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BRD4 Bromodomain Gene Rearrangement in Aggressive Carcinoma with Translocation t(15;19)

2001; Elsevier BV; Volume: 159; Issue: 6 Linguagem: Inglês

10.1016/s0002-9440(10)63049-0

1525-2191

Christopher A. French, I Miyoshi, Jon C. Aster, Ichiro Kubonishi, Todd G. Kroll, Paola Dal Cin, Sara O. Vargas, Antonio R. Pérez‐Atayde, Jonathan A. Fletcher,

Chromatin Remodeling and Cancer

Translocation t(15;19)(q13;p13.1) defines a lethal midline carcinoma arising adjacent to respiratory tract in young people. To characterize molecular alterations responsible for the distinctly aggressive biological behavior of this cancer, we mapped the chromosome 15 and 19 translocation breakpoints by fluorescencein situ hybridization (FISH) and Southern blotting. To evaluate preliminarily the frequency, anatomical distribution, and histological features of t(15;19) cancer, we developed a FISH assay for paraffin sections. Our findings reveal a novel oncogenic mechanism in which the chromosome 19 translocation breakpoint interrupts the coding sequence of a bromodomain gene, BRD4. These studies implicateBRD4 as a potential partner in a t(15;19)-associated fusion oncogene. In addition, we localized the chromosome 15 breakpoint to a 9-kb region in each of two cases, thereby identifying several candidate oncogenes which might represent theBRD4 fusion partner. FISH evaluation of 13 pediatric carcinomas revealed t(15;19) in one of four sinonasal carcinomas, whereas this translocation was not detected in thymic (n = 3), mucoepidermoid (n = 3), laryngeal (n = 2), or nasopharyngeal (n = 1) carcinomas. Our studies shed light on the oncogenic mechanism underlying t(15;19) and provide further evidence that this highly lethal cancer arises from respiratory mucosa. Translocation t(15;19)(q13;p13.1) defines a lethal midline carcinoma arising adjacent to respiratory tract in young people. To characterize molecular alterations responsible for the distinctly aggressive biological behavior of this cancer, we mapped the chromosome 15 and 19 translocation breakpoints by fluorescencein situ hybridization (FISH) and Southern blotting. To evaluate preliminarily the frequency, anatomical distribution, and histological features of t(15;19) cancer, we developed a FISH assay for paraffin sections. Our findings reveal a novel oncogenic mechanism in which the chromosome 19 translocation breakpoint interrupts the coding sequence of a bromodomain gene, BRD4. These studies implicateBRD4 as a potential partner in a t(15;19)-associated fusion oncogene. In addition, we localized the chromosome 15 breakpoint to a 9-kb region in each of two cases, thereby identifying several candidate oncogenes which might represent theBRD4 fusion partner. FISH evaluation of 13 pediatric carcinomas revealed t(15;19) in one of four sinonasal carcinomas, whereas this translocation was not detected in thymic (n = 3), mucoepidermoid (n = 3), laryngeal (n = 2), or nasopharyngeal (n = 1) carcinomas. Our studies shed light on the oncogenic mechanism underlying t(15;19) and provide further evidence that this highly lethal cancer arises from respiratory mucosa. Characteristic recurrent translocations are common in sarcomas and lymphomas/leukemias but are uncommon in epithelial neoplasms. Highly effective and biologically rational therapies have been developed for some leukemias and sarcomas, often enabled by the cell biology insights obtained from the cloning and functional characterization of translocation-associated oncogenes. For example, chronic myeloid leukemia is characterized by a t(9;22) fusion gene encoding the BCR-ABL oncoprotein, resulting in constitutive ABL tyrosine kinase activity. Dramatic therapeutic responses have been achieved in this disease using the ABL ATP-binding pocket inhibitor, Gleevec.1Druker BJ Lydon NB Lessons learned from the development of an abl tyrosine kinase inhibitor for chronic myelogenous leukemia.J Clin Invest. 