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Ann Oncol. 2017 April; 28(4): 890–897.
Published online 2017 February 14. doi:  10.1093/annonc/mdw686
PMCID: PMC5378225

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, chromoplexy, complex genomic rearrangement, mutational signature, bromodomain and extra terminal

Introduction

Nuclear protein in testis (NUT) midline carcinoma (NMC) is a rare lethal malignancy, which was first described in the literature in 1991 [1, 2]. NMC shows heterogeneous clinicopathologic characteristics [3]; it occurs without gender predilection in a wide range of ages at diagnosis from newborns to the 70s with a peak incidence in the late teenage years [3]. It usually presents with poorly differentiated carcinoma with a variable degree of squamous differentiation and tends to occur in the midline anatomical structures, most frequently in the upper aerodigestive tract and anterior mediastinum; however, cases originating from the bladder, salivary glands, and iliac bone have also been described [3]. NMCs are frequently diagnosed with metastatic disease and refractory to chemotherapy with extremely poor patient survival [4]. Because of the heterogeneous presentation overlapping with other malignancies and the aggressive clinical course, NMC cases could have been frequently misclassified as other malignancies [3]; thus, the true incidence is unknown, and it has only recently emerged as an independent diagnostic category defined by the characteristic genomic rearrangements [5].

The genetic hallmarks of NMCs are the rearrangement of the NUT gene with a set of partner genes. Interchromosomal translocations between chromosomes 15q and 19p are frequently observed [1, 2], which generates a fusion oncogene Bromodomain containing 4 (BRD4)−NUT [6, 7]. Briefly, the BRD4NUT fusion oncoprotein results in a global disruption of gene expression programs through extensive chromatin remodeling [8], overexpression of MYC [9] and sequestration of the acetylated (activated) form of p53 [10]. Other partners of the NUT gene rearrangement, i.e. BRD3 [7] or NSD3 [11], are functionally related with BRD4, indicating that the recruitment of NUT to the chromatin through the bromodomain and extra terminal (BET) family proteins is necessary for the pathogenesis of NMCs. In accordance with their functional importance, the use of small molecular inhibitors of BET family proteins suggested a new therapeutic avenue for NMCs [12].

Except for the pathognomonic gene rearrangements, however, the comprehensive mutational landscape of NMC has been unexplored. Here we present the first whole-genome and transcriptome sequences of NMC from two newly diagnosed NMC cases and a previously reported NMC cell line [13]. In-depth analysis of the somatic mutational profile provides insight into the mechanisms of BRD3/4NUT rearrangements and the characteristics of the original cells.

Materials and methods

Establishment of patient-derived cell lines

The protocol of this study was reviewed and approved by the Institutional Review Board of Seoul National University Hospital (#H-1512-083-728). This study was conducted in the observation of the Helsinki Declaration of the World Medical Association. Pleural effusions obtained after written informed consent were centrifuged, washed, and resuspended in growth media and then cultured as previously described [14]. Through this process, SNU-2972-1 and SNU-3178S cells were established from the NMC1 and NMC2 patients, respectively.

High-throughput sequencing and analysis of somatic mutations

Genomic DNA and mRNA were extracted from the early batch SNU-2972-1, SNU-3178S, their matched peripheral blood samples and from Ty-82 cells. Detailed procedures of high-throughput sequencing and somatic mutation calling were described in a separate document (supplementary text, available at Annals of Oncology online).

Cytogenetic characterization and fluorescence in situ hybridization

Conventional cytogenetic examination using Giemsa banding was done for the SNU-2972-1 and SNU-3178S cells. More than 20 metaphase cells were analyzed, and their karyotypes were assessed according to the International System for Human Cytogenetic Nomenclature 2013.

M-FISH and bacterial artificial chromosome (BAC)-FISH were done as previously described [15]. A human 24-color chromosomal painting probe set was used for the M-FISH. For analysis, 10 randomly selected metaphase cells were karyotyped based on the M-FISH and DAPI-banding patterns. For detailed analyses of particular rearrangement segments, human BAC clones were selected based on their genomic positions in the reference genome. Detailed information on the BAC clones used in our experiments is summarized in supplementary Table S1, available at Annals of Oncology online.

Analysis of mutational signatures

Decomposition of mutational signatures from consensus substitution sets of individual cases was done with deconstructSigs package [16] and further confirmed with an in-house algorithm (based on 30 mutational signatures; http://cancer.sanger.ac.uk/cosmic/signatures).

