Mesenchymal chondrosarcomas are fast-growing tumors that account for 2-10% of primary chondrosarcomas. Cytogenetic information is restricted to 12 cases that did not show a specific aberration pattern. Recently, two fusion genes were described in mesenchymal chondrosarcomas: a recurrent HEY1-NCOA2 found in tumors that had not been cytogenetically characterized and an IRF2BP2-CDX1 found in a tumor carrying a t(1;5)(q42;q32) translocation as the sole chromosomal abnormality. Here, we present the cytogenetic and molecular genetic analysis of a mesenchymal chondrosarcoma in which the patient had two histologically indistinguishable tumor lesions, one in the neck and one in the thigh. An abnormal clone with the G-banding karyotype 46,XX,add(6)(q23),add(8)(p23),del(10)(p11),+12,-15[6] was found in the neck tumor whereas a normal karyotype, 46,XX, was found in the tumor of the thigh. RT-PCR and Sanger sequencing showed that exon 4 of HEY1 was fused to exon 13 of NCOA2 in the sample from the thigh lesion; we did not have spare material to perform a similar analysis of the neck tumor. Examining the published karyotypes we observed numerical or structural aberrations of chromosome 8 in the majority of the karyotyped mesenchymal chondrosarcomas. Chromosome 8 was also structurally affected in the present study. The pathogenetic mechanisms behind this nonrandom involvement are unknown, but the presence on 8q of two genes, HEY1 and NCOA2, now known to be involved in mesenchymal chondrosarcoma tumorigenesis is, of course, suggestive.

Cellular angiofibroma is a rare benign mesenchymal neoplasm with morphological and immunohistochemical similarities to spindle cell lipoma. Karyotypic information on cellular angiofibroma is restricted to one case only which showed loss of material from chromosomes 13 and 16. A few other studies using fluorescence in situ hybridization showed deletions of the RB1 and FOXO1 loci, both of which are located in chromosome band 13q14. We present here cytogenetic data on two cellular angiofibromas with an abnormal karyotype.

We describe two cases of extraskeletal osteochondroma in which chromosome bands 12q14~15 were visibly rearranged through a pericentric inv(12). Molecular analysis of the first tumor showed that both transcript 1 (NM_003483) and transcript 2 (NM_003484) of HMGA2 were expressed. In the second tumor, the inv(12) detected by karyotyping had resulted in an HMGA2-SOX5 fusion transcript in which exons 1-3 of HMGA2 were fused with a sequence from intron 1 of SOX5. The observed pattern is similar to rearrangements of HMGA2 found in several other benign mesenchymal tumors, i.e., disruption of the HMGA2 locus leaves intact exons 1-3 which encode the AT-hook domains and separates them from the 3'-terminal part of the gene. Our data therefore show that a subset of soft tissue osteochondromas shares pathogenetic involvement of HMGA2 with lipomas, leiomyomas and other benign connective tissue neoplasms.

Spindle cell tumors are clinically heterogeneous but morphologically similar neoplasms that can occur anywhere, mostly in adult patients. They are treated primarily with surgery to which is often added adjuvant or neoadjuvant radiation. Sub-classification of spindle cell sarcomas requires integration of histology, clinicopathological parameters, immunohistochemistry, cytogenetics (including fluorescence in situ hybridization) and/or molecular genetics. Some of the tumor subtypes are characterized by the presence of distinct chromosomal translocations and fusion genes. When no signs of differentiation are seen, the diagnosis by exclusion becomes undifferentiated spindle cell sarcoma. Cytogenetic, RNA sequencing and RT-PCR analyses were performed on a case of spindle cell sarcoma. The karyotype of the primary tumor was 46,X,del(X)(p?11p?22), der(12)(12pter→12q?22::12q?15→q?22::16p11→16pter),-16,+r(12). MDM2 was found amplified in both the primary tumor and a meta-stasis. RNA-Seq of the primary tumor identified four fusion genes, PTGES3-PTPRB, HMGA2-DYRK2, TMBIM4-MSRB3 and USP15-CNTN1, in which all the partner genes map to the q arm of chromosome 12. In material from the metastasis, RT-PCR detected the PTGES3-PTPRB, HMGA2-DYRK2 and TMBIM4-MSRB3 whereas no USP15-CNTN1 fusion transcript was found. Because MDM2 amplification and the fusion transcripts PTGES3-PTPRB, HMGA2-DYRK2 and TMBIM4-MSRB3 were found both in the primary tumor and in the metastasis, they are components of the same clone and may be involved both in initiation and progression of the tumor. Which of them is pathogenetically primary remains unknown.

