Karolin Hansén Nord

Somatic mosaic IDH1 and IDH2 mutations are associated with enchondroma and spindle cell hemangioma in Ollier disease and Maffucci syndrome

Ollier disease and Maffucci syndrome are non-hereditary skeletal disorders characterized by multiple enchondromas (Ollier disease) combined with spindle cell hemangiomas (Maffucci syndrome). We report somatic heterozygous mutations in IDH1 (c.394C>T encoding an R132C substitution and c.395G>A encoding an R132H substitution) or IDH2 (c.516G>C encoding R172S) in 87% of enchondromas (benign cartilage tumors) and in 70% of spindle cell hemangiomas (benign vascular lesions). In total, 35 of 43 (81%) subjects with Ollier disease and 10 of 13 (77%) with Maffucci syndrome carried IDH1 (98%) or IDH2 (2%) mutations in their tumors. Fourteen of 16 subjects had identical mutations in separate lesions. Immunohistochemistry to detect mutant IDH1 R132H protein suggested intraneoplastic and somatic mosaicism. IDH1 mutations in cartilage tumors were associated with hypermethylation and downregulated expression of several genes. Mutations were also found in 40% of solitary central cartilaginous tumors and in four chondrosarcoma cell lines, which will enable functional studies to assess the role of IDH1 and IDH2 mutations in tumor formation.

Fusion of the AHRR and NCOA2 genes through a recurrent translocation t(5;8)(p15;q13) in soft tissue angiofibroma results in upregulation of aryl hydrocarbon receptor target genes.

Soft tissue angiofibroma is a recently delineated tumor type of unknown cellular origin. Cytogenetic analysis of four cases showed that they shared a t(5;8)(p15;q13). In three of them it was the sole change, underlining its pathogenetic significance. FISH mapping suggested the involvement of the aryl hydrocarbon receptor repressor (AHRR) and nuclear receptor coactivator 2 (NCOA2) genes in 5p15 and 8q13, respectively. RT‐PCR revealed in‐frame AHRR/NCOA2 and NCOA2/AHHR transcripts in all four cases. Interphase FISH on paraffin‐embedded tissue from 10 further cases without cytogenetic data showed that three were positive for fusion of AHRR and NCOA2. While AHRR has never been implicated in gene fusions before, NCOA2 is the 3′‐partner in fusions with MYST3 and ETV6 in leukemias and with PAX3 and HEY1 in sarcomas. As in the previously described fusion proteins, NCOA2 contributes with its two activation domains to the AHRR/NCOA2 chimera, substituting for the repressor domain of AHRR. Because the amino terminal part of the transcription factor AHRR, responsible for the recognition of xenobiotic response elements in target genes and for heterodimerization, shows extensive homology with the aryl hydrocarbon receptor (AHR), the fusion is predicted to upregulate the AHR/ARNT signaling pathway. Indeed, global gene expression analysis showed upregulation of CYP1A1 as well as other typical target genes of this pathway, such as those encoding toll‐like receptors. Apart from providing a diagnostic marker for soft tissue angiofibroma, the results also suggest that this tumor constitutes an interesting model for evaluating the cellular effects of AHR signaling.

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. Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk

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.

Loss of chromosomes is the primary event in near-haploid and low-hypodiploid acute lymphoblastic leukemia

Loss of chromosomes is the primary event in near-haploid and low-hypodiploid acute lymphoblastic leukemia

Concomitant deletions of tumor suppressor genes MEN1 and AIP are essential for the pathogenesis of the brown fat tumor hibernoma

Hibernomas are benign tumors with morphological features resembling brown fat. They consistently display cytogenetic rearrangements, typically translocations, involving chromosome band 11q13. Here we demonstrate that these aberrations are associated with concomitant deletions of AIP and MEN1, tumor suppressor genes that are located 3 Mb apart and that underlie the hereditary syndromes pituitary adenoma predisposition and multiple endocrine neoplasia type I. MEN1 and AIP displayed a low expression in hibernomas whereas the expression of genes up-regulated in brown fat—PPARA, PPARG, PPARGC1A, and UCP1—was high. Thus, loss of MEN1 and AIP is likely to be pathogenetically essential for hibernoma development. Simultaneous loss of two tumor suppressor genes has not previously been shown to result from a neoplasia-associated translocation. Furthermore, in contrast to the prevailing assumption that benign tumors harbor relatively few genetic aberrations, the present analyses demonstrate that a considerable number of chromosome breaks are involved in the pathogenesis of hibernoma.