Results

Clinical history of patients and establishment of NMC cell lines

We investigated two newly diagnosed patients with NMC. In both cases, the definitive diagnosis was made by confirming the high expression of NUT protein with immunohistochemical staining of the biopsy tissue (anti-NUT antibody C52B1; Cell Signaling Technology). Patient 1 was a 34-year-old male with a 10 pack-year history of smoking and was diagnosed with sinonasal and intrathoracic NMC with left pleural effusion. His tumor was resistant to two different combination chemotherapy regimens: three cycles of paclitaxel and carboplatin, and one cycle of cyclophosphamide, doxorubicin and vincristine. The treatment-refractory pleural effusion, which was drained a few days before death was later established as a NMC cell line, SNU-2972-1. Patient 2 was a 33-year-old never-smoking female who was diagnosed with NMC in the lung with multiple bone metastases. The SNU-3178S cell line was established from her pleural effusion at the initial presentation. Her tumor was initially stabilized by the combination chemotherapy of paclitaxel and carboplatin but eventually progressed after five cycles of treatment. The overall survival durations from the diagnosis of the two patients were 4 and 8 months, respectively.

Giemsa band karyotyping revealed that these two NMC cell lines do not have the canonical t(15;19) karyotype (supplementary Figure S1, available at Annals of Oncology online). SNU-2972-1 had a near-diploid karyotype with a translocation between chromosomes 15 and 21, and an insertion of chromosomal 15 segments into chromosome 19. The SNU-3178S cells had a complex karyotype of t(9;15) with hyperdiploidy of chromosomes 1, 5, 8, 12 and X. These microscopic chromosomal rearrangements could be further characterized at nucleotide resolution with whole-genome and transcriptome sequencing. To minimize the in vitro culture-induced mutations, we sequenced the early batches of SNU-2972-1 (denoted as NMC1) and SNU-3178S (NMC2) together with their matched peripheral blood samples as controls.

Complex rearrangements involving BRDNUT oncogenes

We first investigated large structural variations (SVs) including genomic rearrangements and DNA copy number alterations. With en suite bioinformatics analyses and visual inspections, we detected 49 and 33 highly confident cancer-specific SVs from NMC1 and NMC2, respectively (supplementary Table S2, available at Annals of Oncology online). The allele-specific copy number analysis of NMC1 and NMC2 showed highly concordant copy number profiles compared with their karyotypes (supplementary Table S3, available at Annals of Oncology online). Interestingly, the rearrangements in the vicinity of the NUT gene were highly complex in both tumors consisting of many inter- and intra-chromosomal breakpoints with no evidence of local DNA copy number alterations. A careful manual reconstruction of these rearranged segments revealed a remarkably similar “closed chain” pattern of complex genomic rearrangements across multiple chromosomes (Figure 1), previously described as chromoplexy [17]. Inspection of breakpoint pairs which were likely generated from single DNA double strand breakages revealed frequent >10-bp deletions as described previously [17]; these deletions may reflect the DNA resection during the repair process. In NMC1, 24 genomic breakpoints, clustered in several hotspots in chromosomes 1, 15, 19 and 21, were rejoined in a balanced but disordered manner to form a closed chain of 12 rearrangements. The complex event finally rearranged BRD4 to the upstream of NUT with an intervening 31-kb-sized insertion segment from chromosome 15 (Figures 1A and and2A).2A). These findings were validated by multiplex fluorescence in situ hybridization (M-FISH; supplementary Figure S2, available at Annals of Oncology online) and FISH using individual paint probes (supplementary Figure S3, available at Annals of Oncology online). The mRNA sequencing revealed the expression of the rearranged genome: exon 11 of BRD4 was conjoined with exon 2 of NUT generating an in-frame fusion gene (Figure 2B) that skips exon 12 of BRD4, a short intervening gene NOP10 in the opposite strand, and exon 1 of NUT. We also identified an alternative splicing event generating an independent, out-of-frame BRD4NUT transcript using an intervening cryptic exon (supplementary Figure S4, available at Annals of Oncology online), but the pathogenic significance would be minimal. Similarly, in NMC2, we identified 12 genomic breakpoints involved in the BRD3NUT rearrangement. Those breakpoints were located in chromosomes 3, 9 and 15 also generating a closed chain without local DNA copy number changes. Exon 11 of BRD3 was truncated and connected with intron 4 of NUT (Figures 1B and and3A);3A); this rearrangement generated fusion transcripts conjoining exon 10 of BRD3 with exon 5 of NUT (Figure 3B). However, both ends of the rearranged DNA segment encompassing the BRD3NUT were conjoined with a non-specific AATGG (centromeric) repeat, which made it difficult to reconstruct the complete pattern of the complex rearrangements using sequencing data only. Therefore, we further investigated the genome of the NMC2 cells with M-FISH (supplementary Figure S5, available at Annals of Oncology online) and BAC clones (BAC-FISH). Finally, we identified that the rearranged NUT gene was inserted into the repetitive region of the short arm of the acrocentric chromosome 15 (Figure 4). Of note, many rearrangements of submicroscopic segments (<10 Mb), including the DNA segment with the rearranged NUT gene in this case, were not apparent in conventional karyotyping. All the characteristics of these complex chromosomal rearrangements, i.e. the closed-chain pattern of rearrangement, clustered location of breakpoints, no evidence of segmental loss, no significant copy number change at the rearrangement junctions, and little sequence microhomology at the rearrangement breakpoints, indicate that the oncogenic BRD3/4NUT rearrangements can be attributed to a single catastrophic event rather than a gradual accumulation, during the life history of the most recent common ancestor (MRCA) cell of the NMCs [15].

