signature=1c79d58d04498553c591967102afe603,Mutation signature analysis identifies increased mutation...

Tobacco mutation signatures associated with DNA adduct formation are significantly enhanced in laryngeal SCC compared with all other head and neck SCC

In our recent examination of mutation signatures in HNSCC arising in non-sun exposed sites, we observed striking sub-site specificity to the presence of tobacco smoke-associated mutations (COSMIC signature 4, the signature associated with tobacco exposure1A and Supplementary Fig. S1A). Larynx SCC have a significantly greater proportion of COSMIC signatures 4 and 5 compared with oral cavity and oropharynx SCC, as well as a concomitant significant reduction in signature 1 compared with oral cavity and oropharynx SCC, and a significant reduction in COSMIC signature 2 compared with oropharynx (Fig. 1A). No difference was seen in signature weight comparing oral cavity and oropharynx SCC. Overall, 53% of 278 TCGA (The Cancer Genome Atlas) HNSCC samples1B) for which 82% of larynx SCC (59/72 tumors) were positive, while only 44% of oral cavity or oropharynx SCC were positive (90/204 tumors). The total number of mutations was also significantly greater in larynx compared with the major anatomical sub-site classification for oral cavity and oropharynx, however, this number was greatly influenced by those larynx tumors which were positive for tobacco-associated mutations (signature 4, Fig. 1B,C and Supplementary Fig. S1B). When HPV status is considered there is a statistical difference between HPV positive and HPV negative HNSCC for total mutations, signature 4 and signature 5 mutations (Supplementary Fig. S2A), however this difference was dependent on larynx tumors where only 1 from 72 is HPV positive (Supplementary Fig. S2B,C). The number of tobacco-associated signature 4 mutations per tumor for those tumors that were signature 4 positive (>0 mutations attributed to signature 4) was significantly higher in larynx (mean 108 signature 4 mutations, n = 59) compared with other sub-sites (mean 15 signature 4 mutations, n = 90; oral cavity mean of 15.5 signature 4 mutations, oropharynx mean of 13.1 signature 4 mutations), or HPV positive tumors (mean 9.4 signature 4 mutations, n = 12) (Fig. 1B and Supplementary Fig. S2). Overall, mutation burden in larynx was greater than other sub-classes of HNSCC and this was dependent on those larynx samples with signature 4 mutations (Fig. 1 and Supplementary Fig. S2). Complete signature assignation per sample is presented as both total numbers and percentage/weight in Supplementary Fig. S3.

Figure 1

COSMIC signature 4 contributes to significantly higher mutation burden in larynx compared with oral cavity and oropharynx SCC. (A) Pie charts show the proportion of all single nucleotide mutations attributed to each of the six COSMIC mutation signatures identified in head and neck SCC for each of the three major sub-sites. n = total number of individual tumors for each sub-site. Signatures 1, 2, 4, 5, 13, and 18, are derived from version 2 of COSMIC mutational signatures. Signatures 1 and 5 are of unknown etiology and associated with age, signature 2 and 13 are associated with APOBEC mutagenesis, signature 4 is associated with tobacco smoke exposure, and signature 18 is associated reactive oxygen species. Matrix to the right of pie charts shows statistical significance of individual COSMIC signature weight (normalized to mutation number) comparing larynx with oral cavity and oropharynx tumors. No significance was seen comparing signature weight between oral cavity and oropharynx tumors for any of the six signatures. n.s. = no significance. (B) Box and whisker graphs show total number of single nucleotide mutations as well as those attributed to signature 4 and signature 5, identified in each of the major sub-sites of HNSCC. (C) Total mutations stratified by the presence (positive) or absence (negative) of signature 4 mutations. x = mean. *p 

Larynx SCC are dominated by smokers or recently reformed smokers compared with other sub-sites of head and neck SCC

We next compared the incidence of smoking within the TCGA HNSCC data set and between the three major sub-sites of HNSCC: larynx, oral cavity and oropharynx (only 2 hypopharynx samples are included and so were not analyzed). As might be expected, larynx SCC, where more signature 4 mutations are present, had a greater proportion of current smokers or recently reformed smokers compared with oral cavity and oropharynx (p 

