Author Affiliations: Departments of Head and Neck Surgery (Drs Zevallos, Li, and Sturgis), Plastic Surgery (Dr Hanasono), and Epidemiology (Drs Li, Wei, and Sturgis), The University of Texas M. D. Anderson Cancer Center, and Bobby R. Alford Department of Otolaryngology–Head and Neck Surgery, Baylor College of Medicine (Dr Zevallos), Houston.
Glutathione-S-transferase (GST) enzymes play a role in scavenging endogenous oxidants. Altered enzyme activity results from inherited polymorphisms, which results in increased oxidative stress. This study explores the role of GST polymorphisms as modifiers of surgical complications in patients undergoing head and neck microvascular reconstruction.
Patients newly diagnosed as having head and neck cancer and undergoing microvascular reconstruction were selected. Polymerase chain reaction was used to determine GST genotypes. Demographic factors, treatment, and postoperative complications were reviewed. Multivariate logistic regression analysis was used to estimate the risk of surgical complications associated with variant genotypes.
A total of 107 patients were evaluated. Surgical and medical complication rates were 44.9% and 25.2%, respectively. The variant Val allele at the GSTP1 105 codon was associated with a significantly increased risk of surgical complications (P = .046; adjusted relative risk, 1.7; 95% confidence interval, 1.1-2.6). There was no significant association between the other GST variant genotypes and surgical complications. No association was noted between variant GST genotypes and the risk of medical complications.
GSTP1 codon 105 polymorphism may be a marker for risk of wound complications in head and neck microvascular reconstruction.
The glutathione-S-transferase (GST) enzymes are xenobiotic phase II enzymes responsible for the detoxification of various smoking-derived carcinogens and other environmental toxins.1 Several polymorphisms in the GST gene families have been described and have been shown to result in decreased or absent enzyme activity. These polymorphisms have been associated with multiple human cancers, including lung, head and neck, and bladder carcinomas.2- 4 In addition to metabolism of exogenous carcinogens, GST enzymes play an important role in protection from oxidative stress, and several studies1,5 have demonstrated an upregulation of these enzymes in the presence of oxygen-derived free radicals. Glutathione-S-transferase enzymes metabolize byproducts of oxidative damage to cell membranes.6 Absent or altered enzyme activity caused by GST gene polymorphisms has also been shown to result in increased oxidative stress and an increased susceptibility to inflammatory diseases.7,8
Three main classes of cytosolic GST enzymes exist: μ, θ, and π, with corresponding genes GSTM1, GSTT1, and GSTP1, respectively. Functional polymorphisms have been identified in each of these genes, which result in allelic variants that have absent or significantly decreased enzyme activity.1 Null genotypes in the GSTM1 and GSTT1 genes have been associated with increased cancer risk,9 as well as with a decreased ability to catalyze the detoxification of reactive oxygen species and lipid peroxidation products.10 Single nucleotide polymorphisms at the 105 and 114 codons of GSTP1 have also been described and result in decreased GST π enzyme activity.11 As the most common GST enzyme in epithelial tissues, such as skin and mucosa,1,12 decreased GST π activity secondary to an allelic variant may have important implications in surgical wound healing.
Oxygen-derived free radicals can result in multiple pathophysiologic changes that adversely affect wound healing.13 Oxygen-derived free radical damage is of particular importance in patients undergoing microvascular reconstruction, in whom flaps are subjected to ischemia and reperfusion injury. Ischemic time, or the time between removal of the flap from the donor site and reestablishment of blood flow (reperfusion), is an inevitable component of any microvascular reconstruction. When restoration of blood flow occurs, cytotoxic oxidant formation results in a series of injurious events.14- 16 Endogenous free radical scavengers, including GST, offset and afford some protection from reperfusion injury; however, patients with a genetic predisposition secondary to limited antioxidant activity may be at higher risk for complications of wound healing.
We explored the role of GSTM1, GSTT1, and GSTP1 polymorphisms as modifiers of surgical complications in patients undergoing head and neck free-flap reconstruction. We hypothesized that altered or absent GST activity secondary to gene polymorphisms may result in increased oxidative stress in the perioperative period, thereby contributing to poor wound healing and an increased rate of surgical complications.