Figure 1.
Chromoplexy generating the BRD3/4NUT 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, ...
Figure 2.
Pattern of chromosomal rearrangement involving the BRD4NUT 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 ...
Figure 3.
Pattern of chromosomal rearrangement involving the BRD3NUT rearrangement and its transcription in NMC2. (A) Genomic breakpoints and the three derivative chromosomes in NMC2 are described. (B) BRD3NUT rearrangement and its transcriptional ...
Figure 4.
Identification of BRD3NUT rearrangement located in the chromosome 15 by BAC-FISH in NMC2. To visualize the BRD3NUT rearrangement of which the genomic location is unclear in whole-genome sequencing, we chose BAC clones corresponding to ...

We wondered whether complex SVs rather than simple balanced reciprocal translocations underlay the NUT rearrangements even in the NMC cases with a simple t(15;19) karyotype. We sequenced the whole genome of Ty-82, one of the oldest and well-characterized NMC cell lines [13]. Interestingly, we found that the interchromosomal BRD4NUT rearrangement was not simple but was combined with a local inversion and copy number amplification (from 2 to 3 copies) of a small (~300 bp) intervening DNA segment (supplementary Figure S6, available at Annals of Oncology online). The local copy number change and sequence microhomology at the rearrangement junctions indicated that this complex rearrangement was likely generated by the microhomology-mediated break-induced replication mechanism, which has been frequently observed in complex genomic rearrangements [18].

Somatic mutations and their mutational signatures

We next examined somatic point mutations including base substitutions and short indels. We identified 3368 and 3168 somatic base substitutions in NMC1 and NMC2, respectively (supplementary Table S4, available at Annals of Oncology online), with a mutation rate higher than 1.1/Mb. The crude mutation rate of NMC was higher than expected [19]; the burden of mutation in both NMC cases was comparable to other cancer types of different tissues, for example, breast cancers (median number of substitutions = 3492 from 560 whole-genome sequences) [20], low-grade gliomas and B-cell lymphomas [21]. To investigate the mutational processes that have been operative in the NMC cells, we analyzed the mutational signatures of NMC1, NMC2 and Ty-82 (Figure 5) [21]. A large portion of somatic base substitutions were attributable to signature 1 (dominantly C:G > T:A substitution at a CpG sequence context due to endogenous methyl-cytosine deamination) and signature 5 (dominantly T:A > C:G transition; unknown mechanism) in all three samples (53.8%, 69.4% and 74.7% of substitutions in NMC1, NMC2 and Ty-82, respectively). The proportion is comparable or even larger when compared with many other cancer types, i.e. 49.2% in breast cancers [20]. These two mutational signatures are known to have molecular clock-like properties, which may generate mutations throughout life at a more-or-less constant rate [22]. A recent study demonstrated that annual mutation rates of adult stem cells of colon, small intestine and liver were very similar: ~40 point mutations (most of them were attributable to signatures 1 and 5) per year [23]. Overall, prevalent somatic mutations of those two molecular clock-like signatures in our NMCs (1811 for NMC1 and 2199 for NMC2) despite the short chronological ages at diagnosis (NMC1 and NMC2 were 34 and 33 years old, respectively, at diagnosis and Ty-82 was established from a 22-year-old female patient [13]) may represent either distinct physiology of NMC origin cells (i.e. higher number of cell division compared with other adult stem cells) or activation of clock-like mutational processes after malignant transformation.

