Consistent Rearrangement of Chromosomal Band 6P21 with Generation of Fusion Genes JAZF1/PHF1 and EPC1/PHF1 in Endometrial Stromal Sarcoma

Research Article

Consistent Rearrangement of Chromosomal Band 6p21 with Generation of Fusion Genes JAZF1/PHF1 and EPC1/PHF1 in Endometrial Stromal Sarcoma

Francesca Micci,1 Ioannis Panagopoulos,4 Bodil Bjerkehagen,2 and Sverre Heim1,3

Departments of 1Cancer Genetics and 2Pathology, The Norwegian Radium Hospital; 3Faculty of Medicine, University of Oslo, Oslo, Norway; and 4Department of Clinical Genetics, University Hospital, Lund, Sweden

Abstract Little is known about the genetic background of ESS as only 32 Endometrial stromal sarcomas (ESS) represent <10% of all such tumors have been karyotyped and reported scientifically uterine sarcomas. Cytogenetic data on this tumor type are (6–8). The pattern of rearrangements thus detected is nevertheless limited to 32 cases, and the karyotypes are often complex, clearly nonrandom with particularly frequent involvement of but the pattern of rearrangement is nevertheless clearly chromosome arms 6p and 7p (7). Recently, a specific translocation nonrandom with particularly frequent involvement of chro- t(7;17)(p15;q21) leading to the fusion of two zinc finger genes, mosome arms 6p and 7p. Recently, a specific translocation juxtaposed with another zinc finger (JAZF1) and joined to JAZF1 t(7;17)(p15;q21) leading to the fusion of two zinc finger genes, (JJAZ1), was described in a subset of ESS (9). Both genes, the JAZF1 at 7p15 and JJAZ1 at 17q21, contain sequences encoding zinc finger juxtaposed with another zinc finger (JAZF1) and joined to JAZF1 (JJAZ1), was described in a subset of ESS. We present motifs characteristic of DNA-binding proteins. The gene fusion three ESS whose karyotypes were without the disease-specific results in expression of a tumor-specific mRNA transcript V V t(7;17) but instead showed rearrangement of chromosomal containing 5 -JAZF1 and 3 -JJAZ1 sequences but retaining the zinc band 6p21, twice as an unbalanced t(6p;7p) and once as a finger motifs from both genes. Because wild-type JAZF1 is three-way 6;10;10 translocation. All three tumors showed expressed in normal endometrium, it has been suggested that the specific rearrangement of the PHD finger protein 1 (PHF1) JAZF1/JJAZ1 fusion gene creates a chimeric protein that disrupts gene, located in chromosomal band 6p21. In the two tumors transcription in a lineage-specific manner (9). with t(6;7), PHF1 was recombined with the JAZF1 gene from Several sarcoma-specific translocations exist, but some of them 7p15, leading to the formation of a JAZF1/PHF1 fusion gene. display considerable molecular diversity both in terms of intragenic variant fusions and the involvement of alternative translocation The third tumor showed a t(6p;10q;10p) as the sole karyotypic abnormality, leading to the fusion of PHF1 with another partners in pathogenetically equivalent variant translocations partner, the enhancer of polycomb (EPC1) gene from 10p11; (10, 11). Accordingly, the failure