Frequent Fusion of the JAZF1 and JJAZ1 Genes in Endometrial Stromal Tumors
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Frequent fusion of the JAZF1 and JJAZ1 genes in endometrial stromal tumors Jason I. Koontz*†, A. Lee Soreng*†, Marisa Nucci†, Frank C. Kuo*†, Patrick Pauwels‡, Herman van den Berghe‡, Paola Dal Cin†, Jonathan A. Fletcher‡, and Jeffrey Sklar*†§ *Division of Molecular Oncology, †Department of Pathology, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; and ‡Center for Human Genetics, University of Leuven, B-3000 Leuven, Belgium Communicated by Stanley J. Korsmeyer, Dana–Farber Cancer Institute, Boston, MA, March 16, 2001 (received for review December 19, 2000) Endometrial stromal tumors are divided into three types: benign trial stromal tumors as well. However, the incidence of this fusion stromal nodules, endometrial stromal sarcomas, and undifferenti- appears to be reduced among ESSs that might be classified as ated endometrial sarcomas. A variety of cytogenetic abnormalities high-grade. involving chromosome 7 have been reported in endometrial stro- mal sarcomas, including a recurrent t(7;17)(p15;q21). We have Materials and Methods identified two zinc finger genes, which we have termed JAZF1 and Tissues and Cells. Initial molecular genetic studies were per- JJAZ1, at the sites of the 7p15 and 17q21 breakpoints. Analyses of formed on four cases of low-grade ESS found to have the tumor RNA indicate that a JAZF1͞JJAZ1 fusion is present in all types t(7;17)(p15;q21)(see Table 1 for the full karyotypes). In case of endometrial stromal tumors; however, the fusion appears to be BWH-42, the tissue was obtained from a metastatic retroperi- rarer among endometrial stromal sarcomas that would be consid- toneal mass in a 58-yr-old woman who had undergone a hyster- ered high-grade according to certain classification schemes. These ectomy for a low-grade ESS 16 years earlier; in case BWH-665, findings suggest that the less malignant endometrial stromal from a metastatic vaginal mass in a 69-yr-old woman who had tumors may evolve toward more malignant types, but that some undergone a hysterectomy for a low-grade ESS 18 years earlier; endometrial stromal sarcomas with relatively abundant mitotic and in both case LU-550 and case LU-965, from primary uterine activity may compose a biologically distinct group. tumors in 41-yr-old women. Cells cultured directly from the tumors were grown in RPMI medium 1640 supplemented with ndometrial stromal tumors of the uterus constitute a spec- 20% FCS, Mito ϩ Serum Extender (Becton Dickinson), and Etrum of neoplasms that exhibit varying degrees of malig- bovine pituitary extract (Becton Dickinson) under standard nancy and can present a number of challenges with respect to conditions. diagnosis and classification (1). Stromal nodules lie at the benign end of this spectrum of neoplasms. These nodules consist of Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromo- well-circumscribed tumors composed of uniform cells resem- somes (BACs), and cDNAs. YACs and BACs were purchased from bling those of normal endometrial stroma during the prolifera- Research Genetics (Huntsville, AL). Methods for growth of tive phase of the menstrual cycle. Endometrial stromal sarcomas yeast and bacteria, as well as procedures for isolation of YAC (ESSs) are malignant neoplasms that occupy the middle of the and BAC DNA, were as previously described (14, 15). cDNAs spectrum and are histologically similar to stromal nodules except were identified in lambda bacteriophage libraries prepared from ͞ for infiltration of the myometrium and or vascular invasion. human fetal brain (Stratagene) and human umbilical vein en- Tumors that depart significantly in histologic appearance from dothelial cells (HUVEC) (16) by hybridization of probes to normal endometrial stroma are referred to as undifferentiated plaque lifts on nylon membranes. Phage DNA was isolated from endometrial sarcomas (or undifferentiated uterine sarcomas) clones in the HUVEC library according to standard methods and represent the most malignant end of this tumor spectrum. (17), and phagemid cDNA clones were rescued from the brain In the older literature, all malignant endometrial stromal library according to the supplier’s instructions. Plasmids con- tumors were categorized as ESSs and were subclassified into taining expressed sequence tag cDNAs (ESTs) were purchased low-grade and high-grade types (2, 3). High-grade ESSs were from Genome Systems (St. Louis). Plasmid DNA was purified distinguished from low-grade ESSs by an increased frequency of from bacteria following standard protocols (18). mitoses (Ͼ10 per high power microscopic field) and were generally assumed to have a worse prognosis. More recently, it Fluorescence in Situ, Southern Blot, and Northern Blot Hybridization. has been argued that the number of mitoses within ESSs is DNA probes for fluorescence in situ hybridization (FISH) were largely irrelevant to outcome, which is said to be almost exclu- labeled by nick translation with biotin-tagged nucleoside triphos- sively a function of stage at diagnosis (4–6). phates by using the BioPrime labeling