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). ...

Interestingly, we found a C:G > A:T substitution-dominant mutational signature (signature 29), which was known as tobacco chewing-related [21], in NMC1 (accounting for 23.4% of the somatic substitutions in the case). Because the smoking carcinogen induces a G > T transversion mutation only in the epithelial cells that have direct exposure to the carcinogen from tobacco [21], we conclude that the ancestral cells of NMC1 were located in tissues directly exposed to tobacco smoke at the pre-neoplastic stage. In the NMC2 genome, we found no evidence of tobacco-induced mutations, which was consistent with the never-smoking history of the patient. 16.7% of the substitutions were attributable to the unknown signature 8, and 7.3% were APOBEC-induced mutations (signatures 2 and 13) in NMC2. Ty-82 showed the highest percentage of signature 1 and 5 mutations (74.7%), which was consistent with the prolonged history of this cell line since 1992.

Of the >3000 somatic base substitutions in the NMC1 and NMC2 genomes, 18 and 24 changed the amino acids of protein-coding genes, respectively. However, no canonical oncogenes or tumor suppressor genes were affected. A total of 200 and 222 short indels were identified from NMC1 and NMC2, but only 3 from NMC1 (none of them were in the cancer-related genes), and none from NMC2 were located in the protein-coding sequences. These suggest that the functional contribution of somatic point mutations for neoplastic transformation is limited in the NMCs.

Discussion

Chromoplexy is a unique pattern of complex genomic rearrangement first described in prostate cancers. Although the detailed molecular mechanism is currently unknown, it is notable that the chromoplexy frequently involves cancer driver genes in some cancer types, for example, ~60% of prostate cancer cases harbor chromoplexy affecting one or more prostate cancer-related genes including TMPRSS2ERG rearrangements [17]. In the present study, we found chromoplexy events generating the bona fide oncogene BRD3/4NUT although not in all cases. It warrants further investigation whether previously described NMC cases with a non-canonical karyotype also involve chromoplexy in their BRD3/4NUT rearrangements. We speculate that the involvement of a large number of DNA segments with highly expressed genes and remarkable absence of segmental loss may suggest the pathogenesis related with transcriptional process and involvement of recombinogenic enzymes as described for TMPRSS2ETS rearrangements in a previous study [24]. Frequent involvement of cancer-related genes in chromoplexy may suggest this could be a common, and efficient way to generate driver oncogenic rearrangements, although they look highly complex.

It has been postulated that the origin cell of NMCs might be the primordial epithelial cells, most prevalent in the late teenage years in the midline anatomical structures [19]. If this is the case, our findings suggest that these primordial cells are likely to acquire many point mutations from endogenous cellular processes. However, the neoplastic transformation is mainly due to the pathognomonic NUT rearrangements obtained by a stochastic, single catastrophic process.

From the whole-genome and transcriptome sequencing of the NMC cases, this study provides insight on the pathogenesis of this devastating disease. Sequencing of a large cohort of NMCs may not be feasible because of the rarity of this tumor and also because of the unavailability of fresh tissues which are optimal for high-throughput sequencing. International collaborative efforts will be necessary for the comprehensive understanding of the pathogenesis and for the development of effective therapeutic modalities.

Funding

This work was supported by Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), which was funded by the Ministry of Health & Welfare (HI14C1234; T.M.K., HI16C2387; Y.S.J.), a fellowship from the National Research Foundation of Korea (NRF-2013H1A2A1032691; J.K.L.), The Molecular Cytogenetics Core Facility at the Wellcome Trust Sanger Institute and Sanger investigators are funded by the Wellcome Trust (grant number WT098051; F.Y.). The dataset of this study is available at European Genome-phenome Archive (EGA; https://www.ebi.ac.uk/ega/home) with accession number EGAS00001001934.

Disclosure

The authors have declared no conflicts of interest.

Supplementary Material

Supplementary Data

Footnotes

Both authors contributed equally to this work.

