Helene Barcelo

Mitochondrial Copy Number is Associated with Colorectal Cancer Risk

Background: Mitochondria are eukaryotic organelles responsible for energy production. Quantitative changes in human mitochondrial DNA (mtDNA) copy number have been implicated in various cancer types. Data from prospective cohort studies on mtDNA copy number and colorectal cancer risk have been lacking. Methods: We evaluated the association between mtDNA copy number in peripheral blood and colorectal cancer risk in a nested case–control study of 422 colorectal cancer cases (168 cases with pre-diagnostic blood and 254 cases with post-diagnostic blood) and 874 controls who were free of colorectal cancer among participants of the Singapore Chinese Health Study. The relative mtDNA copy number was measured using real-time PCR. Unconditional logistic regression methods were employed to examine the association between mtDNA copy number and colorectal cancer risk. Results: There was a U-shaped relationship between the relative mtDNA copy number and colorectal cancer risk. Compared with the 2nd quartile, the OR (95% confidence intervals) for subjects in the lowest and highest quartiles of relative mtDNA copy numbers were 1.81 (1.13–2.89) and 3.40 (2.15–5.36), respectively (Pcurvilinearity <0.0001). This U-shaped relationship was present in both men and women, similar for colon cancer and rectal cancer, and independent of the timing of blood draw with regard to cancer diagnosis. Conclusions: This is the first prospectively designed study to show a U-shaped association between the relative mtDNA copy number and risk of colorectal cancer. Impact: The findings of the present study support that mtDNA may play a critical role in the colorectal carcinogenesis in humans. Cancer Epidemiol Biomarkers Prev; 21(9); 1574–81. ©2012 AACR.

Mitochondrial DNA Copy Number Is Associated with Breast Cancer Risk

Mitochondrial DNA (mtDNA) copy number in peripheral blood is associated with increased risk of several cancers. However, data from prospective studies on mtDNA copy number and breast cancer risk are lacking. We evaluated the association between mtDNA copy number in peripheral blood and breast cancer risk in a nested case-control study of 183 breast cancer cases with pre-diagnostic blood samples and 529 individually matched controls among participants of the Singapore Chinese Health Study. The mtDNA copy number was measured using real time PCR. Conditional logistic regression analyses showed that there was an overall positive association between mtDNA copy number and breast cancer risk (Ptrend = 0.01). The elevated risk for higher mtDNA copy numbers was primarily seen for women with <3 years between blood draw and cancer diagnosis; ORs (95% CIs) for 2nd, 3rd, 4th, and 5th quintile of mtDNA copy number were 1.52 (0.61, 3.82), 2.52 (1.03, 6.12), 3.12 (1.31, 7.43), and 3.06 (1.25, 7.47), respectively, compared with the 1st quintile (Ptrend = 0.004). There was no association between mtDNA copy number and breast cancer risk among women who donated a blood sample ≥3 years before breast cancer diagnosis (Ptrend = 0.41). This study supports a prospective association between increased mtDNA copy number and breast cancer risk that is dependent on the time interval between blood collection and breast cancer diagnosis. Future studies are warranted to confirm these findings and to elucidate the biological role of mtDNA copy number in breast cancer risk.

No association between mitochondrial DNA copy number and colorectal adenomas

Despite previously reported associations between peripheral blood mtDNA copy number and colorectal cancer, it remains unclear whether altered mtDNA copy number in peripheral blood is a risk factor for colorectal cancer or a biomarker for undiagnosed colorectal cancer. Though colorectal adenomas are well‐recognized precursor lesions to colorectal cancer, no study has evaluated an association between mtDNA copy number and colorectal adenoma risk. Hence, we investigated an association between peripheral blood mtDNA copy number and incident, sporadic colorectal adenoma in 412 colorectal adenoma cases and 526 cancer‐free controls pooled from three colonoscopy‐based case–control studies that used identical methods for case ascertainment, risk factor determination, and biospecimen collection. We also evaluated associations between relative mtDNA copy number and markers of oxidative stress, including circulating F2‐isoprostanes, carotenoids, and fluorescent oxidation products. We measured mtDNA copy number using a quantitative real time polymerase chain reaction (PCR). We used unconditional logistic regression to analyze the association between mtDNA copy number and colorectal adenoma risk after multivariable adjustment. We found no association between logarithmically transformed relative mtDNA copy number, analyzed as a continuous variable, and colorectal adenoma risk (odds ratio = 1.02, 95%CI: 0.82–1.27; P = 0.86). There were no statistically significant associations between relative mtDNA copy number and other markers of oxidative stress. Our findings, taken together with those from previous studies, suggest that relative mtDNA copy number in peripheral blood may more likely be a marker of early colorectal cancer than of risk for the disease or of in vivo oxidative stress.