to detect the JAZF1/JJAZ1 fusion EPC1 has hitherto not been associated with neoplasia. The transcript in ESS not showing the 7;17 translocation (7, 12) suggests PHF1 gene encodes a protein with two zinc finger motifs the existence of pathogenetic, cytogenetic, and molecular variants whose involvement in tumorigenesis and/or tumor progres- of the ESS-specific t(7;17) and JAZF1/JJAZF1. Indicative of the sion has not been reported before, but its rearrangement same possibility is also the fact that chromosomal band 7p15 was found rearranged in 5 of the 32 ESS with karyotypic changes but clearly defines a new pathogenetic subgroup of ESS. (Cancer without the aforementioned 7;17 translocation (8). The second Res 2006; 66(1): 107-12) most common cytogenetic abnormality registered in ESS involves chromosomal band 6p21, rearranged in eight tumors; this too Introduction hints at pathogenetic, cytogenetic, and molecular genetic ESS- specific features that have not yet been sufficiently examined and Malignant tumors mimicking the differentiation of endometrial understood. stromal cells, endometrial stromal sarcoma (ESS), are rare and We therefore subjected to detailed analysis three ESS showing account for <10% of uterine sarcomas. ESS were traditionally rearrangement of chromosomal band 6p21. In all tumors, specific divided into low-grade and high-grade tumors according to the involvement of the PHD finger protein 1 (PHF1) gene in 6p21 could classification of Norris and Taylor (1), but because the so-called be shown. PHF1 was found recombined with two different partners, high-grade ESS usually lack specific differentiation and often with the JAZF1 gene in the two tumors showing a 6p;7p rearrange- present little or no histologic resemblance to endometrial stroma, ment and with the enhancer of polycomb (EPC1) gene from 10p11 they are now mostly referred to as undifferentiated endometrial in the third tumor, which had a 6;10;10 translocation as the sole sarcomas (2). ESS therefore consist of only low-grade ESS and karyotypic abnormality. variants thereof, regardless of the mitotic index, as long as the lesional cells resemble nonneoplastic proliferative endometrial stroma (3–5). Materials and Methods Tumors. Samples from three surgically removed ESS were subjected to histologic and genetic analyses. The patient of case 1 was a 33-year-old woman who underwent hysterectomy after a biopsy of a uterine tumor had Requests for reprints: Francesca Micci, Department of Cancer Genetics, The shown an ESS. The tumor cells were small and spindle shaped with a Norwegian Radium Hospital, 0310 Oslo, Norway. Phone: 47-2-293-4436; Fax: 47-2-293- mitotic count of 0.1 per high-power field. Only small areas with fibrous or 5477; E-mail: [email protected]. I2006 American Association for Cancer Research. myxoid tissue were seen. The tumor infiltrated extensively through the doi:10.1158/0008-5472.CAN-05-2485 uterine wall and into the parametrium and vessels (Fig. 1). The patient of www.aacrjournals.org 107 Cancer Res 2006; 66: (1). January 1, 2006