All patients with newly diagnosed and previously untreated stage II to stage IV tumors requiring ablative surgery and microvascular free-flap reconstruction were chosen from a database of approximately 1200 consecutive patients previously enrolled in a prospective molecular epidemiological study of head and neck squamous cell carcinoma. Each patient previously donated approximately 30 mL of blood for preoperative biomarker testing at the time of initial recruitment. These preexisting blood samples were used to perform genotyping in patients who met the criteria for the current study. A leukocyte pellet obtained from the buffy coat by centrifugation of 1 mL of whole blood was used for genomic DNA extraction according to manufacturer's instructions (Qiagen Inc, Valencia, California). A multiplex polymerase chain reaction (PCR) assay was used to determine the presence or absence of the GSTM1 and GSTT1 polymorphisms, and a PCR-based restriction fragment length polymorphism analysis was used to test for the GSTP1 polymorphisms.
Medical charts for each of the eligible patients were reviewed to obtain clinical staging, treatment, and comorbidity information. Staging was classified according to TNM criteria. Medical comorbidities were classified according to a modification of the Kaplan Feinstein comorbidity index (the 27-item Adult Comorbidity Evaluation comorbidity index [ACE-27]), which reflects the presence of related comorbidities as “none” to “mild,” “moderate,” or “severe.”17 This comorbidity index has been demonstrated to be an independent prognostic factor for patients with head and neck cancer.17,18 In addition, smoking history and alcohol exposure were assessed using epidemiologic questionnaires completed by all patients on enrollment to the parent molecular epidemiologic study. Patients who had smoked more than 100 cigarettes in their lifetimes were classified as smokers, and those who had quit smoking more than 1 year prior to enrollment in the study were classified as former smokers. Patients who used alcohol at least once a week for more than 1 year were classified as drinkers, and those who had quit such alcohol use more than 1 year prior to enrollment were classified as former drinkers. Treatment information, including site and extent of resection and type of flap used, was also recorded. All cases were reviewed for the development of surgical complications at both the donor and recipient sites, including wound infection, wound dehiscence, hematoma, seroma, fistula formation, flap failure, partial flap loss, and flap thrombosis. The development of medical complications was also recorded for each patient, including pneumonia, myocardial infarction, cardiac arrhythmias, and renal failure. Patients who received prior radiotherapy to the head and neck were excluded from this study.
Crude relative risks and 95% confidence intervals (CIs) were calculated to estimate the risk of wound complications associated with each demographic, exposure, or clinical variable. The GST genotypes were first analyzed as a dichotomized variable, with 0 being the null genotype (for GSTM1 and GSTT1) or variant genotype (homozygous and heterozygous variant genotypes for GSTP1) and 1 being the other genotype. Univariate analysis was performed to calculate crude relative risks and 95% CIs for various strata for the GSTM1, GSTT1, and GSTP1 genotypes. Adjusted relative risks with 95% CIs were calculated to estimate the risk of surgical complications associated with variant genotypes using multivariate logistic regression analysis. A multivariate logistic regression model was built using the variables that had prognostic potential suggested by the univariate analysis (P < .25). Owing to epidemiological and clinical considerations in building the model, age, sex, and race/ethnicity were always retained in the main effects and final multivariate model. We used a stepwise search strategy to build the multivariate models, for which a threshold level of 0.25 for the likelihood ratio test was used as a cutoff to determine whether a variable could be entered into, or removed from, the regression model. All tests were 2-sided, and a P = .05 was preset as the level of statistical significance. All analyses were performed using SAS statistical software (SAS Inc, Cary, North Carolina).
A total of 107 patients were identified who met the criteria for this study. The mean age of patients was 56.1 years, and 28% of patients were female. Age, sex, race/ethnicity, tobacco use, alcohol use, and comorbidity distributions for all patients included in this study are listed in Table 1. With 1 exception, there were no statistically significant differences in crude surgical complication risk when the data were segregated by demographic/exposure/comorbidity variables, although the association between male sex was significant (P = .049; crude relative risk, 1.5; 95% CI, 1.0-2.3). Distribution in site of primary tumor, stage, treatment received, and free-flap reconstruction used for all patients are presented in Table 2. Patients with tumors of the hypopharynx and larynx had a significantly lower risk of surgical complications (P = .03; crude relative risk, 0.4; 95% CI, 0.2-1.1) than patients treated for oral cavity and oropharyngeal tumors.