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)

GRM1 is upregulated through gene fusion and promoter swapping in chondromyxoid fibroma

Glutamate receptors are well-known actors in the central and peripheral nervous systems, and altered glutamate signaling is implicated in several neurological and psychiatric disorders. It is increasingly recognized that such receptors may also have a role in tumor growth. Here we provide direct evidence of aberrant glutamate signaling in the development of a locally aggressive bone tumor, chondromyxoid fibroma (CMF). We subjected a series of CMFs to whole-genome mate-pair sequencing and RNA sequencing and found that the glutamate receptor gene GRM1 recombines with several partner genes through promoter swapping and gene fusion events. The GRM1 coding region remains intact, and 18 of 20 CMFs (90%) showed a more than 100-fold and up to 1,400-fold increase in GRM1 expression levels compared to control tissues. Our findings unequivocally demonstrate that direct targeting of GRM1 is a necessary and highly specific driver event for CMF development.

Gene expression and single nucleotide polymorphism array analyses of spindle cell lipomas and conventional lipomas with 13q14 deletion

Spindle cell lipomas (SCL) are circumscribed, usually s.c. tumors that typically occur on the posterior neck, shoulder, and back of middle aged men. Cytogenetically, almost all SCL are characterized by deletions of chromosome arm 13q, often in combination with loss of 16q. Deletions of 13q are seen also in approximately 15% of conventional lipomas. Through single nucleotide polymorphism (SNP) array analyses, we identified two minimal deleted regions (MDR) in 13q14 in SCL. In MDR1, four genes were located, including the tumor suppressor gene RB1. MDR1 in SCL overlapped with the MDR detected in conventional lipomas with 13q14 deletion. In MDR2 in SCL there were 34 genes and the two microRNA (miRNA) genes miR‐15a and miR‐16‐1. Global gene expression analysis was used to study the impact of the deletions on genes mapping to the two SCL‐associated MDR. Five genes (C13orf1, DHRS12, ATP7B, ALG11, and VPS36) in SCL and one gene (C13orf1) in conventional lipomas with 13q‐deletions were found to be significantly underexpressed compared with control tissues. Quantitative real‐time PCR showed that miR‐16‐1 was expressed at lower levels in SCL than in the control samples. No mutations were found at sequencing of RB1, miR‐15a, and miR‐16‐1. Our findings further delineate the target region for the 13q deletion in SCL and conventional lipomas and show that the deletions are associated with down‐regulated expression of several genes, notably C13orf1, which was the only gene to be significantly down‐regulated in both tumor types.

Fusions involving protein kinase C and membrane-associated proteins in benign fibrous histiocytoma

Benign fibrous histiocytoma (BFH) is a mesenchymal tumor that most often occurs in the skin (so-called dermatofibroma), but may also appear in soft tissues (so-called deep BFH) and in the skeleton (so-called non-ossifying fibroma). The origin of BFH is unknown, and it has been questioned whether it is a true neoplasm. Chromosome banding, fluorescence in situ hybridization, single nucleotide polymorphism arrays, RNA sequencing, RT-PCR and quantitative real-time PCR were used to search for recurrent somatic mutations in a series of BFH. BFHs were found to harbor recurrent fusions of genes encoding membrane-associated proteins (podoplanin, CD63 and LAMTOR1) with genes encoding protein kinase C (PKC) isoforms PRKCB and PRKCD. PKCs are serine-threonine kinases that through their many phosphorylation targets are implicated in a variety of cellular processes, as well as tumor development. When inactive, the amino-terminal, regulatory domain of PKCs suppresses the activity of their catalytic domain. Upon activation, which requires several steps, they typically translocate to cell membranes, where they interact with different signaling pathways. The detected PDPN-PRKCB, CD63-PRKCD and LAMTOR1-PRKCD gene fusions are all predicted to result in chimeric proteins consisting of the membrane-binding part of PDPN, CD63 or LAMTOR1 and the entire catalytic domain of the PKC. This novel pathogenetic mechanism should result in constitutive kinase activity at an ectopic location. The results show that BFH indeed is a true neoplasm, and that distorted PKC activity is essential for tumorigenesis. The findings also provide means to differentiate BFH from other skin and soft tissue tumors. This article is part of a Directed Issue entitled: Rare cancers.