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Shorter telomere length is associated with increased ovarian cancer risk in both familial and sporadic cases
  1. Beatriz Martinez-Delgado1,2,
  2. Kira Yanowsky1,2,
  3. Lucia Inglada-Perez2,3,
  4. Miguel de la Hoya4,
  5. Trinidad Caldes4,
  6. Ana Vega2,5,
  7. Ana Blanco2,5,
  8. Teresa Martin6,
  9. Rogelio Gonzalez-Sarmiento7,
  10. Maria Blasco8,
  11. Mercedes Robledo2,3,
  12. Miguel Urioste1,2,
  13. Honglin Song9,
  14. Paul Pharoah9,10,
  15. Javier Benitez1,2
  1. 1Human Genetics Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
  2. 2Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain
  3. 3Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain
  4. 4Oncology Laboratory, Hospital Clinico San Carlos, Madrid, Spain
  5. 5Fundación Pública Galega de Medicina Xenómica-SERGAS, Grupo de Medicina Xenómica-USC, IDIS, Santiago de Compostela, Spain
  6. 6Servicio de Oncología, Hospital Universitario de Salamanca, Salamanca, Spain
  7. 7IBMCC, Universidad de Salamanca-CSIC, Salamanca, Spain
  8. 8Telomeres and Telomerase Group, Spanish National Cancer Centre (CNIO), Madrid, Spain
  9. 9Department of Oncology, University of Cambridge, Cambridge, UK
  10. 10Department of Public Health and Primary Care, University of Cambridge, Cambridge, UK
  1. Correspondence to Dr Beatriz Martinez-Delgado, Human Genetics Group, Spanish National Cancer Research Centre (CNIO), Melchor Fernandez Almagro 3, Madrid 28029, Spain; bmartinez{at}cnio.es

Abstract

Background Alterations in telomere maintenance mechanisms leading to short telomeres underlie different genetic disorders of ageing and cancer predisposition syndromes. It is known that short telomeres and subsequent genomic instability contribute to malignant transformation, and it is therefore likely that people with shorter telomeres are at higher risk for different types of cancer. Recently, the authors demonstrated that the genes BRCA1 and BRCA2 are modifiers of telomere length (TL) in familial breast cancer. The present study analysed TL in peripheral blood leucocytes of hereditary and sporadic ovarian cancer cases, as well as in female controls, to evaluate whether TL contributes to ovarian cancer risk.

Methods TL was measured by quantitative PCR in 178 sporadic and 168 hereditary ovarian cases (46 BRCA1, 12 BRCA2, and 110 BRCAX) and compared to TL in 267 controls.

Results Both sporadic and hereditary cases showed significantly shorter age adjusted TLs than controls. Unconditional logistic regression analysis revealed an association between TL and ovarian cancer risk with a significant interaction with age (p<0.001). Risk was higher in younger women and progressively decreased with age, with the highest OR observed in women under 30 years of age (OR 1.56, 95% CI 1.34 to 1.81; p=1.0×10−18).

Conclusion These findings indicate that TL could be a risk factor for early onset ovarian cancer.

  • Ovarian cancer
  • BRCA1 and BRCA2 genes
  • Telomeres
  • risk factors
  • case-control study
  • cancer: breast
  • genome-wide
  • genetics
  • microarray
  • microRNA
  • genetic epidemiology
  • thyroid disease
  • clinical genetics
  • diagnostics
  • genetic screening/counselling
  • dermatology
  • cancer: endocrine
  • oncology
  • molecular genetics
  • cancer: head and neck
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Introduction

Telomeres are structures responsible for maintaining chromosome stability through two main complexes: the telomerase complex that elongates the telomeres, and the shelterin complex that protects and caps the chromosome ends.1 These complexes are formed by several proteins and we currently know that mutations in some of the genes encoding these proteins can result in disorders associated with telomere shortening.2 Dyskeratosis congenita is one of the best known examples; it is a telomere disease characterised by, among others, bone marrow failure, abnormal skin manifestations, and premature ageing.3 Other examples are idiopathic pulmonary disorder, acute myelogenous leukaemia, and aplastic anaemia.

