The loss of chromosome 12q12-q13 is a novel finding in ACC. Such loss was the only detectable DNA copy number change in three tumors in this study, suggesting that a gene in this region plays a special role in ACC tumorigenesis. Further investigation of chromosome 12 by LOH analysis showed the overall loss on chromosome 12 to be similar to that found by CGH and that this loss likely includes a locus telomeric to D12S1301 since only 6 of 10 tumors with LOH included D12S1301. The finding of 12q loss by CGH analysis alone for sample 12 also leads us to believe that the loss may be telomeric to D12S1301. Interestingly, using the D12S391 marker, there was a high level of loss on the p arm of chromosome 12, suggesting that this loss may involve a relatively large area of chromosome 12 or perhaps, there is more than one consensus region residing on this chromosome — one at 12p11-13 and the other(s) at 12q. The fact, that LOH analysis alone showed loss on the p arm for four tumors (samples 9, 13, 14, and 19), suggests that this area may be involved by deletions too small for identification by CGH. The potential for loss on the q arm of chromosome 12 may perhaps extend further than that shown by LOH analyses because the two 12q markers are non-informative for a number of tested samples. The small microsatellite panel used in this study served only as an independent confirmation of the finding by CGH of deletions on chromosome 12. Further LOH studies using higher-density allelotyping of microsatellite markers are required to conclusively determine the number and localization of consensus regions on chromosome 12.
Although a recent literature review of 283 CGH studies showed that chromosome 12 deletions were not frequent in any of the 73 tumor entities analyzed [21
], one study using microsatellite LOH assays found evidence of genetic deletion at 12q in salivary gland pleomorphic adenomas and in the adenoma component of carcinoma ex-pleomorphic adenoma [22
]; the authors concluded that LOH at 12q may identify a subset of adenomas with the potential to progress to carcinoma. Although the majority of carcinomas arising in pleomorphic adenomas showed loss distal to the 12q12-q13 region, some of the deletions did extend to this locus, and in two cases, this region was the sole area of deletion. Interestingly, a translocation of 12q12-q13 in a case of myoepithelioma, a rare benign salivary gland neoplasm that shares ACC features of myoepithelial differentiation, has been reported [23
]. A similar translocation has also been shown in renal oncocytoma [24
]. These findings, together with our results, suggest that an unidentified gene with tumor-suppressor activity might be present at chromosome 12q12-q13. It is possible that this unknown gene might have functional importance for salivary gland tumors with myoepithelial differentiation. Further studies are required to determine the extent to which chromosome 12, and more specifically the region telomeric to the D12S1301
marker, may play in the development or progression of salivary gland tumors.
The CGH results of the present study, showing losses at 6q23-qter, are in agreement with the results of earlier cytogenetic and LOH findings in ACC [5,10
], and further support the notion that deletions of genetic material at 6q may play an essential role in the development of this neoplasm. The chromosome 6q23-qter cytogenetic location includes the tumor-suppressor gene PLAGL1/LOT1/ZAC
at 6q24.3. This gene was discovered to be downregulated during the transformation of a rat ovarian epithelial cell line [25
], and was subsequently shown to be downregulated in primary human ovarian and breast carcinomas and cell lines [26,27
]. In cell culture experiments, PLAGL1/LOT1/ZAC
has been shown to have anti-proliferative and pro-apoptotic regulatory activities [28
]. Therefore, further testing of PLAGL1/LOT1/ZAC
as a potential candidate for tumor-specific deletion, mutation, and/or transcriptional down-regulation in ACC is required.
The frequent losses we observed in 13q21-q22 have not previously been reported in ACC. This genetic region is known to be deleted in several types of cancer, such as carcinomas of the lung, breast, prostate, and head and neck, as well as in tumors of bone (for review, see Ref. [29
]). This genetic location is considerably distal to the RB1
locus (13q14), and may indicate the presence of a novel tumor-suppressor gene.
Gains in chromosome 19, frequently observed in the present collection of ACC, have been reported in several other types of carcinoma, including pancreatic [30,31
], ovarian [32
], and esophageal cancer [33
]. In contrast to our findings, an earlier microsatellite LOH study by Johns et al.
], using two markers on 19q — D19S210
, reported a 40% rate of allelic loss in 10 ACCs. Our analysis of ACC using seven microsatellite markers (including the two 19q markers used in the previous study) failed to detect chromosomal loss, supporting the conclusions drawn from our CGH analysis. This discrepancy may be attributed to differences in interpretation of LOH assays, where allelic imbalances that occur in amplification events are interpreted as deletions.
The finding of a larger number of genetic changes in grade 3 tumors than in grades 1 and 2 neoplasms supports the hypothesis that more genetic alterations are required for tumor progression. Grade 3 ACC tumors are more poorly differentiated than grade 1 or 2 cancers in terms of tubule formation, and tend to be more aggressive clinically. The observation of a greater number of genetic abnormalities associated with more aggressive forms of neoplasia has been documented in other tumors [34
Although specific DNA copy number abnormalities were detected in ACC, a much lower overall number of DNA copy number changes were seen compared with other epithelial neoplasms arising in the upper and lower aerodigestive tract, including nasopharyngeal carcinoma [35
], head and neck squamous carcinoma [36,37
], and small cell and non-small cell carcinomas of the lung [38–41
]. Only a few ACCs (largely those that were grade 3) had the complexity of DNA copy number changes usually present in carcinomas of the aerodigestive tract. Our results showing limited areas of genetic loss in ACC are corroborated by a previous low-density allelotype analysis, which also did not detect many frequent deletion events [9
]. The fact, that DNA copy number and cytogenetic abnormalities are less frequent in ACC, may indicate that a smaller number of genes are involved in the development of this neoplasm than is typical of most carcinomas. An alternative explanation for lack of DNA copy number abnormalities is the presence of the DNA replication error phenotype, for which an inverse correlation with number of cytogenetic abnormalities and genetic deletions has been found in colorectal carcinoma [42
]. However, in our previous [43
] and ongoing studies using microsatellite allelotyping of ACC, no widespread micro-satellite instability has been detected, as would be expected with the replication error phenotype. Hence, we favor the hypothesis that ACC develops as a result of fewer and more specific genetic alterations.
This study is the first global survey of DNA copy number changes in ACC, and points to the existence of possible novel tumor-suppressor genes on 6q, 12q, and 13q. Further LOH studies using higher-density allelotyping of micro-satellite markers are required for chromosome 12 as well as for chromosomes 6 and 13 to fine map regions that may contain, as yet, unidentified tumor-suppressor genes.