymerase chain reaction, and/or mRNA sequencing in selected cases, we found recurrent EWSR1-CREB3L1 fusion transcripts by reverse transcription polymerase chain reaction in 3/10 pure SEF cases and splits and deletions of the EWSR1 and/or CREB3L1 genes by FISH in 6 additional cases. All 5 cases of LGFMS with progression to SEF morphology or hybrid features had FUS-CREB3L2 fusion transcripts. Our results indicate that EWSR1 and CREB3L1 rearrangements are predominant over FUS and CREB3L2 rearrangements in pure SEF, highlighting that SEF and LGFMS are different tumor types, with different impacts on patient outcome.

A novel GTF2I/NCOA2 fusion gene emphasizes the role of NCOA2 in soft tissue angiofibroma development.

Soft tissue angiofibroma is a recently described benign fibrovascular tumor of unknown cellular origin (Mariño-Enrı́quez and Fletcher, 2012). We reported, in this journal, that the t(5;8)(p15;q13) is a recurrent cytogenetic feature in this tumor type and that it results in the formation of a novel, and so far tumor-specific, fusion of the two transcription-associated genes AHRR in 5p15 and NCOA2 in 8q13 (Jin et al., 2012). An unexpected finding was that all four cases that could be analyzed by reverse transcription PCR (RT-PCR) expressed in-frame fusion transcripts from both derivate chromosomes, i.e., both AHRR/NCOA2 and NCOA2/AHRR. Furthermore, by interphase fluorescence in situ hybridization (FISH) analysis of tumor sections, we showed that a substantial subset of soft tissue angiofibromas is negative for the fusion gene. Here, we report the finding of an alternative fusion gene—GTF2I/NCOA2—in a new case of soft tissue angiofibroma, thereby demonstrating that this tumor type is associated with more than one fusion gene and that it is the transcripts in which NCOA2 is the 30 partner that are pathogenetically relevant. The patient was a 41-year-old woman with a tumor in the thigh. The lesion was first diagnosed as a myxofibrosarcoma but further histopathologic and immunohistochemical analyses, prompted by the cytogenetic finding of a t(7;8;14)(q11;q13;q31) as the sole change, disclosed that it was a soft tissue angiofibroma. Metaphase FISH was carried out on metaphase preparations using a previously described break-apart probe for NCOA2 in 8q13 (Jin et al., 2012). This analysis showed that the NCOA2 gene was rearranged, with one signal on the derivative chromosome 7 and one on the derivative chromosome 8. The 7q11 breakpoint was then mapped with a series of bacterial artificial chromosome (BAC) and fosmid probes, revealing a complex rearrangement of chromosome 7 that involved at least three different breakpoints (Table 1). One of the breakpoint regions covered a single gene— GTF2I—that was further analyzed using RT-PCR. Conventional, nested, and semi-nested RTPCR analysis of extracted total RNA, using GTF2I-1214F (50-GGCAATGAAGGCACAGAA AT), GTF2I-2083F (50-CTTGCAACCCTGAAA TGGAT), NCOA2-3807R (30-GCCTCAGAGTCAAGTTCACA), and NCOA2-3332R (30-CAAG TCATCTGGAGAACTGC) primers in all possible combinations resulted in four fragments— amplified by nested PCR using GTF2I-2083F and NCOA2-3332R—varying in size from 150 to 300 bp (Fig. 1A). Sequencing was performed using GTF2I 2083F and NCOA2 3332R. Sequence analyses were unsuccessful for the two smaller bands. However, the two larger bands were 256 and 221 bp sequences that corresponded to two splicing variants of a GTF2I/NCOA2 chimeric transcript. The 256 bp fragment corresponded to an in-frame fusion of part of exon 14 of GTF2I with part of exon 15 of NCOA2 whereas the 221 bp sequence was an outof-frame fusion of part of exon 11 of GTF2I with part of exon 14 of NCOA2 (Fig. 1B). Thus, a GTF2I/NCOA2 fusion transcript was confirmed. The finding of a new alternative 50 partner for NCOA2 suggests that regulatory sequences in NCOA2 have a primary role in the pathogenesis of soft tissue angiofibromas. GTF2I, a multifunctional DNA-binding transcription factor involved primarily TABLE 1. Results of FISH Analysis of the Breakpoint in 7q in a Soft Tissue Angiofibroma with t(7;8;14)

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

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.