Lipomas are the most common soft tissue tumors in adults. They often carry chromosome aberrations involving 12q13~15 leading to rearrangements of the HMGA2 gene in 12q14.3, with breakpoints occurring within or outside of the gene. Here, we present eleven lipomas and one osteochondrolipoma with a novel recurrent chromosome aberration, t(12;18)(q14~15;q12~21). Molecular studies on eight of the tumors showed that full-length HMGA2 transcript was expressed in three and a chimeric HMGA2 transcript in five of them. In three lipomas and in the osteochondrolipoma, exons 1-3 of HMGA2 were fused to a sequence of SETBP1 on 18q12.3 or an intragenic sequence from 18q12.3 circa 10 kbp distal to SETBP1. In another lipoma, exons 1-4 of HMGA2 were fused to an intronic sequence of GRIP1 which maps to chromosome band 12q14.3, distal to HMGA2. The ensuing HMGA2 fusion transcripts code for putative proteins which contain amino acid residues of HMGA2 corresponding to exons 1-3 (or exons 1-4 in one case) followed by amino acid residues corresponding to the fused sequences. Thus, the pattern is similar to the rearrangements of HMGA2 found in other lipomas, i.e., disruption of the HMGA2 locus leaves intact exons 1-3 which encode the AT-hooks domains and separates them from the 3'-terminal part of the gene. The fact that the examined osteochondrolipoma had a t(12;18) and a HMGA2-SETBP1 fusion identical to the findings in the much more common ordinary lipomas, underscores the close developmental relationship between the two tumor types.

RNA-sequencing of a case of acute myeloid leukemia with the bone marrow karyotype 46,XY,t(2;14)(q22;q32)[5]/47,XY,idem,+?4,del(6)(q13q21)[cp6]/46,XY[4] showed that the t(2;14) generated a ZEB2-BCL11B chimera in which exon 2 of ZEB2 (nucleotide 595 in the sequence with accession number NM_014795.3) was fused to exon 2 of BCL11B (nucleotide 554 in the sequence with accession number NM_022898.2). RT-PCR together with Sanger sequencing verified the presence of the above-mentioned fusion transcript. All functional domains of BCL11B are retained in the chimeric protein. Abnormal expression of BCL11B coding regions subjected to control by the ZEB2 promoter seems to be the leukemogenic mechanism behind the translocation.

Leiomyoma of deep soft tissue is a rare type of benign smooth muscle tumor that mostly occurs in the retroperitoneum or abdominal cavity of women, and about which very little genetic information exists. In the present study, eight leiomyomas of deep soft tissue were genetically analyzed. G-banding showed that three tumors carried rearrangements of the long arm of chromosome 12, three others had 8q rearrangements, the 7th tumor had deletion of the long arm of chromosome 7, del(7)(q22), and the 8th had aberrations of chromosome bands 3q21~23 and 11q21~22. The target genes of the 12q and 8q aberrations were HMGA2 and PLAG1, respectively. In the leiomyomas with 12q rearrangements, both HMGA2 and PLAG1 were expressed whereas in the tumors with 8q aberrations, only PLAG1 was expressed. In the cases without 12q or 8q aberrations, the expression of HMGA2 was very low and PLAG1 was expressed only in the case with del(7)(q22). All eight leiomyomas of deep soft tissue expressed MED12 but none of them had mutation in exon 2 of that gene. In two tumors with 12q rearrangements, RPSAP52 on 12q14.3 was fused with non-coding RNA (accession number XR_944195) from 14q32.2 or ZFP36L1 from14q24.1. In a tumor with inv(12), exon 3 of HMGA2 was fused to a sequence in intron 1 of the CRADD gene from 12q22. The present data together with those of our two previous studies in which the fusions KAT6B-KANSL1 and EWSR1-PBX3 were described in two retroperitoneal leiomyomas carrying a t(10;17)(q22;q21) and a t(9;22)(q33;q12) translocation, respectively, show that leiomyomas of deep soft tissue are genetically heterogenous but have marked similarities to uterine leiomyomas.

