Complex chromosomal rearrangements by single catastrophic pathogenesis in NUT midline carcinoma

Abstract

Background: Nuclear protein in testis (NUT) midline carcinoma (NMC) is a rare aggressive malignancy often occurring in the tissues of midline anatomical structures. Except for the pathognomonic BRD3/4-NUT rearrangement, the comprehensive landscape of genomic alterations in NMCs has been unexplored.

Patients and methods: We investigated three NMC cases, including two newly diagnosed NMC patients in Seoul National University Hospital, and a previously reported cell line (Ty-82). Whole-genome and transcriptome sequencing were carried out for these cases, and findings were validated by multiplex fluorescence in situ hybridization and using individual fluorescence probes.

Results: Here, we present the first integrative analysis of whole-genome sequencing, transcriptome sequencing and cytogenetic characterization of NUT midline carcinomas. By whole-genome sequencing, we identified a remarkably similar pattern of highly complex genomic rearrangements (previously denominated as chromoplexy) involving the BRD3/4-NUT oncogenic rearrangements in two newly diagnosed NMC cases. Transcriptome sequencing revealed that these complex rearrangements were transcribed as very simple BRD3/4-NUT fusion transcripts. In Ty-82 cells, we also identified a complex genomic rearrangement involving the BRD4-NUT rearrangement underlying the simple t(15;19) karyotype. Careful inspections of rearrangement breakpoints indicated that these rearrangements were likely attributable to single catastrophic events. Although the NMC genomes had >3000 somatic point mutations, canonical oncogenes or tumor suppressor genes were rarely affected, indicating that they were largely passenger events. Mutational signature analysis showed predominant molecular clock-like signatures in all three cases (accounting for 54%-75% of all base substitutions), suggesting that NMCs may arise from actively proliferating normal cells.

Conclusion: Taken together, our findings suggest that a single catastrophic event in proliferating normal cells could be sufficient for neoplastic transformation into NMCs.

Keywords: NUT midline carcinoma; bromodomain and extra terminal; chromoplexy; complex genomic rearrangement; mutational signature.

Figure 1.

Chromoplexy generating the BRD3/4−NUT rearrangements. In the upper panels, genomic alterations of two cases (A; NMC1, B; NMC2) are described in circos plots. From the outside in, chromosomal ideograms and their genomic positions in megabases, base substitutions with their variant allelic fractions, smoothened copy numbers, and structural variations are plotted. Genomic rearrangements involved in the chromoplexy are highlighted in blue colors. For base substitutions, we use different colors for indicating different mutational spectra (C > A, blue; C > G, black; C > T, red; T > A, grey; T > C, green; T > G, pink). In the lower panels, the pattern of rearrangement and breakpoints of chromoplexy chains are described. Solid lines represent rearrangements, and dashed lines indicate DNA double strand breaks. >10-bp sized deletions overlying the double strand breaks are indicated as deletion bridges (db). Plus signs indicate rearrangement with reference strand, while minus signs indicate rearrangement with non-reference strand.

Figure 2.

Pattern of chromosomal rearrangement involving the BRD4−NUT rearrangement and its transcription in NMC1. (A) Genomic breakpoints and their rearranged chromosomes in NMC1 are described. (B) Genomic rearrangement (arrows) in the vicinity of NUT gene and its pattern of transcription (purple lines) are represented.

Figure 3.

Pattern of chromosomal rearrangement involving the BRD3−NUT rearrangement and its transcription in NMC2. (A) Genomic breakpoints and the three derivative chromosomes in NMC2 are described. (B) BRD3−NUT rearrangement and its transcriptional pattern are shown.

Figure 4.

Identification of BRD3−NUT rearrangement located in the chromosome 15 by BAC-FISH in NMC2. To visualize the BRD3−NUT rearrangement of which the genomic location is unclear in whole-genome sequencing, we chose BAC clones corresponding to the genomic positions of segment D, E, BRD3, and NUT for individual probe FISH. BAC-FISH visualized the BRD3−NUT rearrangement located in the short arm of acrocentric chromosome 15 (left). All three derivative chromosomes are shown in individual paint probe FISH (right).

Figure 5.

Mutational spectra of NMC cases and contributing signatures. The spectra of base substitutions in three NMC cases defined by the type of substitutions (i.e. C > A or C > T) and their adjacent bases contexts are plotted in histograms (left). The order of 96 trinucleotide contexts is according to the previous study by Alexandrov et al. [32]. From this data, mutational signatures contributing each case were extracted using the deconstructSigs package, and are depicted in bar graphs (right).