ORIGINAL ARTICLE
Influence of genetic background and oxidative stress response on risk of mandibular osteoradionecrosis after radiotherapy of head and neck cancer Daniel Danielsson,1,5 Karl Brehwens, PhD,2 Martin Halle, MD, PhD,3 Michal Marczyk, PhD,4 Alice Sollazzo,2 Joanna Polanska, PhD,4 Eva Munck–Wikland, MD, PhD,7 Andrzej Wojcik, PhD,2,6 Siamak Haghdoost, PhD2* 1
Department of Clinical Science, Intervention and Technology, Division of Ear, Nose, and Throat Diseases, Karolinska Institute, Stockholm, Sweden, 2Department of Molecular Bioscience, Centre for Radiation Protection Research, The Wenner–Gren Institute, Stockholm University, Stockholm, Sweden, 3Department of Molecular Medicine and Surgery, Reconstructive Plastic Surgery, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden, 4Data Mining Group, Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland, 5Department of Oral and Maxillofacial Surgery, Karolinska University Hospital, Stockholm, Sweden, 6Institute of Biology, Jan Kochanowski University, Kielce, Poland, 7Department of Oto-Rhino-Laryngology, Head and Neck Surgery, Karolinska University Hospital and Karolinska Institute, Stockholm, Sweden.
Accepted 24 October 2014 Published online 26 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.23903
ABSTRACT: Background. Osteoradionecrosis (ORN) of the mandible is a severe complication of head and neck radiotherapy (RT) treatment, where the impact of individual radiosensitivity has been a suggested explanation. Methods. A cohort of patients with stage II/III ORN was compared to matched controls. Blood was collected and irradiated in vitro to study the capacity to handle radiation-induced oxidative stress. Patients were also genotyped for 8 single-nucleotide polymorphisms (SNPs) in genes involved in the oxidative stress response. Results. A difference in 8-oxo-7,8-dihydro-20 -deoxyguanosine (8-oxodG) levels was found between the patient cohorts (p 5 0.01). The SNP rs1695 in glutathione s-transferase p1 (GSTP1) was also found to be more frequent in the patients with ORN (p 5 .02). Multivariate analysis
of the clinical and biological factors revealed concomitant brachytherapy plus the 2 biomarkers to be significant factors which influense risk of mandibular osteoradionecrosis after radiotherapy of head and neck cancer. Conclusion. The current study indicates that oxidative stress response contributes to individual radiosensitivity and healthy tissue damage caused C 2015 Wiley Periodiby RT and may be predicted by biomarker analysis. V cals, Inc. Head Neck 38: 387–393, 2016
INTRODUCTION
without recurrence of tumor.”3 ORN can occur any time after RT but commonly occurs within the first few years.3 ORN pathogenesis is not completely understood. The incidence of ORN in mandibular bone ranges between 2.6% and 15%,4 but there is data suggesting that the ORN incidence after RT remains constant.5 Symptoms include pain, severe trismus and local infection, often with orocutaneous fistulae. As ORN progresses, it may lead to pathologic fracture of the affected bone requiring extensive surgical procedures. Patient symptoms are often so severe that quality of life is seriously impaired. Treatment ranges from local debridement and sequestrectomy in mild cases to large resections and reconstructive surgery utilizing free tissue transfer in severe cases. An imbalance in the cellular levels of reactive oxygen species (ROS) can aggravate the process of tissue necrosis.6 This implies that the individual capacity to handle oxidative stress could influence the risk of developing late adverse effects. In fact, it has been shown that irradiated tissues suffer from chronic oxidative stress, but the mechanisms behind the pathogenesis are not yet understood.7 A good indicator of cellular oxidative stress is 8oxo-7,8-dihydro-20 -deoxyguanosine (8-oxo-dG), which is produced when ROS react with deoxyguanosine (dG) in DNA or deoxyguanosine triphosphate in the nucleotide
The use of radiotherapy (RT) to treat malignant tumors affects surrounding healthy tissues, and patients often experience acute and delayed normal tissue adverse effects. Tumor doses are prescribed as not to result in more than 5% severe late toxicites.1 Many factors contribute to risk and severity of adverse effects, among which individual susceptibility has recently been highlighted as an important factor. It has been proposed that patientrelated factors could constitute 80% to 90% of the variation seen in the patient response.2 However, there is a paucity of well-defined phenotypes for individual susceptibility to RT. Mandibular osteoradionecrosis (ORN) is a late adverse effect in patients with head and neck cancer. It is defined as “irradiated bone that becomes devitalized and exposed through the overlying skin or mucosa without signs of healing for a period of more than 3 months,
*Corresponding author: S. Haghdoost, Department of Molecular Bioscience, Centre for Radiation Protection Research, The Wenner–Gren Institute, Stockholm University, 106 91 Stockholm, Sweden. E-mail: [email protected] Daniel Danielsson and Karl Brehwens contributed equally to this work.