Figure 2

Larynx SCC patients are dominated by smokers or recently reformed smokers and SCC from smokers have significantly more signature 4 and signature 5 mutations compared with non-smokers. Percentage of patients stratified by major anatomical sub-site who are either current smokers, recently reformed smokers (within 15 years), historically reformed smokers (for more than 15 years), or never smokers (A). Box and whisker plots show total mutations (B) and mutations attributed to individual signatures in HNSCC stratified by smoking status (C). Signatures 1, 2, 4, 5, 13, and 18, are derived from version 2 of COSMIC mutational signatures. Signatures 1 and 5 are of unknown etiology and associated with age, signature 2 and 13 are associated with APOBEC mutagenesis, signature 4 is associated with tobacco smoke exposure, and signature 18 is associated reactive oxygen species. x = mean. *p 

Signature 5 and signature 4 mutations correlate with smoking status

In line with previous analysis2C). Signature 5 mutation numbers also showed a stepwise reduction from historically reformed smokers (>15 years) and life-long never smokers suggesting a more direct relationship between signature 5 and smoking in HNSCC compared with signature 4 (Fig. 2C).

Signature 5 correlates with age in HNSCC non-smokers

Previous analysis has demonstrated an association with age and the number of signature 1 mutations in all HNSCCS4). We recently showed that tissue-damage associated SCC arising in the skin of patients with the rare blistering disease, recessive dystrophic epidermolysis bullosa (RDEB), show remarkable similarity to HNSCC at the level of mutation signature and transcriptomic analysis, and also show a correlation with age and mutation signature 5 numbers3A).

Figure 3

Signature 5 mutations shows positive correlation with age in non-smokers and are enriched in larynx SCC non-smoking patients who are older than non-smoking patients with oropharynx SCC. (A) Graphs shows the number of signature 5 mutations (y-axis) plotted against the age (x-axis) of all smokers (left graph, n = 170) or non-smokers (right graph, n = 101) with HNSCC. Pearson Correlation r and p values given. (B) Box and Whisker plots show the number of signature 5 mutations identified in HNSCC of non-smokers stratified by major anatomical sub-site. (C) Box and Whisker plots showing age of patients with HNSCC stratified by major anatomical sub-site in smokers (left graph) and non-smokers) right graph). Signature 5 is derived from version 2 of COSMIC mutational signatures, is of unknown etiology, and associated with age in certain cancers and somatic tissues. x = mean *p 

Larynx SCC non-smokers have significantly greater signature 5 mutations and are generally older compared with other HNSCC non-smokers

Comparing the number of signature 5 mutations in non-smokers (defined as lifelong non-smokers or historically reformed, >15 years), larynx had significantly more signature 5 mutations than either oral cavity or oropharynx (Fig. 3B). In contrast to smokers, non-smoking patients with larynx SCC (n = 11) were significantly older than non-smoking patients with oropharyngeal SCC (n = 14), while non-smoking patients with oral cavity SCC were approximately intermediate in age (n = 76) (Fig. 3C). No difference was seen with signature 1 mutations and major anatomical sub-site in non-smokers (data not shown).

Laryngeal SCC have significantly less Ki67 positive nuclei than oral cavity or oropharyngeal SCC

We next compared Ki-67 immuno-reactivity as a marker of tumor cell proliferation in similarly sized SCC excised from the larynx, oral cavity, and the oropharynx, from Thomas Jefferson University Hospital from 2015–2018. Comparison of tumors 2–4 cm in size showed oral cavity and oropharyngeal tumors had significantly greater Ki67 cell positivity per tumor when compared with laryngeal tumors (Fig. 4 and Supplementary Table S1).

Figure 4

Ki-67 immuno-reactivity is increased in oral cavity SCC and oropharynx SCC compared with larynx SCC. (A) Representative larynx SCC section incubated with anti-Ki = 67 antibodies and visualized using the Aperio platform. (B) Representative oral cavity SCC section incubated with anti-Ki = 67 antibodies and visualized using the Aperio platform. (C) Representative oropharynx SCC section incubated with anti-Ki = 67 antibodies and visualized using the Aperio platform. Bar = 400 μM. (D) Graph shows % Ki-67 positive tumor cells quantified using Aperio ImageScope for 19 larynx SCC, 22 oral cavity SCC, and 18 oropharynx SCC. Bar shows mean +/− standard deviation with individual points shown as circles. Blue indicates P16 positive samples. P16 not assessed in 2 larynx SCC and 11 oral cavity SCC. **p