Sequential combination of cytogenetics and RNA-sequencing (RNA-Seq) has been shown to be an efficient approach to detect pathogenetically important fusion genes in neoplasms carrying only one or a few chromosomal rearrangements. We performed RNA-Seq on an acute myeloid leukemia in a 2-year-old girl with the karyotype 46,XX,add(1)(p36), der(2)t(2;3)(q21;q21),del(3)(q21),der(10)t(1;10)(q32;q24),der(16)(2qter-->2q21::16p11-->16q24::16p11-->16pter)[13]/46,XX[2] and identified a cryptic FUS/ERG fusion gene. PCR and direct sequencing verified the presence of the FUS-ERG chimeric transcript in which exon 7 of FUS from 16p11 (nt 904 in sequence with accession number NM_004960 version 3) was fused in frame to exon 8 of ERG from sub-band 21q22.2 (nt 967 in NM_004449 version 4). The FUS-ERG transcript found here has been reported in only two other cases of childhood leukemia, in a 1-year-old boy and an 8-month-old boy, both diagnosed with precursor B cell ALL. The fusion transcript codes for a 497 amino acid residues FUS-ERG protein and, similar to other AML-related FUS-ERG fusion proteins, contains both functional domains (TR1 and TR2) of the transactivation domain of FUS and the ETS domain of ERG. The clinical significance, if any, of the amino acid residues which are coded by the exons 8, 9 and 10 of ERG in the fusion FUS-ERG proteins, remains unclear.

Sarcomas account for 3% of all uterine malignancies and many of them are characterized by acquired, specific fusion genes whose detection has increased pathogenetic knowledge and diagnostic precision. We describe a novel fusion gene, GREB1-NCOA2, detected by transcriptome sequencing and validated by reverse transcriptase polymerase chain reaction and Sanger sequencing in an undifferentiated uterine sarcoma. The chimeric transcript was an in-frame fusion between exon 3 of GREB1 and exon 15 of NCOA2. The fusion is reported here for the first time, but it involves the GREB1 gene, an important promoter of tumor growth and progression, and NCOA2 which is known to be involved in transcriptional regulation. The alteration and recombination of these genes played a role in the tumorigenesis and/or progression of this sarcoma.

Recent cytogenetic and molecular investigations have improved our understanding of endometrial stromal tumors, including sarcomas (ESS), and helped redefine their classification into more pathogenetically meaningful categories. Because much more can be gained through such studies, we add information on another 22 ESS examined by karyotyping, PCR analysis, expression array analysis, and transcriptome sequencing. In spite of the known preference for certain pathogenetic pathways, we found considerable genetic heterogeneity in high-grade (HG) as well as in low-grade (LG) ESS. Not all HG tumors showed a YWHAE-NUTM chimeric transcript and as many as six LGESS showed no hitherto known ESS-related fusions. Among the transcripts identified by transcriptome sequencing and verified by Sanger sequencing, new variants of ZC3H7-BCOR and its reciprocal BCOR-ZC3H7 were identified as was involvement of the CREBBP and MLLT4 genes (both well known leukemia-related genes) in two new fusions. FISH analysis identified a known EPC1-PHF1 fusion which led to the identification of a new variant at the molecular level. The fact that around 70 genes were found differentially expressed, by microarray analysis, when comparing LGESS showing ESS-related fusions with LGESS without such transcripts, underscores the biochemical importance of the observed genetic heterogeneity and hints that new subgroups/entities in LGESS still remain undiscovered. © 2016 The Authors. Genes, Chromosomes & Cancer Published by Wiley Periodicals, Inc.