Under normal conditions, telomeres undergo shortening with each round of DNA replication, and consequently telomeres shorten with ageing.4 Some studies have reported that shorter telomeres in lymphocytes are associated with increased susceptibility to common diseases of ageing and may be predictive of both malignant and non-malignant diseases.5 Associations between telomere length (TL) and cancer have been found for bladder,6 head and neck,5 lung7 and gastric cancers,8 while studies on breast cancer have yielded inconsistent results.9 Regarding ovarian cancer, previous studies have shown that telomeres are shorter and telomerase activity is increased in malignant epithelial ovarian tumour tissues.10 In addition, plasma-derived free DNA of ovarian cases had shorter telomeres than controls,11 and shorter telomeres in peripheral blood leucocytes were associated with risk of serous ovarian adenocarcinoma.12 However, another very recent study found no differences in mean TL in lymphocytes between a group of ovarian cancer cases and controls, although they were able to find a significant association between different single nucleotide polymorphisms (SNPs) in the TERT gene and ovarian cancer risk.13

Regarding familial breast and ovarian cancer (FBOC), we recently demonstrated that familial breast cancer, with or without mutations in the BRCA1 or BRCA2 genes, was characterised by shorter telomeres compared with sporadic breast cancer and the control population.14 These findings suggest that BRCA1 and BRCA2 are telomere modifying genes, probably related to the role they play in telomere maintenance.15

In the present study, we retrospectively analysed TL in blood leucocytes from both hereditary and sporadic ovarian cancer cases, to investigate further the possible role of shorter TL as a risk factor for ovarian cancer. Our study demonstrates that both familial and sporadic ovarian cancer cases have shorter telomeres than controls and that there is a significant association between short telomeres and ovarian cancer risk.

Methods

Samples

Familial ovarian cancer patients were selected from FBOC (familial breast-ovarian cancer) syndrome families from the register of the Familial Cancer Consultation at the CNIO and the Hospital Clinico San Carlos in Madrid, Spain, the Hospital Clinico in Salamanca, Spain, and the Hospital Clinico in Santiago, Spain. All of them fulfilled the high risk criteria for genetic BRCA1/2 testing. Informed consent was obtained from all patients and the research project was approved by the ethics committees of all institutions. Selected patients included 46 cases from families carrying BRCA1 mutations, 12 from families carrying BRCA2 mutations, and 110 from families without a mutation in either of the two BRCA genes (BRCAX). Only one ovarian cancer patient from each family was included in the study. In addition, a series of 178 sporadic ovarian cancer cases from the Departments of Oncology and Public Health and Primary Care, University of Cambridge, UK, were analysed. They belong to an ongoing population based study of ovarian cancer started in 1998.16 Women with a history of breast and ovarian cancer were not included in the study. Control samples were from 267 women without personal or familial antecedents of cancer collected between 2000 and 2005, that had been previously used for the analysis of TL in patients with familial breast cancer.14 The mean age and range of all groups are given in table 1.

Table 1

Mean age and telomere length (TL) in the hereditary and sporadic ovarian cancer groups and controls

TL measurement

Blood DNA from ovarian cancer cases and controls was analysed for mean TL by using a quantitative PCR (qPCR) method previously described.14 Briefly, mean TL was calculated as the ratio (T/S) between telomere repeat copy number (T) and a single copy gene, 36B4 (S). All samples were analysed in triplicate using an ABI 7900HT thermal cycler. Since control and ovarian cancer cases were collected and analysed with difference on time, calibration samples were used to control inter-plate variation of PCR reactions, and the T/S ratio was calculated relative to the calibration sample, taking PCR efficiency into account using standard curves.