Fluorescence in situ hybridization analyses. Different fluorescence in situ hybridization (FISH) analyses were done depending on which types of chromosome aberrations were discovered by G-banding. Multiplex FISH (15) was done on cases 1 and 2; in case 1 on a previously G-banded slide according to the protocol described by Teixeira et al. (16), whereas in case 2, fresh chromosome preparations were available for multiplex FISH. In case 3, locus-specific probes derived from bacterial artificial chromosomes (BAC) and P1 artificial chromosome clones were prepared and hybridized to detect the exact breakpoint of the 10;10 translocation originally thought to be the only chromosomal abnormality present in this tumor (see below). The BAC clones were retrieved from the RPCI-11 Human BAC library and CalTech human BAC library D; the P1 artificial chromosome clones were from the RPCI-4 human P1 artificial chromosome library (P. de Jong libraries, http://bacpac.chori.org/home.htm). They were selected according to physical and genetic mapping data on chromosomes 6, 7, and 10 (see below) reported in the Human Genome Browser at the University of California, Santa Cruz website (May 2005, http://genome.ucsc. edu/). The clones initially used were RP11-414H17 and RP11-74N14 mapping to 10p15 and RP11-34E5 and RP11-7D5 mapping to 10q24. The identification of a three-way translocation involving also chromosome 6 and the redefinition of the breakpoints on 10p and 10q (see below) led us to perform additional analyses using BAC clones RP11-615A19, RP11-513I15, and RP11-754H10, mapping to 6p21 (from centromere to telomere); RP11- 322I2 and RP11-241I20 mapping to 10p11; and 433J16 mapping to 10q21 (resources for Molecular Cytogenetics, Bari, Italy; http://www.biologia. Figure 1. Histologic sections of the three ESS. A, extensive infiltration in the uniba.it/rmc/). In case 2, clones RP11-81H15 and RP4-781A18, mapping to myometrium in case 1. B, vessel infiltration in case 3. C, tumor with a finger-like projection into the surrounding myometrium in case 2. D, extensive 7p15 and covering the JAZF1 locus, and CTD-2307H19, mapping to 6p21 hyalinization with areas of endometrial stromal cells around spiral arteriole-like and covering the PHF1 locus, were used to detect JAZF1/PHF1 fusion at the vessels in case 2. molecular cytogenetic level. The clones were grown in selective media, and DNA was extracted according to standard methods (17). DNA probes were case 2 was a 72-year-old woman with a 2.2-cm large tumor in the anterior directly labeled with a combination of FITC-12-dCTP and FITC-12-dUTP, wall of the uterine corpus. Histologic examination of the operation Texas Red-6-dCTP and Texas Red-6-dUTP (New England Nuclear, Boston, specimen showed a tumor with large areas of hyalinization as well as MA), or indirectly with biotin-dUTP (Molecular Probes, Invitrogen, Carlsbad more cellular areas with spindle cells without atypia. The mitotic count was CA) by nick translation and detected with streptavidin-diethylaminocou- 0.1 per high-power field. The tumor was well demarcated macroscopically marin (Invitrogen). The subsequent hybridization conditions as well as the but microscopically showed one or two finger-like tumor projections detection procedure were according to standard protocols (18). exceeding 3 mm into the surrounding myometrium (Fig. 1). No infiltration Molecular genetic investigations. Tumor tissue adjacent to that used of vessels was seen. The patient of case 3 was a 34-year-old woman who for cytogenetic analysis and histologic examination had been frozen and underwent hysterectomy because a biopsy had shown the presence of stored at 80jC. Total RNA was extracted from the three tumors using ESS. Macroscopic examination showed a polyp-like tumor with a diameter of Trizol reagent according to the manufacturer’s instructions (Invitrogen). 2.5 cm in the uterus. Histologic examination showed a cellular tumor with The primers used for PCR amplification and sequencing are listed small spindle cells without atypia. The mitotic count was 0 to 0.1 per high- in Table 2. cDNA quality was checked in each case using ABL1-specific power field. The tumor showed diffuse infiltration into the myometrium and primers (19). also vessel infiltration. Only small areas with fibrous or myxoid tissue were 3V-Rapid amplification of cDNA ends (RACE)-PCR was done in case 2 present (Fig. 1). with primers JAZF1-182F and JAZF1-357F. 5V-RACE-PCR was done in case 3 G-banding and karyotyping. The specimens intended for cytogenetic with primers PHF1-327R and PHF1-250R (Table 2). In both cases, the cDNA analysis were mechanically and enzymatically disaggregated and short-term synthesis, the RACE-PCR, the subsequent NESTED-PCR were done using cultured according to Mandahl (13). Chromosome preparations were made the BD SMART RACE kit following the manufacturer’s recommendations and G-banded using Wright stain. The subsequent cytogenetic analysis and (BD Biosciences, San Jose CA). The PCR cycles were as follows: 1 minute at karyotypic description followed the recommendations of the International 94jC and 25 cycles of 30 seconds at 94jC, 30 seconds at 60jC, and 3 System for Human Cytogenetic Nomenclature (14). The karyotype of case 1 minutes at 72jC. of the present series (Table 1) was previously reported as an addendum in cDNA was synthesized using 5 Ag of total RNA in 20 AL reaction mixture j Micci et al. (7). [50 mmol/l Tris-HCl (pH 8.3) at 25 C, 75 mmol/L KCl, 3 mmol/L MgCl2,

