BRD4 bromodomain gene rearrangement in aggressive carcinoma with translocation t(15;19)

Abstract

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 fluorescence in 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 implicate BRD4 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 the BRD4 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.

Figure 1.

Histological features of t(15;19) cases 1–3. a: Case 1 is remarkable for sheets of very discohesive undifferentiated cells with scant cytoplasm and prominent, central nucleoli. The tumor was found adjacent to thymus, but as seen here also invaded bronchial seromucinous glands, and was found nearby to undermine and replace the ciliated columnar epithelium (H&E, ×400). b: Case 2 has syncitial sheets of undifferentiated cells with frequent mitoses, single cell necrosis, and percolating lymphocytes giving a lymphoepithelial-like appearance (H&E, ×600). c: Case 3 has prominent nests of undifferentiated cells with focal keratinization (H&E, ×600). Inset: dual-color FISH reveals rearrangement, as evidenced by splitting apart of red-green probe doublet, in paraffin section of this sinonasal carcinoma. d: Immunoperoxidase studies (case 2) show reactivity for pan-keratin (MNF116-alkaline phosphatase, red), indicating epithelial differentiation (hematoxylin counterstain, ×400).

Figure 2.

a: FISH localization of t(15;19) translocation breakpoint within the BRD4-containing cosmid clone R31546. Green and red FISH signals correspond to cosmid clone R31546 and BAC 87 m17, respectively. DAPI counterstain is shown as Giemsa emulation. Normal chromosome 19 has paired red and green signals (top, center), whereas the der(19) has only a green signal (centromeric end of R31546), and the der(15) has a red signal paired with a very weak green signal (telomeric end of R31546). b: Cosmid and BAC map of the 19p13.1 translocation breakpoint region (map framework from Lawrence Livermore National Laboratory). BACs 87m17 and 187l3 delimit the telomeric and centromeric ends of the region, respectively. All clones between the BACs are cosmids. Candidate genes in the region are BRD4 and NOTCH3. Translocation breakpoints in each of two t(15;19) carcinomas were FISH mapped to a 5-kb region, interrupting the BRD4 coding sequence, at the telomeric end of cosmid R31546 (arrows).

Figure 3.

Southern blot of genomic DNAs from t(15;19) cases 1 and 2, and normal lymphocytes (N). Novel restriction fragments are seen in the EcoRI, HindIII, and BamHI t(15;19) cell digests after hybridization to BRD4 (a) and Nop10p (b) probes. c: Exon-intron maps for long and short transcripts of BRD4. The t(15;19) translocation breakpoints interrupt the coding sequence of the long transcript (intron 10), and are approximately 4-kb 3′ to the end of the short transcript. d: Schematic of the BRD4 isoforms: BD, bromodomain; ET, ET domain; S, serine-rich region; P, proline-rich regions; Q, glutamine-rich region; *, kinase-like motifs. Vertical dotted lines define the translocation breakpoint region.