2000; 105: 3-7Crossref PubMed Scopus (815) Google Scholar Most clinically aggressive, widely metastatic, treatment-refractory carcinomas have complex karyotypes, without identifiable recurrent chromosomal translocations. However, it is unclear whether pathognomonic translocations are truly absent in these cancers, or whether they are simply masked by the overall cytogenetic complexity. It is generally assumed that carcinoma cytogenetic complexity is a manifestation of underlying genetic instability, permitting rapid acquisition of the sequential mutations that bestow the collective "aggressive" phenotype on an epithelial progenitor cell. On the other hand, there are several types of carcinoma, typically low-to-medium grade, which have relatively simple karyotypes, and which often feature balanced translocations. For example, recurrent translocations are found in follicular and papillary carcinoma of the thyroid,2Kroll TG Sarraf P Pecciarini L Chen CJ Mueller E Spiegelman BM Fletcher JA PAX8-PPARγ1 fusion oncogene in human thyroid carcinoma.Science. 2000; 289: 1357-1360Crossref PubMed Scopus (745) Google Scholar, 3Sozzi G Bongarzone I Miozzo M Borrello MG Blutti MG Pilotti S Della Porta G Pierotti MA A t(10;17) translocation creates the RET/PTC2 chimeric transforming sequence in papillary thyroid carcinoma.Genes Chromosomes Cancer. 1994; 9: 244-250Crossref PubMed Scopus (81) Google Scholar, 4Nakata T Kitamura Y Shimizu K Tanaka S Fujimori M Yokoyama S Ito K Emi M Fusion of a novel gene, ELKS, to RET due to translocation t(10;12)(q11;p13) in a papillary thyroid carcinoma.Genes Chromosomes Cancer. 1999; 25: 97-103Crossref PubMed Scopus (113) Google Scholar, 5Corvi R Berger N Balczon R Romeo G RET/PCM-1: a novel fusion gene in papillary thyroid carcinoma.Oncogene. 2000; 19: 4236-4242Crossref PubMed Scopus (76) Google Scholar and in papillary renal cell carcinoma,6Kardas I Denis A Babinska M Gronwald J Podolski J Zajaczek S Kram A Lubinski J Limon J Translocation (X;1)(p11.2;q21) in a papillary renal cell carcinoma in a 14-year-old girl.Cancer Genet Cytogenet. 1998; 101: 159-161Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar none of which are particularly aggressive clinically. Balanced translocation t(15;19) has been reported in a small number of carcinomas in children and young adults, and the clinical correlations with this translocation are striking.7Kubonishi I Takehara N Iwata J Sonobe H Ohtsuki Y Abe T Miyoshi I Novel t(15;19)(q15;p13) chromosome abnormality in a thymic carcinoma.Cancer Res. 1991; 51: 3327-3328PubMed Google Scholar, 8Kees UR Mulcahy MT Willoughby ML Intrathoracic carcinoma in an 11-year-old girl showing a translocation t(15;19).Am J Pediatr Hematol Oncol. 1991; 13: 459-464Crossref PubMed Scopus (121) Google Scholar, 9Lee AC Kwong YI Fu KH Chan GC Ma L Lau YL Disseminated mediastinal carcinoma with chromosomal translocation (15;19): a distinctive clinicopathologic syndrome.Cancer. 1993; 72: 2273-2276Crossref PubMed Scopus (83) Google Scholar, 10Dang TP Gazdar AF Virmani AK Sepetavec T Hande KR Minna JD Roberts JR Carbone DP Chromosome 19 translocation, overexpression of Notch3, and human lung cancer.J Natl Cancer Inst. 2000; 92: 1355-1357Crossref PubMed Scopus (218) Google Scholar, 11Vargas SO French CA Faul PN Fletcher JA Davis IJ Dal Cin P Perez-Atayde AR Upper respiratory tract carcinoma with chromosomal translocation 15;19: evidence for a distinct disease entity of young patients with a rapidly fatal course.Cancer. 