This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Orlow, I. Articles by Rusch, V. W. Search for Related Content PubMed PubMed Citation Articles by Orlow, I. Articles by Rusch, V. W. Social Bookmarking                            What's this?

DNA Damage and Repair Capacity in Patients With Lung Cancer: Prediction of Multiple Primary Tumors

Irene Orlow, Bernard J. Park, Urvi Mujumdar, Himali Patel, Puiki Siu-Lau, Brian A. Clas, Robert Downey, Raja Flores, Manjit Bains, Nabil Rizk, Gemma Dominguez, Jen Jani, Marianne Berwick, Colin B. Begg, Mark G. Kris, Valerie W. Rusch

From the Departments of Epidemiology and Biostatistics, Surgery, and Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY; and the University of New Mexico, Albuquerque, NM

Corresponding author: Irene Orlow, PhD, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, Box 353, New York, NY 10021; e-mail: orlowi{at}mskcc.org

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 
Purpose Patients who survive one occurrence of non–small-cell lung cancer (NSCLC) are at higher risk of a second malignancy. Capacity to repair damaged DNA may modulate individual susceptibility to develop lung cancer. Therefore, we evaluated constitutive and induced DNA damage, and repair capacity, in patients with multiple NSCLCs (cases) and compared the results to those obtained in patients with a single NSCLC (controls).

Patients and Methods One hundred eight cases and 99 controls matched by age, sex, and time since diagnosis were studied. DNA damage was assessed on peripheral blood lymphocytes by the comet assay before and after exposing cells to a tobacco-derived carcinogen, using the tail moment and the tail intensity as measures to assess baseline damage, induced damage and repair capacity.

Results Constitutive DNA damage, benzo(a)pyrene diol epoxide–induced damage, and repair after BPDE-induced damage were all significantly higher in cases than in controls. These results were confirmed in regression analyses adjusted for potential confounders.

Conclusion DNA damage as measured by the comet assay is associated with the development of multiple primary tumors in individuals with NSCLC.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 
Lung cancer is the second most frequent cancer in both males and females in the United States after breast and prostate cancer; and the most common cause of death from cancer in men and women ages 40 and 60 years, respectively.¹ A major etiologic factor is exposure to tobacco smoke. However, only a fraction of heavy smokers develop lung cancer. It is probable that people differ in their susceptibility to tobacco smoke-inflicted damage and that their interindividual differences, attributed to heritable traits, modulate the risk of lung cancer.²

Regardless of the etiology, the high incidence and poor prognosis of lung cancer make this disease a major health problem worldwide. Patients who survive one primary cancer are at considerably higher risk of a second malignancy. Current evidence suggests that the annual incidence of second primary lung cancers is in the region of 1% to 2%.^3,4 A second primary lung cancer is potentially curable by surgical resection if discovered early and if the initial primary is also cured. Distinguishing patients who are at higher risk to develop multiple lung tumors is of great clinical relevance. Moreover, risk factors that are identified for a second primary lung cancer are highly likely to be risk factors for lung cancer in general. Thus, studies of factors that differ significantly between patients with second primary lung cancer and patients with a solitary lung cancer are convenient vehicles for identifying new risk factors for the disease.⁵ This study design has been utilized, for example, in studies of contralateral breast cancer.^6,7

There is considerable evidence that individual variation in the ability to limit DNA damage from endogenous and exogenous carcinogens contributes to cancer risk.^8-10 A number of epidemiologic studies in which a variety of global measures of DNA repair capacity were used found that individuals with lung cancer displayed a higher rate of spontaneous and carcinogen-induced chromosomal aberrations than the healthy controls.^11-14 Similarly, patients with lung cancer displayed a lower DNA repair capacity than controls,^13,15 which in the presence of a deficient cell cycle checkpoint increased the risk for developing lung cancer even further.¹⁴ Paz-Elizur et al¹⁶ evaluated a more specific aspect of repair. The enzymatic 8-oxoguanine DNA N-glycosylase (OGG) activity was significantly decreased in patients with lung cancer and the risk of developing non–small-cell lung cancer (NSCLC) for smokers with low OGG1 activity was observed to be 34- to 124-fold higher than for smokers with a normal OGG1 activity.