Table 1. Karyotypic data on the three ESS based on G-banding and FISH analysis

Case no. G-banding karyotype FISH karyotype

1 46,XX,2,6,add(7)(p15),+2mar[11]/46,XX[4] 46,XX,inv(2)(p21q37),der(6)del(6)(p21)t(6;7)(q21;p15), der(7)t(6;7)(p21;p15)del(6)(q21) 2 46,X,-X,del(2)(q36),add(3)(q29),6, del(6)(q21),add(7)(p22), 47,X,der(X)t(X;16)(?;?),+2,add(2)(q11), ins(2;22)(q31;q11q13),der(3)ins(3;13) der(14)t(1;14)(q25;q32),+mar[5] (p24;q?22q32)t(3;6)(q28;q22), der(6)t(3;6)(q28;q22),del(6)(p11), der(7)(7qter!7p15::6?::15?::3?::13?::15?::6?::X?::6?::3?),der(14)t(1;14)(q25;q32) 3 46,XX,t(10;10)(q24;p15)[13]/46,XX[2] 46,XX,t(6;10;10)(p21;q22;p11)

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Table 2. Primers used for PCR and sequencing

Designation Sequence Position Gene Accession no.

EPC1-1651F 5V-CGCGGTGGAAGGGTCTTACTGGA-3V 1651-1673 EPC1 NM_025209 EPC1-1860F 5V-GCCTCGGACACCATCCCTACATGA-3V 1860-1883 EPC1 NM_025209 JAZF1-182F 5V-GCAGCCAACCTATGTTGCCCTGAG-3V 182-205 JAZF1 NM_175061 JAZF1-357F 5V-CCACAGCAGTGGAAGCCTTA´-3 335-354 JAZF1 NM_175061 JJAZ1-843R 5V-CCGGGTTTTGTTTGATTGAGG-3V 848-868 JJAZ1 NM_015355 PHF1-250R 5V-AAGCTGGGTCCCAAAGTGAGGAGG-3V 250-273 PHF1 NM_024165 PHF1-327R 5V-AGCCCATCAGTCCATCTGGCCAG-3V 327-349 PHF1 NM_024165

10 mmol/L DTT, 1 mmol/L each deoxynucleotide triphosphate, 20 units specific primer combinations for JAZF1-182F and JJAZ1-885R RNase inhibitor (RNA guard, Amersham Biosciences, Piscataway, NJ), (Table 2; ref. 7), but no specific transcript was found. 0.5 pmol/L random hexamers, and 400 units Moloney murine leukemia To test if tumors 1 and 2 (i e., those with cytogenetic aberrations virus reverse transcriptase (Invitrogen)]. The reaction was carried out at of 7p15) presented some other fusion transcript than the JAZF1/ 37jC for 60 minutes, heated for 5 minutes at 65jC, then kept at 4jC JJAZ1, we did 3V-RACE-PCR analysis for the JAZF1 gene. This until analysis. The PCR amplification was done using 1 AL of the cDNA as template in investigation detected a specific transcript in which the JAZF1 gene 50 AL reaction volume containing 20 mmol/L Tris-HCl (pH 8.4) at 25jC, 50 was fused with the PHF1 gene, and the fusion was then confirmed