system (Life Technolo- Diagnosis and classification of endometrial stromal tumors gies). Hybridization was performed on slides of metaphase has until now been based primarily on histologic criteria. In chromosomes prepared according to methods of Fletcher and recent years, specific genetic alterations identified in different colleagues (15, 19). Procedures for Southern blot and Northern types of human tumors have provided useful diagnostic markers, blot hybridization have been described (15). led to insights into the basic biology of both neoplastic and normal tissues, and increasingly contributed to the development of rational forms of cancer therapy (7–9). With these consider- Abbreviations: ESS, endometrial stromal sarcoma; YAC, yeast artificial chromosome; BAC, ations in mind, we have characterized the DNA and genes bacterial artificial chromosome; EST, expressed sequence tag; FISH, fluorescence in situ surrounding the breakpoints of a recurrent chromosomal trans- hybridization; RT-PCR, reverse transcription–PCR; STS, sequence-tagged site; I-PCR, location, the t(7;17)(p15;q21), reported in several cases of inverse PCR. low-grade ESS (10–13). We have found that recombination at §To whom reprint requests should be addressed at: Harvard Medical School, Brigham and Women’s Hospital, Department of Pathology, 75 Francis Street, Thorn 605, Boston MA these breakpoints results in fusion of two previously unknown 02115. E-mail: [email protected]. genes, which we have termed JAZF1 and JJAZ1. Fusion of these The publication costs of this article were defrayed in part by page charge payment. This genes appears to be common in low-grade ESSs but is not limited article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. to these neoplasms and can be found in other types of endome- §1734 solely to indicate this fact. 6348–6353 ͉ PNAS ͉ May 22, 2001 ͉ vol. 98 ͉ no. 11 www.pnas.org͞cgi͞doi͞10.1073͞pnas.101132598 Downloaded by guest on October 1, 2021 Table 1. Karyotypes of low-grade ESSs initially studied sue section adjacent to those used for extraction of RNA was Case Karyotype examined histologically. BWH-42 46, XX, t(7;13)(p15;p13), t(7;17)(p15;q21) Determination of Nucleotide Sequence and Computer-Assisted Anal- BWH-665 46, XX, t(7;17)(p15;q21) ysis of Derived Sequence. Sequence data were obtained by using LU-550 46, XX, t(7;17)(p15;q21) the Amplicycle Sequencing Kit (Perkin–Elmer) and analyzed by LU-954 45, XX, Ϫ7, t(7;17)(p15;q21) using the National Center for Biotechnology Information (NCBI) BLAST server and the GCG software package (Genetics Computer Group, Madison, WI). Iterated sequence was sup- Inverse PCR (I-PCR) and Reverse Transcription (RT)-PCR. I-PCR was pressed in analyses for genes by use of the REPEATMASKER2 performed following protocols previously described (15). For software program available at the MRC Mammalian Genetics RT-PCR, total RNA was transcribed into cDNA with Super- Unit WWW server (http:͞͞www.mgu.har.mrc.ac.uk͞). Peptides script II (Life Technologies), using either 7AntisenseOuter or were analyzed by using software available at the Expert Protein 17AntisenseOuter as a primer (see below). The resulting cDNA Analysis System (ExPASy) WWW server (http:͞͞www. was subjected to two rounds of PCR using first the ‘‘Outer’’ expasy.ch͞). and then the ‘‘Inner’’ set of primers. The JAZF1 primers were 7SenseOuter 5Ј-CCACAGCAGTGGAAGCCTTA-3Ј, Results 7AntisenseOuter 5Ј-GCTTCTCTTCCCCTCCATTCAT-3Ј, Identification of a YAC Spanning the Chromosome 7 Breakpoint. FISH 7SenseInner 5Ј-ATCACCCCCTCCTCTTCATT-3Ј, and 7Anti- was performed on metaphase preparations from the tumor of senseInner 5Ј-GGACTCATCGCTGTCCGACT-3Ј. The JJAZ1 case BWH-42, using as probes YACs from the Whitehead Centre primers were 17SenseOuter 5Ј-GTTACTCGGCCTCCTCCTC- d’E´tude du Polymorphisme Humain (CEPH) megaYAC contig CTC-3Ј, 17AntisenseOuter 5Ј-GGTTCAAATTCATTACTG- 7.3 in the region of the HOXA gene cluster (Fig. 1a). FISH with GAAACTGC-3Ј, 17SenseInner 5Ј-GAGCTTTTCCTCCAG- YAC y830D2 produced hybridization signals limited to both GCCTTTG-3Ј, and 17AntisenseInner 5Ј-CCGGGTTTTGTT- derivative 7 (der7) chromosomes in this tumor, whereas the TGATTGAGG-3Ј. Specific primers for glyceraldehyde 3-phos- analysis with YAC y816F12 demonstrated hybridization to the phate dehydrogenase (5Ј-CACATCGCTCAGACACCATG-3Ј der13 and der17 chromosomes. FISH with YACs y910G4 and and 5Ј-GCCATGGAATTTGCCATGGG-3Ј) were used to as- y929H6 demonstrated hybridization signals on the der17 and the sess the quality of the input RNA. der7 of the t(7;13). These results indicated that the chromosome For RT-PCR of RNA from formalin-fixed, paraffin- 7 breakpoints of both the t(7;17) and the t(7;13) of BWH-42 map embedded tissue, 10 serial 10-m tissue sections were deparaf- within the 7.3 contig between YACs y830D2 and y816F12 but finized with three washes in 10 ml of xylene, washed twice with that the breakpoints are separated by more than 1 megabase. 100% EtOH, and then digested for 16 h at 60°C in 2 ml 1ϫ Furthermore, the chromosome 7 breakpoint in the t(7;17) lies digestion solution (20 mM Tris⅐HCl, pH 8.0͞20 mM between DNA contained within y830D2 and y910G4.