JAZF1/JJAZ1 Gene Fusion in Endometrial Stromal Sarcomas

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1 Journal of Molecular Diagnostics, Vol. 7, No. 3, August 2005 Copyright American Society for Investigative Pathology and the Association for Molecular Pathology JAZF1/JJAZ1 Gene Fusion in Endometrial Stromal Sarcomas Molecular Analysis by Reverse Transcriptase-Polymerase Chain Reaction Optimized for Paraffin-Embedded Tissue Andelko Hrzenjak,* Farid Moinfar,* Fattaneh A. Tavassoli, Bettina-Strohmeier,* Marie-Luise Kremser,* Kurt Zatloukal,* and Helmut Denk* From the Institute of Pathology,* Medical University of Graz, Graz, Austria; and the Department of Pathology, Yale University, New Haven, Connecticut Endometrial stromal tumors are rare uterine neoplasms including benign stromal nodules, low-grade endometrial stromal sarcomas (ESS), and undifferentiated endometrial sarcomas (UES), the latter representing the most aggressive form. Morphological characteristics and cytogenetic abnormalities are heterogeneous, making diagnosis difficult. Recently, a gene fusion on chromosome 7 that includes two zincfinger genes (JAZF1 and JJAZ1) has been discovered in these tumors. Hitherto only 31 cases, described by three different research groups, have shown JAZF1/ JJAZ1 fusion in 50% of all analyzed low-grade ESSs whereas it is less frequent in UESs. In this study we analyzed 20 ESS and 2 UES cases using two-step reverse transcriptase-polymerase chain reaction optimized for formalin-fixed, paraffin-embedded tissue. In our subset of samples, the JAZF1/JJAZ1 fusion transcript occurred in 80% of analyzed ESS cases and in none of two UES cases. In comparison to published data, our results identified the JAZF1/JJAZ1 gene fusion more frequently in endometrial stromal tumors than hitherto presumed. This cytogenetic abnormality was not present in normal endometria, leiomyomas, or leiomyosarcomas or in lung, gastric, or hepatic carcinomas, indicating its specificity for endometrial stromal tumors. In combination with other established methods, accurate reverse transcriptase-polymerase chain reaction analysis of JAZF1/JJAZ1 gene fusion may be useful in diagnosing difficult or unusual ESS/UES cases. (J Mol Diagn 2005, 7: ) Endometrial stromal tumors are very rare uterine neoplasms, comprising 15% of uterine sarcomas and 1% 388 of all uterine malignancies. 1 According to the new World Health Organization classification these tumors are divided into low-grade endometrial stromal sarcomas (ESS) and undifferentiated endometrial sarcomas (UES). Low-grade ESSs show less aggressive clinical courses than their undifferentiated counterparts. 2,3 UESs are characterized by high-grade nuclear atypia (anaplastic mesenchymal tumor cells), numerous atypical mitotic figures, and rapidly progressive disease. Whereas in the past the mitotic activity was used as one of criteria for low-/high-grade classification, nowadays its relevance has fallen out of favor. The feature mostly used for diagnosis and classification is morphological resemblance to normal endometrial stroma. However, the heterogeneity of ESS and UES makes the diagnosis and investigation of these malignancies much more complicated. Recently, Amant and co-workers 4 stressed that even today the differential diagnosis of ESS and UES is still problematic. In the last few years immunohistochemical and cytogenetic methods became increasingly important in solving these diagnostic problems. Histochemical markers such as CD and h-caldesmon 8,9 are widely used but because they are not exclusively expressed in ESSs and in healthy endometrium the diagnosis can still not be made on this basis alone. As already reported by many authors CD10 is expressed in the vast majority of the ESS/UES cases, whereas ESSs/UESs are mainly negative for h-caldesmon, with exception of some complex tumors involving also a smooth muscle cell component. Cytogenetic studies and chromosomal analysis have shown that chromosomal changes in ESS and UES are also heterogeneous. 4 Among others, one of the most frequently found aberration seems to be the t(7;17)(p15; q21) chromosomal translocation that has been found in 50% of all reported cases. As already described by different authors this is clearly a nonrandom chromosomal aberration Recently this translocation be- Supported by the Lore-Saldow research fund. Accepted for publication March 4, This paper is dedicated to the memory of Mrs. Lore Saldow. Address reprint requests to Andelko Hrzenjak, Ph.D., Institute of Pathology, Medical University of Graz, Auenbruggerplatz 25, 8036 Graz, Austria. andelko.hrzenjak@klinikum-graz.at.