The overall rate of surgical complications in this series was 44.9%. Surgical complications included fistula formation (13 patients), recipient site wound dehiscence (11 patients), partial flap loss (8), recipient site hematoma (5), donor site dehiscence (6), total flap loss (1), recipient site seroma (2), donor site infection (1), and donor site hematoma (1). Table 3 lists the rate of surgical complications observed in patients with each of the different GSTM1, GSTT1, and GSTP1 genotypes, as well as associated crude and adjusted risk of surgical complications. No significantly increased risk of surgical complications was noted in patients with the null genotypes in the GSTM1 and GSTT1 genes.
The presence of the variant Val allele at GSTP1 105 was associated with a significantly increased risk of complications (P = .046; adjusted relative risk, 1.7; 95% CI, 1.1-2.6). A dose-dependent relationship was noted between complication rate and the presence of the Val variant allele, with patients homozygous for the variant allele having the highest rate of complications (64.7%), followed by heterozygous variant/wild-type (a 48.8% surgical complication rate), and followed by homozygous wild-type (a 34.0% surgical complication rate). No significantly increased risk of complications was noted in patients with the GSTP1 114 variant allele. The risk of surgical complications associated with the presence of variant alleles in the combination GSTM1 and GSTT1, GSTP1 105 and GSTP1 114, and in GSTM1, T1, P1 105 and P1 114 genotypes are shown in Table 4. A significantly increased risk of surgical complications was noted in patients with at least 1 variant allele present in the GSTP1 105 and GSTP1 114 combined genotypes, although this association seemed to be completely driven by the GSTP1 105 genotype.
The overall rate of medical complications in this series was 25.2%. Postoperative medical complications included pneumonia (12 patients), alcohol withdrawal (9 patients), cardiac arrhythmia (3), myocardial infarction (1), stroke (1), and congestive heart failure exacerbation (1). As expected, patients with a moderate to severe ACE-27 score had a significantly higher rate of medical complications (P = .04; crude relative risk, 2.0; 95% CI, 1.1-3.8). Also, patients with overall stage IV tumors were significantly more likely to have postoperative medical complications (P = .03; crude relative risk, 5.6; 95% CI, 0.8-38.6). All other demographic, exposure, comorbidity, tumor, and treatment variables were similar between patients with and without medical complications. No significant association was noted between variant genotypes in the GSTM1, GSTT1, and GSTP1 genes and the risk of postoperative medical complications (data not shown).
This study explores the possible role of GST polymorphisms as biomarkers for surgical complications in patients undergoing microvascular head and neck reconstruction. We demonstrate that surgical complications are common in patients undergoing head and neck microvascular reconstruction, with fistula formation and recipient site wound dehiscence as the most common complications in this series. We found that the presence of GSTP1 105 allelic variant encoding for decreased GST π enzyme activity was associated with an almost 2-fold risk of developing surgical complications in this subset of patients. No association was noted between the risk of surgical complications and null allelic variants in the GSTM1 and GSTT1 genes or the GSTP1 114 polymorphism, and none of the GST polymorphisms studied were associated with medical complications.
Surgical complications are common in patients undergoing head and neck microvascular reconstruction, occurring in up to 53% of cases.19 Some factors that have been described as possible predictors of surgical complications in patients undergoing head and neck microvascular reconstruction include current smoking, age older than 65 years, preoperative hemoglobin level lower than 11 g/dL (110 g/L), an American Society of Anesthesiologists score of 3 or 4, and preoperative radiotherapy.19,20 Comorbidities, including hypertension and cerebrovascular disease, have also been described as possible predictors.21- 23
In this study, the surgical complication rate was consistent with previously published reviews of patients undergoing head and neck microvascular reconstruction19 (44.9%). We attempted to account for potential confounding variables to the development of surgical complications in this subset of patients. Although male sex was significantly associated with surgical complications, this possible confounding variable was controlled for using multivariate analysis. None of the patients received preoperative radiotherapy or had undergone prior head and neck surgery, 2 other important factors associated with surgical complications in patients undergoing head and neck free-flap reconstruction.24 Also, no significant association with preoperative comorbid conditions as measured by a validated comorbidity scale (ACE-27) was noted.
