Mitochondrial DNA Copy Number Variation as a Potential Predictor of Renal Cell Carcinoma

Background Peripheral blood mitochondrial DNA (mtDNA) copy number alteration has been suggested as a risk factor for several types of cancer. The aim of the present study was to assess the role of peripheral blood mtDNA copy number variation as a noninvasive biomarker in the prediction and early detection of renal cell carcinoma (RCC) in a cohort of Egyptian patients. Methods Quantitative real-time polymerase chain reaction (qPCR) was used to measure peripheral blood mtDNA copy numbers in 57 patients with newly diagnosed, early-stage localized RCC and 60 age- and sex-matched healthy individuals as a control group. Results Median mtDNA copy number was significantly higher in RCC cases than in controls (166 vs. 91, p<0.001). Increased mtDNA copy number was associated with an 18-fold increased risk of RCC (95% confidence interval: 5.065-63.9). On receiver operating characteristic curve analysis, it was found that mtDNA could distinguish between RCC patients and healthy controls, with 86% sensitivity, 80% specificity, 80.3% positive predictive value and 85.7% negative predictive value at a cutoff value of 108.5. Conclusions Our results showed that increased peripheral blood mtDNA copy number was associated with increased risk of RCC. Therefore, RCC might be considered as part of a range of potential tumors in cases with elevated blood mtDNA copy number.

INTRODUCTION AND OBJECTIVES: Peripheral blood mitochondrial DNA (mtDNA) is suggested as a risk factor for several types of cancer. Previous studies have assessed the association between mtDNA and renal cell carcinoma (RCC), however, contradictory results were obtained. The aim of the present study was to assess the role of peripheral blood mtDNA in prediction and early detection of renal cell carcinoma in a cohort of Egyptian patients.
METHODS: The study enrolled 57 patients with early localized RCC (TNM stage I and II), confirmed by histopathological examination. They were consecutively recruited between August 2012 and March 2015 after excluding patients with advanced RCC (TNM stage III and IV) (to omit the effect of disease progression on mtDNA content), patients with other malignancies, family history of kidney cancer, recurrent RCC, and patients who received neoadjuvant therapy. Sixty healthy individuals with matching age and sex were included as control. The Ethics Committee approved this study and all subjects gave informed consent.Relative mtDNA copy number was measured using quantitative real-time PCR assay. Mitochondrial DNA (mtDNA) copy number was determined relative to the nuclear gene (HBB gene) using the formula -2DDCt .
RESULTS: Fifty seven patients (36 males, 21 females) diagnosed with early localized RCC were included: 50 clear RCC,5 papillary RCC and 2 chromophobe RCC cases. The mean age was 60.14 AE 6.83 years. Median mtDNA copy number was significantly higher in RCC cases than controls (166 vs 91, P<0.001). The role of mtDNA copy number as a risk factor for RCC was evaluated using unconditional logistic regression analysis. The median mtDNA content of the control group (91) was used as a cutoff value to analyze mtDNA copy number. It was found that patients with mtDNA content higher than 91 had a significant increase in RCC risk of 18 fold than those with lower levels with OR (odds ratio) of 18.0 (95% CI ¼ 5.065-63.9) in univariate analysis and an adjusted OR of 18.9 (95% CI ¼ 5.11-70.11) in multivariate analysis (after adjusting for age, gender, smoking status, hypertension and body mass index). The diagnostic value of mtDNA content in early detection of RCC was further assessed. Using receiver operating curve (ROC curve) analysis, it was found that mtDNA can detect RCC at a cutoff value of 108.5 with 86% sensitivity, 80% specificity, 80.3 % positive predictive value and 85.7% negative predictive value.
CONCLUSIONS: Increased mtDNA copy number could be used as a potential independent predictor of RCC risk. In addition, it may serve as a promising non-invasive biomarker for early detection of RCC. METHODS: An increase in the clear cytoplasm caused using adipogenic induction media (Lonza) in Caki-1 and Caki-2 cells. Human PCR array (Qiagen) was conducted for comparison of gene expressions which are related to adipogenesis and mitochondria. Western blotting and confocal microscopy were utilized to observe the protein expressions for mitochondria biogenesis. JC-1 dye staining (Cayman) to measure mitochondrial membrane potential was performed to observe mitochondrial function.
RESULTS: Clear cytoplasm was found to increase more in Caki-1 cells than in Caki-2. The increases of clear cytoplasm in Caki-1 cells were induced up-regulation of adipogenesis-related genes. In particular, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1a) which promotes mitochondrial biogenesis demonstrated an 11-fold increase (Fig. 1A). This up-regulation of PGC1a genes led increases in genes of voltage-dependent anion channels and cytochrome C oxidase subunit IV, ultimately promoting mitochondrial biogenesis (Fig. 1B, C). Also, in Caki-1 cells with increased clear cytoplasm, a mitochondrial healthy state was maintained with no impact on membrane potential, which is a significant role of mitochondria for cell's homeostasis ( Fig. 2A). Such activation of PGC1a was found to be caused by increased phosphorylation of AMPactivated protein kinase (AMPK) (Fig. 2B, C).