It is well known that KAs as well as SCCs develop preferentially on sun-exposed areas of the skin, and that actinic keratosis as a result of sun damage is a precursor lesion for SCC (Elder et al., 2005
). The reported frequencies of malignant transformation within actinic keratosis are debated, but recent data indicate a significant frequency of transformation, possibly higher than 10% (Mittelbronn et al., 1998
). Genetic studies have demonstrated a high frequency of loss of heterozygosity in actinic keratosis (Rehman et al., 1994
). Contrary to SCCs, no such precursor lesion has been recognized for KAs, which undergo complete regression in almost all cases (Weedon, 2003
). There are thus distinct differences in biological characteristics of these two types of lesions that may be reflecting abrogations of different signaling pathways.
There are only few reports on KAs that have been subjected to CGH analysis (Ashton et al., 2005
), including those from our previous study on 70 lesions, of which 36.4% showed genomic aberrations (Clausen et al., 2002
). We therefore wanted to get a more comprehensive overview of the genomic alterations aquired by KAs and SCCs, analyzing and comparing a total of 132 KAs and 37 SCCs. In KAs the previously reported recurrent gain of genomic material from 9q (18.4%) and losses from 3p (18.4%) and from 9p (20.4%) were still seen in the extended material with high frequency, whereas previously reported gains from 1p and 8q in this study were reduced to 12.2% each. Our data from CGH analysis of 37 cutaneous SCCs is to our knowledge the largest data set to date. The recurrent aberrations in SCCs are mainly different from those in KAs, with gains from 1p (41.9%), 3q(25.8%), 8q(25.8%), 20q(25.8%), 19q(22.6%), and 19p(19.4%), and losses from 3p(19.4%) and 4p(16.1%) being the most common. Of recurrent aberrations only loss of 3p is seen to about the same extent in KAs and SCCs, whereas gains of 3q, 8q, and 19p to a lesser extent also are seen in KAs.
In addition to sporadic reports about limited numbers of cutaneous SCCs analyzed by CGH, one research group has analyzed 15 SCCs and 12 actinic keratosis by CGH (Ashton et al., 2003
). Their conclusion is that numerous genetic imbalances are shared by actinic keratosis and SCC, consistent with the former lesion being a precursor for SCC. The most frequently occurring imbalances of SCCs in our material, that is, gains of 3q, 8q, and 20q, and loss of 3p, are in accordance with results reported previously (Ashton et al., 2003
). Gain of material from 1p was the most recurrent aberration in our study. Scoring of imbalances in this region, however, may be challenged by the presence of artifacts, and it is known that many authors do not score imbalances here (Ashton et al., 2005
). This may suggest that imbalances on 1p in fact are more frequent than reported so far. To overcome the possibility of false results, we have used reverse hybridization experiments to confirm our data, that is, reversed fluorochrome labeling of test and control DNA (Kallioniemi et al., 1994
Many of the aberrations in SCCs found by CGH, that is, +3q, +8q, -3p, are consistent with reports from cytogenetic studies (Ashton et al., 2005
). The present analyses confirm and extend our previous data (Clausen et al., 2002
), showing that more than one-third (36.5%) of KAs harbor genetic imbalances, whereas more than twice as many SCCs (83.8%) showed such imbalances. We now also show that SCCs harbor on average about twice as many aberrations per lesion as KAs with detectable imbalances (6.45 versus
3.08, respectively), and that in addition to generally increased numbers, cutaneous SCCs also harbor significantly higher numbers of specific genetic aberrations than KAs (). The most significant difference is gain of chromosomal material from 20q, which was found in eight SCCs and in only one KA (P
-value 0.0017). Gains from 1p, 14q, and 16q, as well as losses from 4p were also observed significantly more frequently in SCCs than in KAs, whereas losses from 9p were seen significantly more frequently in KAs (Tables and , ). Furthermore, the patterns of recurrent aberrations were also different for the two types of lesions, as illustrated by the dendrogram in .
