Ovarian carcinoma (OC) is the fifth most frequent female cancer type and the fourth most frequent cause of death from cancer among women in Denmark.1, 2 At the time of diagnosis, approximately 70% of patients have advanced disease (International Federation of Gynecology and Obstetrics [FIGO] Stage III or IV). Published 5-year survival rates for patients with OC range from > 80% for patients with Stage I OC to < 20% for patients with Stage III and IV OC.3–5 The high frequency and poor prognosis of OC emphasizes the need for both additional and better prognostic factors.
The molecular genetic events underlying ovarian neoplasms are complex and are understood poorly. It is believed that loss of tumor suppressor gene activity plays an important role in the origin and progression of malignant tumors. A commonly described mechanism leading to the loss of tumor suppressor gene function, originally reported by Knudson,6 requires the occurrence of two events: First, one of two alleles must contain a mutation (somatic or germline), rendering this allele incapable of producing a functional tumor suppressor protein. Second, the remaining allele must be lost, resulting in an overall absence of tumor suppressor gene function.7 This loss may be detected in individuals heterozygous for a marker closely linked to the tumor suppressor gene (loss of heterozygosity [LOH]) and is characteristic of tumor suppressor genes. LOH frequencies up to 80% in OC have been described on multiple chromosomes, most commonly involving chromosomes 3, 6, 9, 11, 17, 18, and 22, suggesting the involvement of many different tumor suppressor genes.7–13 However, limited information on LOH at chromosome X is available, and LOH has been reported in 40–60% of the informative OC cases tested.9, 14, 15 Some studies suggest that LOH is more common in ovarian tumors with more aggressive biologic behavior and in more advanced ovarian tumors.7, 16 Most LOH studies on patients with OC have involved relatively small number of tumors, a wide range of different chromosomal loci, and variable patient characteristics. Furthermore, genetic differences within the study population are possible, which may explain the discordant findings in the published studies. It has been hypothesized that tetranectin (TN) plays a role in proteolytic processes is an important factor in the ability of malignant cells to proliferate, infiltrate normal tissue, and metastasize.17–21 In patients with cancer, the decreases in serum or plasma TN (s/p-TN) levels may reflect the proteolytic activity of the tumor, and low s/p-TN levels may be due to absorption of TN from the blood to the tumor site for the purpose of proteolysis. This hypothesis is supported by the immunohistochemical findings of high extracellular TN concentrations in malignant tumors in combination with low p-TN values and a poor survival for OC patients with this combination.22 Previously, it was reported that TN should be included as a prognostic factor in OC studies evaluating new prognostic markers.23
Based on previous reports that LOH occurs frequently on chromosome 17p and 17q,7, 9, 24 chromosome 18q,25 and chromosomes Xp and Xq,9, 14, 15 we decided to perform LOH analyses with 5 microsatellite markers in 160 Danish women with primary OC: TP53 (17p13.1), CACNLB1 (17q21–q22), D18S58 (18q22–q23), DXS538 (Xp11.21–p21.1), and DXS454 (Xq21-q23). We were able to perform this study using a set of samples from the Malignant Ovarian Cancer Study (“MALOVA”) collection in Denmark.
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- MATERIALS AND METHODS
In the current study, LOH analysis was performed to evaluate the importance of five microsatellite markers from three different chromosomes as prognostic markers for OC. The markers analyzed were TP53 (17p13.1), CACNLB1 (17q21–q22), DXS538 (Xp11.21–p21.1), and DXS454 (Xq21–q23). Results from one of the microsatellite markers, D18S58 (chromosome region 18q22–23) did not correlate with any of the tested clinical variables and, thus, is not discussed further.
The overall frequencies found in this study were lower compared with the frequencies reported in the literature. One explanation may be the use of a stricter definition to confirm LOH. This criteria for LOH was chosen to ensure a low person-to-person variability when scoring LOH. When comparing the observed frequencies in this study with reported frequencies, it is of relevance to consider the different scales used.