In this study we sought to identify the risk of developing multiple primary lung tumors in relation to levels of constitutive and induced DNA damage among patients with a single NSCLC. For this purpose, constitutive, induced, and unrepaired damage were quantified by measuring single strand DNA breaks and abasic sites of untreated and treated peripheral blood lymphocytes using the alkaline comet assay.¹⁷ This assay provides a measure of the net effect of each cell's antioxidant and DNA repair capacity to prevent and repair oxidative damage, the major endogenous source of genomic instability. To evaluate the cells’ sensitivity to the potent tobacco smoke constituent benzo(a)pyrene which is also found in fuel combustion, car exhaust, occupational settings, and contaminated air, cells were exposed to the main metabolic product and protein reactive benzo(a)pyrene diol epoxide (BPDE).¹⁸ In vitro assays indicate that the BPDE-DNA adducts are formed in less than 1 hour, while the removal of the induced damage can take several hours,^19,20 and depending on the position on the DNA, some adducts remain unrepaired.²¹ With the comet assay, the BPDE-modified DNA strands are revealed as fragmented DNA due to alkaline lability, presence of apurinic sites, and cleavage in the DNA at guanine, adenine, or cytosine sites.²² BPDE-DNA adducts are repaired mainly by the nucleotide excision repair pathway, although some unstable adducts may produce depurination that can be repaired by the base excision repair pathway.^23,24 In this study, we hypothesized that endogenous, BPDE-induced, and unrepaired damaged DNA as measured by the comet assay would be higher in patients who develop multiple primaries than in those who develop single primary tumors.

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 
Study Subjects
All participants were recruited at Memorial Sloan-Kettering Cancer Center (New York, NY) and gave written informed consent to participate in this patient-control study approved by the institutional review board. One hundred eight individuals diagnosed with a second primary cancer (cases) and 99 individuals diagnosed with a first primary lung cancer (controls) participated, matched by age (± 5 years), sex, and time since diagnosis. The distinction between a second primary tumor and a metastasis from the first primary was established as previously described by Martini and Melamed.²⁵ Briefly, patients met the criteria for having multiple primary lesions if both lung tumors had a different histology, or if the histology was the same but the second tumor originated from a carcinoma in situ, or was located in a different lung or lobe with no evidence of lymphatics common to both and no extrapulmonary metastases at the time of diagnosis. All participants donated blood and completed a questionnaire that provided detailed information on demographic data, exposure to tobacco smoke, occupational exposures, family history of cancer, dietary and exercise habits, medications, reproductive history, and past treatments for noncancer conditions. In addition, clinicopathologic information was collected from the medical records.

Blood Samples
Blood samples were collected in two to three green top heparinized Vacutainer tubes (BD Diagnostics, Franklin Lakes, NJ). These were light protected, and immediately transported at room temperature to the molecular epidemiology laboratory at Memorial Sloan-Kettering Cancer Center. Lymphocytes were isolated with standard procedures (Ficol) and cryopreserved in liquid nitrogen at a concentration of 1 x 10⁶/mL. Ninety-six hours before the comet assay, lymphocyte cultures were established in three T-25 flasks using RPMI-1640-1640 media supplemented with 20% fetal calf serum, antibiotics, and 1.5% phytohemagglutinin in a final volume of 10 mL.

Comet Assay or Single-Cell Gel Electrophoresis
This assay was utilized to measure constitutive genetic instability or baseline damage. Briefly, by this method, one can determine the constitutive or unrepaired DNA damage represented by shorter DNA strands that migrate faster in the electrophoretic field and resemble the tail of a comet. This assay has been used to monitor genotoxic effects of certain compounds, as well as to measure genetic instability associated with certain conditions including cancer.