References

1. Kees 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: 459–464. [PubMed]
2. Kubonishi I, Takehara N, Iwata J et al. Novel t(15;19)(q15;p13) chromosome abnormality in a thymic carcinoma. Cancer Res 1991; 51: 3327–3328. [PubMed]
3. French CA. Pathogenesis of NUT midline carcinoma. Annu Rev Pathol 2012; 7: 247–265. [PubMed]
4. Bauer DE, Mitchell CM, Strait KM et al. Clinicopathologic features and long-term outcomes of NUT midline carcinoma. Clin Cancer Res 2012; 18: 5773–5779. [PMC free article] [PubMed]
5. Travis WD, Brambilla E, Nicholson AG et al. The 2015 World Health Organization classification of lung tumors: impact of genetic, clinical and radiologic advances since the 2004 classification. J Thorac Oncol 2015; 10: 1243–1260. [PubMed]
6. French CA, Miyoshi I, Kubonishi I et al. BRD4-NUT fusion oncogene: a novel mechanism in aggressive carcinoma. Cancer Res 2003; 63: 304–307. [PubMed]
7. French CA, Ramirez CL, Kolmakova J et al. BRD-NUT oncoproteins: a family of closely related nuclear proteins that block epithelial differentiation and maintain the growth of carcinoma cells. Oncogene 2008; 27: 2237–2242. [PubMed]
8. Alekseyenko AA, Walsh EM, Wang X et al. The oncogenic BRD4-NUT chromatin regulator drives aberrant transcription within large topological domains. Genes Dev 2015; 29: 1507–1523. [PubMed]
9. Grayson AR, Walsh EM, Cameron MJ et al. MYC, a downstream target of BRD-NUT, is necessary and sufficient for the blockade of differentiation in NUT midline carcinoma. Oncogene 2014; 33: 1736–1742. [PMC free article] [PubMed]
10. Reynoird N, Schwartz BE, Delvecchio M et al. Oncogenesis by sequestration of CBP/p300 in transcriptionally inactive hyperacetylated chromatin domains. EMBO J 2010; 29: 2943–2952. [PubMed]
11. French CA, Rahman S, Walsh EM et al. NSD3-NUT fusion oncoprotein in NUT midline carcinoma: implications for a novel oncogenic mechanism. Cancer Discov 2014; 4: 928–941. [PMC free article] [PubMed]
12. Stathis A, Zucca E, Bekradda M et al. Clinical response of carcinomas harboring the BRD4-NUT oncoprotein to the targeted bromodomain inhibitor OTX015/MK-8628. Cancer Discov 2016; 6: 492–500. [PMC free article] [PubMed]
13. Kuzume T, Kubonishi I, Takeuchi S et al. Establishment and characterization of a thymic carcinoma cell line (Ty-82) carrying t(15;19)(q15;p13) chromosome abnormality. Int J Cancer 1992; 50: 259–264. [PubMed]
14. Ku JL, Kim KH, Choi JS et al. Establishment and characterization of six human lung cancer cell lines: EGFR, p53 gene mutations and expressions of drug sensitivity genes. Cell Oncol (Dordr) 2011; 34: 45–54. [PubMed]
15. Stephens PJ, Greenman CD, Fu B et al. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell 2011; 144: 27–40. [PMC free article] [PubMed]
16. Rosenthal R, McGranahan N, Herrero J et al. DeconstructSigs: delineating mutational processes in single tumors distinguishes DNA repair deficiencies and patterns of carcinoma evolution. Genome Biol 2016; 17: 31.. [PMC free article] [PubMed]
17. Baca SC, Prandi D, Lawrence MS et al. Punctuated evolution of prostate cancer genomes. Cell 2013; 153: 666–677. [PMC free article] [PubMed]
18. Helleday T, Eshtad S, Nik-Zainal S. Mechanisms underlying mutational signatures in human cancers. Nat Rev Genet 2014; 15: 585–598. [PubMed]
19. French C. NUT midline carcinoma. Nat Rev Cancer 2014; 14: 149–150. [PubMed]
20. Nik-Zainal S, Davies H, Staaf J et al. Landscape of somatic mutations in 560 breast cancer whole-genome sequences. Nature 2016; 534: 47–54. [PMC free article] [PubMed]
21. Alexandrov LB, Nik-Zainal S, Wedge DC et al. Signatures of mutational processes in human cancer. Nature 2013; 500: 415–421. [PMC free article] [PubMed]
22. Alexandrov LB, Jones PH, Wedge DC et al. Clock-like mutational processes in human somatic cells. Nat Genet 2015; 47: 1402–1407. [PMC free article] [PubMed]
23. Blokzijl F, de Ligt J, Jager M et al. Tissue-specific mutation accumulation in human adult stem cells during life. Nature 2016; 538: 260–264. [PubMed]
24. Haffner MC, Aryee MJ, Toubaji A et al. Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements. Nat Genet 2010; 42: 668–675. [PMC free article] [PubMed]

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