A Benign Vascular Tumor With a New Fusion Gene: EWSR1-NFATC1 in Hemangioma of the Bone.

The EWSR1 gene in chromosome band 22q12 is a promiscuous fusion partner involved in a vast array of tumors characterized by gene fusions. In this study, we report the finding of a new fusion gene, EWSR1-NFATC1, in a hemangioma of the bone; genetic rearrangements have not previously been described in this tumor type. Chromosome banding analysis showed a t(18;22)(q23;q12) translocation as the sole change. Fluorescence in situ hybridization mapping suggested the involvement of each of the 2 partner genes, and reverse transcriptase polymerase chain reaction revealed an in-frame EWSR1-NFATC1 transcript. NFATC1 has not previously been shown to be involved in a fusion chimera. However, NFATC2, encoding another member of the same protein family, is known to be a fusion partner for EWSR1 in a subgroup of Ewing sarcoma. Thus, our findings further broaden the spectrum of neoplasms associated with EWSR1 fusion genes, add a new partner to the growing list of EWSR1 chimeras, and suggest that chromosomal rearrangements of pathogenetic, and possibly also diagnostic, significance can be present in benign vascular bone tumors.

Recurrent chromosome 22 deletions in osteoblastoma affect inhibitors of the wnt/beta-catenin signaling pathway.

Osteoblastoma is a bone forming tumor with histological features highly similar to osteoid osteoma; the discrimination between the tumor types is based on size and growth pattern. The vast majority of osteoblastomas are benign but there is a group of so-called aggressive osteoblastomas that can be diagnostically challenging at the histopathological level. The genetic aberrations required for osteoblastoma development are not known and no genetic difference between conventional and aggressive osteoblastoma has been reported. In order to identify recurrent genomic aberrations of importance for tumor development we applied cytogenetic and/or SNP array analyses on nine conventional and two aggressive osteoblastomas. The conventional osteoblastomas showed few or no acquired genetic aberrations while the aggressive tumors displayed heavily rearranged genomes. In one of the aggressive osteoblastomas, three neighboring regions in chromosome band 22q12 were homozygously deleted. Hemizygous deletions of these regions were found in two additional cases, one aggressive and one conventional. In total, 10 genes were recurrently and homozygously lost in osteoblastoma. Four of them are functionally involved in regulating osteogenesis and/or tumorigenesis. MN1 and NF2 have previously been implicated in the development of leukemia and solid tumors, and ZNRF3 and KREMEN1 are inhibitors of the Wnt/beta-catenin signaling pathway. In line with deletions of the latter two genes, high beta-catenin protein expression has previously been reported in osteoblastoma and aberrations affecting the Wnt/beta-catenin pathway have been found in other bone lesions, including osteoma and osteosarcoma.

In-depth genetic analysis of sclerosing epithelioid fibrosarcoma reveals recurrent genomic alterations and potential treatment targets

Purpose: Sclerosing epithelioid fibrosarcoma (SEF) is a highly aggressive soft tissue sarcoma closely related to low-grade fibromyxoid sarcoma (LGFMS). Some tumors display morphologic characteristics of both SEF and LGFMS, hence they are known as hybrid SEF/LGFMS. Despite the overlap of gene fusion variants between these two tumor types, SEF is much more aggressive. The current study aimed to further characterize SEF and hybrid SEF/LGFMS genetically to better understand the role of the characteristic fusion genes and possible additional genetic alterations in tumorigenesis. Experimental Design: We performed whole-exome sequencing, SNP array analysis, RNA sequencing (RNA-seq), global gene expression analyses, and/or IHC on a series of 13 SEFs and 6 hybrid SEF/LGFMS. We also expressed the FUS-CREB3L2 and EWSR1-CREB3L1 fusion genes conditionally in a fibroblast cell line; these cells were subsequently analyzed by RNA-seq, and expression of the CD24 protein was assessed by FACS analysis. Results: The SNP array analysis detected a large number of structural aberrations in SEF and SEF/LGFMS, many of which were recurrent, notably DMD microdeletions. RNA-seq identified FUS-CREM and PAX5-CREB3L1 as alternative fusion genes in one SEF each. CD24 was strongly upregulated, presumably a direct target of the fusion proteins. This was further confirmed by the gene expression analysis and FACS analysis on Tet-On 3G cells expressing EWSR1-CREB3L1. Conclusions: Although gene fusions are the primary tumorigenic events in both SEF and LGFMS, additional genomic changes explain the differences in aggressiveness and clinical outcome between the two types. CD24 and DMD constitute potential therapeutic targets. Clin Cancer Res; 23(23); 7426–34. ©2017 AACR.