2 Modified RT-PCR for JAZF1/JJAZ1 Fusion in ESS 389 came even more interesting because of the JAZF1/JJAZ1 gene fusion found at the sites of the 7p15 and 17q21 breakpoints. Up to now this gene fusion has been found in 50% of all 31 analyzed ESS and UES cases, with higher frequency in ESS. Because of the rarity of these tumors former studies have been performed on relatively small number of samples. Moreover, it was shown that analyses of RNA isolated from formalin-fixed, paraffinembedded material could be quite problematic. Therefore many more samples have to be analyzed and detection assays have to be further optimized to establish reliable statistics. In this work we analyzed 22 endometrial stromal tumors (ESS, n 20; UES, n 2) to investigate the frequency of a JAZF1/JJAZ1 fusion transcript in these malignancies. We established a sensitive protocol for reverse transcriptase-polymerase chain reaction (RT- PCR) with RNA isolated from formalin-fixed, paraffinembedded tissue samples. Here we show that further optimization of JAZF1/JJAZ1 gene fusion detection is possible and necessary to accurately investigate this cytogenetic alteration in endometrial stromal tumors. No correlation of JAZF1/JJAZ1 fusion with CD10 expression and proliferation rate of tumor cells could be detected. Materials and Methods Tumor Material Snap-frozen and formalin-fixed, paraffin-embedded samples were obtained from patients with histologically confirmed ESS (n 20) and UES (n 2). The diagnoses were made on the basis of established criteria. 5,16 Classification of ESS/UES cases was made by two pathologists (F.M. and H.D.) using the new World Health Organization classification on Tumors of the Breast and Female Genital Organs. 17. For cryopreservation tumors were frozen in liquid nitrogen immediately after dissection. Sections of frozen tissue were cut on a Leica CM3050 cryomicrotome (Leica, Vienna, Austria). Formalin-fixed (10%), paraffin-embedded tissue samples were retrieved from the archives of the Departments of Pathology of the Universities of Graz (Graz, Austria), Tehran (Tehan, Iran), and Tabriz (Tabriz, Iran), and Yale University (New Haven, CT). Histological classification: classic type ESS (n 14), variant with prominent sex cord-like pattern (n 2), variant with fibromyxoid pattern (n 1), extra uterine ESS (n 1). Four ESS cases showed increased mitotic activity with 15 mitotic figures per 10 high-power fields. There were also two UES cases with highly atypical tumor cells, numerous atypical mitotic figures, and multiple areas of tumor cell necrosis (necrotic areas were removed by microdissection and excluded from further analyses). Sections of paraffin-embedded tissue (3 m) were cut on a Microm HM 355S microtome (Carl Zeiss, Jena, Germany). As control tissues, snap-frozen nonmalignant endometria (n 10); gastrointestinal carcinomas, lung carcinomas, hepatocellular carcinomas (n 3 for each); leiomyoma (n 5); and leiomyosarcomas (n 5) were used. RNA Extraction Total RNA from frozen tissue was extracted using Trizol (Invitrogen Life Technologies, Los Angeles, CA). Briefly, 50 to 100 mg of tissue were extracted with 1 ml of Trizol, homogenized by passing through a 23-gauge syringe needle, and incubated at room temperature for 15 minutes. After chloroform extraction and precipitation with isopropanol RNA was washed with 80% ethanol, and finally the RNA pellet was dissolved in RNase-free water. Total RNA from formalin-fixed, paraffin-embedded tissue was extracted using the Optimum FFPE RNA isolation kit from Ambion (Austin TX). Briefly, 3 to 5 unstained sections (5 m thick) were prepared, scraped into a tube, and paraffin was removed by extracting twice with xylene for 10 minutes at room temperature. After extraction tissue was collected by centrifugation at 13,000 rpm for 3 minutes. The tissue pellet was subsequently washed with 100%, 90%, and 70% ethanol and after centrifugation ethanol was removed by aspiration and tissue pellets were dried at room temperature. Each sample was incubated with 200 l