In this prospective cohort of women aged 35-74 years, no association was observed between relative telomere length in blood and breast cancer risk. Short telomeres were rather associated with a decreased risk of breast cancer in several subgroups; however, these associations were not significant and showed no indication of linear trend. Our finding of no association between telomere length and breast cancer risk is consistent with recent reports including two prospective investigations [8
]. On the other hand, short telomeres were associated with either an increased [9
] or a decreased [10
] risk of breast cancer in case-control studies where telomere length was quantified in blood after diagnosis of cancer. This could reflect possible reverse causation in case-control studies. It has been reported that individual patients underwent various changes in leukocyte telomere length after completion of adjuvant standard-dose chemotherapy commonly used for breast cancer [19
]. The present prospective study is not subject to potential bias due to reverse causality by treatment effect. Although the follow-up time was relatively short with mean of 460 days in cases in this study, the stratified analysis by time between blood draw and diagnosis showed no difference in the association with telomere length by recency of diagnosis suggesting that telomere length in blood is neither indicator of future breast cancer risk nor a marker for the presence of breast cancer prior to clinical diagnosis.
The present study introduced several innovations aimed at enhancing both accuracy and precision in telomere length measurement. First, we determined individual telomere length in up to 8 technical replicates run across 4 PCR plates in all study samples. We also adopted a single-tube monochrome multiplexing PCR method, which is an improved alternative to the conventional singleplex (two tube) PCR-based method [15
] because the T/S ratio is no longer affected by the pipetting-induced variation in DNA amount[15
]. With this method, however, %CV in our study was not particularly improved from some of the previously reported [11
]. This might be partly explained by different methods calculating %CV found in the literature including computation of %CV based on variability of threshold cycle values [20
], or computation using only the remaining measurements after excluding outliers, with outliers being defined in various manners [11
]. Such approaches would have reduced our reported %CV. For example, we performed sensitivity analyses recalculating individual's average telomere length after excluding technical outliers based on several predefined criteria. While we could reduce %CV as low as 5% under different definition of outlier, we observed that there were very few technical outliers regardless of the methods chosen, and exclusion of the few outliers did not impact the observed association between telomere length and breast cancer (data not shown). Since we found no influence of outlier on the association of interest, we used all the measurements in our analysis and reported conservative %CV based on T/S ratio.
In any studies, a null finding could have been caused by insufficient power. However, our current sample size was previously estimated to be sufficient to detect a significant difference of 0.08 (assuming SD=0,4) in mean relative telomere length between cases and non-cases. The mean difference in telomere length of 0.01(SD=0.37) observed in the present study might have become statistically significant with much bigger sample size, but it is doubtful that such small mean difference, which can be converted to approximately 42 base pairs according to a validation study [21
], would have a biologic implication or any clinical relevance.
Accumulating evidence from experimental studies clearly supports the role of telomere regulation in the development and progression of cancer: unrestrained telomere shortening and activation of telomerase may lead to genomic instability that accelerates accumulation of genetic aberrations for cellular immortalization and cancer development [3
]. Telomere length in blood, which may record a replicative history of hematopoietic stem cells and progenitor cells, has been associated with chronological age, obesity and smoking [22
]. However, our data did not suggest that telomere length in blood is associated with telomere function in distant tissues like breast.
In conclusion, we used prospectively collected blood samples to conduct a case-cohort analysis of telomere length and breast cancer and did not find evidence supporting telomere length in blood as a biomarker for breast cancer risk. It is unlikely that telomere length in blood is an important predictor of breast cancer risk.