To our knowledge, this is the first detailed study large enough to clarify the prognostic value of LOH using microsatellite markers at chromosomes 17, 18, and X. We found that LOH at Xq (DXS454) was associated with reduced survival in patients with OC, as illustrated in both univariate and multivariate analyses, and that LOH at DXS538 (Xp11.21–p21.1) was associated with poor survival in patients with OC, as illustrated in the univariate analysis but not in the multivariate analysis. In this study, we used our selected combination of variables and compared the relative strength of each variable with respect to prognosis.
Disease stage in patients with OC was not found to be an independent prognostic factor in the multivariate Cox analysis. One explanation may be that very few early-stage tumors were informative for both markers. However, 19 early-stage tumors were informative for both markers and were included in the Cox analysis. Therefore, one-third of the included tumors were early-stage OC. However, this is not why disease stage was of no prognostic value in the Cox analysis performed in this study. Because of this finding, it may be important to evaluate the DXS454 marker as a prognostic factor in other, larger studies.
Because OC is a female malignancy, it has been speculated often that there may be a specific role for the X chromosome in ovarian carcinogenesis.9 If a tumor suppressor gene is located on the X chromosome, and if females with a germline mutation in one copy of that tumor suppressor gene experienced nonrandom X chromosome inactivation, then some or all of the tissues of such women may lack the wild-type suppressor gene function. Buller et al. found that nonrandom X chromosome inactivation may be a predisposing factor for developing an invasive ovarian tumor.35, 36 Our study, which was performed on tissues and corresponding blood samples from women with OC, may support this hypothesis; furthermore, our findings suggest that loss of the X chromosome may be a primary or early event in ovarian tumorigenesis, because LOH at DXS454 (Xq21–q23) occurred in both early and late stages of OC. These findings raise the possibility of a tumor suppressor gene locus on the X chromosome (21q–23q) that may have prognostic value.
We have not identified any other studies that investigated the markers DXS454 and DXS538 in patients with OC with respect to survival. Furthermore, due to limitations of sample size, few previous studies included prognostic evaluation of LOH markers at all.
Manderson et al. described LOH on both arms of the X chromosome in 75 patients with OC, among whom 62 patients had Stage III or IV disease.37 Those authors reported a 7% frequency of LOH of Xp compared with 36% at Xq. Buekers et al. investigated LOH on the X chromosome using 6 different microsatellite markers in 81 OC patients with no information of stage and found that LOH was more frequent on Xp, with the highest frequency of LOH demonstrated at the region Xp 22.2-3 (37.7%).15 Shelling et al. described that LOH of Xp was 38%, whereas loss at Xq was 29%.9 In the current study, the frequency of LOH at Xp was 20%, and the frequency of LOH at Xq was 29%, consistent with the most recent of the studies by Manderson et al.37
Different frequencies of LOH can be demonstrated between the various histologic subgroups of OC with borderline tumors that display a lower overall frequency of LOH compared with invasive tumors.13 We observed greater frequencies of LOH in serous adenocarcinoma compared with mucinous adenocarcinoma, findings that are in agreement with earlier published studies13, 16, 25 and that add further to the growing evidence that the pathways of molecular carcinogenesis and progression may differ among the histologic subtypes of OC.
Manderson et al. investigated whether there was significant difference in the frequency of LOH with respect to age. They found one locus at 3p14 for which the frequency of LOH increased significantly with age.37 Pieretti and Turker38 reported that the frequency of LOH of an entire chromosome 17 homologue increased with older age at the time of diagnosis of OC, and Pieretti et al.13 found that patients with chromosome 17 loss were older than patients without this alteration. We found a significant difference between the median age of patients with LOH and patients with retention in the CACNLB1 locus. One possible explanation for this phenomenon may be that LOH of certain chromosomal loci on chromosome 17 are nonrandom events that reflect the contribution of multiple tumor suppressor genes to a more aggressive phenotype, resulting in poorer survival often being noted in women of older age.39
In the current study, LOH at DXS454 appeared to correlate with shorter survival in Danish patients with epithelial OC.