Rationale and objectives
Cancers have a very heterogeneous mutation rate, which can vary by orders of magnitude among different tumor types. Usually, pediatric and hematological cancers have a low mutation rate while tumors influenced by mutagens (exposure to UV-light and tobacco, for example) are at the other end of the spectrum, with high mutation rates. Mutation rate also varies greatly among different tumors of the same type, and this is influenced by length of exposure to a given mutagen or by the particular mutated genes, for example. The mutational spectrums vary among different cancer types as well, and are related not only to the background mutational events happening throughout the life of a cell but also are highly influenced by mutagens and by the repair mechanisms that go into play when mutations arise. BCC is known to be highly influenced by UV-light induced mutagenesis and to have an extremely high mutation rate. Its mutational spectrum is dominated by C>T mutations in a pyrimidine context, the typical profile of UV-light.
The objective of this section of the study is to carry out a complete and accurate determination of the mutation rate in BCC using high confidence somatic variants only, and compare BCC’s mutation rate with that of other tumor types. Furthermore, we aimed at identifying the dominant mutational signatures in BCC and to perform a comparison with those of melanoma, another UV-light induced skin cancer, to identify BCC-specific features.
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We identified a total of 234,770 somatic coding mutations in our 293 sample set. In agreement with previous observations (Jayaraman et al., 2014, Sharpe et al., 2015) the average mutation rate was very high. BCC is, up to date, the tumor type with the highest mutation rate, with 65 mutations/Mb for sporadic tumors and 21 mutations/Mb for tumors of Gorlin syndrome patients. Figure 26 below shows how BCC compares based on mutation rate, in relation to other cancers studied as part of international projects. Its position is consistent with it being a tumor type caused by UV-light exposure, as it is in the far right along with melanoma and lung carcinomas, other tumors caused by the environmental
mutagens UV-light and tobacco, respectively (Lawrence et al., 2013). Tumors from Gorlin syndrome individuals have a lower mutation rate than sporadic tumors as expected, since patients with this condition are germline heterozygous for mutations predisposing to BCC (See introduction) and therefore already have a “first hit”. Only one somatic deleterious event is needed to initiate tumorigenesis in these patients while in sporadic BCCs, two hits are required.
Figure 26. Exonic mutation rate across 27 cancer types, sporadic and Gorlin syndrome BCCs. Each box corresponds to the distribution of mutation rates within a tumor type set (indicated in the x axis), obtained from Lawrence et al., 2013. Gorlin syndrome BCC and sporadic BCC data are from this study, 24 Gorlin syndrome tumors and 108 sporadic BCCs were used.
Tumors are ordered by their median mutation rate in increasing order.
116 of 299 2.2. Mutational profile of BCC
The mutational profile analysis of BCC was carried out in collaboration with Vladimir B.
Seplyarskiy and Dr. Maria A. Andrianova from Lomonosov Moscow State University, Moscow Russia.
The great majority of SNVs (90%) were found to be C>T mutations in a YpC or CpY context (where Y denotes C or T) and 5% of all mutations were dinucleotide substitutions. As previously reported (Jayaraman et al., 2014, Sharpe et al., 2015, Atwood et al., 2015b, Hodis et al., 2012, Martincorena et al., 2015), this type of mutational profile is compatible with UV-light induced mutagenesis in skin.
To study more in detail the mutational mechanisms in BCC, we compared it to cutaneous melanoma, another UV-light induced cancer (Cancer Genome Atlas Network, 2015). We specifically investigated the prevalence of UV-light and oxidative stress-induced mutagenesis as well as the effectiveness of transcription coupled repair (TCR) in BCC. The fraction of UV-light induced mutations is higher in BCC than in melanoma (90% vs 85%;
P<2.2x10-16, chi-square test). We estimated the fraction of mutations with highly specific UV-light signatures (Drobetsky and Sage, 1993, Marionnet et al., 1995, Alexandrov et al., 2013a) of the TpCpC > TpTpC and CpC>TpT type from all C>D and CpC>DpD mutations (where D denotes A, T or G). In BCC, mutations occurred on these contexts 2% (C>D, P<2.2x10-16, chi-square test) and 15% (CpC>DpD, P<2.2x10-16, chi-square test) more frequently than in the case of melanoma.
