Characterization of genetic aberrations in a single case of metastatic thymic adenocarcinoma

A fresh-frozen tissue sample from a 31-year-old man with thymic adenocarcinoma was acquired by gun-biopsy. This study was approved by the Institutional Review Board (IRB) of Seoul National University Hospital (1206–086-414). The patient provided his written informed consent to participate in this study. The informed consent form included the publication of his clinical details and/or clinical images. Genomic DNA and total RNA were extracted by using the QIAamp DNA Micro Kit (Qiagen, Valencia, CA, USA) and PureLink RNA mini kit (ambion), respectively. Genomic DNA from the peripheral blood of the patient was used as a matched normal, which was extracted by the Maxwell 16 LEV Blood DNA Kit (Promega, Madison, WI, USA).

Genomic DNA was randomly fragmented (250-300 bp fragments) and amplified by ligation-mediated polymerase chain reaction (PCR). For WES, DNA was captured by the SureSelectXT Human All Exon V4 + UTR 71 Mb kit (Agilent Technologies, Santa Clara, CA, USA). Libraries for WTS were prepared by the TruSeq RNA Sample Preparation kit (Illumina, San Diego, CA, USA). WES and WTS were performed by the Illumina Hiseq2000 platform (Illumina) and generated 101 bp pair-end reads.

Varscan2 [22] and Mutect [23] were used to identify somatic mutations from WES data. First, we obtained the raw output of somatic single nucleotide variants (SNVs) and indels using Varscan2, and then applied the high-confidence and false-positive filters with options of a variant frequency >=10% and variant read counts >4. In addition, variants in base repeats greater than five were filtered out. Second, we used Mutect to find somatic SNVs and false-positive SNVs were removed by the internal variant filter. Third, we selected somatic SNVs that were detected by both Varscan2 and Mutect while somatic indels were selected independently from Varscan2.

Next, we selected nonsynonymous variants within exonic, splicing and ncRNA exonic regions. All the variants were annotated by ANNOVAR [24]. To prioritize functional variants, mutations within a non-conserved region were filtered out (phastConsElements46way). Common single nucleotide polymorphisms (SNPs) were removed by annotating in 1000 Genomes Projects and dbSNP. False positives by paralogous alignments were filtered out using genomicSuperDups. Lastly, cancer census mutations were annotated using COSMIC (cosmic68).

We utilized two pipelines for copy number analysis using WES data: Varscan2 followed by a circular binary segmentation (CBS) [25] and EXCACVATOR [26]. From Varscan2, we obtained raw bins with a size of 50 - 200 bp, which were delimited by CBS with options of alpha = 0.01, nperm = 10,000, and undo.SD = 3. Next, we applied an additional merging step in which we first excluded focal noises predicted by fewer than 25 copy number bins and then we iteratively merged adjacent segments within the copy number difference < 0.2. For EXCAVATOR, we made targeted regions by merging all the probes of the SureSelectXT Human All Exon V4 + UTR 71 Mb kit. Targeted regions with less than 30X coverage were excluded. We called the HSLM algorithm with options of 1) Omega = 0.1; 2) Theta = 1e-3; and 3) D_norm parameter = 10e6. Segments supported by fewer than ten exons were filtered out.

After the initial segmentation steps of Varscan2 and EXCAVATOR, we classified segments into large-scale somatic copy number alterations (SCNAs; >25% of a chromosome arm) and focal SCNAs (<=25% of a chromosome arm). We used copy number thresholds of −0.25 and 0.3 for large-scale SCNAs and −0.8 and 0.9 for focal SCNAs. We only selected large-scale SCNAs and focal SCNAs (>=10 exons) that were detected by both pipelines. Focal SCNAs (<10 exons) were just estimated using the Varscan2 pipeline because EXCAVATOR has a lower sensitivity for regions with a small number of exons. The 3′ UTR-biased discoveries with a high fraction (>60%) of copy number bins in a 3′ UTR region which was not properly targeted by WES were further filtered out.