2001; 92: 1195-1203Crossref PubMed Scopus (88) Google Scholar All t(15;19) carcinomas were poorly differentiated histologically and extremely aggressive (average survival 18 weeks) with rapid clinical progression despite multi-modality therapies. Hence, this is the first recurring translocation identified in a particularly aggressive form of carcinoma. Characterization of the translocation breakpoints might therefore enable development of models which evaluate "aggressive" clinicopathological features, including invasiveness, high proliferative rate, and metastatic potential in epithelial tumors. In the present report we identify the putative chromosome 19 translocation target, and we also localize chromosome 15 breakpoints to a 9-kb genomic region. Further, we develop a paraffin-based fluorescence in situ hybridization (FISH) assay, and show application of this method in evaluating potential t(15;19) events in pediatric carcinomas. Cytogenetic analysis was performed on Giemsa-banded metaphase spreads as described previously.12Fletcher JA Kozakewich HP Hoffer FA Lage JM Weidner N Tepper R Pinkus GS Morton CC Corson JM Diagnostic relevance of clonal cytogenetic aberrations in malignant soft-tissue tumors.N Engl J Med. 1991; 324: 436-442Crossref PubMed Scopus (275) Google Scholar We established a rapidly growing cell line from a lymph node metastasis in t(15;19) case 1. The primary tumor cells were grown in RPMI 1640 medium (Life Technologies, Inc. (Gibco BRL), Rockville, MD) and 15% fetal bovine serum and were held at confluence in T25 flasks, being fed thrice weekly between passaging. Cells were passed by releasing the confluent monolayers with trypsin and splitting the cells 1:2 to new T25 flasks. The cells grew rapidly and after passage 5 could be passed at 1:4. A cell line was also established from t(15;19) case 2, as reported previously.13Kuzume T Kubonishi I Takeuchi S Takeuchi T Iwata J Sonobe H Ohtsuki Y Miyoshi I Establishment and characterization of a thymic carcinoma cell line (Ty-82) carrying t(15;19)(q15;p13) chromosome abnormality.Int J Cancer. 1992; 50: 259-264Crossref PubMed Scopus (29) Google Scholar Both cell lines were cytogenetically stable, with persistence of the t(15;19) translocation in all cells after more than 20 passages. YACs, BACs, and cosmids were biotin or digoxigenin-labeled by random octamer priming (Life Technologies, Inc. (Gibco BRL), Rockville, MD).14Xiao S Renshaw A Cibas ES Hudson TJ Fletcher JA Novel fluorescence in situ hybridization approaches in solid tumors: characterization of frozen specimens, touch preparations, and cytological preparations.Am J Pathol. 1995; 147: 896-904PubMed Google Scholar Metaphase preparations from cell line cells or control normal lymphocytes were spread on slides and denatured according to standard protocols. Hybridization and washing steps were performed as described.14Xiao S Renshaw A Cibas ES Hudson TJ Fletcher JA Novel fluorescence in situ hybridization approaches in solid tumors: characterization of frozen specimens, touch preparations, and cytological preparations.Am J Pathol. 1995; 147: 896-904PubMed Google Scholar Detection was with FITC-anti-digoxigenin (Roche, Indianapolis, IN) and 594 rhodamine streptavidin (Molecular Probes, Eugene, OR), and nuclei were counterstained with 1 mg/ml 4,6-diamidino-2-phenylindole-dihydrochloride (DAPI). Images were captured using a charge-coupled device camera (Photometrics, Tucson, AZ). FISH mapping of the chromosome 15 and 19 breakpoints was performed by "walking" with YAC, BAC, then cosmid (cosmids not used for chromosome 15) probes inward to the breakpoint region using dual-color, split-apart FISH. Cell line DNA isolation and blotting techniques were performed as described.15Cleary ML Mellentin JD Spies J Smith SD Chromosomal translocation involving the β T-cell receptor gene in acute leukemia.J Exp Med. 1988; 167: 682-687Crossref PubMed Scopus (49) Google Scholar Polymerase chain reaction-generated, gel-isolated DNA probe templates (intron 12 and exon 13 ofBRD4 (GenBank Accession No. AF386649) (see Figure 3b); intron 1 and exon 2 of Nop10p) were labeled by random nanonucleotide priming (Prime-It II, Stratagene, Cedar Creek, TX) and [α-32P]dCTP. Paraffin sections from 13 carcinomas (1975 to present; age range, 3 to 53 years; mean age, 16 years; M/F = 3/10), including 3 thymic, 3 mucoepidermoid (2 parotid gland, 1 lung), 2 laryngeal, 1 nasopharyngeal, and 4 sinonasal from Children's Hospital, Boston, Massachusetts, were examined for the t(15;19) by FISH. Chromosome 15 and 19 breakpoints were evaluated using a dual color, split-apart FISH assay. For chromosome 15, a telomeric YAC, 908c5, and a centromeric YAC, 733c7, were used; for chromosome 19, a telomeric BAC, 87 m17, and a centromeric YAC, 766e7, were used. FISH on paraffin sections was performed using a modification of the technique described by Bull and Harnden.16Bull JH Harnden P Efficient nuclear FISH on paraffin-embedded tissue sections using microwave pretreatment.Biotechniques. 