Current Research Summary, Christopher A. Moskaluk MD, PhD; Henry F. Frierson, Jr. MD; Deptartment of Pathology, University of Virginia
6. Future plans
This technique uses DNA taken from ACC tumor cells and compares it directly with DNA from normal cells, by labeling the two types of DNA with different colors of fluorescent dye. These dyed DNA populations are then used to probe normal human chromosomes, where the DNA sticks to areas of the chromosomes from which they came. By using a special microscope, camera and computer system, we could see differences between tumor and normal DNA by differences in the relative amounts of different colored dye that was present on the chromosomes. Finding such areas gives one important clues about where to look for genes that are important in the development of ACC. We found 3 distinct areas of genetic loss that occurred non-randomly in ACC, on chromosomes 6, 12 and 13. The chromosome 12 deletions are not common in other forms of cancer studied so far, and may point to a gene fairly specific for ACC tumor development.
The results of this study were published in the journal Neoplasia Vol. 3, pp.173-178, 2001.
In order to find common genetic abnormalities that may identify loci of genes involved in the development of adenoid cystic carcinoma (ACC), we investigated DNA copy number changes in 24 of these tumors by comparative genomic hybridization (CGH). Our results indicate that unlike many carcinomas, ACC have relatively few changes in DNA copy number overall. Twenty tumors had DNA copy number changes, which were mostly restricted to a few chromosomal arms. A frequent novel finding was the loss of DNA copy number in chromosome 12q (8 tumors, 33%) with the minimal common overlapping region at 12q12-q13. Deletion in this region has not been reported to be frequent in other types of cancer analyzed by CGH. In addition, deletions in 6q23-qter and 13q21-q22, and gains of chromosome 19 were observed in 25-38% of ACCs. Deletion of 19q, previously reported in a small series of ACC, was not identified in the current group of carcinomas. The current CGH results for chromosomes 12 and 19 were confirmed by microsatellite allelotyping.
These results indicate that DNA copy number losses in 12q may be important in the oncogenesis of ACC and suggest that the 12q12-q13 region may harbor a new tumor suppressor gene.
We have decided to take a closer look at chromosome 6, to try and find genes in this area of the human genome that are involved in the development of ACC. We are using a technique called polymerase chain reaction (PCR) in which we can determine if genetic markers called microsatellite repeats have been lost in an ACC tumor by genetic deletion. The assay of these markers is also called loss of heterozygosity (LOH) analysis. LOH is a higher resolution technique than CGH, but it takes a lot more time and effort. CGH is like flying in an airplane over a country to find out where the mountains are. LOH is like walking through the mountains to find a particular grove of trees. A high resolution deletion map of 58 cases of ACC using microsatellite loss of heterozygosity is complete for chromosome 6. We have identified a consensus area on the distal portion of chromosome 6 spanning a 4.1 Mb stretch (4.1 million base pairs of DNA). By using information available from the Human Genome Project, we have identified a number of genes within and adjacent to this area which have the potential to be tumor suppressor genes, including PLAGL1/LOT1/Zac (6q24-q25), LATS1 (6q24-q25.1), STX11 /TSAP (6q23.1-6q25.3), CX43 (6q21-q23) and LOC57107 (6q23.1-q25.3). The available evidence is most strong for PLAGL1 as a tumor suppressor gene and we have begun single strand conformational analysis of the open reading frame of this gene in our panel of ACC in order to assay for tumor-specific mutations. Finding such mutations is essentially the genetic proof that a gene is involved in the tumorigenesis of a specific cancer type.
We have completed a medium resolution mapping phase of deletions in chromosome 12. Data generated from alleleotyping 16 polymorphic microsatellite markers in paired tumor and normal samples of 58 cases of ACC shows 43% overall loss on chromosome 12.
This loss is consistent with that found using comparative genomic hybridization. Results from microsatellite loss of heterozygosity analysisalso indicate that a large region of loss occurs on the q arm of chromosome 12. In addition, a 9 Mb region of deletion has also been identified on the p arm of chromosome 12 indicating that one or more tumor suppressor genes may reside on this chromosome. This 9 Mb region contains a number of potential tumor suppressor genes including TNFRSF1A (tumor necrosis factor receptor superfamily, member 1A), NOL1 (proliferating cell nucleolar protein p120), LPRP (lacrimal proline rich protein), PTPN6 (protein tyrosine phosphatase, non-receptor type 6), PRH2/PRH1 (acidic parotid salivary proline rich protein) and PRB3/PRB1 (basic parotid salivary proline rich protein/ glycoprotein).
Manuscripts with the chromosome 6 and 12 deletion mapping are currently being prepared.
We have used microarray chip analysis of small portions of nucleotides to survey the expression of approximately 9,000 genes in each of five normal salivary glands, 15 human ACC, and one ACC cell line. In this technique we find out what genes are active by extracting RNA from cells and identifying the RNA species by their stickiness to specific areas in the microarray. Genes are active if they are transcribed into RNA which is then usually translated into protein. We performed this study, as the molecular alterations that underlie the development and progression of ACC are poorly characterized, and we wanted to determine which genes are overexpressed in ACC compared with the normal salivary glands from which they are derived. We found that the gene expression profile of ACC was very different from that of the normal glands, and we determined the top 30 genes that were highly expressed in ACC. These genes encode proteins having various functions that are important in the growth and differentiation of the tumors. We also compared the gene expression profile of ACC with that for the top 10 carcinomas that cause the deaths of patients in the U.S.A. We found that using these genes ACC could easily be distinguished from the other cancers (with only a couple of exceptions).The genes overexpressed in ACC also point to molecules that one day may become specific targets of novel chemotherapeutic approaches. A manuscript with the microarray results is currently being reviewed for publication.
We have contacted members of the SEER program at the National Cancer Institute to study their records of ACC patients. SEER stands for Surveillance, Epidemiology, and End Results, and it is the most authoritative source of information on cancer incidence and survival in the United States. Drs. Tim Cote and Margaret McCusker at SEER have been extremely helpful and interested in collating and analyzing their ACC data. 1,729 cases have been identified, and they are currently analyzing patient outcomes compared to a variety of parameters.
The results of these analyses are being prepared for publication.
The screening of gene mutations in candidate genes in the chromosome 6 region will continue. If no mutations are found in obvious candidate genes, the mapping effort will be expanded to more tumors in order to obtain more deletion events that may serve to further narrow the consensus location, thus more specifically pinpoint specific genes. The chromosome 12 data will be analyzed for consensus areas, and candidate tumor suppressor genes will be sought in the Human Genome Database. Strong candidate genes will be analyzed for tumor-specific mutations as for candidate genes on chromosome 6. The gene expression data from the oligonucleotide microarray will be analyzed for clinically significant associations, and a subset of the data will be validated with reverse transcription -polymerase chain reaction assays of tumor specimens.