Gene fusion analysis using WES data was performed on regions captured by the SureSelectXT Human All Exon V4 + UTR 71 Mb kit (>30X coverage). We used FACTERA [27] for inter-gene fusions with options of minimum split reads = 30 and minimum discordant reads = 10. We removed fusion events located in repeated regions from RepeatMasker and GenomicSuperDups. To detect fusion transcripts, we used defuse [28] with default options and filtered out fusion events supported by fewer than 50 split reads. Transcript fusion events located in repeated regions were also excluded. The read-through event was further confirmed by using MapSplice [29].

We validated seven somatic mutations (six SNVs and a small insertion) and a gene fusion event via Sanger sequencing. Genomic positions and designed primers were summarized in Additional file 1: Table S1. PCR was implemented using h-Taq DNA polymerase (genes with somatic mutations) and Dr. MAX DNA polymerase (the fusion gene) on DNA Engine Tetrad 2 Peltier Thermal Cycler. PCR products were purified and prepared for Sanger sequencing with the BigDye Terminator v3.1 Cycle Sequencing Kit. They were sequenced by the ABI PRISM 3730XL Analyzer.

A 31-year-old man presented with chest tightness and discomfort for two weeks’ duration. Positron emission tomography/computed tomography (PET/CT) revealed an anterior mediastinal mass with metastatic lesions at the left supraclavicular lymph node and pelvic bone (Fig. 1a). Chest X-ray and CT showed the 11.9 cm × 5.7 cm main mass occupying the anterior mediastinum (Fig. 1b, c). Serum levels of CEA and CA19–9 were elevated (37.5 ng/ml and 64.9 U/ml, respectively), although AFP and beta-hCG were normal. Abdominal CT additionally excluded an upper gastrointestinal origin. He was treated with a combination chemotherapy, which included doxorubicin, cyclophosphamide, and cisplatin. And then he was treated with another combination regimen consisting of etoposide, ifosfamide, and cisplatin. After three months since the start of first treatment, systemic metastases were developed in the thoracic and lumbar spine, mediastinal lymph nodes, and pelvis (Fig. 1d). The main mass was enlarged with metastatic lung nodules (Fig. 1e). The patient received laminectomy because of the significant cord compression by tumor extension at the T10 and L2 vertebrae (Fig. 1f). CD117 (c-kit) staining was positive in metastatic bone tumors, indicating metastasis from the thymic carcinoma origin.

A needle biopsy confirmed the involvement of carcinoma with extracellular mucin pools and mucin-producing epithelial cells. (Fig. 2a, b). IHC staining was negative for TTF1 and HER2 (Fig. 2c, d), but positive for CDX2, CK7, and CK20 (Fig. 2e–g). Focal positivity for CD5 was shown in lymphoid cells (Fig. 2h). These histological features and IHC staining patterns were consistent with previous reports of thymic mucinous adenocarcinoma.

From WES data of the patient, we detected total 55 somatic mutations in exonic regions and 23 mutations of them were synonymous SNVs (Additional file 2: Table S2). Functional mutations were prioritized by several reducing processes (see Methods). As a result, we obtained 19 SNVs and a 6-base pair insertion (Table 1). The 6-base pair insertion was found in FAT1. FAT1 also harbored a somatic SNV (G > A) near the 6-base pair insertion in the same allele (Additional file 3: Figure S1). FAT1 is a tumor suppressor gene involved in the Wnt signaling, and FAT1 inactivation has been reported to promote tumor growth [30]. Next, we investigated WTS data to find transcribed variants among 20 somatic variants. WTS supported seven somatic variants (APH1A, RNASEL, TNFSF15, NOL6, TNEM3, GZF1, and TP53), which showed similar allele frequencies between WES and WTS (Table 1). We validated six cancer-related genes (RNASEL, PEG10, TNFSF15, TP53, TGFB2, and FAT1) using Sanger sequencing (Additional file 4: Figure S2).

