Exome and genome sequencing of nasopharynx cancer identifies NF-κB pathway activating mutations

Abstract

Nasopharyngeal carcinoma (NPC) is an aggressive head and neck cancer characterized by Epstein-Barr virus (EBV) infection and dense lymphocyte infiltration. The scarcity of NPC genomic data hinders the understanding of NPC biology, disease progression and rational therapy design. Here we performed whole-exome sequencing (WES) on 111 micro-dissected EBV-positive NPCs, with 15 cases subjected to further whole-genome sequencing (WGS), to determine its mutational landscape. We identified enrichment for genomic aberrations of multiple negative regulators of the NF-κB pathway, including CYLD, TRAF3, NFKBIA and NLRC5, in a total of 41% of cases. Functional analysis confirmed inactivating CYLD mutations as drivers for NPC cell growth. The EBV oncoprotein latent membrane protein 1 (LMP1) functions to constitutively activate NF-κB signalling, and we observed mutual exclusivity among tumours with somatic NF-κB pathway aberrations and LMP1-overexpression, suggesting that NF-κB activation is selected for by both somatic and viral events during NPC pathogenesis.

Figure 1. Somatic mutation rates and mutational signatures of NPC tumours identified by WES.

(a) NPC tumours have significantly lower rate of non-synonymous mutations as compared with HPV-positive HNSCC and EBV-positive stomach adenocarcinoma (TCGA, USA). (b) The mutational burden, stratified by the top, mid and low quartile counts per patient, correlated with overall and disease-free survival in NPC patients (N=70 NPC patients with primary tumours). (c) Mutation signatures of 70 primary and 27 recurrent or metastatic NPC cases reveal a primarily C to T transition signature. (d) The top COSMIC cancer mutation signatures in NPC.

Figure 2. The genomic landscape of NPC.

For each patient (each column), recurrently altered genes (rows) with mutations and CNVs are shown. Significantly mutated genes (identified using the MutSigCV 2.0 algorithm; q<0.1, left panel) are ordered by q value, with additional genes near significance are also shown. Recurrent gene copy changes across the patients are summarized on the right panel. For each patient, the number of mutations and CNVs are shown on the top panels, as well as the sample origin, latent-membrane protein 1 (LMP1) expression, smoking status, clinical staging and tumour type.

Figure 3. Recurrent somatic mutations in NPC.

(a) Mapping of mutation sites of CYLD, TRAF3, NFKBIA, NLRC5, TP53, HLA-A, MED12L, NRAS and PIK3CA from this NPC cohort. Functional domains of the altered proteins are based on UniProt database. (b) Non-synonymous mutations in chromatin remodelling genes and PI3K/MAPK activating genes in NPC. Primary NPCs are shown in light blue boxes, local recurrent and metastatic tumours in pink and green boxes, respectively.

Figure 4. Copy number alterations in NPC.

(a) By WES, global chromosomal gains (shown in red) and losses (shown in blue) across 97 NPC exomes from unique Asian NPC patients (rows) show recurrent arm-level CNV events. (b) Genome-wide view of copy number aberrations in 15 NPC tumours with WGS. Homozygous deletion regions containing cancer genes such as CDKN2A, FHIT, SMARCC1, CYLD are indicated. Amplification of 11q13 containing CCND1 is shown in HKNPC-075T and HKNPC-084T.

Figure 5. CYLD and TRAF3 alterations in NPC.

(a) Additional genetic alterations of CYLD and TRAF3 in NPC detected by fluorescent in-situ hybridization (FISH). Left: Five additional cases of CYLD rearrangements and 1 case of CYLD homozygous deletion were identified by FISH using a CYLD break-apart probe and 1 case of CYLD tandem repeat was confirmed by Sanger sequencing (Supplementary Figs 7 and 8). Right: Seven additional cases of TRAF3 gene rearrangements were also identified using TRAF3 break-apart probes. (b) Left: CYLD gene rearrangement as revealed by WGS. A circos plot showing the CYLD gene rearrangement (HKNPC-088T, confirmed by FISH). A scale bar representing 1 μm is shown in all FISH pictures. Right: TRAF3 gene rearrangement as revealed by WGS. A circos plot showing the TRAF3 gene rearrangement (HKNPC-089T, confirmed by FISH). (c) Transient transfection of CYLD wild-type (WT) gene into an EBV-positive NPC cell line, C666-1 resulted in significant growth inhibition at day 5 (7 × 10⁴ cells in 4% FBS, P=0.0022, n=6). Similar results were obtained in three independent experiments. Expression of CYLD mutants (p.S600F and frameshift mutant p.AL527fs) revealed a loss-of-function of the tumour suppressive activity versus CYLD WT gene in C666-1 cells. CYLD WT and mutant protein expressions are shown underneath. (d) CYLD WT expression, but not the mutants, inhibited the anchorage-independent growth ability of C666-1 cells in soft-agar colony formation assay. C666-1 cells were infected with retroviral vectors expressing the CYLD WT gene and the CYLD mutants. Colonies were stained with 0.1% indonitrotetrazolium chloride and counted, and presented as a bar graph, which showed cumulative results of five independent experiments (n=15). (e) CYLD WT expression was able to reduce NF-κB activity in C666-1 cells in complete culture medium containing 10% FBS. Expression of CYLD mutants was not able to suppress NF-κB activity in C666-1 cells. The NF-κB activity was measured using the luciferase/Renilia assay (Promega, USA) with the Cignal NF-κB Pathway Reporter gene system (Qiagen, USA). A cumulative plot of five independent experiments (total n=19) showing changes in NF-κB-dependent luciferase activity in CYLD stable versus EGFP-Ctrl cells.

Figure 6. Genomic aberrations in NF-κB pathways in NPC.

Mutual exclusivity between LMP1 overexpression and NF-κB somatic alterations in NPC tumours (P=0.00014). LMP1 staining of representative tumours are also shown.

Figure 7. Genomic aberrations of MHC class I genes in NPC.

(a) A summary of genetic alterations in MHC class I genes, including HLA-A, HLA-B, B2M and NLRC5. (b) NPC patients with MHC class I gene mutations have poorer overall survival and disease-free survival (P=0.003, and P<0.0001, respectively; Log-rank test on 70 primary NPC cases).

Figure 8. Pathway diagram summarizing deregulation of signalling pathways and transcription factors in NPC.

Key affected pathways, components and inferred functions, are summarized in the main text. The frequency (%) of genetic alterations for NPC tumours are shown. Pathway alterations including homozygous deletions, amplifications and somatic mutations are shown. Activated and inactivated pathways/genes, and activating or inhibitory symbols are based on predicted effects of genome alterations and/or pathway functions.