1999; 26 (422): 416-418PubMed Google Scholar Briefly, formalin-fixed, 4-μm thick paraffin-embedded sections were baked overnight (60°C) and deparaffinized in xylene. Slides were immersed in 100 mmol/L Tris-base, 50 mmol/L EDTA, and incubated at 100°C in a temperature-controlled microwave for 30 to 60 minutes. Tissue digestion was performed with pepsin solution (Digest-All III, Zymed, San Francisco, CA), applied directly to the slides at 37°C for 10 minutes, and postfixed in 10% formaldehyde for 1 minute. The biotin and digoxigenin-labeled YAC DNA and Cot1 mixture was diluted in a 50% formamide solution containing 10% dextran sulfate, applied and coverslipped onto the slide. The slide was denatured on a flatbed polymerase chain reaction machine at 94°C for 3 minutes. Hybridization and washing steps were performed as described14Xiao S Renshaw A Cibas ES Hudson TJ Fletcher JA Novel fluorescence in situ hybridization approaches in solid tumors: characterization of frozen specimens, touch preparations, and cytological preparations.Am J Pathol. 1995; 147: 896-904PubMed Google Scholar and signal detection was as described above. Clinicopathological features for all known t(15;19) carcinomas are summarized in Table 1, and are remarkable for poor survival and a treatment-refractory course. The histologies of the t(15;19) cancers evaluated in this study are shown in Figure 1. Cases 1 and 2 provided the cell lines used for mapping studies. The tumor in case 1 arose adjacent to thymus and respiratory epithelium in a 22-year-old female. The tumor in case 2 arose from the epiglottis in a 13-year-old female.Table 1Clinicopathological Features of t(15:19)+ CarcinomasCase no.Age/sexUndifferentiated cellsDifferentiationKeratinSurvival (weeks)TherapyReference no.1*A cell line derived from this case was used for mapping studies described herein.22/F+Sq+14CR72*A cell line derived from this case was used for mapping studies described herein.13/F++38CR11312/F+Sq+13CR411/F+Sq+18CR855/M+Sq/G+8C9634/F+NS+NSNS10Average Survival Was 18 Weeks. C, chemotherapy; G, glandular; NS, not specified; R, radiation; Sq, squamous.* A cell line derived from this case was used for mapping studies described herein. Open table in a new tab Average Survival Was 18 Weeks. C, chemotherapy; G, glandular; NS, not specified; R, radiation; Sq, squamous. FISH mapping analyses are summarized in Figure 2. The chromosome 19p13.1 cosmid clone R31546 was split by the t(15;19) breakpoints in cases 1 and 2. The split cosmid FISH signal was approximately 80:20, with the smaller signal being on the telomeric side of the breakpoint. This localization is consistent with involvement of the 3′ end of the BRD4 coding sequence. The chromosome 15q13 BAC clone 122p18 was also split approximately 50:50 by the translocation breakpoints in t(15;19) cases 1 and 2. BAC 122p18 contains three genes, Nop10p,Golgin-67, and KCC3 which have not yet been localized relative to the breakpoints. Southern hybridization with a probe to BRD4 intron 12 and exon 13 revealed rearranged fragments in t(15;19) cases 1 and 2 (Figure 3a). A restriction map of the translocation breakpoint region was created by evaluating the cosmid R31546 sequence using Webcutter, Baylor College of Medicine Sequence Utilities. These correlations narrowed the translocation breakpoints to a 3.5-kb region delimited by BamHI andHindIII restriction sites at cosmid