An acute myeloid leukemia was suspected of having a t(8;16)(p11;p13) resulting in a KAT6A-CREBBP fusion because the bone marrow was packed with monoblasts showing marked erythrophagocytosis. The diagnostic karyotype was 46,XY,add(1)(p13),t(8;21)(p11;q22),der(16)t(1;16)(p13;p13)[9]/46,XY[1]; thus, no direct confirmation of the suspicion could be given although both 8p11 and 16p13 seemed to be rearranged. The leukemic cells were examined in two ways to find out whether a cryptic KAT6A-CREBBP was present. The first was the "conventional" approach: G-banding was followed by fluorescence in situ hybridization (FISH) and reverse transcription PCR (RT-PCR). The second was RNA-Seq followed by data analysis using FusionMap and FusionFinder programs with special emphasis on candidates located in the 1p13, 8p11, 16p13, and 21q22 breakpoints. FISH analysis indicated the presence of a KAT6A/CREBBP chimera. RT-PCR followed by Sanger sequencing of the amplified product showed that a chimeric KAT6A-CREBBP transcript was present in the patients bone marrow. Surprisingly, however, KATA6A-CREBBP was not among the 874 and 35 fusion transcripts identified by the FusionMap and FusionFinder programs, respectively, although 11 sequences of the raw RNA-sequencing data were KATA6A-CREBBP fragments. This illustrates that although many fusion transcripts can be found by RNA-Seq combined with FusionMap and FusionFinder, the pathogenetically essential fusion is not always picked up by the bioinformatic algorithms behind these programs. The present study not only illustrates potential pitfalls of current data analysis programs of whole transcriptome sequences which make them less useful as stand-alone techniques, but also that leukemia diagnosis still relies on integration of clinical, hematologic, and genetic disease features of which the former two by no means have become superfluous.

Lipomas are common benign soft tissue tumors whose genetic and cytogenetic features are well characterized. The karyotype is usually near- or pseudodiploid with characteristic structural chromosomal aberrations. The most common rearrangements target the high mobility group AT-hook 2 (HMGA2) gene in 12q14.3, with breakpoints occurring within or outside of the gene locus leading to deregulation of HMGA2. The most common fusion partner for HMGA2 in lipoma is lipoma-preferred partner (3q27), but also other genes frequently recombine with HMGA2. Furthermore, truncated HMGA2 transcripts are recurrently observed in lipomas. The present study describes 5 lipomas carrying the translocation t(4;12)(q27~28;q14~15) as the sole chromosomal anomaly, as well as 1 lipoma in which the three-way translocation t(1;4;12)(q21;q27~28;q14~15) was identified. Molecular analyses performed on 4 of these cases detected 4 truncated forms of HMGA2. In 3 tumors, the HMGA2 truncated transcripts included sequences originating from the chromosomal sub-band 4q28.1. Notably, in 2 of these cases, the fourth exon of HMGA2 was fused to transposable elements located in 4q28.1.

Juxtaposition of two different genes or gene parts due to chromosomal rearrangement is a well-known neoplasia-associated pathogenetic mechanism. The detection and characterization of such tumorigenic fusions is of great importance both research-wise, diagnostically because they may be specific for distinct tumor entities, and because they may serve as therapeutic targets for antioncogenic drugs that interact directly with the molecular changes responsible for neoplastic transformation.At present, more than 10,000 fusion transcripts have been reported in different types of neoplasia, with one tenth of them being identified in squamous cell carcinomas (SCC) of different locations. No recurrent fusion gene has to date been identified in SCC of the vulva.We performed high-throughput paired-end RNA-sequencing of 12 vulvar SCC and found two recurrent fusions with the STIP1-CREB3L1 and ZDHHC5-GPR137 being present in two tumors each. The transcripts were detected only in the tumor samples, not in normal vulvar tissue from healthy donors used as control. The CREB3L1 and ZDHHC5 genes encode proteins involved in transcription suggesting that the chimeras may alter downstream events in their respective pathways. Expression analysis of the CREB3L1 gene showed the presence of two distinct groups of tumors, one having fusion and downregulation of the gene and the other showing upregulation of CREB3L1.

Acute erythroid leukemia was diagnosed in a 4-month-old boy. Cytogenetic analysis of bone marrow (BM) cells showed a t(11;20)(p11;q11) translocation. RNA extracted from the BM was sequenced and analyzed for fusion transcripts using the software FusionMap. A ZMYND8-RELA fusion was ranked first. RT-PCR and direct sequencing verified the presence of an in frame ZMYND8-RELA chimeric transcript. Fluorescence in situ hybridization showed that the ZMYND8-RELA was located on the p12 band of der(11); therefore a cytogenetically invisible pericentric inversion in chromosome 11 must have taken place besides the translocation. The putative ZMYND8-RELA fusion protein contains the Zinc-PHD finger domain, a bromodomain, a PWWP domain, a MYND type of zinc finger of ZMYND8, and the entire RELA protein, indicating that it might act leukemogenically by influencing several cellular processes including the NF-kappa-B pathway.