Associations of mitochondrial polymorphisms with sporadic colorectal adenoma

Somatic mutations in mitochondrial DNA have been reported in colorectal adenomatous polyps (adenomas), the precursors to most colorectal cancers. However, there are no reports of associations of germline variation in mitochondrial DNA with adenoma risk. We investigated associations of germline polymorphisms in the displacement loop (D‐loop) and non‐D‐loop region of the mitochondrial genome with incident, sporadic colorectal adenoma in three pooled colonoscopy‐based case‐control studies (n = 327 adenoma cases, 420 controls) that used identical methods for case and risk factor ascertainment. We sequenced a 1124 bp fragment to identify all genetic variation in the mitochondrial D‐loop region, and used the Sequenom platform to genotype 64 tagSNPs in the non‐D‐loop region. We used multivariable unconditional logistic regression to estimate associations of the polymorphisms with adenoma. The odds ratios (OR) for associations of four polymorphisms in the HV1 region (mt16294, mt16296, mt16278, mt16069) with adenoma were 2.30, 2.63, 3.34, and 0.56, respectively; all 95% confidence intervals (CI) excluded 1.0, however, after correction for multiple comparisons, none of the findings remained statistically significant. Similar results were found for six polymorphisms in the non‐D‐loop region. In the HV1 region poly C tract, relative to those with 5 repeats, the ORs for those with fewer or more repeats were, respectively, 2.29 (95%CI 1.07‐4.89) and 0.63 (95%CI 0.36‐1.08), but repeat numbers in the HV2 region were not associated with adenoma. These findings suggest that mitochondrial D‐loop HV1 region polymorphisms may be associated with colorectal adenoma risk and support further investigation.

Effect of delayed cell processing and cryopreservation on immunophenotyping in multicenter population studies

Variability induced by delayed cell processing and cell cryopreservation presents unique challenges for immunophenotyping in large population studies. We conducted a pilot study to evaluate the effect of delayed cell processing and cryopreservation on cell percentages obtained by immunophenotyping. We collected blood from 20 volunteers and compared the effect of (a) delayed cell processing up to 72 h (b) cryopreservation and (c) the combined effect of delayed cell processing and cryopreservation on immunophenotyping of 31 cell subsets that included several subsets of T, B, Natural Killer (NK) cells, monocytes and dendritic cells using both whole blood collected in EDTA tubes and peripheral blood mononuclear cells collected in CPT tubes. We found the delayed cell processing up to 72 h or cryopreservation alone did not significantly affect the percentages T cells, dendritic cells or monocytes but significantly increased the percentage of B cells and NK cells (p for trend ≤0.01) but. However combination of delayed cell processing up to 72 h and cryopreservation significantly increased the percentage of T cells as compared to cells processed immediately (p for trend <0.0001) while a delayed cell processing followed by cryopreservation decreased the percentage of NK cells (p for trend <0.0001). Total B-cells increased significantly with a 24-48 h delay in cell processing and cryopreservation but not at 72 h. The percentages of monocytes and dendritic cells remained unaffected by the combination of delayed cell processing and cryopreservation. These findings suggest that immunophenotyping of several immune cell subsets can be successfully implemented in large population studies as long as blood is processed within 48 h of biospecimen collection though some cell subsets may be more susceptible to a combination of delayed cell processing and cryopreservation.