We performed the comet assay under alkaline conditions as described by Singh et al¹⁷ with minor modifications. Briefly, 10 microliters of a cell suspension containing approximately 11,000 cells were mixed with 100 µL of 0.5% low-melting-point agarose in phosphate-buffered saline, kept at 37°C in a dry-bath incubator, spread on CometSlides HT slides (Trevigen, Gaithersburg, MD). The agarose solidified at 4°C for 10 minutes. Cells were lysed for 1 hour at 4°C in a freshly prepared lysis buffer (pH 10) containing 2.5 M NaCl, 100 mmol/L EDTA, 100 mmol/L Trizma base, 10% DMSO, and 1% Triton X-100. Slides were then rinsed three times in 0.4 M Tris-HCl (pH 7.5) for 5 minutes to remove detergents and salts and placed on a horizontal electrophoretic unit without power for 30 minutes in freshly prepared alkaline buffer (300 mmol/L NaOH and 1 mmol/L EDTA, pH > 13) at 4°C. Electrophoresis was carried out in a TECA2222 electrophoresis unit (Ellard Instrumentation Ltd, Monrow, WA) during 30 minutes at 25 volts, adjusting the current to 295 to 300 mAmp and with a constant recirculating flow of 100 mL/min. All these steps were carried out in the dark. Finally, the slides were rinsed in neutralization buffer (0.4 M Tris-HCl, pH 7.5) for 5 minutes three times, fixed in cold 100% ethanol for 15 minutes and air dried. To control the comet assay performance, each run included an internal laboratory control comprised of a pool of lymphocytes. In addition, room temperature and humidity levels were recorded.

Response to Induced Damage and DNA Repair Capacity
A modification of the basic alkaline comet assay was introduced in order to test the cells’ response to and their capacity to repair after in vitro induced damage. In addition to the baseline comet assay described earlier, we grew two culture flasks with cells that were exposed to 0.25 µmol/L BPDE. After 1 hour of incubation, all cells were washed with fresh media. Cells from one flask were processed for the comet assay in order to measure the sensitivity to BPDE while cells from the second flask were allowed to recover from the damage for 24 hours and then processed in order to measure the DNA repair capacity. The BPDE (CAS registry number 58,917 to 67-2) was purchased from the National Cancer Institute Chemical Carcinogen Repository (Midwest Research Institute, Kansas, MO), dissolved in DMSO (Sigma Chemical Co, St Louis, MO) at a concentration of 2 mmol/L, and stored at –20°C in the dark to prevent photo-oxidation.

Scoring of DNA Damage
Immediately before imaging analysis, slides were stained with 10 µL of a 1 µg/mL ethidium bromide solution for 5 minutes. Observations were made at 200x magnification using a fluorescent microscope (Olympus BX51; Olympus, Center Valley, PA) connected to a CoHu 4915-2010 CCD camera (Poway, CA). One hundred consecutive cells (50 from each duplicate slide) were randomly selected with care to avoid borders, starting with the center of the slide, and quantified with the Komet 5.5 software (Andor Technology, Belfast, United Kingdom). The extent of the damage was quantified by the tail moment (TM) and the tail intensity (TI). The most commonly reported parameter is the TM, defined as the product of the percentage of DNA in the comet tail and the distance between the means of the tail and head fluorescence distributions, where mean is the profile center of gravity, divided by 100.²⁶ The TM is expressed in arbitrary units. The TI is most useful as it bears a linear relationship to the density of abasic sites and DNA breaks. In addition, it is relatively unaffected by threshold settings, allows discrimination of damage over the widest possible range (0% to 100%), and gives an impression of the comet's appearance. The TI is defined as percentage of DNA (fluorescent) in the tail. Final results were expressed as the mean, median, and standard deviation of TMs and TIs of 100 cells from two duplicate slides. For quality control assurance, independent intra- and inter-observer readings were compared between two readers, for a total of 25 samples. For the analysis of sensitivity to BPDE, the relative induced damage (RID) was calculated as:

and for the analysis of repair kinetics, the percentage of residual DNA damage (RD%) after exposure to BPDE was calculated as follows²⁷:

where t is the time elapsed after incubating cells with BPDE (t0, 0 hours; t1, 24 hours), DNA damage at time t0 corresponds to the damage found immediately after replacing BPDE with fresh media, and DNA damage at time t1 corresponds to the DNA damage found after allowing the cells to recover from the BPDE induced damage.

