BMC Cancer 2009, 9:113 http://www.biomedcentral.com/1471-2407/9/113 point to a specific lineage ε belonging to haplogroup F3b that thrives in Taiwan [27], whereas the mutation pair for C16256T, C16270T testifies to a frequent European line- age ζ from haplogroup U5a. Since the former mutations are reflected in the mtDNA from buccal cells and the latter mutations in the mtDNA from lymphocytes, the presence of these six mutations could be explained by a single sam- ple-mixing event in the laboratory: contamination of the buccal sample ζ with the buccal sample ε at the amplifica- tion step for the "Dloop1" fragment. The cartoon of Figure 2 thus reconstructs a possible scenario that could explain this contamination event. Given the amplicon ranges [26] and the frequencies of the changes observed, one can attempt to provide a most par- simonious explanation of the mutational pattern. Besides the "Dloop1" amplicon, the long amplicon "GR" (9827– 10629) covers several mutations from lineages α, β, γ, and δ. The amplicon "ND5.2" (12949–13738) covers muta- tions from lineages α, β, and δ. Three further amplicons then cover 930, 4529, 15884 and 15944del, respectively. This means that a few amplicons have been drawn from the wrong lineages in at least four cases (assuming the simplest scenario that the order of the nucleotides at 10034 and 13105 should rather be reversed in that table). The precise recombination events are impossible to recon- struct unambiguously in the absence of haplotype infor- mation but the signal for artificial recombination is beyond doubt. Moreover, we cannot assume that all dis- tinguishing mutations were actually recorded, since it has been stated that TTGE can detect many but not all muta- tions [28]. The expected percentage of missed mutations in the case of mtDNA does not seem to have been deter- mined systematically. Therefore the full amount of con- tamination and falsely assigned amplicons may be larger here, especially with amplicons that are distinguished only by a single mutation. The pattern of lineage mixing that nonetheless emerges from our Figure 3 gets indirect support from Table 1 of Tan et al. [12] in that the most dramatic amount of alto- gether 40 differences is clustered in just 8 of the 42 smok- ers. This, however, does not imply that 1–3 mtDNA alterations between matched samples would not be suspi- cious. For instance, the alterations other than A2444C recorded for the non-smokers could all signify the con- trast between haplogroup L2 and other lineages. The number of wrongly assigned amplicons in the non- smoker group likely did not exceed one per individual. An artifactual status of the findings in Tan et al. [12] is also reflected by the extremely uneven distribution of seeming mtDNA alterations across the different amplicons, which is a typical feature of mixing up different mtDNA lineages at some but not all amplification steps. Another ampli- con-specific feature is the seeming length variation of homopolymeric A or C tracts, which was recorded only for the amplicon "SD" (7234–7921), although several other notorious tracts exist elsewhere in the molecule. Conclusion Neither of the two studies published in the journal Car- cinogenesis which we have reassessed here can provide any convincing indication of an increased amount of somatic point mutations in smokers or cancerous tissues, since the reported variation shows the typical imprint of mosaicism of amplicons caused by inadvertent contami- nation and sample confusion Once a problematic mtDNA study is published, the authors of the study and the reader are in very unequal positions: Whereas the former possess the samples and know all the data on which the article was built, the unconvinced reader who has to make do with the meagre portion of the data that is revealed in the article can only base his arguments on reconstructed haplotypic data (as in the two cases that we have re-examined here). Artifactual patterns differ from the natural ones in that they usually mirror basal parts of the mtDNA phylogeny whence they can easily be detected by comparison with published mtDNA classification trees [29]. In contrast, the natural instabilities are expected to result from a stochas- tic mutation process that randomly hits nucleotide posi- tions along the mtDNA molecule and does not just run down and climb up the mtDNA phylogeny. On the other hand, one cannot a priori preclude the possibility that some damage may exist in the form of an elevated amount of large deletions in mtDNAs of exposed cells. However, results would always come under suspicion of experimen- tal error and thus warrant revision whenever single exposed tissues or tumoral samples showed an atypical pattern of (seeming) instabilities. It is then not only the kind of mutation involved (whether highly or lowly mutable), but also the combination of these mutations in haplotypes and, moreover, the multiple appearance of such 'yin-yang' haplotypes (sensu e.g. [30,31]) in several individuals, as in the case of Prior et al. [11], that render the results highly dubious. A study on brain tumors that compared matched cancer- ous/non-cancerous tissues and was executed under foren- sic sequencing conditions [3,32] did not find any remarkable mtDNA alterations: the vast majority of changes in the two hypervariable segments of the control region concerned the homopolymeric C tract, and in no case among 69 patients was more than one substitution found [33]. A similar pattern can also be seen in the study on esophageal cancer by Tan et al. [34] (which was dupli- cated from [35]): in the entire mtDNA of 20 matched Page 4 of 8 (page number not for citation purposes)