A Practical Cryopreservation and Staining Protocol for Immunophenotyping in Population Studies

Large population‐based cohort studies, through their prospective collection of a broad range of health information, represent an invaluable resource for novel insights into the pathogenesis of human diseases. Collection and cryopreservation of viable cells from blood samples is becoming increasingly common in large cohorts as these cells are a valuable resource for immunophenotyping and functional studies. The cryopreservation of peripheral blood mononuclear cells (PBMCs), thawing, and immunophenotyping protocols used to immunophenotype 9938 participants in the Health and Retirement Study (HRS) are described. The extensive quality control involved in a large‐scale immunophenotyping epidemiological study is also outlined. The existing literature on the effect of cryopreservation on various immune cell subsets including T, B, NK cells, monocytes, and dendritic cells is provided.

Abstract 4281: Mitochondrial DNA copy number, urinary isoprostanes and colorectal cancer risk

Previous epidemiological studies on mitochondrial DNA (mtDNA) copy number and risk of colorectal cancer have yielded mixed results. One study reported a positive association, another reported an inverse association, and a third, a U-shaped association of mtDNA copy number with colorectal cancer risk. Since oxidative stress is one of the proposed underlying mechanisms that may link mtDNA copy number variation to colorectal cancer risk, we conducted a matched case-control study of incident colorectal cancer nested within the Singapore Chinese Health Study. Pre-diagnostic blood and urine samples were obtained from 448 colorectal cancer cases and 869 age- and sex-matched controls. The mtDNA copy number in peripheral white cells was measured using real time PCR and urinary F2-isoprostanes were quantified using a liquid chromatography mass spectrometry based method. Conditional logistic regression was used to estimate associations of mtDNA copy number and urinary F2-isoprostanes with incident colorectal cancer. The odds ratios (OR) and 95% confidence intervals (95% CI) for those in the highest relative to those in the lowest quartiles of relative mtDNA copy numbers and urinary F2-isoprostanes were 1.02 (95% CI 0.72 - 1.43; p for trend = 0.56) and 1.11 (95% CI 0.78-1.59; p for trend = 0.35) respectively. Urinary F2-isoprostanes levels were not associated with the mtDNA copy number among the controls (r = 0.03; p = 0.33). The results of this prospective study suggest that white blood cell mtDNA copy number or urinary levels of F2-isoprostanes in biospecimens collected several years prior to the diagnosis of colorectal cancer may not be associated with risk of developing colorectal cancer. Citation Format: Bharat Thyagarajan, Renwei Wang, Woon-Puay Koh, Helene Barcelo, Jennifer Adams-Haduch, Weihua Guan, Roberd M. Bostick, Jian-Min Yuan, Myron D. Gross. Mitochondrial DNA copy number, urinary isoprostanes and colorectal cancer risk. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4281.

Abstract 3277: A low plasma mitchondrial DNA copy number is associated with increased breast cancer risk