Our results clearly show that chromosomal instability is significantly more frequent in cutaneous SCCs than in KAs. The presence of different imbalances in the genome of the two types of lesions may be correlated to their different biological behavior, and suggests different pathogenetic pathways for their developments. This is also consistent with a previous study reporting that loss of heterozygosity was found more frequently in SCCs than in KAs, with fractional allelic losses of 32.0 and 1.3%, respectively (Waring et al., 1996
The most significant difference between genetic imbalances in KAs and SCCs in this study was the high degree of gains of 20q in SCCs. 20q is a region which is frequently amplified in breast as well as colorectal cancer (Tanner et al., 1994
; Ried et al., 1996
). Amplification of the consensus region 20q13 is seen in about 70% of colorectal cancers and is associated with increased S-phase fraction as well as bad prognosis (De Angelis et al
). Candidate oncogenes localized to 20q13 are ZNF217
, which is associated with immortality in mammary epithelial cells (Nonet et al., 2001
), and STK6/STK15
which codes for Aurora A, a kinase important for segregation of chromosomes during mitosis and which is also linked to tumorigenesis and development of aneuploidy (Goepfert et al., 2002
). Detailed genetic analysis on these genes will clarify their possible involvement in SCCs. Recurrent losses from 4p as well as from 4q are also seen in SCCs in the present study, and allelic losses from chromosome 4 arms have been shown to correlate with tumor aggressivness in colorectal cancer (Arribas et al., 1999
). Imbalances of the above-mentioned chromosomal regions are hardly ever seen in KAs, suggesting that such aberrations may affect genes inducing infiltrative and metastatic potential of SCCs. Interestingly, the most frequently occurring recurrent aberration in SCCs is gain on chromosome arm 1p, which also, although to a lesser extent, occurs in KAs. The protooncogene JUN
is located at 1p32-31 and encodes for a transcription factor that has been reported to be overexpressed in human cancers. JUN
regulates cell cycle progression as well as apoptosis, and it is therefore possible that proliferation of SCC as well as of KA may be stimulated through 1p amplification and subsequent JUN
overexpression. It is also possible that JUN
amplification in KAs may be related to apoptosis as demonstrated during regression. In contrast to the recurrent aberrations discussed above, loss of 9p was seen significantly more frequently in KAs than in SCCs. It is known that the tumor suppressor gene CDKN2A
) which is inhibiting cell cycle traverse, is located to 9p21. Many reports mention 9p deletion as an early aberration that is seen in histomorphologically normal tissues, including nasopharyngeal mucosa, and premalignant tissues (Chan et al., 2002
; Stoehr et al., 2005
). Thus, 9p deletion may be associated with growth advantage in areas with a retained architectural tissue hierarchy like in KAs, whereas other aberrations may give selective growth to tissues with disrupted architecture. An obvious trait of KAs is the maintenance of symmetric architecture with well-differentiated epithelial strands, which may have fundamental implication for the surveillance of the neoplastic cells.
Other overlapping recurrent imbalances of KAs and SCCs are gains from 3q, 8q, 9q, and 19p, and loss at 3p (). Many different genes from these regions may be altered and stimulate uncontrolled proliferation, one possibility is the protooncogene C-myc localized to 8q24.
CGH is a screening method with the advantage that it allows for simultaneous detection and mapping of amplified, duplicated, or deleted chromosomal regions of the total genome of tumors in one hybridization procedure (Kallioniemi et al., 1992
). Limitations are that balanced aberrations, such as translocations and inversions, will not be detected, and that the hybridization target (the normal human metaphase chromosomes) limits the resolution of the method. Deleted regions must be as large as 5-10 Mb to be detected, whereas amplified regions may be detected down to 1-2 Mb if the degree of amplification is sufficiently large (Forozan et al., 1997
). This implies that the percentages of lesions with genetic aberrations, as well as the number of aberrations per lesion reported here, are minimum values. Methods with higher resolution, such as comparative hybridization to genomic arrays (array CGH) and positional hybridization, will be able to reveal more specific genetic aberrations that may serve to further differentiate between KA and SCC.
If we assume that KA and SCC are biologically separate lesions with different pathogenetic background, the presence of overlapping genetic imbalances detected by CGH must be considered. First of all, many of the aberrations detected are probably without influence on tumor progression and cell death. Secondly, it is well known that sun exposure through UV irradiation is important for development of KAs as well as for SCCs. Thus, many of the overlapping aberrations may be due to DNA damage and selection mechanisms caused by the common influence of UV. The observation that genetic aberrations are seen more frequently and are more numerous in SCC than in KA, may result in abrogation of signaling pathways in SCC that are intact in KA, the latter possibly facilitating apoptosis and spontaneous regression of KA.