Analysis of genetic alterations in uterine leiomyomas and leiomyosarcomas by comparative genomic hybridization.

Uterine leiomyomas are the most prevalent tumor type in women of reproductive age and are the most common reason for hysterectomies. Although uterine leiomyomas are considered to be benign, they are a major public health concern for women. In contrast, leiomyosarcomas are rare but highly malignant uterine tumors. They may arise in uteri with preexisting leiomyomas and histologically sometimes resemble leiomyomas, thus causing controversy about whether leiomyosarcomas arise within leiomyomas. In this study, we used comparative genomic hybridization (CGH) to identify genetic alterations unique to each tumor type and alterations that are common between the two tumors. We analyzed 14 cases of uterine leiomyomas and eight cases of uterine leiomyosarcomas. Only two of the 14 leiomyomas exhibited genetic alterations, and those were restricted to gains on chromosomes 14 and 19 and losses on chromosomes 1 and 4. In addition, 68 leiomyomas were examined for loss of heterozygosity on chromosomes 1 and 4, and only three tumors exhibited any losses. In contrast, all eight leiomyosarcomas showed gains and losses of DNA by CGH, and in many cases multiple changes were observed. The most commonly observed genetic aberration, occurring in five tumors, was gains on both arms of chromosome 1, suggesting that this chromosome contains loci involved in the development of leiomyosarcoma. Our results do not provide evidence for the progression from benign leiomyoma to malignant leiomyosarcoma. Moreover, the large number of random chromosomal alterations in the leiomyosarcomas suggests that increased genetic instability plays a role in the formation of these tumors.

Homozygous deletions but no sequence mutations in coding regions of p15 or p16 in human primary bladder tumors.

Chromosome 9p21 appears to harbor a tumor suppressor gene, as evidenced by deletions in this region in a variety of human primary tumors and cell lines. To map the deletion at 9p21 in bladder tumors, we analyzed DNA from 28 tumor and normal pairs at five microsatellite markers that flank the region occupied by the putative tumor suppressor genes p16 and p15. Loss of heterozygosity (LOH) at the markers human interferon (HIFN) alpha and D9S171, which are adjacent to the p15 and p16 loci, was detected in 41% and 33%, respectively, of informative cases of bladder tumors. No sequence mutations were detected in exons 1 or 2 of either p15 or p16 in any of the bladder tumors. Three sequence-tagged site markers in the region bordered by HIFN alpha and D9S171 were used to further map the deleted region by multiplex polymerase chain reaction with the HIFN gamma maker (on chromosome 12) as a control for amplification. Six of 11 tumors with LOH at surrounding markers had homozygous deletions of the marker c5.1, which is located within the p16 gene; and two tumors appeared to have homozygous deletions within p15 (RN1.1) but not p16 (c5.1). A recently identified microsatellite marker, p16-CA-1, located 16 kb distal to p16, proved valuable in defining the minimal deletion involved in these bladder tumors. Five tumors exhibited homozygous deletions of this marker but not HIFN alpha and two tumors showed LOH at this marker and homozygous deletion of p16. Although these data could not be used to identify p16 or p15 as the definitive tumor suppressor gene in this region that is involved in bladder carcinogenesis, they suggest that homozygous deletion is a common mechanism of loss of tumor suppressor gene function in this region.

Homozygous deletions at chromosome 9p21 and mutation analysis of p16 and p15 in microdissected primary non-small cell lung cancers.

Loss of heterozygosity on chromosome 9p has been detected in many primary human tumors and cell lines, suggesting that this chromosomal arm harbors one or more tumor suppressor genes. The recently cloned p16 and p15 genes, mapped to 9p21, are likely candidates for such tumor suppressors. To map the deletion at chromosome 9p21 in non-small cell lung tumors, we analyzed DNA from 25 tumors and matching normal DNAs at six microsatellite markers that flank the region occupied by the p16 and p15 genes. Loss of heterozygosity of at least one microsatellite marker on chromosome 9p21 was detected in 13 (52%) of 25 tumors, including one tumor that exhibited homozygous deletion of both human IFNalpha and D9S171. Six tumors analyzed by a comparative multiplex PCR technique showed homozygous deletions of the sequence tag site marker c5.1 (within p16). Screening for mutations in p16 and p15 revealed one tumor with a non-sense mutation in exon 2 of p16, but no mutations were detected in p15 in any of the tumors. Thus, in these analyses approximately one-half of the non-small cell lung tumors had loss of heterozygosity at chromosome 9p21, and of these tumors, one-half had homozygous deletions of the region that includes p16. This appears to confirm the importance of a locus in this region critical to growth control in lung. The apparent lack of other mutations in p16 and p15 in the tumors with loss of heterozygosity leaves open the possibility of an unidentified gene in this region that may function as a tumor suppressor.

Endothelium specific Weibel-Palade bodies in a continuous human cell line, EA.hy926.

Weibel-Palade bodies are ultrastructurally defined organelles found only in vascular endothelial cells. Because endothelium in corpo is very dispersed, isolation and further characterization of this organelle has been dependent on increasing the number of cells in culture. However, primary isolates of endothelial cells have a limited replication potential and tend to senesce in culture. In this report, EA.hy926, a continuously replicating cell line derived from human endothelium, is shown to contain Weibel-Palade bodies. Electron micrographs demonstrate the ultrastructural characteristics of these tissue-specific organelles and their cytoplasmic distribution in EA.hy926 cells. Von Willebrand factor, which has been shown to exist in Weibel Palade bodies, is demonstrated by immunofluorescence in discrete rod-shaped organelles whose size, shape, and distribution are consistent with that of Weibel-Palade bodies in primary endothelial cell cultures. Rapid release of von Willebrand factor can be induced by calcium ionophore, and large multimeric forms of the protein are found in EA.hy926 cells. These two properties are consistent with the function currently ascribed to Weibel Palade bodies: storage of multimerized von Willebrand factor. Thus ultrastructural, immunologic, and functional data establish the existence of this as yet poorly understood tissue-specific organelle in a continuous, vigorously replicating human cell line.