of proteinase K working solution (100 l of proteinase K digestion buffer plus 10 l of proteinase K; 60 U/ l) at 60 C overnight. RNA was further isolated as recommended by the manufacturer and eluted with 20 l of preheated (75 C) elution solution. Each RNA sample was treated with DNase I (2 U per sample) at 37 C for 30 minutes, DNase I was inactivated with DNase I inactivation reagent supplied by the manufacturer, and RNA samples were immediately used for RT-PCR or stored at 80 C until further use. Reverse Transcription Total RNA, extracted from frozen and/or formalin-fixed, paraffin-embedded tissue sections, was reverse-transcribed using Superscript II reverse transcriptase (200 U/ l; Invitrogen). Reactions were performed in a final volume of 20 l using buffer provided by the manufacturer, additionally containing 10 mmol/l dntps, 40 U/ l of RNase inhibitor (Invitrogen), 0.3 l (5 nmol/l) of random hexamers, and 1 g of RNA. Reaction conditions were as follows: 65 C, 5 minutes; 42 C, 90 minutes; 90 C, 15 minutes. Detection of the JAZF1/JJAZ1 gene fusion was performed by reverse-transcriptase polymerase chain reaction (RT-PCR) or by nested PCR. PCR Analysis Sequences of synthetic oligonucleotides used in this study are summarized in Table 1 [the primer numbers indicate the position of the first 5 nucleotide for each primer in the cdna sequences for JAZF1 (NM ), JJAZ1 (BC015704), and GAPDH (BC083511)]. GAPDH For RT-PCR of the control gene 2 l of cdna were mixed with 28 l of Mastermix (10 pmol/l GAPDH forward primer, 10 pmol GAPDH reverse primer, 15 l Universal

3 390 Hrzenjak et al Table 1. Oligonucleotides Used for PCR Optimization Purpose Primer Sequence Product length (bases) Nested PCR outer round JAZF1-308-FW 5 -tcgagggaatgtgtccactc JJAZ1-753-RV 5 -ccgggttttgtttgattgagg-3 Nested PCR inner round JAZF1-753-RV 5 -ccacagcagtggaagcctta JJAZ1-586-RV 5 -tctgagtttggtgatggctta-3 Nested PCR inner round JAZF1-335-FW 5 -ccacagcagtggaagcctta JJAZ1-730-RV 5 -aggattcaaaggcacctgct-3 PCR short product JAZF1-369-FW 5 -ccacccatcaccccctcct-3 93 JJAZ1-400-RV 5 -tgctatgagattccgagttc-3 PCR GAPDH GAPDH-35-FW 5 -ccacatcgctcagacaccat-3 71 GAPDH-105-RV 5 -accaggcgcccaatacg-3 Mastermix, and ddh 2 Oat30 l). RT-PCR was performed with the following cycling profile: 1 cycle of 50 C for 2 minutes followed by 1 cycle of 10 minutes at 95 C, 1 cycle of 15 seconds at 95 C, and 50 cycles of 1 minute at 60 C. JAZF1/JJAZ1 Three l of cdna were mixed with 28 l of Mastermix (6 mmol/l dntps, 45 mmol/l MgCl 2,3 l of10 PCR buffer, 6 pmol/l forward primer, 6 pmol/l reverse primer, ddh 2 Oat30 l, and 0.12 l of Ampli Taq). RT-PCR for JAZF1/JJAZ1 was performed with the following cycling profile: initial denaturation at 94 C for 30 seconds, followed by 40 cycles of 30 seconds at 94 C, 30 seconds at 57 C, 20 seconds at 72 C, and a final extension for 10 minutes at 72 C. PCR products were checked on 3% TAE-agarose gel and visualized by ethidium bromide staining. JAZF1/JJAZ1 Nested PCR For outer round 2 l of cdna were mixed with 28 l of Mastermix (6 mmol/l dntps, 45 mmol/l MgCl 2,3 l of 10 PCR buffer, 6 pmol/l forward primer, 6 pmol/l reverse primer, ddh 2 Oat30 l, and 0.12 l of Ampli Taq). Outer PCR was run with the following cycling profile: initial denaturation at 94 C for 30 seconds, followed by 40 cycles of 30 seconds at 94 C, 30 seconds at 57 C, 20 seconds at 72 C, and a final extension for 10 minutes at 72 C. For inner round 5 l of outer reaction were mixed with 20 l of Mastermix (4 mmol/l dntps, 30 mmol/l MgCl 2,2 l of10 PCR buffer, 4 pmol/l forward primer, 4 pmol/l reverse primer, ddh 2 Oat20 l, and 0.08 l of Ampli Taq). The inner PCR was run under the same conditions as described for the outer round. Final products were checked on 3% TAE-agarose gel and visualized by ethidium-bromide staining. Reactions omitting the reverse transcriptase (no-rt) as well as reactions omitting the RNA were run routinely to monitor cross contamination of RNA samples. DNA Sequencing After electrophoresis PCR products were extracted from