R31546 nucleotides 6433 and 9945, respectively. Hence, two independent genomic mapping methods, FISH and Southern blotting, demonstrated localization of the chromosome 19 translocation breakpoints to the 3′ end of the BRD4 coding sequence in each of two t(15;19) cancers. There are two major isoforms of BRD4 encoded by alternate splicing products of 3179 nucleotides (GenBank Accession No. XM009302), and 5198 nucleotides (GenBank Accession No. AF386649). The t(15;19) breakpoints map between intron 10 and intron 13 of the longer BRD4 transcript (Figure 3b). This is consistent with a breakpoint splitting the coding sequence of the longer BRD4 transcript approximately in half, while leaving the short transcript unaltered. At the protein level, the translocation is predicted to result in separation of the BRD4 N-terminal end, containing bromodomains, kinase-like motifs, and a serine-rich domain, from the C-terminal glutamine-rich and proline-rich domains (Figure 3c). Southern hybridization with a probe to intron 1 and exon 2 ofNop10p revealed rearranged fragments in each of two t(15;19) cancers (Figure 3b). This observation localizes the chromosome 15q13 breakpoint to a ∼9-kb region containing the Nop10p candidate gene. The proximity of two other known genes,Golgin-67 and KCC3, to this region, remains to be determined. FISH screening of 13 paraffin-embedded, supradiaphragmatic pediatric carcinomas revealed a sinonasal carcinoma with chromosome 19p13.1 and 15q13 rearrangements (Figure 1c and Table 1, case 3), consistent with t(15;19). The remaining 12 carcinomas, including 3 thymic, 3 mucoepidermoid, 2 laryngeal, 1 nasopharyngeal, and 3 sinonasal, lacked chromosome 15 or 19 rearrangements. We describe herein the molecular characterization of t(15;19)(q13;p13.1), which is the cytogenetic hallmark of a particularly aggressive form of carcinoma in children and young adults. Our studies implicate the bromodomain-encoding gene, BRD4, as the chromosome 19 target of the t(15;19)(q13;p13.1) translocation, and they also localize the chromosome 15 breakpoints to a 9-kb genomic region. Notably, the BRD4 breakpoints in each of two t(15;19) cancers interrupt the BRD4 coding sequence, and these findings are strong evidence for a BRD4 fusion oncogene mechanism. This is in distinction to recurrent cancer translocations associated with non-fusion oncogenes, which generally involve the untranslated or flanking regions of those gene(s). Examples of non-fusion oncogenic mechanisms include those targeting c-myc, Bcl-2, and cyclin D1 in non-Hodgkin's lymphomas, where the translocation breakpoints are scattered and vary by as much as 400 kb among tumors whose cytogenetically determined translocations are indistinguishable.17Cleary ML Smith SD Sklar J Cloning and structural analysis of cDNAs for Bcl-2 and a hybrid Bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation.Cell. 1986; 47: 19-28Abstract Full Text PDF PubMed Scopus (1080) Google Scholar, 18Johnston JM Carroll WL c-myc hypermutation in Burkitt's lymphoma.Leuk Lymphoma. 1992; 8: 431-439Crossref PubMed Scopus (27) Google Scholar, 19Dalla-Favera R Ye BH Lo Coco F Chang CC Cechova K Zhang J Migliazza A Mellado W Niu H Chaganti S Chen W Rao PH Parsa NZ Louie DC Offit K Chaganti RSK BCL-6 and the molecular pathogenesis of B-cell lymphoma.Cold Spring Harb Symp Quant Biol. 1994; 59: 117-123Crossref PubMed Scopus (24) Google Scholar Of note, there are no reported examples of recurrent balanced translocation breakpoints which localize within the coding sequence of a gene, but which fail to alter the function of that gene. Our evidence, albeit based on analysis of only two t(15;19) carcinomas, is that the chromosome 19 genomic breakpoints cluster within a 3.5-kb region in the BRD4 coding sequence. Notably, there is precedent for involvement of bromodomain genes, eg,CBP and p 300, in fusion oncogenes.20Giles RH Dauwerse JG Higgins C Petrij F Wessels JW Beverstock GC Dohner H Jotterand-Bellomo M Falkenburg JH Slater RM van Ommen GJ Hagemeijer A van der Reijden BA Breuning MH Detection of CBP rearrangements in acute myelogenous leukemia with t(8;16).Leukemia. 