KCl, 1.25 mmol/L MgCl2, 0.2 mmol/L of each deoxynucleotide triphosphate, using combinations of specific primers for the aforementioned 1 unit platinum Taq polymerase (Invitrogen), and 0.5 Amol/L of each of the genes. In case 1, two PCR fragments were present, of 170 and 250 forward and reverse primers used. The JAZF1/PHF1 fusion transcript was bp, respectively, whereas in case 2, a single 450-bp PCR fragment detected using JAZF1-182F and PHF1-250R and JAZF1-182F and PHF1-327R was detected (Fig. 3) using a specific JAZF1-182F/PHF1-250R primer combinations, whereas the EPC1/PHF1 transcript was detected primer combination. Direct sequencing of the transcripts revealed using EPC1-1651F and PHF1-327R and EPC1-1860F and PHF-250R primer that the fragments from case 1 were JAZF1/PHF1 chimeric frag- combinations. The PCR was done with the following cycles: 5 minutes at ments, both containing a sequence of intron 2 of JAZF1 (Fig. 3). 94jC and 30 cycles of 1 minute at 94jC, 1 minute at 60jC, and 1 minute at Fragment 1 contained an open reading frame for the JAZF1/PHF1 72jC, and a final extension for 10 minutes at 72jC. The PCR products were analyzed by electrophoresis through 1.2% agarose gel, stained with ethidium fusion, whereas fragment 2 was an out-of-frame JAZF1/PHF1 bromide, and purified using the QIAquick gel extraction kit (Qiagen, Hilden, fusion. In case 2, direct sequencing revealed that the fragment Germany). Both strands were subjected to sequence analysis using the contained sequences from intron 3 of JAZF1 fused with sequences ABI Prism BigDye terminator cycle sequencing ready reaction kit (Perkin- of a noncoding region from PHF1 intron 1 (Fig. 3). The fusion Elmer, Applied Biosystems, Foster City, CA). For cases 1 and 2, the JAZF1- transcript had an open reading frame. FISH analysis of this case 182F and PHF1-250R primers were used, whereas for case 3, EPC1-1860F with two JAZF1-specific and one PHF1-specific probe unequivo- and PHF1-250R were used. The analysis was done on the Applied Bio- cally showed that the PHF1 gene was fused with the JAZF1 gene systems Model 3100-Avant DNA sequencing system. The BLAST software (Fig. 2). (http://www.ncbi.nlm.gov/BLAST) was used for computer analysis of In the third ESS, FISH with locus-specific probes, RP11-414H17 sequenced data. and RP11-74N14 mapping to 10p15 and RP11-34E5 and RP11-7D5 mapping to 10q24, identified a t(6;10;10)(p21;q22;p11) as the sole Results karyotypic abnormality (Fig. 2). We again used locus-specific probes to better characterize the breakpoints of the translocation, Cases 1 and 2 showed complex karyotypes, which could not be but due to lack of material, we could not find out which BAC described completely after G-banding analysis only; therefore, clones spanned the breakpoints. In addition, we tested for possible multiplex FISH was used to identify the chromosomes involved in cryptic rearrangements between chromosomes 6 and 7 by reverse different rearrangements (Table 1; Fig. 2). In case 3, FISH with transcription-PCR using a JAZF1/PHF1-specific primer combina- locus-specific probes was done to identify the breakpoint positions tion, but no specific transcript was identified. The PHF1 gene, on the short and long arms of chromosome 10, as a t(10p;10q) was located in chromosomal band 6p21, was then tested for involve- thought to be the only rearrangement based on the G-banded ment in this tumor using 5V-RACE-PCR with primers for the karyotype. The FISH analysis, however, showed that the probe from PHF1gene. A specific transcript was detected identifying a fusion 10p11 mapped to a cytogenetically seemingly normal 6p; thus, between the EPC1 and PHF1 genes. Direct sequencing of this eventually, a three-way translocation t(6p;10q;10p) was identified transcript revealed in-frame fusion of exon 10, codon 581, of the (Table 1; Fig. 2). None of the three tumors showed the disease- EPC1 mRNA to exon 2, 17 bp upstream the ATG, of the PHF1 specific 7;17 translocation. However, as all three ESS presented mRNA (Fig. 4). rearrangements involving chromosome arm 6p and in cases 1 and 2 in recombination with 7p15, we decided to study these tumors at the molecular level to identify possible variant partners of the Discussion JAZF1 gene located in 7p15 and already known to be rearranged in All three ESS examined in this report showed rearrangement of the subgroup of ESS with t(7;17) (9, 12). chromosomal band 6p21 but no disease-specific t(7;17); that was RNA of good quality was extracted from fresh frozen samples of the reason why these tumors were selected for study. In cases 1 and all three ESS. To investigate for a cryptic rearrangement of 2, 6p21 was recombined with 7p15, whereas in case 3, the chromosomes 7 and 17, reverse transcription-PCR was done using recombination was with chromosomal band 10p11. Molecular www.aacrjournals.org 109 Cancer Res 2006; 66: (1). January 1, 2006