agarose gel using Millipore Ultrafree-MC mini column (Millipore, Bedford, MA). PCR amplification for direct sequencing of PCR products was performed under following conditions: 50 C, 2 minutes; 95 C, 10 minutes; 95 C, 15 seconds; 60 C, 1 minute; 55 cycles. Immunohistochemistry Formalin-fixed, paraffin-embedded tissues were cut into 3- m-thick sections on Microm HM 355S microtome (Carl Zeiss) and mounted on precoated glass slides. The sections were deparaffinized, rehydrated, and rinsed in ddh 2 O. Immunohistochemical reactions for CD10 and Ki-67 were performed using a standardized automated procedure on a Ventana Benchmark (Ventana, Tucson, AZ) with an iview DAB kit (Ventana) as recommended by the manufacturer. For Ki-67 immunohistochemistry we used anti-ki-67, clone 2, mouse monoclonal IgG 1 (Ventana, No ) antibody. For CD10 mouse monoclonal IgG 1 (NCL-CD10-270) from DAKO (Copenhagen, Denmark) was used (dilution, 1:50). Second antibodies and developing solutions were components of the iview DAB kit. Negative controls, omitting the first antibody, were performed under the same conditions. Results JAZF1/JJAZ1 RT-PCR Primer sets used in this study are described in Materials and Methods section. JAZF1/JJAZ1 transcript of 457 bases was detected in two of three cryopreserved tumor samples (Figure 1A, lanes 23 and 24). Nested PCR with RNAs isolated from formalin-fixed, paraffin-embedded material of the same cases (Figure 1A, lanes 10 and 11) and from other paraffin-embedded samples resulted in no product. The same results were observed using a primer set for a 333-base product (data not shown). RT-PCR amplification of a 93-base product specific for JAZF1/JJAZ1 fusion resulted in positive reaction in 16 of 20 analyzed low-grade ESSs (80%) and in 0 of 2 analyzed UESs (Figure 1B, Table 2). The sequence was proved by direct sequencing and breakpoint identical to that previously described has been detected in all 16 cases. As a quality control for RNA isolated from paraffinembedded material a 71-base GAPDH fragment was amplified in all analyzed samples. Controls omitting reverse

4 Modified RT-PCR for JAZF1/JJAZ1 Fusion in ESS 391 CD10 Immunohistochemistry Among all 22 cases of ESS and UES, weak (1 ), moderate (2 ), strong (3 ), and very strong (4 ) reactivity was observed in 3 (13.6%), 3 (13.6%), 7 (31.8%), and 8 (36.4%) cases, respectively. One ESS case (4.5%) was CD10-negative, as determined in few independent experiments. Both UES cases showed very strong CD10 reactivity (4 ). Thirteen of twenty cases (65%) representing low-grade ESS exhibited strong (3 /4 ) CD10 immunoreactivity (Table 2). As shown in Figure 2 both intensity and localization of CD10 positivity vary considerably between different tumors. In most cases membrane-associated CD10 reactivity was observed but in some cases staining was rather diffuse and more prominent in the cytoplasm of tumor cells. Figure 1. Amplification of JAZF1/JJAZ1 fusion transcript in 22 different ESSs. Nested PCR and RT-PCR were performed as described in Materials and Methods. A: Amplifications of large JAZF1/JJAZ1 transcripts (457 bases) were successful on RNA isolated from frozen tissue but not on RNA isolated from paraffin-embedded tissues. B: Small bands visible on the upper gel represent primer oligonucleotides. Amplification of the small transcript (93 bases) was successful on RNA isolations from frozen and formalin-fixed, paraffin-embedded tissues. Two high-grade UES cases are underlined. Frozen samples 23, 24, and 25 correspond to paraffin-embedded samples 10, 11, and 12, respectively. St., 100-bp DNA leader. transcriptase or the RNA were always negative, thus excluding the possibility of cross contamination. Normal endometria (n 10); leiomyomas and leiomyosarcomas (n 5 for each group); as well as hepatocellular carcinomas, gastric tumors, and lung carcinomas (n 3 for each group) were all negative for JAZF1/JJAZ1 gene fusion (Table 3). Discussion ESSs are very rare uterine malignancies and this rarity is the main limiting factor for accurate molecular and cytogenetic analysis. A variety of chromosomal translocations have been reported in these tumors confirming not only their morphological but also cytogenetic heterogeneity. 