Statistical Analysis
Differences in distribution between the patients with multiple NSCLC (cases) and those with single primary NSCLC (controls) with regard to age at consent and other lung cancer risk factors were determined using ² and t test statistics. Analysis of variance was conducted to estimate differences in the logarithms of the measures of DNA damage between subgroups (eg, sex, age). The t test was used to compare the differences between cases and controls in terms of log DNA damage measured by the TM and the TI. Linear regression analyses were conducted to adjust these comparisons for potential confounders using the SAS software, version 9.1 (SAS Institute, Cary NC). All statistical tests were two sided.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 
A total of 108 patients with multiple primary lung cancer (cases) were recruited, along with 99 patients with a single primary lung cancer (controls). All patients were recruited at Memorial Sloan-Kettering Cancer Center between March 2003 and November 2006. The majority of patients were white, non-Hispanic (89%) with the remaining being Asian, Hispanic, black, or other. Sixty-three of 108 second primary tumors were diagnosed within 2 years of the initial primary. Cases and controls were similar with respect to age at first diagnosis, age at consent, sex, histology, and smoking history (Table 1).

View larger version (37K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. (A) Constitutive, (B) benzo(a)pyrene diol epoxide (BPDE)–induced, and (C) unrepaired DNA damage in patients with non–small-cell lung cancer (NSCLC). Left: bar graphs show the distribution of DNA damage among patients with single (blue) and multiple (yellow) primary tumors. Right: representative nucleoids and median tail intensity (TI) values obtained with the comet assay (200x). Note the extent and intensity of the tails in cases 16 and 21 (arrows).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

Our genetic measures were obtained with the comet assay, also known as single-cell gel electrophoresis, one of the current standard methods for assessing DNA damage. The assay integrates the effects of exposure to exogenous and endogenous genotoxins due to both the amount of damaging agent, and the individual's metabolic and DNA repair capacities. The analysis of constitutive DNA damage in circulating lymphocytes indicates that cases have a reduced capacity to limit endogenous damage than controls (Table 3 and Fig 1A). As the most important etiologic risk factor for lung cancer is tobacco smoke, we also considered it appropriate to challenge the patients’ cells to BPDE in order to measure the sensitivity to the compound and the capacity to repair the induced damage. For this measure, we also found an association between DNA damage and the development of multiple tumors in NSCLC patients (Figs 1B and 1C). Linear regression indicates that these differences are statistically significant even when controlling for age, sex, smoking habits, and treatment status (Table 3).

This findings are consistent with those from previous studies in that constitutive DNA damage and capacity to repair damaged DNA may modulate an individual's susceptibility to develop NSCLC. Specifically, others have found that, when compared with unaffected individuals, patients with lung cancer were characterized by higher rate of spontaneous chromosomal aberrations,¹¹ elevated endogenous single-stranded breaks,¹² higher sensitivity to gamma-radiation, bleomycin, and BPDE,^12-14, and lower DNA repair capacity.¹⁵

In our study, we did not detect significant differences in the comet assay measures by age, histologic subtypes, or family history. However, men showed increased endogenous and induced DNA damage as well as higher levels of unrepaired DNA than females, regardless of case-control status. Sex differences in the sensitivity to tobacco carcinogens and in the susceptibility to lung cancer have been reported.^15,28-31 Several authors have hypothesized that women are at higher risk to develop lung cancer than men of the same age and smoking history. However, evidence from several large prospective studies found no such differences.^32-34 Women with lung cancer survive their disease longer than men.^35-38 This sex difference in survival may be due to the overall levels of damaged DNA. Future studies should examine in more depth sex differences in the repair capacity of patients with lung cancer.

Our study has some limitations. Use of postdiagnostic samples for assessing the predictive value of phenotype assays, such as the comet assay, is not ideal because the assay outcome may be affected by disease status or treatment. In fact, 18 of our study participants received chemotherapy, radiation therapy, or a combination of both. Treatment occurred within 5 years before recruitment. Levels of DNA damage were higher in individuals treated with chemotherapy, followed by those with radiation therapy (data not shown). To further characterize this type of measure as a potential biomarker of lung cancer development, the study should be replicated with prediagnostic samples collected in a prospective study. Nevertheless, in this pilot study after adjustment for confounding factors including treatment status the difference between cases and controls remained significant. These measures of DNA damage may prove to be helpful markers of risk for the development of multiple tumors, and for the development of de novo NSCLC. A further potential limitation is the possibility that some of the subsequent primaries may be clonal products of the initial tumor (ie, metastases). This issue is an active topic of research, and the literature to date suggests that lung tumors designated as second primaries by the Martini-Melamed criteria are predominantly, but not exclusively, independent occurrences of the disease.^39-41 Clearly, it is probable that some of our cases may be misdiagnosed metastases. However, this phenomenon would only have the effect of attenuating any observed differences between patients and controls.

