Purpose: Mitochondria are highly susceptible to oxidative damage. Although mitochondrial function decreases with oxidative damage, overall mitochondrial DNA (mtDNA) content increases to compensate for general mitochondrial dysfunction. We performed quantitative polymerase chain reaction for genes specific to mitochondrial and nuclear genomes to investigate relative mitochondrial abundance in a spectrum of dysplastic head and neck lesions.Experimental Design: DNA from mild, moderate, and severe dysplasias, as well as invasive tumors and normal mucosal cells, was extracted. Using quantitative polymerase chain reaction, mitochondrial to nuclear DNA ratios were determined by quantification of cytochrome c oxidase subunit 1 (CoxI) and ␤-actin genes.Results: Mean CoxI/␤-actin DNA ratios for mild, moderate, and severe premalignant lesions were 0.0529, 0.0607, and 0.1021, respectively. The mean ratio for the normal mucosal cells contained in saliva was 0.0537, whereas the mean ratio for tumors was 0.1667. As a whole, our experimental model demonstrated significance (P ‫؍‬ 0.0358). Comparisons between individual categories showed borderline significance when compared with the normal group, with P values of 0.0673, 0.0747, and 0.0824 for moderate and severe dysplasia and invasive tumor, respectively. Conclusions:Head and neck squamous cell carcinomas arise through premalignant intermediates and may be merely morphologic manifestations of accumulated genetic alterations. In keeping with this molecular tumor progression model, our study shows that mtDNA increases according to histopathologic grade, a phenomenon that may be a feedback mechanism that compensates for a generalized decline in respiratory chain function. Therefore, high mtDNA content may be another marker of genetic alteration, a measure of relative DNA injury, and a surrogate measure of histopathologic grade.

Alterations of the mitochondrial DNA (mtDNA) have been described in human tumors and in other tissues in association with smoking exposure.We did quantitative PCR of cytochrome c oxidase I (Cox I) and cytochrome c oxidase II (Cox II) genes on oral rinse samples obtained from 94 patients with primary head and neck squamous cell carcinoma (HNSC) and a control group of 656 subjects. Mitochondrial DNA/nuclear DNA in saliva from HNSC patients and controls in relationship to smoking exposure, ethanol intake, and tumor stage were examined. Mean levels of Cox I and Cox II in saliva samples were significantly higher in HNSC patients: Cox I, 0.076 [95% confidence interval (95% CI), 0.06-0.09] and Cox II, 0.055 (95% CI, 0.04-0.07) in comparison with controls Cox I, 0.054 (95% CI, 0.05-0.06), P < 0.0001 and Cox II, 0.046 (95% CI, 0.04-0.05), P = 0.003 (t test). MtDNA levels were elevated in primary tumors when compared with matched, pretreatment saliva and significant correlation was noted (Cox I, r = 0.30, P = 0.005 and Cox II, r = 0.33, P = 0.002, respectively, Pearson's correlation). On univariate analysis, smoking, age, HNSC diagnosis, and advanced stage of HNSC were associated with higher level of mtDNA content in saliva. Multivariate analysis showed a significant and independent association of HNSC diagnosis, age, and smoking with increasing mtDNA/nuclear DNA for Cox I and Cox II. mtDNA content alteration is associated with HNSC independently of age and smoking exposure, can be detected in saliva, and may be due to elevation in mtDNA content in primary HNSC.

Background: Tumor-specific molecular alterations in surgical margins have been shown to predict risk of local recurrence. However, assays used for these analyses are time-consuming and therefore cannot be used in the intraoperative setting. Objective: To detect and quantify tumor-specific methylated promoter sequences in surgical margins in a time frame suitable for intraoperative use. Design: A novel quantitative methylation-specific polymerase chain reaction (QMSP) protocol. Methods: A total of 13 patients with head and neck squamous cell carcinoma (HNSCC) were initially characterized for molecular alterations in their tumor at the time of biopsy. Six primary tumors were found to harbor promoter hypermethylation for p16 and O 6-methylguanine-DNA-methyltransferase (MGMT) genes. Rapid QMSP was then used to identify promoter hypermethylation of these genes in the surgical margins. Results were compared with standard intraoperative histologic frozen section analysis and with conventional QMSP. Results: Using our rapid QMSP assay, we found that 3 patients had methylation-positive margins. Tumor margins from 2 patients were methylated for p16 alone, and margins from 1 patient were methylated for p16 and MGMT simultaneously. Molecular margin analysis was completed in less than 5 hours, a time frame appropriate for selected major HNSCC resections that require combined primary tumor resection, cervical lymphadenectomy, and complex reconstruction. This technique was comparable in sensitivity to conventional QMSP. Conclusion: Rapid molecular margin analysis using QMSP is feasible and may be performed intraoperatively in selected patients with HNSCC that requires extensive resection.

An increase in mitochondrial DNA (mtDNA) content and decline in mitochondrial function occurs with aging and in response to DNA-damaging agents, including tobacco smoke. We did a cross-sectional study and quantified changes in mtDNA content in a population of individuals with varied smoking and alcohol exposure. Age, smoking history, ethanol intake, and other demographic data were characterized for 604 individuals participating in a screening study for smoking-related upper aerodigestive malignancy. Total DNA was extracted from exfoliated cells in saliva. DNA from a nuclear gene, b-actin, and two mitochondrial genes, cytochrome c oxidase I and II (Cox I and Cox II), were quantified by real-time PCR. mtDNA content was correlated with age, exposure history, and other variables using multivariate regression analyses. A significant increase (P < 0.001) in mtDNA content was noted in smokers (31% and 29% increase for Cox I and Cox II, respectively) and former smokers (31% and 34%) when compared with never smokers. This association persisted after adjustment for other significant factors including age, alcohol drinking, and income (P < 0.001). Increased mtDNA content was positively associated with pack-years of smoking (P = 0.02). Despite an average smoking cessation interval of 21 years in former smokers, tobacco cessation interval was not statistically significantly associated with mtDNA content. Smoking is associated with increased mtDNA content in a dose-dependent fashion. Mitochondrial DNA alterations in response to smoking persist for several decades after smoking cessation, consistent with long-term, smoking-related damage. (Cancer Epidemiol Biomarkers Prev 2006;15(1):19 -24)

Purpose and Experimental Design: Alterations in mitochondrial DNA (mtDNA) sequence and content have been described in human tissues and tumors in association with smoking exposure. We did quantitative PCR analysis of cytochrome c oxidase (Cox) I and Cox II genes to measure changes in mtDNA content in pretreatment and posttreatment salivary rinses obtained from 76 patients undergoing surgical resection for primary head and neck squamous cell carcinoma. We also examined the relationship between changes in mtDNA content and postoperative radiation therapy, smoking exposure, alcohol intake, and other clinical characteristics. Results: Overall, mtDNA content in posttreatment saliva was significantly decreased. The mean change for Cox I was À0.21 [95% confidence interval (95% CI), À0.44 to 0.01, P = 0.06] and for Cox II was À0.31 (95% CI, À0.55 to À0.08, P = 0.01). Patients in the radiation therapy group exhibited a significant decrease compared with the nonradiated group (P = 0.03 for Cox I; P = 0.05 for Cox II). In addition, significant decreases in Cox I (À0.71; 95% CI, À1.17 to À0.25, P = 0.005) and Cox II (À0.65; 95% CI, À1.17 to À0.13, P = 0.02) were found in never-smoking patients but not in former or current smokers. Conclusion: Our data suggest that salivary mtDNA content is decreased in never smokers and in response to radiation therapy after primary surgical resection.