Statistical analysis

Age adjusted TL was calculated as the difference between the observed and the predicted values using the straight line of best fit defined in controls. Differences in age adjusted TL in the different groups were tested by t tests and nominal two sided p values <0.05 were considered statistically significant; Pearson's coefficient of correlation between age adjusted TL and age of ovarian cancer onset was estimated for hereditary and sporadic cancer. Statistical calculations were performed using SPSS V.17.0 (SPSS Inc).

For risk estimation, all sporadic and hereditary ovarian cases were analysed together, as no significant difference was found between their mean age adjusted TLs. The association between TL and ovarian cancer risk was assessed using unconditional logistic regression comparing age adjusted TL in cases and controls, including both TL and age as continuous variables as well as in different age intervals, and the interaction term between these two variables. TL units were rescaled to 0.1 u.

Results

TL in ovarian cancer patients

Mean age and TL of the different groups is shown in table 1. Controls were aged between 23 and 70 years and showed the expected decline in TL with age (r=−0.37). Shorter mean TL was found in all ovarian cancer groups (mean TL 0.63), compared to control women (mean TL 0.91) (table 1 and supplementary figure 1). Both familial and sporadic ovarian cases showed shorter telomeres than controls. After adjusting for age, significantly shorter TL was found in affected individuals from BRCA1 and BRCAX families than in the control population (p=0.005 and p=0.001, respectively) (table 1). Although ovarian cancer cases from BRCA2 families also had a shorter mean TL than controls, there was a limited number of BRCA2 cases and age adjusted TL was not statistically significantly different to controls (p=0.179). Age adjusted TL in blood leucocytes from sporadic ovarian cancer patients was also significantly shorter than in controls (p=1×10−5). However, TLs in sporadic cases were not significantly different from hereditary ovarian cases (p=0.449).

TL and ovarian cancer risk

Given that both familial and sporadic ovarian cancer cases presented shorter telomeres than the control population, we attempted to determine if there was a correlation between TL and age of ovarian cancer onset. A positive correlation between TL and age of onset (r=0.36, p=3.1×10−12) was found, where cases with the shortest telomeres tended to be diagnosed earliest. This correlation was significant both for hereditary and sporadic cases (r=0.32, p=1.5×10−5 and r=0.47, p=1.7×10−11, respectively) (supplementary figure 2).

Next, we assessed the interaction between TL and age of ovarian cancer. Strong evidence for an interaction between TL and age was found (OR 1.012, 95% CI 1.006 to 1.018; p=1×10−5). Further analysis showed that women of the same age with a shorter TL (Δ=0.1) generally had a higher risk for developing ovarian cancer than those with a longer TL. The risk associated with short telomeres was higher in younger women and progressively reduced as age increased (figure 1A). Women under 60 years with short telomeres appeared to be at increased risk of ovarian cancer, with a maximum estimated OR of 1.56 (95% CI 1.34 to 1.81; p=1.0×10−18) observed in women under 30 years that decreased to 1.07 (95% CI 1.00 to 1.14; p=9.7×10−16) for women between 50–60 years. Results for the older women (>60 years old) were not statistically significant, but they showed the same tendency (figure 1B).

Figure 1

Telomere length (TL) associated risk of ovarian cancer as a function of age. (A). OR estimates are higher in younger women and progressively decrease as the age of cancer onset increases. Lines correspond to the ORs and the 95% CI. (B) Specific mean OR values at different age groups and p values of the association between TL and ovarian cancer. Number of ovarian cancer cases and controls in each interval of age is shown.