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2006 American Association for Cancer Research. Cancer Research investigation revealed that the PHF1 gene in 6p21 was split in all that modifies local chromatin structure and establishes a heritable three tumors. PHF1 encodes a protein with significant sequence repression state at particular loci. The JAZF1/PHF1 chimeric fusion similarity to the protein coded for by the Drosophila polycomblike in case 1 led to an open reading frame containing 63 amino acid (PCL) gene. The region of similarity between PHF1 and PCL residues from JAZF1, 27 additional amino acid residues from includes the sequences coding for the two PHD fingers, the region intronic sequences in frame with the initial methionine in PHF1, between them, and sequences 3V-terminal to the PHD fingers (20). and the entire PHF1 protein consisting of 567 amino acids. This The Drosophila polycomb group of genes (PcG) has been shown to gives a predicted fusion protein of 657 amino acids. In case 2, the be required for the maintenance of repression of a number of key fusion led to an open reading frame containing the first 126 amino developmental regulatory genes, including the homeotic genes acid residues from JAZF1 and 54 additional residues from intronic (20–22). PcG proteins are thought to form a multimeric complex sequences from both genes, in frame with the initial methionine in PHF1, and the entire PHF1 protein sequences. This gives a predicted chimeric protein of 727 amino acids. The putative proteins in both cases retain one zinc finger domain from the JAZF1 gene and the two zinc finger domains from PHF1.In addition, as a result of the t(6;7) the entire coding region of PHF1 is brought under the control of the JAZF1 promoter. The specific functions of the JAZF1 and PHF1 and why they are involved in a neoplasia-specific gene fusion are not directly apparent, but the fact that regions in their sequences encode zinc finger motifs suggests that deregulation of normal transcription processes in the tumor cells may be the crucial result. In two tumors, cases 1 and 2, the PHF1 gene was fused with the JAZF1 gene from 7p15. The involvement of JAZF1 in ESS has previously been reported by Koontz et al. (9) but then with another translocation partner, the JJAZ1 gene from 17q21. Both JAZF1 and JJAZ1 encode zinc finger proteins. In this fusion transcript, too, the chimeric mRNA retains zinc finger motifs from both genes. Because wild-type JAZF1 is expressed in normal endometrium, it has been suggested that the JAZF1/JJAZ1 fusion creates a chimeric protein that disrupts transcription in a lineage-specific manner (9); however, the role of the JAZF1 gene in the regulation of its downstream products and its involvement in tumorigenesis have not been clarified. JAZF1 has been shown to interact physically and specifically with TAK1, which is a positive as well as a negative regulator of transcription. JAZF1 acts as a strong repressor of DR1- dependent transcriptional activation by TAK1 (23). In case 3, the PHF1 gene was fused with the EPC1 gene from 10p11. EPC1 is the paralogue of the E(Pc) enhancer of polycomb gene of Drosophila, which is a member of the polycomb group of genes. E(Pc) is a chromatin protein of limited distribution which presumably participates in the regulation of specific gene loci and thus may have an indirect effect on nuclear architecture (24). Using homology cloning and FISH techniques, the human paralogue of this gene was identified and mapped to 10p11 (24, 25); however, no mutations of this gene have previously been described that may hint at its function in humans. This is the first demonstration that EPC1 is involved in a human malignancy. The EPC1/PHF1 chimeric fusion led to an open reading frame containing 581 amino acid residues from EPC1, six additional amino acid residues upstream from the initial methionine, and the entire PHF1 protein sequences consisting of 567 amino acids, in total, 1,154 amino acids in the Figure 2. G-banding and FISH analyses of the three ESS. For detailed predicted chimeric protein. In this case, as in cases 1 and 2, the karyotypic description, see Table 1. Arrows, breakpoints of rearranged entire coding region of PHF1 came under a different promoter; that chromosomes. Normal chromosomes are included to facilitate comparison. is, it is regulated via the EPC1 promoter; therefore, altered PHF1 A, partial G-banding (left) and multiplex FISH (right) karyogram from case 1. B, partial karyogram from case 2: G-banding (left), multiplex FISH (middle), and regulation may possibly contribute to the pathogenetic effect of the FISH cohybridization on the derivative chromosome 7 of clones RP11-81H15 fusion gene. The specific functions of the EPC1 and PHF1 and the (red signal) and RP4-781A18 (green signal) for the JAZF1 gene and clone CTD2307H19 (blue signal) for the PHF1 gene (right). C, partial G-banding reason for their involvement in a gene fusion associated with karyogram from case 3. D, G-banding (left) and FISH image (right) of the same neoplasia are not directly apparent. Whether EPC1 is involved in metaphase spread from case 3. Hybridization with locus-specific probes other neoplastic contexts has to be further studied in a larger series RP11-74N14 (green signal) mapping to 10p15 and RP11-34E5 (red signal) mapping to 10q24. Clone RP11-74N14 produces a specific hybridization of cases, possibly of different neoplasias but having cytogenetic signal to 6p. rearrangement of 10p11 in common.