In summary, these results suggest that elevated levels of damaged DNA are associated with the development of multiple NSCLC tumors in patients with NSCLC. If replicated in a large prospective study, relatively simple functional assays such as this could contribute to the identification of individuals with increased cancer susceptibility and may prove useful for follow-up for patients diagnosed with their initial NSCLC.

	AUTHORS’ DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 
The author(s) indicated no potential conflicts of interest.

	AUTHOR CONTRIBUTIONS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 
Conception and design: Irene Orlow, Marianne Berwick, Colin B. Begg, Mark G. Kris, Valerie W. Rusch

Financial support: Colin B. Begg, Mark G. Kris

Administrative support: Irene Orlow, Urvi Mujumdar, Marianne Berwick, Colin B. Begg, Valerie W. Rusch

Provision of study materials or patients: Irene Orlow, Bernard J. Park, Puiki Siu-Lau, Robert Downey, Raja Flores, Manjit Bains, Nabil Rizk, Mark G. Kris, Valerie W. Rusch

Collection and assembly of data: Irene Orlow, Urvi Mujumdar, Himali Patel, Puiki Siu-Lau, Brian A. Clas, Jen Jani, Mark G. Kris

Data analysis and interpretation: Irene Orlow, Bernard J. Park, Urvi Mujumdar, Himali Patel, Brian A. Clas, Colin B. Begg, Mark G. Kris

Manuscript writing: Irene Orlow, Urvi Mujumdar, Colin B. Begg

Final approval of manuscript: Irene Orlow, Bernard J. Park, Urvi Mujumdar, Himali Patel, Puiki Siu-Lau, Brian A. Clas, Robert Downey, Raja Flores, Manjit Bains, Nabil Rizk, Gemma Dominguez, Jen Jani, Marianne Berwick, Colin B. Begg, Mark G. Kris, Valerie W. Rusch

	ACKNOWLEDGMENTS

 
We thank Pampa Roy, Diana Tommasi, Javier Cotignola, and Henry Benjamin for their assistance with sample processing; Hetal Bhingradia, Kathleen Meneses, Alain Vincent, and Linette Lee for assisting with patient accrual; Pamela Ginex for patient accrual and initial stages of the protocol development; Jill Kleczko for clinical data management; Amanda Hummer for assistance with statistical analyses; and Narendra Singh, MD, and Marie Vasquez for useful discussions regarding the comet assay.

	NOTES

 
Supported by the Society of Memorial Sloan-Kettering Cancer Center through their annual appeal and Steps for Breath.

Presented in part at the 41st Annual Meeting of the American Society of Clinical Oncology, Orlando FL, May 13-17, 2005; and at the 11th World Lung Cancer Meeting, Barcelona, Spain, July 3-6, 2005.

Authors’ disclosures of potential conflicts of interest and author contributions are found at the end of this article.

	REFERENCES

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION AUTHORS' DISCLOSURES OF... AUTHOR CONTRIBUTIONS REFERENCES

 
1. Jemal A, Siegel R, Ward E, et al: Cancer statistics, 2007. CA Cancer J Clin 57:43-66, 2007[Abstract/Free Full Text]

2. Ezzati M, Henley SJ, Lopez AD, et al: Role of smoking in global and regional cancer epidemiology: Current patterns and data needs. Int J Cancer 116:963-971, 2005[CrossRef][Medline]

3. Rubins J, Unger M, Colice GL: Follow-up and surveillance of the lung cancer patient following curative intent therapy: ACCP evidence-based clinical practice guideline. Chest 132:355-367, 2007[CrossRef]