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

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.

Inflammatory leiomyosarcoma is a distinct tumor characterized by near-haploidization, few somatic mutations, and a primitive myogenic gene expression signature

Inflammatory leiomyosarcoma is a soft-tissue tumor resembling conventional leiomyosarcoma, but with a prominent intrinsic inflammatory component. Previous studies have suggested that inflammatory leiomyosarcoma differs genetically from leiomyosarcoma, but in-depth analyses are lacking. Here we provide a comprehensive picture of the genome and transcriptome of inflammatory leiomyosarcoma by combining cytogenetic, single-nucleotide polymorphism array, mRNA-sequencing, and whole-exome sequencing data. The results show that inflammatory leiomyosarcoma has a specific genetic profile characterized by near-haploidization with or without subsequent whole-genome doubling. Consistently, both parental copies of chromosomes 5 and 22 are preserved. Apart from recurrent mutation of the NF1 gene, additional somatic events that could serve as driver mutations were not found at either the nucleotide or the genome level. Furthermore, no fusion transcripts were identified. Global gene expression profiling revealed particularly prominent differential expression of genes, including ITGA7, MYF5, MYF6, MYOD1, MYOG, and PAX7, involved in muscle development and function, providing strong argument for grouping inflammatory leiomyosarcoma with myogenic sarcomas, rather than with myofibroblastic lesions. Combined with previously published data, there are now 10 cases of inflammatory leiomyosarcoma with confirmed near-haploid genotype. These patients differ from leiomyosarcoma patients in being younger (median 41 years), showing a male predominance (9:1), and few relapses (1 of 8 informative patients). Thus, the clinical, morphological, and genetic data provide compelling support for inflammatory leiomyosarcoma being a distinct subtype of myogenic tumors.

Soft tissue angiofibroma: Clinicopathologic, immunohistochemical and molecular analysis of 14 cases

Soft tissue angiofibroma is rare and has characteristic histomorphological and genetic features. For diagnostic purposes, there are no specific antibodies available. Fourteen lesions (6 females, 8 males; age range 7‐67 years) of the lower extremities (12) and trunk (2) were investigated by immunohistochemistry, including for the first time NCOA2. NCOA2 was also tested in a control group of other spindle cell lesions. The known fusion‐genes (AHRR‐NCOA2 and GTF2I‐NCOA2) were examined using RT‐PCR in order to evaluate their diagnostic value. Cases in which no fusion gene was detected were additionally analysed by RNA sequencing. All cases tested showed nuclear expression of NCOA2. However, this was not specific since other spindle cell neoplasms also expressed this marker in a high percentage of cases. Other variably positive markers were EMA, SMA, desmin and CD34. STAT6 was negative in the cases tested. By RT‐PCR for the most frequently observed fusions, an AHRR‐NCOA2 fusion transcript was found in 9/14 cases. GTF2I‐NCOA2 was not detected in the remaining cases (n = 3). RNA sequencing revealed three additional positive cases; two harbored a AHRR‐NCOA2 fusion and one case a novel GAB1‐ABL1 fusion. Two cases failed molecular analysis due to poor RNA quality. In conclusion, the AHRR‐NCOA2 fusion is a frequent finding in soft tissue angiofibroma, while GTF2I‐NCOA2 seems to be a rare genetic event. For the first time, we report a GAB1‐ABL1 fusion in a soft tissue angiofibroma of a child. Nuclear expression of NCOA2 is not discriminating when compared with other spindle cell neoplasms.