1997; 11: 2087-2096Crossref PubMed Scopus (63) Google Scholar By contrast, bromodomain genes have not been implicated in non-fusion, translocation-associated, cancer cytogenetic mechanisms. The translocation breakpoints identified thus far in t(15;19) cancers divide the BRD4 long isoform into two pieces wherein the N-terminal component contains chromatin-binding bromodomains,21Winston F Allis CD The bromodomain: a chromatin-targeting module?.Nat Struct Biol. 1999; 6: 601-604Crossref PubMed Scopus (225) Google Scholar predicted kinase activity,22Dey A Ellenberg J Farina A Coleman AE Maruyama T Sciortino S Lippincott-Schwartz J Ozato K A bromodomain protein, MCAP, associates with mitotic chromosomes and affects G(2)-to-M transition.Mol Cell Biol. 2000; 20: 6537-6549Crossref PubMed Scopus (237) Google Scholar and serine-rich potential transactivation or co-repressor domains (Figure 3c).23Chen C Agnes F Gelinas C Mapping of a serine-rich domain essential for the transcriptional, antiapoptotic, and transforming activities of the v-Rel oncoprotein.Mol Cell Biol. 1999; 19: 307-316Crossref PubMed Scopus (21) Google Scholar The C-terminal component has a glutamine-rich domain with potential transactivation function,24Escher D Bodmer-Glavas M Barberis A Schaffner W Conservation of glutamine-rich transactivation function between yeast and humans.Mol Cell Biol. 2000; 20: 2774-2782Crossref PubMed Scopus (53) Google Scholar and a proline-rich domain with potential roles in protein-protein interaction.25Alexandropoulos K Cheng G Baltimore D Proline-rich sequences that bind to Src homology 3 domains with individual specificities.Proc Natl Acad Sci USA. 1995; 92: 3110-3114Crossref PubMed Scopus (251) Google Scholar Further studies are needed to determine whether functional t(15;19) BRD4 oncoprotein(s) use one or both of these BRD4 regions. An understanding of BRD4 oncogenic mechanisms will likely require characterization of the functional differences between the native short and long BRD4 isoforms. For example, relative levels of isoform expression might determine tissue-specific and developmentally relevant programs of cell proliferation or differentiation. Characterization of translocation-associated perturbation of the long BRD4 isoform, and likely sparing of the short isoform, may provide clues to mechanisms of normal and oncogenic BRD4 function. In particular, it is possible that the short isoform may play an inhibitory role by occupying chromosomal positions that would otherwise be available to the long isoform. In this sense, the t(15;19) translocation could function to inhibit BRD4 long isoform function by a dominant negative mechanism. Within 40 kb telomeric of BRD4 is another candidate oncogene, NOTCH3 (Figure 2b), which Dang et al10Dang TP Gazdar AF Virmani AK Sepetavec T Hande KR Minna JD Roberts JR Carbone DP Chromosome 19 translocation, overexpression of Notch3, and human lung cancer.J Natl Cancer Inst. 2000; 92: 1355-1357Crossref PubMed Scopus (218) Google Scholar have implicated as a potential transcriptionally up-regulated target in a single case of t(15;19) carcinoma. Although the family member, NOTCH1, is a translocation target in human leukemia,26Ellisen LW Bird J West DC Soreng AL Reynolds TC Smith SD Sklar J TAN-1, the human homolog of the Drosophila notch gene, is broken by chromosomal translocations in T lymphoblastic neoplasms.Cell. 1991; 66: 649-661Abstract Full Text PDF PubMed Scopus (1459) Google Scholar we have evaluated NOTCH3 expression by Northern blotting in t(15;19) versus control carcinomas, and have not observed translocation-associated positional effects onNOTCH3 expression (CA French, JA Fletcher, unpublished data). Therefore, although minor NOTCH3 positional effects cannot be excluded, our evidence highlights BRD4 rearrangement