4. Trousse D, Barlesi F, Loundou A, et al: Synchronous multiple primary lung cancer: An increasing clinical occurrence requiring multidisciplinary management. J Thorac Cardiovasc Surg 133:1193-1200, 2007[Abstract/Free Full Text]

5. Begg CB, Berwick M: A note on the estimation of relative risks of rare genetic susceptibility markers. Cancer Epidemiol Biomarkers Prev 6:99-103, 1997[Abstract]

6. Bernstein JL, Langholz B, Haile RW, et al: Study design: Evaluating gene-environment interactions in the etiology of breast cancer - the WECARE study. Breast Cancer Res 6:R199-214, 2004[CrossRef][Medline]

7. Begg CB, Hummer AJ, Mujumdar U, et al: A design for cancer case-control studies using only incident cases: Experience with the GEM study of melanoma. Int J Epidemiol 35:756-764, 2006[Abstract/Free Full Text]

8. Berwick M, Vineis P: Markers of DNA repair and susceptibility to cancer in humans: An epidemiologic review. J Natl Cancer Inst 92:874-897, 2000[Abstract/Free Full Text]

9. Spitz MR, Wei Q, Dong Q, et al: Genetic susceptibility to lung cancer: The role of DNA damage and repair. Cancer Epidemiol Biomarkers Prev 12:689-698, 2003[Free Full Text]

10. Vineis P: Individual susceptibility to carcinogens. Oncogene 23:6477-6483, 2004[CrossRef][Medline]

11. Wei Q, Gu J, Cheng L, et al: Benzo(a) pyrene diol epoxide-induced chromosomal aberrations and risk of lung cancer. Cancer Res 56:3975-3979, 1996[Abstract/Free Full Text]

12. Zhang H, Buchholz TA, Hancock D, et al: Gamma-radiation-induced single cell DNA damage as a measure of susceptibility to lung cancer: A preliminary report. Int J Oncol 17:399-404, 2000[Medline]

13. Rajaee-Behbahani N, Schmezer P, Risch A, et al: Altered DNA repair capacity and bleomycin sensitivity as risk markers for non-small cell lung cancer. Int J Cancer 95:86-91, 2001[CrossRef][Medline]

14. Wu X, Roth JA, Zhao H, et al: Cell cycle checkpoints, DNA damage/repair, and lung cancer risk. Cancer Res 65:349-357, 2005[Abstract/Free Full Text]

15. Wei Q, Cheng L, Amos CI, et al: Repair of tobacco carcinogen-induced DNA adducts and lung cancer risk: A molecular epidemiologic study. J Natl Cancer Inst 92:1764-1772, 2000[Abstract/Free Full Text]

16. Paz-Elizur T, Krupsky M, Blumenstein S, et al: DNA repair activity for oxidative damage and risk of lung cancer. J Natl Cancer Inst 95:1312-1319, 2003[Abstract/Free Full Text]

17. Singh NP, McCoy MT, Tice RR, et al: A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184-191, 1988[CrossRef][Medline]

18. Lodovici M, Luceri C, Guglielmi F, et al: Benzo(a)pyrene diolepoxide (BPDE)-DNA adduct levels in leukocytes of smokers in relation to polymorphism of CYP1A1, GSTM1, GSTP1, GSTT1, and mEH. Cancer Epidemiol Biomarkers Prev 13:1342-1348, 2004[Abstract/Free Full Text]

19. Li D, Firozi PF, Wang LE, et al: Sensitivity to DNA damage induced by benzo(a) pyrene diol epoxide and risk of lung cancer: A case-control analysis. Cancer Res 61:1445-1450, 2001[Abstract/Free Full Text]

20. Speit G, Hanelt S, Helbig R, et al: Detection of DNA effects in human cells with the comet assay and their relevance for mutagenesis. Toxicol Lett 88:91-98, 1996[Medline]

21. Hanelt S, Helbig R, Hartmann A, et al: A comparative investigation of DNA adducts, DNA strand breaks and gene mutations induced by benzo[a]pyrene and (+/-)-anti-benzo[a]pyrene-7,8-diol 9,10-oxide in cultured human cells. Mutat Res 390:179-188, 1997[Medline]

22. Sage E, Haseltine WA: High ratio of alkali-sensitive lesions to total DNA modification induced by benzo(a)pyrene diol epoxide. J Bio Chem 259:11098-11102, 1984[Abstract/Free Full Text]