We then estimated the efficiency of TCR through the analysis of the ratio of UV-light induced mutations occurring on the transcribed or on the non-transcribed strand (Hanawalt et al., 1994). For TpCpC > TpTpC mutations, this ratio was lower in BCC than in melanoma (0.59 vs 0.71, P<2.2x10-16, chi-square test), and a similar pattern was observed for the genes expressed in both melanoma and BCC (0.56 vs 0.68, P<2.2x10-16, chi-square test), suggesting that TCR is more effective in BCC than in melanoma. Moreover, the effect of TCR was even stronger in BCC when only highly expressed genes were taken into account (0.21 vs 0.29, P<2.2x10-16, chi-square test) (Figure 27a).
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Figure 27. Mutational signatures in BCC compared to melanoma. a. The effect of transcription coupled repair as a function of gene expression in BCC. Tumor type specific gene expression levels were used for BCC (this study) and cutaneous melanoma from Guan et al., 2015.
Genes were grouped into 5 equal size bins by their level of expression. 95% confidence intervals for the ratio between the two binomial distributions were calculated using the R function riskscoreci(). b. Fraction of each nucleotide at the 5 prime position to C>A mutations. c.
Dinucleotide mutation frequencies in BCC and melanoma.
a b
c
C>A mutations, which may occur due to either oxidative stress (Besaratinia et al., 2007) or UV-light exposure (Brash, 2015) were investigated. We first selected a subset of C>A mutations in the RpCpR context (where R denotes A or G) that are only mildly affected by UV-light because cytosine residues in the RpCpR context are not involved in the formation of photodimers when exposed to UV-light (Hu et al., 2015). In this case, C>A changes may be enriched in mutations caused by oxidation of guanine. As expected, C>A mutations were enriched on the transcribed strand in both BCC and melanoma (1.26 and 1.27 times more, respectively; in both tumor types P<0.001, chi-square test), suggesting that mutations indeed occurred in guanines, which were then repaired by TCR on the transcribed strand.
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We next selected a subset of C>A mutations in CpC, the context of UV-light induced mutagenesis where these mutations are mostly enriched (Figure 27b). We have observed no statistically significant strand bias of C>A mutations in BCC (1.09, P=0.10, chi-square test) but conversely in melanoma there was a significant excess of C>A mutations on the non-transcribed strand (0.89, P<0.001, chi-square test). Considering the effect of TCR, it is possible that C>A mutations in a CpC context in BCC represent a mixture of mutations in cytosine and guanine residues while in melanoma, cytosine mutations are more frequent.
In line with this observation, the fraction of C>A mutations in the RpCpR context (depleted from UV-light induced mutations) was twice as high in BCC than in melanoma (21% in melanoma vs. 10% in BCC, while expectations are 19%). Furthermore, the fraction of C>A mutations in a CpC context (enriched in UV-light induced mutations) was 1.6 times higher in melanoma than in BCC (53% in melanoma vs. 35% in BCC, while expectations are 28%).
These data suggest that the mechanism of generation of C>A mutations is different between BCC and melanoma. In BCC, they predominantly occur due to oxidation of guanine, while in melanoma, the contribution of UV-light induced mutagenesis targeting cytosine residues is more important.
Additionally, we find that among dinucleotide mutations, the fraction of CpC>TpT mutations was over 80% in both BCC and melanoma but that the fraction of CpC>ApA dinucleotide mutations was 86-fold higher in melanoma (Figure 27c).
A summary figure showing the mutational profile of BCC in a per-sample basis can be found in Figure 28.
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Figure 28. Mutational spectrum of BCC.Each column corresponds to one sample. Each color corresponds to a different mutation type, as indicated below the figure (Y= C or T, R= A or G). The fraction of each mutation type can be found on the y axis. Sporadic BCCs are on the left and BCCs from Gorlin syndrome individuals to the right, the number of tumors inside each category used to produce this figure is in parenthesis.
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