as the major event responsible for t(15;19)-mediated epithelial cell transformation. We have narrowed the 15q13 breakpoint to a 9-kb region containingNop10p. This highly conserved gene encodes a core small nucleolar ribonucleoprotein (snoRNP) which is essential for snoRNP function,27Henras A Henry Y Bousquet-Antonelli C Noaillac-Depeyre J Gelugne JP Caizergues-Ferrer M Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs.EMBO J. 1998; 17: 7078-7090Crossref PubMed Scopus (198) Google Scholar and which potentially interacts with snoRNAs and the RNA subunit of telomerase.28Pogacic V Dragon F Filipowicz W Human H/ACA small nucleolar RNPs and telomerase share evolutionarily conserved proteins NHP2 and NOP10.Mol Cell Biol. 2000; 20: 9028-9040Crossref PubMed Scopus (195) Google Scholar It is not yet known whether Nop10p participates with BRD4 in a t(15;19) fusion oncoprotein, and there are several other genes within 100 kb of Nop10p which are alternate targets of the translocation. These includeGolgin-67 and the KCl transporter, KCC3. Golgin-67 encodes a Golgi apparatus-associated protein with a coiled-coil, proline-rich domain and a leucine zipper at its N-terminal end, both potential dimerization or protein-protein interaction domains that might contribute to transforming activity in a fusion oncogene.29McWhirter JR Galasso DL Wang JY A coiled-coil oligomerization domain of Bcr is essential for the transforming function of Bcr-Abl oncoproteins.Mol Cell Biol. 1993; 13: 7587-7595Crossref PubMed Scopus (376) Google Scholar, 30Xiao S McCarthy JG Aster JC Fletcher JA ZNF198-FGFR1 transforming activity depends on a novel proline-rich ZNF198 oligomerization domain.Blood. 2000; 96: 699-704Crossref PubMed Google Scholar, 31Chaplin T Bernard O Beverloo HB Saha V Hagemeijer A Berger R Young BD The t(10;11) translocation in acute myeloid leukemia (M5) consistently fuses the leucine zipper motif of AF10 onto the HRX gene.Blood. 1995; 86: 2073-2076PubMed Google Scholar The t(15;19) FISH assay described herein is a straightforward method for identification of t(15;19) cancers in archival materials. Therefore, this assay will be useful in establishing clinicopathological correlations, and it will be particularly important to determine whether t(15;19) carcinomas are uniformly associated with the devastating clinical course of those reported to date. Our initial FISH-based screen demonstrates that the t(15;19) is not vanishingly rare in pediatric cancers. Rather, the t(15;19) was detected in 1 of 13 such carcinomas, a sinonasal primary with squamous cell differentiation. The clinical course in this patient, in whom rapid local disease progression was accompanied by widespread bony metastases, was highly unusual for sinonasal squamous cell carcinoma but entirely in keeping with the t(15;19) syndrome. Notably, the FISH assays failed to show t(15;19) in three thymic carcinomas. This finding, together with our observation of t(15;19) in epiglottic and sinonasal primaries, demonstrates that thymic origin, which has been posited in several previous reports, fails to account for all t(15;19) carcinomas. It remains to be determined whether any t(15;19) carcinomas are convincingly thymic, but we favor the possibility that most such cases arise from the respiratory tract. In sum, t(15;19) carcinoma is a uniquely devastating disease, typically arising in children or young adults, and associated with a distinctive molecular mechanism involving BRD4 rearrangement. Further clinicopathologic and molecular characterizations of this disease will likely provide important insights in the fields of pathology, molecular oncology, and cancer biology. We thank Dr. Sheng Xiao and Dr. Andrew Weng for their valuable suggestions, Dr. Linda Ashworth, of the Lawrence Livermore National Laboratory (LLNL), for generously providing all cosmids used in this work, Dr. Ann Anderson for providing paraffin material, and Ms. Michelle Taffaro for her technical assistance.