23. Sancar A: DNA repair in humans. Annu Rev Genet 29:69-105, 1995[CrossRef][Medline]

24. Friedberg EC: How nucleotide excision repair protects against cancer. Nat Rev Cancer 1:22-33, 2001[CrossRef][Medline]

25. Martini N, Melamed MR: Multiple primary lung cancers. J Thorac Cardiovasc Surg 70:606-612, 1975[Abstract]

26. Olive PL, Banath JP, Durand RE: Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay. Radiat Res 122:86-94, 1990[Medline]

27. Marcon F, Andreoli C, Rossi S, et al: Assessment of individual sensitivity to ionizing radiation and DNA repair efficiency in a healthy population. Mutat Res 541:1-8, 2003[Medline]

28. Risch HA, Howe GR, Jain M, et al: Are female smokers at higher risk for lung cancer than male smokers? A case-control analysis by histologic type. Am J Epidemiol 138:281-293, 1993[Abstract/Free Full Text]

29. Harris RE, Zang EA, Anderson JI, et al: Race and sex differences in lung cancer risk associated with cigarette smoking. Int J Epidemiol 22:592-599, 1993[Abstract/Free Full Text]

30. Zang EA, Wynder EL: Differences in lung cancer risk between men and women: Examination of the evidence. J Natl Cancer Inst 88:183-192, 1996[Abstract/Free Full Text]

31. Henschke CI, Miettinen OS: Women's susceptibility to tobacco carcinogens. Lung Cancer 43:1-5, 2004[CrossRef][Medline]

32. Bach PB, Kattan MW, Thornquist MD, et al: Variations in lung cancer risk among smokers. J Natl Cancer Inst 95:470-478, 2003[Abstract/Free Full Text]

33. Bain C, Feskanich D, Speizer FE, et al: Lung cancer rates in men and women with comparable histories of smoking. J Natl Cancer Inst 96:826-834, 2004[Abstract/Free Full Text]

34. Prescott E, Osler M, Hein HO, et al: Gender and smoking-related risk of lung cancer: The Copenhagen Center for Prospective Population Studies. Epidemiology 9:79-83, 1998[CrossRef][Medline]

35. Fu JB, Kau TY, Severson RK, et al: Lung cancer in women: Analysis of the national Surveillance, Epidemiology, and End Results database. Chest 127:768-777, 2005[CrossRef][Medline]

36. Ferguson MK, Skosey C, Hoffman PC, et al: Sex-associated differences in presentation and survival in patients with lung cancer. J Clin Oncol 8:1402-1407, 1990[Abstract]

37. Visbal AL, Williams BA, Nichols FC III, et al: Gender differences in non-small-cell lung cancer survival: An analysis of 4,618 patients diagnosed between 1997 and 2002. Annals Thoracic Surg 78:209-215, 2004[CrossRef]

38. Henschke CI, Yip R, Miettinen OS: Women's susceptibility to tobacco carcinogens and survival after diagnosis of lung cancer. JAMA 296:180-184, 2006[Abstract/Free Full Text]

39. Chang YL, Wu CT, Lin SC, et al: Clonality and prognostic implications of p53 and epidermal growth factor receptor somatic aberrations in multiple primary lung cancers. Clin Cancer Res 13:52-58, 2007[Abstract/Free Full Text]

40. van Rens MT, Eijken EJ, Elbers JR, et al: p53 mutation analysis for definite diagnosis of multiple primary lung carcinoma. Cancer 94:188-196, 2002[CrossRef][Medline]

41. Hiroshima K, Toyozaki T, Kohno H, et al: Synchronous and metachronous lung carcinomas: Molecular evidence for multicentricity. Pathol Int 48:869-876, 1998[Medline]

Submitted June 26, 2007; accepted January 9, 2008.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Facebook    Reddit    Technorati    Twitter    What's this?

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Save to my personal folders Download to citation manager Rights & Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Orlow, I. Articles by Rusch, V. W. Search for Related Content PubMed PubMed Citation Articles by Orlow, I. Articles by Rusch, V. W. Social Bookmarking                            What's this?