Intramuscular myxoma is a benign soft tissue tumor about which very limited genetic information exists. We studied 68 intramuscular myxomas by means of chromosome banding analysis finding abnormal karyotypes in 21 of them. The most clearly nonrandom involvement was of chromosome 8 which was found gained in seven tumors (+8 was the sole change in five myxomas) and structurally rearranged in another two. Since mutation of the gene GNAS (20q13) has been implicated in the pathogenesis of both solitary and hereditary multiple myxomas, we assessed the transcription and mutation status of this gene in five tumors from which we had suitable RNA. All five intramuscular myxomas expressed biallelic transcripts. The mutated GNAS allele found in one tumor was also biallelically transcribed. In none of the five myxomas were maternally expressed transcripts detected. Collectively, the data suggest that intramuscular myxomas have acquired genetic abnormalities that often include chromosome 8 changes but may also involve alterations of GNAS. To what extent these aberrations are pathogenetically important, remains uncertain.

Retroperitoneal leiomyoma is a rare type of benign smooth muscle tumor almost exclusively found in women and with histopathological features similar to uterine leiomyomas. The pathogenesis of retroperitoneal leiomyoma is unclear and next to nothing is known about the cytogenetics and molecular genetics of the tumor. Here we present the first cytogenetically analyzed retroperitoneal leiomyoma. It had a t(10;17)(q22;q21) as the sole chromosomal abnormality. Using RNA-Sequencing and the 'grep' command to search the fastq files of the sequence data we found that the translocation resulted in fusion of the genes KAT6B (10q22) with KANSL1 (17q21). RT-PCR together with direct (Sanger) sequencing verified the presence of a KAT6B-KANSL1 fusion transcript. No reciprocal KANSL1-KAT6B transcript was amplified suggesting that it was either absent or unexpressed. The KAT6B-KANSL1 fusion transcript consists of exons 1 to 3 of KAT6B and exons 11 to 15 of KANSL1, is 3667 bp long, has a 1398 bp long open reading frame, and codes for a 466 amino acid residue protein. The corresponding KAT6B-KANSL1 protein contains the NEMM domain (including the linker histone H1/H5, domain H15) of KAT6B and the PEHE domain of KANSL1. The function of the fusion protein might be regulation of transcription with an affinity for chromatin (linker histone H1/H5) and interaction with the HAT domain of KAT8 (PEHE domain). The tumor expressed HMGA2 and HMGA1 even though 12q14-15 and 6p looked normal by G-banding analysis. The tumor also expressed MED12 in the absence of exon 2 mutations. Overall, the data show that the examined retroperitoneal leiomyoma resembles a subset of uterine leiomyomas in terms of histology and genetics.

Formation of fusion genes is pathogenetically crucial in many solid tumors. They are particularly characteristic of several mesenchymal tumors, but may also be found in epithelial neoplasms. Ovarian carcinomas, too, may harbor fusion genes but only few of these were found to be recurrent with a rate ranging from 0.5 to 5%. Because most attempts to find specific and recurrent fusion transcripts in ovarian carcinomas focused exclusively on high-grade serous carcinomas, the situation in the other carcinoma subgroups remains largely uninvestigated as far as fusion genes are concerned. We performed transcriptome sequencing on a series of 34 samples from ovarian tumors that included borderline, clear cell, mucinous, endometrioid, low-grade and high-grade serous carcinomas in search of fusion genes typical of these subtypes. We found a total of 24 novel fusion transcripts. The PCMTDI-CCNL2 fusion transcript, which involves a member of the cyclin family, was found recurrently involved but only in endometrioid carcinomas (4 of 18 tumors; 22%). We also found three additional fusion transcripts involving genes belonging to the cyclin family: ANXA5-CCNA2 and PDE4D-CCNB1 were detected in two endometrioid carcinomas, whereas CCNY-NRG4 was identified in a clear cell carcinoma. The recurrent involvement of CCNL2 in four fusions and of three other genes of the cyclin family in three additional transcripts hints that deregulation of cyclin genes is important in the pathogenesis of ovarian carcinomas in general but of endometrioid carcinomas particularly.