A C > A substitution encoding p.C176F in TP53 was detected with the highest allele frequency. The p.C176F mutation of TP53 was previously reported in the COSMIC database [31], which showed high occurrences of the p.C176F variant in thoracic tumors such as lung, esophagus, and upper aerodigestive tract (Additional file 5: Table S3). Besides the TP53 variant, an A > G substitution (p.V184A) and C > T substitution (p.R211C) were found in TNFSF15 and TGFB2, respectively, which are cancer-related immunocytokine genes. TNFSF15 encodes TNF-like ligand 1A (TL1A), which costimulates T cells with highly regulated expression [32]. TGFB2 is a member of the TGFB family, which has tumor promoting functions enhancing the epithelial-mesenchymal transition and evasion of immunity [33]. Especially, the p.R211C mutation occurred in the propeptide domain of TGFB2 (Additional file 6: Figure S3), which is responsible for maintaining the latency of TGFB2 [34]. Point mutations (R218C, C223R, and C225R) in the propeptide of TGFB1 have been reported to increase the active TGFB1 level [35, 36]. Similarly, the p.R211C mutation of TGFB2 might affect the latency of TGFB2.

We analyzed SCNAs using Varscan2 and EXCAVATOR (see Methods) and identified 14 large-scale SCNAs, which included four large-scale amplification (chr5p, chr12p, chr7, and chr20) and 10 large-scale deletions (chr3p, chr4p, chr6q, chr8p, chr9p, chr10p, chr15q, chr17p, chr21q, and chr16). Especially, chr4p, chr8p, and chr10p deletions and chr12p amplifications were noticeable (log2 ratio < −0.75 and > 0.75 each). Thymic carcinomas have shown more frequent arm-level SCNAs than thymomas [37, 38]. Similarly, thymic adenocarcinoma showed a considerable number of arm-level SCNAs.

Copy number analysis uncovered complex copy number states of chromosome 8 (Fig. 3a). With the loss of the whole p arm of chromosome 8 (log2 ratio = −0.77), the end of the q arm showed stepwise copy number increases. The most highly amplified region (log2 ratio = 1.89) included NDRG1 and MYC. With complex copy number states, an intrachromosomal rearrangement (head-to-tail, tandem duplication type) was found between MCM4 and SNTB1 (Additional file 7: Table S4). Breakpoints of the head-to-tail fusion (chr8:48,878,682 and 121,816,370) were found coincidently near copy number breakpoints (chr8:48,878,526 and 125,164,614) (Fig. 3a). The head-to-tail fusion with loss of chr8p possibly implied monosomy 8 with duplicated chr8q arms. The fusion sequence between MCM4 exon 8 and SNTB1 intron 1 was derived from the head-to-tail fusion (Fig. 3b). The Integrative Genomics Viewer (IGV) [39] showed more than 50 clipped reads at each SV breakpoint exclusively in the tumor sample (Additional file 8: Figure S4), which indicates that the fusion sequence was newly derived in the tumor and attained more than 100X coverage. The fusion sequence was further validated by Sanger sequencing (Fig. 3b). MCM4 is involved in the MCM2-MCM7 complex essential for DNA replication licensing, and a MCM4 mutation (F345I) was reported to cause mammary adenocarcinomas in mice [40]. The MCM4 fusion event may result in dysregulated DNA replication, while the function of the MCM4 fusion gene is not yet known.

We identified total five focal SCNAs (Additional file 9: Table S5). Three SCNAs out of the five focal SCNAs were detected in chr8q24.21–22, chr8q24.3, and chr6p21.32. (>10 exons). Two focal SCNAs were found in exon 1 to 5 of MUC16 and exon 6 of GPR112 (<10 exons) (Additional file 10: Figure S5). The focal deletion of chr6p21.32 was found with a slight copy number increase of chr6p (Fig. 4a). All the 29 copy number bins consecutively supported the focal deletion with a log2 ratio of −1.2664 (Fig. 4b), whose value suggests a single-copy loss. The deleted region included HLA-DR, HLA-DQ and DLA-DO alleles that encode HLA class II molecules, which are highly expressed on thymic epithelial cells in normal regulating CD4+ T cell immunity.

