Telomere length in different histologic types of ovarian carcinoma with emphasis on clear cell carcinoma.

The mean patient age was 57 years (range 23 –88 years). Most of the ovarian carcinoma specimens were from primary tumors but in seven cases tissue from both primary and recurrent tumors was available for comparison ( Table 1 ). All patients with high-grade serous carcinomas were stage III or IV. The median follow-up time for 196 cases with available follow-up information was 34 months (range: 0.1 to 166.8). The telomere index in all samples analyzed is shown in Figure 1 and representative images of telomere FISH in different types of ovarian carcinoma are shown in Figure 2 . Comparing the four different ovarian carcinoma histologic types (low-grade serous, high-grade serous, low-grade endometrioid and clear cell carcinoma), the mean telomere index in clear cell carcinoma was significantly higher than that in other histologic types in combination ( Figure 1 ; P = 0.007). Similarly, if the telomere index (≤1 vs >1) was used as a cutoff for the binary analysis, clear cell carcinoma had more cases with longer telomeres (index >1) than the other histologic subtypes (12 of 56 vs 9 of 164, respectively; P = 0.01, Fisher’s exact test). In contrast, neither low-grade serous nor high-grade serous and low-grade endometrioid carcinoma demonstrated a significant difference compared with the other ovarian carcinoma types ( P > 0.05). We also analyzed the prognostic significance of telomere index in all the histologic types. After adjusting for age and clinical stage, the overall hazard ratio for death was 4.93 (95% CI: 1.64 to 14.86, P = 0.005) for clear cell carcinomas with a telomere index >1 as compared with those with telomere index ≤1 ( Figure 3 ). The other cancer subtypes did not show similar findings ( P > 0.05). We have reported that subdividing ovarian clear cell carcinomas into cystic and adenofibromatous reveals differences in a number of clinicopathological features including their association with endometriosis, histologic patterns, stage distribution and clinical behavior. 25 We therefore correlated the telomere index in cystic vs adenofibromatous clear cell carcinomas in 50 cases but there was no statistically significant difference between them ( P = 0.955). Among 99 high-grade serous carcinomas, there were seven cases in which tissue from the primary and first recurrence in the same patient was available for analysis. Recurrent high-grade serous carcinomas tended to have shorter telomeres than the primary tumors, although the difference was not statistically significant ( P = 0.09). All seven primary tumors except one (patient 4) had shorter telomeres in the recurrent tumors ( Table 2 ).

In view of the importance of telomere function in carcinogenesis, we hypothesized that measurement of telomere length might provide clues to the pathogenesis of different types of ovarian carcinoma. In this study, we focused on telomere length, not telomerase expression level and activity because telomere length, in contrast to telomere expression, in mammalian cells is regulated by a complex and dynamic process involving both telomerase-dependent and independent mechanisms. 26 We found that ovarian clear cell carcinomas contained on average significantly longer telomeres than high-grade serous, low-grade serous and low-grade endometrioid carcinomas, a finding that confirms the distinct molecular genetic differences of the different ovarian carcinoma types and underscores the unique nature of ovarian clear cell carcinoma. Although ovarian clear cell carcinoma represents <10% of ovarian carcinoma in the United States, it occurs more frequently in Asian women and, in fact, its incidence appears to be increasing in this population. 27 , 28 As compared to high-grade (conventional) serous carcinoma, women with clear cell carcinoma present at a younger age and at an earlier clinical stage. 27 , 29 , 30 Also compared with high-grade serous carcinoma, advanced stage clear cell carcinoma is not as sensitive to conventional cytotoxic chemotherapeutic agents. 31 The molecular genetic alterations that underlie the development of ovarian clear cell carcinoma are now beginning to emerge. The most common molecular genetic changes that have been identified thus far are a somatic inactivating mutation of ARID1A , 32 , 33 an activating mutation of PIK3CA 34 and deletion of PTEN , a tumor suppressor gene involved in the PIK3CA signaling pathway, 35 , 36 which support the role of an aberrant PIK3CA pathway in the development of clear cell carcinoma. In addition, single nucleotide polymorphism array analysis has identified frequent amplification of the ZNF217 (zinc finger protein 217) locus and deletion of the CDKN2A/2B locus in clear cell carcinoma, suggesting that pathways involving these two genes are also important in the development of this tumor. 37 Although both clear cell and endometrioid carcinomas are derived from endometriosis 35 and share some molecular genetic features such as inactivating mutations of ARID1A and activating mutations of PIK3CA , they adopt different molecular programs for their development, as is evident by their distinctly different morphologic phenotype. For example, activating mutations in β-catenin and microsatellite instability, which occur frequently in endometrioid carcinoma, have only rarely been detected in clear cell carcinoma. 2 The results from the current study provide additional evidence that telomere length in ovarian clear cell carcinoma is distinctly different from the other histologic types of ovarian carcinoma. It is also worth noting that clear cell carcinoma in contrast to low-grade serous and low-grade endometrioid carcinomas, has a high level of inter-tumoral heterogeneity of telomere length as evidenced by the wide standard error ( Figure 1 ), suggesting that there are subsets of clear cell carcinoma based on different telomere lengths. Telomerase inhibition enhances the response to anticancer drugs 38 and telomere length may prove to be a useful prognostic feature in combination with other immunohistochemical markers. For example, IGF2BP3 (IMP3) expression has been reported to be an independent marker of reduced disease-specific survival in clear cell carcinoma, but not in high-grade serous or endometrioid carcinomas of the ovary. 39 Similarly, enhanced expression of Annexin A4 in clear cell carcinoma and its association with chemoresistance to carboplatin has been recently reported. 40 The lack of correlation of telomere length and clinical outcome in other types of ovarian carcinoma is intriguing. Although the finding suggests that the role of telomere length in clear cell carcinoma is different from other histologic types, more cases, particularly of low-grade serous and low-grade endometrioid carcinomas must be studied in a prospective manner to validate this conclusion. Another unrelated but interesting observation was that recurrent high-grade serous carcinomas tend to have shorter telomeres than the primary tumor but the number of cases in our study was small and therefore additional cases must be studied to confirm these findings. In summary, this report is the first comprehensive investigation of telomere length among the major types of ovarian carcinoma. Using quantitative telomere FISH, we are able to precisely measure the mean relative telomere length and correlate it with various histologic types of ovarian carcinoma and with clinical outcome. Our findings showed that clear cell carcinomas have longer telomeres compared with the other histologic types of ovarian carcinoma. Additionally, longer telomeres in clear cell carcinoma were associated with poor outcome suggesting that aberrations in telomere length, in particular lengthening, may have an important role in the development and progression of this neoplasm.