4 A subgroup of endometrial stromal tumors is characterized by the presence of a specific gene fusion in which two zinc finger genes (JAZF1 and JJAZ1) are fused as a result of t(7;17)(p15;q21) translocation. 13 Although this aspect is very interesting, only a limited number (31 cases in total) of these tumors has been investigated so far These published cases comprise 5 stromal nodules as a benign form of this neoplasm, 19 low-grade ESSs (different subtypes), and 7 high-grade tumors (2 of Table 2. General Characterization of Tumors and RT-PCR Detection of JAZF1/JJAZ1 Gene Fusion Case no. Tissue source Tumor type Morphology/histological subtype CD10 staining JAZF1/JJAZ1 RT-PCR 1 Paraffin-embedded ESS Classic type* 1 2 Paraffin-embedded ESS Classic type 2 3 Paraffin-embedded ESS Classic type 1 4 Paraffin-embedded ESS Classic type 3 5 Paraffin-embedded ESS Highly increased MR 3 6 Paraffin-embedded ESS Classic type 4 7 Paraffin-embedded ESS Partly epithelioid and sex cord-like pattern, 1 highly increased MR 8 Paraffin-embedded ESS Classic type 4 9 Paraffin-embedded ESS Fibromyxoid variant 4 10 Frozen/paraffin-embedded ESS Classic type 4 11 Frozen/paraffin-embedded ESS Highly increased MR 3 12 Paraffin-embedded UES Severe atypia 4 13 Paraffin-embedded ESS Classic type 4 14 Frozen/paraffin-embedded ESS Sex cord-like pattern, high MR 3 15 Paraffin-embedded ESS Classic type 2 16 Paraffin-embedded ESS Classic type 4 17 Paraffin-embedded ESS Classic type 0 18 Paraffin-embedded ESS Extra uterine, ovarian tumor 3 19 Paraffin-embedded ESS Classic type 3 20 Paraffin-embedded ESS Classic type 2 21 Paraffin-embedded ESS Classic type 3 22 Paraffin-embedded UES Severe atypia 4 *Classic type, usual morphology with small uniform tumor cells showing mild atypia and closely resembling endometrial stromal cells. MR, mitotic rate (highly increased MR more than 15 mitotic events per 10 high-power fields were found).

5 392 Hrzenjak et al Figure 2. A C: Examples of low-grade ESS with intense and diffuse immunoreaction for CD10. Note the infiltrative growth of the tumor into the surrounding myometrium (A) and a very strong (4 ), membrane-associated positive staining (B). D F: Examples of low-grade ESS with moderate (2 ) and/or heterogeneous reactivity for CD10. Note heterogeneity of CD10 staining in different ESS samples. them were classifiable as UES). An overview of published data in Table 2 shows that there are considerable discrepancies in those data. Whereas Koontz and colleagues 13 showed that the JAZF1/JJAZ1 fusion transcript was present in all low-grade ESSs (n 5) and in 50% of high-grade tumors, Huang and colleagues 15 found it in only 23% of all analyzed ESS cases. In fact four of five (80%) classic ESSs analyzed by Huang and colleagues 15 were JAZF1/JJAZ1-positive, but different ESS variants, including sex cord-like and fibromyxoid patterns, were all negative. Whether these discrepancies can be explained by differences in the sensitivity of assays used, by RNA quality, or by other still unknown factors should be elucidated by analysis of more tumor samples. In the present study we investigated 22 primary endometrial stromal tumors and 10 nonneoplastic endometria (proliferate and secretory phases). The diagnosis of all 22 tumor cases was histologically confirmed and as shown in Table 1, 20 of them were classified as ESS and 2 as UES. The latter showed highly atypical tumor cells and numerous atypical mitotic figures. Although the relevance of mitotic rate has fallen out of favor as criteria to define high-grade ESSs, in our set of samples the number of atypical mitotic figures was predominantly increased in both UES cases. That corresponds well to data published by our group previously. 18 Increased mitotic rate was also observed in two classic ESS cases and two cases with sex cord-like pattern (Table 1).