Discussion

TL decreases during lifetime and is variable among individuals of the same age. There is increasing evidence that telomere shortening can contribute to cancer development by inducing genomic instability. It is therefore possible that individuals with shorter telomeres have an increased risk of cancer compared with those with longer telomeres. Although there are controversies regarding the role of TL in breast and colorectal cancer risk,17 an association between shorter TL and increased risk was observed for other cancer types including bladder, oesophageal, gastric, head and neck, ovarian, renal, and overall incident cancer.17 In our present retrospective study the results are in concordance with a prior case–control study by Mirabello et al, who observed significant association between TL and increased risk of serous ovarian carcinoma, especially in poorly differentiated carcinomas.12 A recent study revealed controversial results as it did not find a significant association between TL and ovarian cancer risk.13 More work is required to clarify whether inconsistencies among studies could be due to differences in assays used for TL measurements, retrospective versus prospective study, sample size, influence of chemotherapy or other factors that may affect TL such as smoking status or obesity.18 Furthermore, we included here novel data about TL in familial forms of ovarian cancer. Our study reveals that not only sporadic but also hereditary ovarian cancer cases exhibit shorter telomeres. Since short TL has been mainly associated with poorly differentiated ovarian carcinomas,12 and ovarian tumours arising in FBOC families are more often high grade serous carcinomas involving aggressive and genomically unstable tumours,19 it would be of interest to explore the risk associated with different histological ovarian tumour subtypes to better understand the role of TL in the aetiology of ovarian carcinomas. It is possible that critically short telomeres may induce chromosome instability and play a role in tumour formation.

An interaction between age adjusted TL and age was observed. Significant correlation was seen between these variables in both hereditary and sporadic cases. In addition, regression analysis showed a significant risk association of TL and ovarian cancer, which progressively decreases with age, from an estimated OR of 1.56 for women younger than 30 years, 1.36 between 30–40 years, 1.21 between 40–50 years, to an OR of 1.07 between 50–60 years. This suggests that TL could be a risk factor that might be modifying the age of ovarian cancer development.

Although some studies have highlighted the importance of chemotherapy treatment for TL,20 information regarding chemotherapy was not available in the present study. However, a previous study of TL in ovarian cancer cases did not find a difference between cases that did or did not undergo chemotherapy before sample collection.12 Previous results from our group on retrospectively analysed breast cancer cases carrying BRCA1/2 mutations and their asymptomatic carrier sisters also suggested that treatment did not affect the TL.14 Since affected and unaffected carriers had shorter telomeres than the control population, we concluded that BRCA1/2 mutations were modifiers of TL.14 We can now extend this conclusion to familial ovarian cancer cases, because they also presented shorter telomeres in lymphocytes. Shorter telomeres than controls were also found in sporadic ovarian cases, suggesting that other genetic or environmental factors may be acting as modifiers of TL. The same occurs with non-BRCA1/2 familial tumours, where shorter telomeres were found. In that case either the same modifying factors as in sporadic cases, or the involvement of other genes involved in telomere maintenance responsible for susceptibility to ovarian cancer, would be acting as telomere modifiers in these families. We cannot rule out the possibility that the observed association between TL and ovarian cancer risk is affected by a potential interaction between TL and other risk factors for ovarian cancer, such as the number of pregnancies, lifetime ovulatory cycles or hormone replacement therapy, which have not been considered in the present study.

Although larger studies are necessary to confirm these findings, it seems that short telomeres could be a risk factor for ovarian cancer in general, and for younger women in particular. Measuring TL in surrogate tissues such as peripheral blood lymphocytes would be of interest in the future for clinical application to detect women at higher risk for developing ovarian cancer as well as other cancer types.

Acknowledgments

We thank Roger Milne for support with the statistical analysis.

References

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Supplementary materials

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Footnotes

  • Funding This work was supported by Genetic Counseling Programme in Hereditary Cancer (Junta de Castilla y León) grant number FIS PI10/00219 (RGS); the Spanish Association Against Cancer (AECC), FIS PI08-1120 and by Red Tematica de investigacion Cooperativa en Cancer grant number RETICC RD06/0020/1060 (JB), and RETICC 06/0020/0021 (TC and MH); by the Xunta de Galicia grant number 10PXIB 9101297PR and Fundación Mutua Madrileña (AV).

  • Competing interests None declared.

  • Patient consent Obtained.

  • Ethics approval The ethics approval was provided by the ethics committee of the Instituto de Salud Carlos III, Spain.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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