Previous studies have shown that plasma mtDNA can be separated into two fractions; a supernatant form that is cell-free and a pelleted form that is thought to be platelet associated. These fractions of plasma mtDNA should be measured in studies evaluating the role of plasma mtDNA as biomarkers for early detection or susceptibility to various cancers. Though one case-control study has shown lower plasma mitochondrial DNA (mtDNA) copy number to be associated with increased breast cancer risk this study did not evaluate the two fractions of plasma mtDNA. Hence, the role of plasma mtDNA in determining breast cancer risk was investigated further. We conducted a case-control study of 29 breast cancer cases and 28 cancer free controls to evaluate the association between (a) plasma mtDNA copy number and (b) peripheral blood leucocyte mtDNA copy number and breast cancer risk. Plasma obtained from the breast cancer cases and controls was centrifuged further at 18,000 g for 10 minutes to separate the plasma into the supernatant and pelleted fractions. Using flow cytometry, we stained the pelleted fraction of the plasma with Annexin V and CD61 to determine the cellular source of the plasma pellet. DNA from the two plasma fractions and the buffy coat was extracted using the Qiagen DNAesy DNA extraction kit. Nuclear DNA (18s) and mitochondrial DNA (ND1) copy numbers were measured using quantitative real time PCR. We used a two-sample independent t test to evaluate the difference in mtDNA in the two plasma fractions and mtDNA copy number in peripheral blood leucocytes among breast cancer cases and controls. The plasma pellet fraction predominantly contained particles that were 1-3 microns in size that stained positively for both Annexin V and CD61 indicating that these particles were platelet derived microparticles. The mtDNA in the plasma pellet accounted for 96% of all the plasma mtDNA seen in both cases and controls. Both the plasma supernatant mtDNA (2601034 mtDNA copies/ml of plasma vs. 4700446 mtDNA copies/ml of plasma; p = 0.004) and plasma pellet mtDNA (67787069 mtDNA copies/ml of plasma vs. 112883929 mtDNA copies/ml of plasma; p = 0.001) were significantly lower in the breast cancer cases as compared to controls. Peripheral blood leucocytes mtDNA copy number was not associated with breast cancer risk (4.27 vs. 3.73; p=0.14). There was no correlation between mtDNA in peripheral blood and the various fractions of plasma mtDNA (r= -0.08 - 0.07; p = 0.55 - 0.63). This is the first study to demonstrate platelet derived microparticles as the source of the plasma pellet mtDNA. This study shows that both the supernatant and pelleted fractions of plasma mtDNA are associated with breast cancer risk. Peripheral blood leucocytes mtDNA was not associated with breast cancer risk and was not correlated with plasma mtDNA. Future studies are needed to confirm these associations and evaluate possible mechanisms through which plasma mtDNA copy number is associated with breast cancer risk. Citation Format: Bharat Thyagarajan, Helene Barcelo, Kristin E. Anderson, Karen Swenson, Heather Nelson, Myron D. Gross. A low plasma mitchondrial DNA copy number is associated with increased breast cancer risk. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3277. doi:10.1158/1538-7445.AM2014-3277

Genetic variants in DNA repair pathways are not associated with disease progression among multiple myeloma patients

DNA damage induced by high dose melphalan and autologous transplantation is repaired by the nucleotide excision repair (NER) and base excision repair (BER) pathways. We evaluated the association between single nucleotide polymorphisms (SNPs) (n=311) in the NER and BER pathways and disease progression in 695 multiple myeloma patients who underwent autologous transplantation. None of the SNPs were associated with disease progression. Pathway based analyses showed that the NER pathway had a borderline association with disease progression (p=0.09). These findings suggest that common variation in the NER and BER pathways do not substantially influence disease progression in multiple myeloma patients.

Abstract 1339: Mitochondrial DNA copy number is prospectively associated with breast cancer risk.

A previous case-control study reported a positive association between mitochondrial DNA (mtDNA) copy number in peripheral blood and breast cancer risk. However, data on mtDNA copy number and breast cancer risk in prospective cohorts is lacking. We evaluated the prospective association between mtDNA copy number and breast cancer risk in the Singapore Chinese Health Study. We conducted a nested case-control study of 183 incident breast cancer cases with pre-diagnostic blood samples and 529 individually matched control women who were free of breast cancer. The mtDNA copy number was measured using real time PCR. Conditional logistic regression methods were employed to examine the association between mtDNA copy number and breast cancer risk. Overall, there was no association between mtDNA copy number and breast cancer after adjustment for potential confounders (p=0.37). Among participants who donated a blood sample within 3 years of breast cancer diagnosis women in the second and third tertile of mtDNA copy number had a significantly increased breast cancer risk as compared to those in the lowest mtDNA copy number tertile (odds ratio (95% confidence interval): 1.84 (0.93-3.64) and 2.13 (1.09-4.17) respectively; p for trend = 0.03). There was no association between mtDNA copy number and breast cancer risk among women who donated a blood sample ≥ 3 years before breast cancer diagnosis (p for trend = 0.73). This study supports a prospective association between increased mtDNA copy number and breast cancer risk that is dependent on the time interval between blood collection and breast cancer diagnosis. Citation Format: Bharat Thyagarajan, Renwei Wang, Helene Barcelo, Heather Nelson, Woon Puay Koh, Jian Min Yuan. Mitochondrial DNA copy number is prospectively associated with breast cancer risk. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1339. doi:10.1158/1538-7445.AM2013-1339