BMC Cancer 2009, 9:113 http://www.biomedcentral.com/1471-2407/9/113 kFMniugotuwartnieopn3asthliswteaydsbiyn Tthaen mett DalN.[A12p]htyhlaotgjeoninytly highlight well- Mutations listed by Tan et al. [12] that jointly high- light well-known pathways in the mtDNA phylogeny. For symbols, see legend to Figure 1. Recurrent mutations are underlined. when reporting the results from our study on mtDNA mutations in OSCC. PCR products were sequenced multi- ple times (in both directions) and only consistent muta- tions described. When we submitted our manuscript to Carcinogenesis one of the reviewers instructed that, before mutations could be authenticated, PCR products would have to be generated using high fidelity PCR to minimize the likelihood of error. Thus, despite having already repli- cated PCR and sequencing a number of times for each sample we had to confirm our results by repeating this process once more and further submit this data. The sec- ond reviewer demanded that we 'repeat PCR and sequenc- ing of all positive samples independently to exclude the possibility of contamination'. Thus, the mutations we reported were done so with a high degree of confidence and we can clearly state that Carcinogenesis demonstrated a high degree of caution prior to acceptance of our results. Our sequence data were available at all times during the review process. In our study of OSCC cases six patients (out of thirty) individually showed a single base substitution between tumour and normal tissue within a 473 bp region of the ND2 gene. These mutations were observed at three nucle- otides: 4580 (1 patient), 4769 (1 patient) and 4197 (4 patients). The frequencies of these mutations and pattern, according to Bandelt and Salas, reflect a hypothetical sam- ple mixing. They conclude that 'it is very unlikely that the recorded haplotypic variation presents authentic somatic variation, which would be expected to hit positions from the entire fragment randomly'. This is a conclusion drawn on only a single nucleotide per sample and an assumption that the nucleotide target specificity of mutagens is sur- prisingly random. Bandelt and Salas go on to conclude that somatic mutations observed in the D-Loop of OSCC in our study are also due to sample mixing and that muta- tion patterns observed at nucleotides 146, 152 and 186 are incompatible with features of a 'natural mutational process'. We are intrigued as to what the natural muta- tional process may be in oral epithelial cells of cigarette smokers. They go on to state that 'the natural instabilities are expected to result from a stochastic mutation process that randomly hits nucleotide positions along the mtDNA molecule...'. If we adopt this rationale then we will always discard those legitimate somatic mutations that occur at polymorphic sites (it is ironic too that polymorphic sites are actually markers of mutation events sometime in the past). We are surprised that we are expected to believe that mtDNA somatic mutations would be found at random positions in the genome particularly when the genome of a smoker is exposed to constituents of cigarette smoke. Cigarette smoke contains over 4000 chemicals [41] many of which are known to be mutagenic. There are literally thousands of publications for mutagenicity assays high- lighting the fact that mutagens cause base substitutions which do not appear randomly in a target gene but dem- onstrate sequence specificity. As an example we consider the polycyclic aromatic hydrocarbon (PAH) carcinogen (±) anti-7β,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahyd- robenzo [a]pyrene (BPDE) found in cigarette smoke that preferentially targets CG dinucleotides (unmethylated and methylated) at a high rate in the supF gene using the supF mutational assay [42]. There is also a strong correla- tion between BPDE adduct sites and mutation hotspots in the TP53 gene in lung cancer [43]. A search of the litera- ture would reveal that many other mutagens specifically display other types of sequence context. The exposure of mtDNA to mutagens from cigarette smoke could thus result in a non-random mutation spectrum. The non-ran- Page 6 of 8 (page number not for citation purposes)