¹Department of Dermatology, University Hospital Maastricht, Maastricht, The Netherlands. E-mail: mauricevansteensel@gmail.com

TO THE EDITOR

It was with considerable surprise that we read the recent publication by Shulman et al. (2006) in the March 2006 issue of the JID. In that, the authors employ X-inactivation and loss of heterozygosity (LOH) studies at 9q22.3 to support their hypothesis that basal cell carcinomas (BCCs) arising at distinct anatomical sites in one affected individual are of monoclonal origin (Shulman et al., 2006). We think that their interpretation and experimental approach are largely invalid, mainly for three reasons.

First, BCC grows slowly and rarely or never metastasizes (Williams, 2003). This latter fact and common sense make it highly unlikely that this tumor would discontinuously spread to anatomically distinct body sites far beyond its point of origin. If multifocal BCCs could indeed arise as the result of separate LOH events in a single tumor cell clone, the entire anatomical region encompassing the different sites would be expected to carry a PTCH mutation or other pre-existent genetic defect (Reifenberger et al., 2005). For PTCH, this would constitute a type 2 segmental manifestation of basal cell nevus syndrome, analogous to what has been recently described for other autosomal dominantly inherited genodermatoses as, for example, Hailey–Hailey disease (Poblete-Gutierrez et al., 2004). The authors do not deliver any such molecular evidence to strengthen their argument. Patients should have been screened for the presence of germline PTCH mutations.

Second, the way in which the authors designed their LOH studies seems inadequate to us in proving their theory. We do not dispute that the chromosomal region on 9q22.3 harboring the PTCH gene plays a crucial role in any LOH study addressing BCC, but the PTCH gene is outside of the genomic interval defined by STSA002A37 and D9S287 (Figure 1). Therefore, LOH at one or both markers does not necessarily imply LOH of the PTCH gene and may be entirely irrelevant to tumor development. The gene itself should have been sequenced in tumor material. Finding recurrent mutations in distinct BCCs would have supported the authors' hypothesis.

Figure 1.

Marker location relative to the PTCH gene. (http://genomce.ucsc.edu).

Full figure and legend (494K)

Additionally, the fact that the same two polymorphic markers on 9q22.3 studied by Shulman et al. are lost in 20 out of 30 tumors does not prove that all these BCCs are of monoclonal origin. We believe that chance loss of the same allele in 20 out of 30 tumors is quite possible, particularly when considering that this merely occurred in seven patients out of 15, with one of those patients accounting for no less than four out of 20 tumors studied. The two markers are at 2 Mb distance (Figure 1). Thus, it is not possible to exclude that chance recombination events have contributed to Shulman et al.'s findings. Additional markers should have been typed to resolve this matter. Also, the authors did not consider that the genomic structure of the region in which the markers reside could cause preferential loss of one marker. LOH predominantly occurs at D9S287. When looking at the sequence, this marker is close to a LINE repeat. It is known that such genomic elements will increase the frequency of recombination (Zhou et al., 2001), which may explain the observation of preferential LOH at D9S287. In addition, the authors did apparently not take into consideration that other genes and chromosomal loci have already been demonstrated to be important players in the molecular pathogenesis of BCCs (Ling et al., 2001; Reifenberger et al., 2005).

Third, we think that the pattern of X-inactivation is neither a reliable nor a convincing indicator of clonality, although we are aware that X-inactivation based clonality assays for evaluation of human epithelial tumors have been utilized in the past and are well documented (for a review see e.g. Chuaqui et al., 1998). Extreme lyonization forms a considerable limitation of these assays, because it can mimic clonal derivation of cells (Kristiansen et al., 2005). Therefore, skewed lyonization in healthy females represents the major disadvantage of X-chromosome-based clonality assays as employed by Shulman et al. (Busque et al., 1996; El Kassar et al., 1998). Because most techniques are based on the difference in DNA methylation between active and inactive X-chromosomes, incomplete DNA digestion may occur, giving an unreliable clonality result (El Kassar et al., 1998). The X-inactivation pattern is established during early embryogenesis (Happle, 1985). Of note, the authors performed their studies in predominantly elderly women, with just two out of 20 patients being younger than 60 (Shulman et al., 2006). With aging, acquired skewing occurs in normal females and is present in 38% of women over the age of 60 years (Busque et al., 1996), whereas truly random lyonization is rare (El Kassar et al., 1998). Whether acquired skewing is a consequence of stem cell depletion, true clonal tumor expansion, growth advantage conferred by parental-specific X-chromosomes, or other unknown mechanisms has not been elucidated yet. Unfortunately, the authors do not address the putative influence of skewing on the outcome of their study, although the broad range of variation conferred by this mechanism might provide a better explanation for their data. A further disadvantage of X-chromosome inactivation studies of tumors is that they are only informative when interpreted in the context of the clonal composition of the surrounding normal tissue and correlated with an adequate sample number of normal control tissue from age-matched individuals (Novelli et al., 2003). We feel that the latter aspect was also insufficiently addressed by Shulman et al. because they did not incorporate age-matched controls for lyonization. Another important factor, often ignored in such studies, is the distribution of X-inactivated cells in tissues. Taking into consideration that lyonization occurs early in development, many of the progeny of a single embryonic stem cell are grouped together in the adult, forming patches. As polyclonality can only be demonstrated at the borders of X-inactivation patches, not only the patch size is crucial in determining the chance of demonstrating polyclonality but also the number of tumors that need to be examined to exclude polyclonality (Novelli et al., 2003) – one more aspect not considered by the authors.

In conclusion, we believe that the authors did not deliver sufficient evidence to support their theory that anatomically and temporally distinct BCCs can originate from one single tumor cell clone.