Genetic integrity is maintained, in part, by the architecture of telomeres. By capping the ends of chromosomes, telomeres prevent nucleolytic degradation, end-to-end fusion, and irregular recombination. Telomere shortening might lead to chromosomal instability. We previously developed a laser scanning cytometry based quantitative fluorescence in situ hybridization assay (Q-FISHLSC) to assess telomere length in individual cells and showed that telomeres in peripheral blood lymphocytes (PBLs) were significantly shorter in patients with tobacco-related cancers than in matched controls. In this study, we aim to further refine this assay, expand our kidney cancer study and extend our analysis to subpopulations of lymphocytes. We cultured PBLs in the presence of phytohemagglutinin to expand T cell population and then isolated CD4+ and CD8+ T cells using a conventional immunomagnetic separation method. We modified our Q-FISHLSC assay, incorporated 9 internal controls to normalize telomere length, and applied to a case-control study with 65 renal cell carcinoma (RCC) patients and 65 age (±1 year), sex, and ethnicity matched controls. We found that the telomere length was significantly shorter in cases than in controls. The normalized telomere length in cases and controls was 0.84 ± 0.15 vs. 0.95 ± 0.18 for CD4+ cells (P=0.001); 0.80 ± 0.21 vs. 0.95 ± 0.22 for CD8+ cells (P<0.001); and 0.88 ± 0.25 vs. 0.99 ± 0.22 for overall PBLs (P=0.011). There was no significant difference between the telomere length of CD4+ and that of CD8+ T cells either in controls (P=0.15) or in cases (P=0.85). After adjusted by age, sex, ethnicity, and smoking status and using the 75th percentile of telomere length in the controls as a cutoff point, the short telomeres in CD4+ T cells was associated with a significantly increased risk for RCC (OR=3.08, 95% CI= 1.14-8.34). After stratifying by the quartiles of the telomere length distribution in the controls, a dose response association between telomere length and RCC risk was discovered. Compared to individuals with the highest quartile of telomere length, the ORs for those with the 3rd quartile, 2nd quartile and the lowest quartile were 1.81 (95% CI 0.54-6.08), 2.15 (95% CI 0.67-6.91) and 5.41 (95% CI 1.78-16.4), respectively (P for trend <0.01). Similar trends were observed in CD8+ T cells and overall PBLs. Our data further strengthened the hypothesis that telomere shortening is a genetic predisposing factor for cancer, but not due to different subpopulations of immune cells resulting from differential clonal expansion. Incorporating sufficient internal controls to normalize the telomere length enhanced the reproducibility of Q-FISHLSC assay. Supported by grants CA98897 and CA85576.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]