We obtained copy number profiles as measured by array comparative genomic hybridization (aCGH) from tumors arising in individuals with multiple independent cutaneous squamous cell carcinomas (SCCs) or keratocanthomas (KAs) (intra-group) and copy number profiles of SCCs and KAs of separate individuals (inter-group). 305 independent tumor samples from 181 patients were included in this initial study (). As previously reported 
, focal genomic aberrations were rare in these tumors and DNA copy number aberrations consisted mostly of the loss or gain of whole chromosome arms. As the resolution of copy number changes using aCGH is around 1Mb, it is possible that we missed focal amplifications or deletions in this study.
Frequency of aCGH aberrations in skin tumors.
We compared aCGH profiles between the three types of skin tumors in our study, SCC, SCC in situ
(Bowen's Disease) and keratocanthoma. There were no statistically significant differences in frequency of clone loss or gain between the SCC and keratocanthoma profiles; however there were several loci which showed differences between the SCC in situ
profiles and profiles from the other two tumor types (data not shown). Because of this, we focused our comparative analysis on SCCs only. Our data set included 222 SCCs from 135 individuals. From 25 of those individuals, three or more SCCs (median
4.2; range 3–6) were analyzed to compare the intra-group and inter-group similarities of DNA copy number changes.
We found a significantly higher concordance of chromosomal aberrations in SCCs within than between patients [two-sided T-test p-values: 6.97×10−8](). Interestingly, certain chromosomal regions (4q, 11q, and 17q) were preferentially affected by this concordance (individual arm p-values<0.05; ). The intra-group correlation coefficients (ICC) for the array elements of these regions were compared but did not allow narrowing the genomic region to specific loci within these regions. This is not unexpected, considering that most of the tumors showed copy number changes affecting large genomic regions, such as entire chromosomal arms or chromosomes.
aCGH profiles of independent tumors from two individuals.
Comparison of somatic changes within versus across individuals.
We rationalized that any inherited variants that promote cancer in an allele-specific manner would result in allele-specific DNA copy number changes reflected by preferential loss or gain of one specific chromosome in the tumors of an individual patient. By contrast, dosage events affecting genes that promote cancer in allele-independent manner, e.g. loss of CDKN2A
or gain of MYC
, were expected to display random somatic alterations of either allele 
. To determine the presence of allele-specific changes occurring within tumors of individual patients, we performed loss of heterozygosity analyses of 45 microsatellite markers covering 14 chromosomal regions that were chosen based on the frequency of aberration as measured by aCGH and without prior knowledge of regions showing more similarity within versus across patients. 270 tumors from 65 individuals with a minimum of three independent tumors were included in this analysis. The constitutional genotype was determined from DNA extracted from blood leukocytes of each patient. Allelic imbalance was defined as a tumor to normal DNA allelic ratio of greater than 1.5 or less than 0.67. Statistical analyses for preferential imbalance were conducted for individuals who were heterozygous for a given marker and had two or more tumors showing imbalance; two examples are illustrated in . Thirteen markers representing eight different genomic regions showed significant skewing towards one allele as determined by a Bayesian/frequentist approach (Text S1
). Markers demonstrating significant preferential allelic imbalance mapped to chromosomal locations 3p24, 3q21-26, 5q23, 7p12-21, 7q31, 8q24, 9p21, 11q24, and 18q22 (, ). These data indicate that the increased similarity of copy number changes within individuals is at least in part due to inherited variation within the same region as the copy number change.
Loci with and without evidence of preferential allelic imbalance.
Preferential allelic imbalance studies.
The next question we addressed was whether variations in any known tumor susceptibility genes were driving allele-specific imbalance at the loci identified through our studies. Several genome-wide association studies (GWAS) have been performed for multiple cancers including breast, prostate, colon, and melanoma 
. Variants at 8q24 identified via GWAS have been associated for cancer risk for multiple cancer types 
. To determine if any of these were candidates for the observed allele-specific imbalances at 8q24, we tested three variants, rs13281615
, and rs6983267
, for allele-specific imbalances in matched normal and tumor DNAs from individuals with SCC. Of these, only rs13281615
showed statistically significant evidence of allelic skewing (). Of 35 heterozygous tumors showing imbalance for rs13281615
, 28 of them showed an imbalance in favor of the A allele while only 7 showed an imbalance favoring the G allele (p-value 0.012). A second SNP, rs6983267
, showed a similar trend that did not reach statistical significance (p-value 0.157). These data raise the possibility that rs13291615
may be a candidate susceptibility allele for SCC. Our results suggest that the use of preferential allelic imbalance may be an efficient approach to map susceptibility variants in specific clinical settings.
8q24 variants tested for preferential allelic imbalance.