RNA-sequencing was performed on three tenosynovial giant cell tumors (TSGCT) in an attempt to elicit more information on the mechanisms of CSF1 expression in this tumor type. A novel CSF1-S100A10 fusion gene was found in a TSGCT that carried the translocation t(1;1)(q21;p11) as the sole karyotypic abnormality. In this fusion gene, the part of CSF1 coding for the CSF1 protein (exons 1-8 in sequences with accession nos. NM_000757 and NM_172212) is fused to the 3'-part of S100A10. Since the stop codon TAG of CSF1 is present in it, the CSF1-S100A10 fusion gene's predominant consequence seems to be the replacement of the 3'-untranslated region (UTR) of CSF1 (exon 9; nt 2092-4234 in sequence with accession no. NM_000757 or nt 2092-2772 in NM_172212) by the 3'-end of S100A10 (exon 3; nt 641-1055 in sequence with accession no. NM_002966). In the other two TSGCT, a novel CSF1 transcript was detected, the same in both tumors. Similar to the occurrence in the CSF1-S100A10 fusion gene, the novel CSF1 transcript 3'-UTR is replaced by a new exon located ~48 kb downstream of CSF1 and 11 kb upstream of AHCYL1. Although only 3 TSGCT were available for study, the finding in all of them of a novel CSF1-S100A10 fusion gene or CSF1 transcript indicates the existence of a common pathogenetic theme in this tumor type: the replacement of the 3'-UTR of CSF1 with other sequences.

Mesenchymal chondrosarcomas (MCs) account for 3-10% of primary chondrosarcomas. The cytogenetic literature includes only ten such tumours with karyotypic information and no specific aberrations have been identified. Using a purely molecular genetic approach a HEY1-NCOA2 fusion gene was recently detected in 10 of 15 investigated MCs. The fusion probably arises through intrachromosomal rearrangement of chromosome arm 8 q. We report a new case of MC showing a t(1;5)(q42;q32) as the sole karyotypic aberration. Through FISH and whole transcriptome sequencing analysis we found a novel fusion between the IRF2BP2 gene and the transcription factor CDX1 gene arising from the translocation. The IRF2BP2-CDX1 has not formerly been described in human neoplasia. In our hospital's archives three more cases of MC were found, and we examined them looking for the supposedly more common HEY1-NCOA2 fusion, finding it in all three tumours but not in the case showing t(1;5) and IRF2BP2-CDX1 gene fusion. This demonstrates that genetic heterogeneity exists in mesenchymal chondrosarcoma.

Whole transcriptome sequencing was used to study a small round cell tumor in which a t(4;19)(q35;q13) was part of the complex karyotype but where the initial reverse transcriptase PCR (RT-PCR) examination did not detect a CIC-DUX4 fusion transcript previously described as the crucial gene-level outcome of this specific translocation. The RNA sequencing data were analysed using the FusionMap, FusionFinder, and ChimeraScan programs which are specifically designed to identify fusion genes. FusionMap, FusionFinder, and ChimeraScan identified 1017, 102, and 101 fusion transcripts, respectively, but CIC-DUX4 was not among them. Since the RNA sequencing data are in the fastq text-based format, we searched the files using the "grep" command-line utility. The "grep" command searches the text for specific expressions and displays, by default, the lines where matches occur. The "specific expression" was a sequence of 20 nucleotides from the coding part of the last exon 20 of CIC (Reference Sequence: NM_015125.3) chosen since all the so far reported CIC breakpoints have occurred here. Fifteen chimeric CIC-DUX4 cDNA sequences were captured and the fusion between the CIC and DUX4 genes was mapped precisely. New primer combinations were constructed based on these findings and were used together with a polymerase suitable for amplification of GC-rich DNA templates to amplify CIC-DUX4 cDNA fragments which had the same fusion point found with "grep". In conclusion, FusionMap, FusionFinder, and ChimeraScan generated a plethora of fusion transcripts but did not detect the biologically important CIC-DUX4 chimeric transcript; they are generally useful but evidently suffer from imperfect both sensitivity and specificity. The "grep" command is an excellent tool to capture chimeric transcripts from RNA sequencing data when the pathological and/or cytogenetic information strongly indicates the presence of a specific fusion gene.