Patients undergoing microvascular reconstructive surgery are particularly susceptible to wound complications secondary to increased oxidative stress as a result of ischemia-reperfusion injury.16 Oxidative stress can cause apoptosis of wound fibroblasts,25 direct DNA damage, and lipid peroxidation damage to cell membranes.6 Previous research on the association of GSTP1 allelic variants and the risk of increased oxidative stress support the findings in this study. Allelic variants in the GSTP1 gene result in decreased GST π enzyme activity, and have been found to result in a decreased cellular response to oxidative stress.25 At the GSTP1 105 codon, the homozygous Ile /Ile has been shown to have the greatest enzyme activity, followed by heterozygous Ile /Val, followed by Val /Val.11 Given the predominance of the GST π enzyme in epithelial cells such as skin and mucosa, GSTP1 allelic variants may have lead to decreased ability to combat oxidative stress during wound healing and an increased rate of wound complications noted in this study.
Null variants in the GSTM1 and GSTT1 genes were not associated with an increased risk of surgical complications in this study. These genes are upregulated in the liver,1 underscoring their important role in xenobiotic metabolism. When compared with GSTP1, the GSTM1 and GSTT1 genes may not be as important in the wound healing process. In addition, we did not find a significant association between the risk of postoperative medical complications and any of the GST genotypes, suggesting that these GST polymorphisms do not have major influence on the pathogenesis of medical complications.
There are several limitations associated with this study. The sample size is relatively small for molecular epidemiological analysis, and a larger number of patients may yield interesting associations between other GST variant genotypes and the risk of surgical complications. Also, although we included self-reported data on smoking exposure, there were no objective measures of tobacco use, such as exhaled carbon monoxide measurements or serum cotinine levels.26 Other confounding variables that are not accounted for in this analysis relate to intraoperative events and flap characteristics, including ischemic time, total surgery time, use of anticoagulation, and surgical complexity. However, several large published series21- 23 of head and neck free-flap complications have either not reviewed or failed to demonstrate a consistent association between these free tissue transfer characteristics and the development of surgical complications.
This study introduces the possible role of the GSTP1 105 polymorphism as a marker for increased risk of wound complications in patients with head and neck cancer undergoing microvascular reconstruction. It is well known that the development of surgical complications is multifactorial, and we acknowledge that in certain cases the complications noted in this series are not directly related to altered GST enzyme activity. Although we demonstrated a significant association between surgical complications and a single variant GSTP1 genotype, the future implementation of genotype markers such as GSTP1 will be to create a personalized preoperative risk profile based on multiple genetic markers as well as patient- and treatment-related factors. This genotype risk profile could be used clinically to aid in perioperative decision-making. Surgeons treating patients with the GTSP1 105 polymorphism may elect a pedicled flap reconstruction as opposed to free tissue transfer or make efforts to limit ischemia time. Furthermore, patients could be counseled more aggressively on the importance of smoking cessation based on their genetic predisposition to oxidative stress. This study serves as a first step toward developing a more comprehensive preoperative assessment in patients undergoing major oncologic resection and reconstruction.
Correspondence: Erich M. Sturgis, MD, MPH, Department of Head and Neck Surgery, The University of Texas M. D. Anderson Cancer Center, Unit 441, 1515 Holcombe Blvd, Houston, TX 77030 (email@example.com).
Accepted for Publication: February 27, 2010.
Author Contributions:Study concept and design: Zevallos, Hanasono, and Sturgis. Acquisition of data: Zevallos, Li, Wei, and Sturgis. Analysis and interpretation of data: Zevallos, Li, Wei, and Sturgis. Drafting of the manuscript: Zevallos and Wei. Critical revision of the manuscript for important intellectual content: Hanasono, Li, and Sturgis. Statistical analysis: Zevallos, Li, Wei, and Sturgis. Obtained funding: Zevallos, Wei, and Sturgis. Administrative, technical, and material support: Wei. Study supervision: Hanasono.
Financial Disclosure: None reported.
Funding/Support: This study was funded by the American Academy of Otolaryngology/Head and Neck Surgery CORE-American Academy of Facial Plastic and Reconstructive Surgery Leslie Bernstein resident research grant (principal investigator, Dr Zevallos).
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Figure e18.1-4. Common (Wild-Type) Allele and 4 Types of Genetic Polymorphisms
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