6 Modified RT-PCR for JAZF1/JJAZ1 Fusion in ESS 393 Table 3. Summary of RT-PCR Data Previously Reported by Other Authors Combined with Our Data Obtained with 22 Endometrial Stromal Tumors Tumor type Total cases Positive cases Percent positive Reference Stromal nodule * Low-grade ESS (different subtypes) , 4 Our data (different subtypes) High-grade tumors UES Our data (UES) Normal endometrium Our data Leiomyomas Leiomyosarcomas *, Mean value of all data already published by other authors (see references) for indicated tumor type. Data presented in this study are marked in bold. Two high-grade cases were classified by Koontz et al 13 as undifferentiated endometrial sarcoma. One of two UES cases was JAZF1/JJAZ1-positive. Endometrial stromal tumors are very rare and formalinfixed, paraffin-embedded archival material is more readily accessible than frozen tumor tissue. We performed RT-PCR on RNA extracted from formalin-fixed, paraffin-embedded material and from frozen tissue. In three cases we were able to analyze RNA isolated from both paraffin-embedded and from cryopreserved material in parallel and to compare those results. Direct comparison of those data showed that there are difficulties in detecting JAZF1/JJAZ1 gene fusion transcript using RNA isolated from paraffin-embedded material. It is well known that the quality of RNA from paraffin-embedded material is often very critical for such type of experiments and the size limit for efficient amplification of a distinct RNA segment is often very low. This problem was addressed by Huang and colleagues, 15 who found that in their material more than two thirds of analyzed cases were negative for this gene fusion. In addition to RNA quality they also speculated on the existence of other variants of JAZF1/JJAZ1 transcripts as a possible explanation. Therefore, we used different sets of primers to get transcripts of different sizes. With some sets of primers (see Materials and Methods) we also performed nested PCR to further increase the specificity and sensitivity of the assay. Using two different sets of primers we were able to detect JAZF1/JJAZ1 gene fusion (fragment size, 457 bases) on RNA isolated from frozen material of two tumor cases but not on RNA isolated from paraffin-embedded material of the same tumors. The attempts to amplify a fragment containing 333 bases using nested PCR ended with the same results when RNA from paraffin-embedded tissue was used (data not shown). Concerning the size of the amplification products the logical conclusion was that the RNA quality was not sufficient for this purpose. The third cryopreserved tumor, which was an UES, was negative for JAZF1/JJAZ1 gene fusion independently of primer set used and tissue preservation. Huang and co-authors 15 succeeded to amplify larger fragments in a limited number of paraffin-embedded tumor samples of different histological subtypes using nonnested PCR, whereas other samples were difficult to analyze and/or resulted in no product. Because in some cases the amplification of the control gene (eg, PGK) was also impossible, the absence of JAZF1/JJAZ1 fusion transcript was ascribed to the limited RNA preservation in formalin-fixed, paraffin-embedded tissue. Interestingly, we made the observation that in a subset of cases the detection of JAZF1/JJAZ1 gene fusion was not possible, although the amplification of the control gene with similar PCR product size was unproblematic. This might be explained by differences in the stability of specific RNA molecules. To exclude the possibility that these cases were false-negative because of differences in the stability of the RNA coding for JAZF1 and JJAZ1 genes we amplified a smaller fragment containing 93 bases of the JAZF1/JJAZ1 gene fusion sequence. To our surprise in many samples that were negative after amplification of larger fragments (both for 457 and 333 bases) we were able to unambiguously detect the JAZF1/JJAZ1 fusion transcript when amplifying a 93-base product by nonnested PCR. By direct sequencing we confirmed the presence of specific JAZF1/JJAZ1 fusion transcript in all positive cases. In all 16 positive ESS/UES cases the same breakpoint in the JAZF1/JJAZ1 fusion sequence was detected. This breakpoint is identical to that previously described by other authors, in which G-435 from the JAZF1 sequence is followed by A-468 from the JJAZ1 sequence. Summarizing data published by others and data presented in this study the same breakpoint sequence has been found so far in all JAZF1/JJAZ1-positive tumor cases. This suggests that this breakpoint is highly conserved. Although functional relevance of the potential fusion protein could have not been shown until now, high conservation of this sequence suggests that this translo-