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Figure 3. Detection of the JAZF1/PHF1 chimeric transcripts in ESS cases 1 and 2. Total RNA was reversely transcribed and cDNA used as template in PCR amplification with specific primer combinations. A, chimeric transcript was amplified and identified in two fragments from case 1 by two primer combinations (JAZF1-182F and PHF1-250R, lane 2; JAZF1-182F and PHF1-327R, lane 3); 100-bp DNA ladder (lane 1); no RNA in the cDNA synthesis (lane 4). The two alternative splicing variants for the reciprocal JAZF1/PHF1 from case 1 (B). cDNA and deduced amino acid sequence of the fragment 1 (the shortest from case 1, 170 bp), which gives an in-frame fusion transcript. cDNA and deduced amino acid sequence of fragment 2 (the longest from case 1, 250 bp), which gives an out-of-frame sequence. C, chimeric transcript was amplified in the ESS of case 2 by three primer combinations (JAZF1-182F and PHF1-250R, lane 3; JAZF1-182F and PHF1-327R, lane 4; JAZF1-182F and PHF1-intronR, the latter primer was used to amplify sequence from the intronic region, lane 5; chimeric transcript of the ABL gene used as an internal control, lane 6; 1-kb DNA ladder, lane 1; 100-bp DNA ladder, lane 2; no RNA in the cDNA synthesis, lane 7). D, cDNA and deduced amino acid sequence of the fragment from case 2. Arrows, junction of the JAZF1 and PHF1 genes; vertical lines, transition between intron and exon and/or exon/exon.

The orientation of the genes, PHF1, JAZF1, and EPC1, found the case also for ESS, where three different and seemingly rearranged in these three tumors, was unexpected and worthy of disease-specific fusion transcripts have now been identified further comment: JAZF1 was oriented from the centromere (5V)to (ref. 9; present study). The fusion transcripts hitherto shown in the telomere (3V; i e., opposite of the PHF1 gene). These opposite ESS all encode zinc finger domains and therefore have the orientations preclude the generation of a fusion gene by a simple possibility to deregulate the transcription of a number of genes. balanced translocation, and yet a fusion product between the two Further studies of JAZF1/JJAZ1, JAZF1/PHF1, and EPC1/PHF1 and genes was shown; similarly, opposite orientations of the involved their downstream effects are necessary to provide us with infor- genes has also been described in a subset of Ewing tumors by mation that may help not only to design better and more specific Desmaze et al. (26). A similar situation occurred for the EPC1 and diagnostic tools, but eventually, also to generate medicines that PHF1 genes: the orientation of EPC1 was from the centromere (5V) counteract the protein-level effect(s) of these cancer-specific to the telomere (3V), whereas that of the PHF1 was from the fusion genes. telomere (5V) to the centromere (3V). This indicates the presence of additional and cryptical genomic rearrangements, possibly an Acknowledgments inversion, in the chromosomal region containing the EPC1 gene, Received 7/15/2005; revised 9/14/2005; accepted 9/21/2005. whose detection was beyond the cytogenetic level. Unfortunately, Grant support: Norwegian Cancer Society, COST Action B-19 (Molecular we did not have spare material to perform FISH analysis with cytogenetics of solid tumors: Short-term scientific mission programme), and Gunnar Nilsson’s Cancer Foundation. locus-specific probes to test this hypothesis. The costs of publication of this article were defrayed in part by the payment of page A recurrent pathogenetic theme among mesenchymal tumors charges. This article must therefore be hereby marked advertisement in accordance is that a fusion gene is generated by disease-specific trans- with 18 U S.C. Section 1734 solely to indicate this fact We thank Prof. Mariano Rocchi (Department of Genetics, University of Bari, Bari, locations found in the cells of the tumor parenchyma resulting in Italy) for kindly providing most of the BAC clones used in this study (resources for the generation of chimeric transcription factors. This is clearly Molecular Cytogenetics, http://www.biologia.uniba.it/rmc/). www.aacrjournals.org 111 Cancer Res 2006; 66: (1). January 1, 2006

Figure 4. Detection of the EPC1/PHF1 chimeric transcript in ESS from case 3. A, chimeric transcript was amplified with two primer combinations (EPC1-1860 and PHF1-250R, lane 3; EPC1-1651F and PHF1-327R, lane 4; 1-kb DNA ladder, lane 1; 100-bp DNA ladder, lane 2;no RNA in the cDNA synthesis, lane 5). B, partial chromatogram showing the junction of the EPC1/ PHF1 genes. C, cDNA and deduced amino acid sequence of the fusion transcript. Arrows, junction of the EPC1 and PHF1 genes.

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