We investigated WTS data to identify transcription-mediated fusion genes. We found two fusion transcripts: FABP2-C4orf3 and CTBS-GNG5 (Additional file 11: Table S6). The sequence of the CTBS-GNG5 fusion transcript showed a junction exactly between the end of CTBS exon 6 and the beginning of GNG5 exon 3 (Additional file 12: Figure S6). The observation suggested an intergenic splicing from the read-through transcript between CTBS and GNG5. Splice-junction alignment confirmed 76 reads derived from the fusion between CTBS exon 6 and GNG5 exon 3 (Additional file 12: Figure S6). The same fusion sequence between CTBS exon 6 and GNG5 exon 3 has been found in several cancer types and its role in the growth inhibitory function has been demonstrated [41].

For functional enrichment analysis, we integrated somatic SNVs and indels, focal SCNAs, and fusion genes. Because focal SCNAs whose sizes were larger than 1 Mbp included an excessive number of genes, we only selected cancer-census genes (MYC and NDRG1) annotated by the COSMIC database from the focal SCNAs (>1 Mbp). Consequently, we obtained a total set of 39 genes (Additional file 13: Table S7). Using BiNGO [42] (BH-corrected, P < 0.00001), we found 52 GO terms in the biological process (Fig. 5a and Additional file 14: Table S8). Remarkably, 19 out of 39 genes (48.7%) were annotated to the immune system process (Fig. 5b). Most of the enriched GO terms were related with T cell signaling pathways. Additionally, KEGG and Reactome enrichment analysis [43] (BH-corrected, P < 0.00001) showed over-representations of autoimmune diseases, chronic infections, and immunological signaling pathways (Additional file 15: Table S9).

A recent study reported a higher incidence of somatic mutations in thymic carcinomas than that in thymomas (average of 43.5 and 18.4, respectively) [37]. Furthermore, another study reported that thymic carcinomas exhibited more somatic mutations than thymomas in cancer-related genes, especially in TP53 [44]. The case of thymic adenocarcinoma had 55 somatic mutations including the TP53 variant. The incidence of somatic mutations seems to be consistent with previous reports of thymic carcinomas. We compared somatic mutations in thymic adenocarcinoma with somatic mutations previously discovered in 32 thymic carcinomas (Fig. 6a) [37, 45]. Five genes (TP53, PBRM1, MYT1L, SPTA1, and FAT1) were recurrently mutated between thymic adenocarcinoma and carcinomas. Recurrent mutations in chromatin remodeling genes were reported in thymic carcinomas [45], but no mutation in chromatin remodeling genes (SETD2, KDM6A, MLL2, and MLL3) was found in thymic adenocarcinoma. In addition, we compared SCNAs between thymic adenocarcinoma and 35 thymic carcinomas previously analyzed by array CGH [37, 38, 46]. Five amplifications (chr5p, chr8q, chr12p, chr20p, and chr20q) and six deletions (chr3p, chr6q, chr9p, chr16q, and chr17p) were recurrent (>15%) in thymic carcinomas and adenocarcinoma (Fig. 6b). The focal MYC amplification was remarkable in thymic adenocarcinoma while thymic carcinomas showed broad chr8q amplifications. Chr6p and chr6q deletions, which have been recurrent in TETs [21, 47], were frequent (26 and 37% each) in thymic carcinomas. Instead of an arm-level deletion of chr6p, the focal deletion of HLA class II alleles with the slight amplification of chr6p was found in thymic adenocarcinoma, which was the same genotype with the other case of thymic adenocarcinoma [2].