7 394 Hrzenjak et al cation could play an important role in the ESS/UES pathogenesis. Some discrepancies to data already published are furthermore present: Huang and colleagues 15 find that fibromyxoid variant (n 3), as well as sex cord-like variant (n 1) were all negative for JAZF1/JJAZ1 gene fusion. In our study we analyzed one fibromyxoid ESS that was JAZF1/JJAZ1-positive and two ESS cases showing sex cord-like pattern, one of which was also positive. This is the first evidence that these ESS subtypes can also contain JAZF1/JJAZ1 gene fusion, at least in a subset of samples. Whether some ESS subtypes, eg, fibromyxoid subtype and mixed smooth muscle variant, should be considered as histogenetically distinct entities, as nicely discussed by Huang and colleagues, 15 can because of the low number of samples not be answered yet. Both of analyzed UES cases were JAZF1/JJAZ1-negative. Therefore, in correlation with data published by Koontz and colleagues 13 the discrepancy between ESS and UES furthermore exist, showing lower proportion of this gene fusion in UESs (Table 2). This may indicate different pathogenesis of these two tumor groups and supports growing number of arguments showing that low-grade ESSs and UESs may present two separate entities. 19 It has to be stressed that UES cases are even rarer than ESS, so it is very difficult to analyze enough cases for proper statistical evaluation. In agreement with data published by others, JAZF1/ JJAZ1 gene fusion was not detectable in a total of 10 nonneoplastic endometria showing that this fusion is a tumorspecific event. Hepatocellular carcinomas, gastric tumors, and lung carcinomas (n 3 for each group) analyzed by us were all negative for JAZF1/JJAZ1 gene fusion (data not shown). Only two uterine cellular leiomyomas were analyzed so far and although both of them were negative, it is difficult to make a precise conclusion on such a small number of samples. 15 In our study we analyzed 10 samples of other mesenchymal uterine tumors; leiomyomas and leiomyosarcomas (n 5 for each group). All of them were negative for JAZF1/JJAZ1 gene fusion. Therefore, in agreement with published data, our results clearly support the evidence that this gene fusion is specific for endometrial stromal tumors. The question whether this fusion can also be found in some other types of mesenchymal tumors containing similar chromosomal rearrangements has yet to be answered by analyzing a larger group of different samples. Although all but one tumor of our series showed positive immunoreaction for CD10, no correlation between the CD10 reactivity and the presence of JAZF1/JJAZ1 gene fusion existed. However, combination of these two methods may be valuable for the diagnosis and better characterization of unusual ESSs. In summary, in this work we show that in our group of samples the JAZF1/JJAZ1 gene fusion is present in 80% of ESSs of classic histology and in a subset of different histological ESS subtypes. This suggests that the presence of JAZF1/JJAZ1 fusion transcript is more frequent than hitherto presumed. In addition we report here an optimization of RT-PCR to exclude false-negative results that may, at least in part, be ascribed to the instability and/or poor quality of RNA isolated from formalin-fixed, paraffin-embedded tissue. Further studies, especially on UES cases, should enable more general conclusions and answer the question whether JAZF1/JJAZ1 fusion contributes to the initiation of the neoplastic processes involved in ESS/UES. Acknowledgments We thank Dr. Ali Dastranj Tabrizi from University of Tabriz (Iran) and Dr. Narges Isadi-Moud from University of Tehran (Iran) for providing some tumor samples. References 1. Mansi JL, Ramachandra S, Wiltshaw E, Fisher C: Endometrial stromal sarcomas. Gynecol Oncol 1990, 36: Norris HJ, Taylor HB: Mesenchymal tumors of the uterus. I. A clinical and pathological study of 53 endometrial stroma tumors. Cancer 1966, 19: Chang KL, Crabtree GS, Lim-Tan SK, Kempson RL, Hendrickson MR: Primary uterine endometrial stromal neoplasms. A clinicopathologic study of 117 cases. Am J Surg Pathol 1990, 14: Amant F, Moerman P, Cadron I, Neven P, Berteloot P, Vergote I: The diagnostic problem of endometrial stromal sarcoma: report of six cases. Gynecol Oncol 2003, 90: Chu P, Arber DA: Paraffin-section detection of CD10 in 505 nonhematopoietic neoplasms. Frequent expression in renal cell carcinoma and endometrial stromal sarcoma. 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