A total of 219 ovarian carcinomas from 212 patients were included in this study. There were 106 high-grade serous carcinomas, 26 low-grade serous carcinomas, 56 clear cell carcinomas and 31 low-grade endometrioid carcinomas of the ovary. There were seven patients whose primary and recurrent high-grade serous carcinomas were available for analysis. This study was approved by the Institutional Review Board at the Johns Hopkins Medical Institutions. All tumor paraffin blocks were retrieved from the archival files of the Johns Hopkins Hospital except low-grade endometrioid and clear cell carcinomas, which were obtained from the National Taiwan University Hospital. The carcinomas were arranged in tissue microarrays to minimize the potential technical inconsistency related to in situ hybridization. Four micrometer thick paraffin sections were prepared for telomere fluorescence in situ hybridization. The clinical information including age, ethnic background, clinical stage, primary vs recurrent tumor were obtained from medical records. Telomere length in different types of ovarian carcinoma were assessed by telomere fluorescence in situ hybridization (FISH) using a method previously described. 23 Briefly, de-paraffinized slides were hydrated through a graded series of ethanol followed by deionized water plus 0.1% Tween-20. Slides were then placed in citrate buffer, steamed for 14 min (Black and Decker Handy Steamer Plus; Black and Decker, Towson, MD, USA) and then placed in PBS with Tween (PBST) for 5 min. Twenty-five μl of a Cy3-labeled telomere-specific peptide nucleic acid (PNA) (0.3-μg/ml PNA in 70% formamide, 10 mmol/l Tris, pH 7.5, 0.5% B/M Blocking reagent) was applied to the samples and incubated at 83 °C for 4 min. Slides were then moved to a dark, closed chamber for hybridization at room temperature for 2 h. After two washes in a solution of 70% formamide, 10 mmol/l Tris, pH 7.5, 0.1% albumin, slides were counterstained with 4′-6-diamidino-2-phenylindole (DAPI). The PNA probe complementary to the mammalian telomere repeat sequence (5′-CCCTAACCCTAACCCTAA-3′) was obtained from Applied Biosystems (Framingham, MA), with an N -terminal covalently linked to Cy3 fluorescent dye. As a positive control for hybridization, a FITC-labeled PNA probe (5′-ATTCGTTGG AAACGGGA-3′) specific for human centromeric DNA repeats (CENP-B binding sequence) was also included in the hybridization solution. 24 To measure the relative changes in telomere length, slides were imaged with a Nikon 50i epifluorescence microscope equipped with fluorescence excitation/emission filters for different (fluorophores) (Omega Optical). Grayscale images were captured using Nikon NIS-Elements software and an attached Photometrics Cool snap EZ digital camera. For presentation purposes, images were pseudo-colored and merged. Quantitative FISH was performed on the grayscale images using a custom plug in ( http://bui2.win.ad.jhu.edu/telometer/ ) written for the program Image J ( http://rsbweb.nih.gov/ij/ ). Quantitative telomere FISH was used to determine telomere length in tumor cells and associated stromal cells. The telomere fluorescence intensity was calculated as (total Cy3 intensity)/(total 4′,6-diamidino-2-phenylindole intensity) for each sampled nucleus; thus, compensating for ploidy differences or differing nuclear fraction due to microtomy. Approximately 30 representative nuclei were analyzed for tumor cells and tumor stromal cells as described. 23 The relative mean telomere length for tumor and stromal cells was calculated based on fluorescence signal from each case. The telomere index was then defined as the ratio of relative mean telomere length of tumor cells/stromal cells from each specimen. Telomere characteristics of the primary tumors, including mean cancer cell ratio, mean stromal cell ratio and telomere index among the four histologic types were compared using ANOVA. Comparisons between individual histologic types were performed using the Wilcoxon rank-sum test when the telomere index was treated as a continuous variable and the Fisher’s exact test when the telomere index was treated as a categorical variable. The telomere index among matched pairs of primary and recurrent tumor tissue from the same patient was also compared using the Wilcoxon sign-rank test. Kaplan–Meier survival curves of both telomere index categories were plotted for each tumor type. A stratified log-rank test was used to test equality of the survivor functions. Hazard ratios for risk of dying were estimated using Cox regression models stratified by histological type and adjusted for age and stage at diagnosis among those in whom followup was available. For this analysis, the telomere index was treated as a binary variable, grouped as either ≤ 1 or > 1. Greater than 1 reflecting a larger average telomere length in the carcinoma cells than the stromal cells. Proportionality of the models was also assessed. P = 0.05 was considered statistically significant. Stata 9 (College Station, TX, USA) was used to carry out the analyses. Correlation of the telomere index and specific histological features (for example cystic vs adenofibromatous clear cell carcinomas) was performed using Kruskal– Wallis test.