KEY WORDS: serum 8-oxo-7,8-dihydro-20 -deoxyguanosine (8-oxodG), glutathione s-transferase p1 (GSTP1) mutation, oxidative stress, radiotherapy, rs 1695, head and neck, osteoradionecrosis
HEAD & NECK—DOI 10.1002/HED
MARCH 2016
387
DANIELSSON ET AL.
pool.8 The 8-oxo-dG is excised from the DNA by the OGG1 protein belonging to the base excision repair pathway.9 Oxidized deoxyguanosine triphosphate is removed by the nucleotide pool sanitization mechanism10 by which it is dephosphorylated to 8-oxo-7,8-dihydro-20 -deoxyguanosine monophosphate (by MTH1) and further to 8-oxo-dG by 8-oxo-7,8-dihydro-20 -deoxyguanosine monophosphatase and subsequently excreted to the extracellular matrix.11 Thus, the levels of stress induced extracellular 8-oxo-dG, both under in vitro and in vivo conditions, can be regarded as a marker of the capacity to handle oxidative stress.12 Elevated levels of oxidative stress as measured by urinary and/or cellular 8-oxo-dG were observed in patients with radiosensitivity syndromes, including ataxia telangiectasia and Fanconi anemia.13,14 Indeed, in a recent study, it was shown that patients with breast cancer who developed skin reactions to RT had a lower level of in vitro radiation-induced 8-oxo-dG in blood serum and urine than patients without side effects.15 Possibly, 8-oxo-dG levels could be affected by low penetrance, high frequency point mutations (singlenucleotide polymorphisms [SNPs]) in genes involved in the oxidative stress response. In the present study, we compared the levels of radiation-induced 8-oxo-dG in serum in a unique patient group receiving treatment for stage II/III ORN with that of a closely matched control group. The patients were also genotyped for 8 SNP in genes involved in the oxidative stress response. Results from these endpoints were analyzed in conjunction with clinical data using multivariate analysis and an ORN risk model was constructed. The results suggest that reduced levels of 8-oxo-dG and the SNP rs1695 in glutathione s-transferase p1 (GSTP1) are associated with a risk of developing ORN.
MATERIALS AND METHODS This retrospective study was conducted with permission from the Stockholm Regional Ethical Committee, according to the Declaration of Helsinki.
Study cohort Approximately 280 cases of patients with head and neck cancer undergo treatment at the Karolinska University Hospital in Stockholm yearly. Treatment modalities include surgery, external RT or RT in combination with brachytherapy, and/or chemotherapy. Patients who develop ORN are referred to the Department of Oral and Maxillofacial Surgery at the Karolinska University Hospital for treatment. In 2008, according to clinic records, a total of 64 patients could be found with the diagnosis ORN. All patients alive were asked to participate in the study. Thirty-seven accepted, 16 were already deceased, and 11 declined. There were no significant differences between the patients who participated or declined. With assistance of the Stockholm Regional Cancer Center, 37 patients with head and neck cancer (controls) were recruited. The controls were matched for tumor site, irradiation dose, TNM classification and sex. The inclusion criterion was the absence of ORN over a time period between RT and recruitment, equal or longer than for the matched case. ORN stage was classified according to 388
HEAD & NECK—DOI 10.1002/HED
MARCH 2016
Schwartz and Kagan.16 All patients diagnosed with ORN were classified as stage II or III. In the ORN(1) group, 29 patients had dental extractions identified as a direct cause to onset of ORN and for 8 patients the cause was unknown. In the ORN(-) group, 24 patients had dental extractions either associated with infection screening before RT or any time post-RT. Nine patients had not had any teeth extracted post-RT and in 4 cases there were no data available. The patients were given a median tumor dose of 68 Gy delivered in 2 Gy fractions, 5 times per week. A few patients received brachytherapy and/or chemotherapy. A summary of the relevant cohort characteristics is presented in Table 1. Based on a 3D dose planning system, the highest accepted dose in the mandible was 100% 6 5% for patients in both groups and the dose plans were approved by the responsible radiotherapists, oncologists, and physicists to ensure no over dosage to the mandible.
Measurement of extracellular 8-oxo-7,8-dihydro-20 deoxyguanosine Morning blood was collected by venipuncture in 3 tubes without anticoagulant (Vacutainer; Sarstedt, N€umbrecht, Germany), allowed to coagulate, and the samples were then kept on ice. Within a few hours, 2 of the samples were irradiated on ice with either 5 mGy (15 mGy/h) or 2 Gy (0.40 Gy/min) from a 137Cs source. All 3 samples were subsequently kept in a 37 C water bath for 20 minutes followed by 40-minute incubation in a humidified tissue culture incubator (37 C; 5% CO2). After incubation, the samples were put on ice for 15 to 20 minutes, followed by centrifugation at 250 3 g, 5 C, for 20 minutes in order to separate the coagulate and serum. This experimental design has also been used in our previously published studies.12,15,17,18 All samples were coded and analyzed blind. The 8-oxodG was measured using enzyme-linked immunosorbent assay (ELISA), as described previously.12,17 The ELISA kit was provided by Health Biomarkers Sweden AB. Briefly, 800 lL blood serum was purified using a C18 solid phase extraction column (Varian, CA), as described previously.19 This step is necessary to remove products other than 8-oxo-dG that could cross-react with the monoclonal antibody. The purified samples were freeze-dried, reconstituted, and the filtration step was repeated. The final volume was adjusted to 600 lL. Then, 90 lL of the purified sample were mixed with 50 lL of primary antibody against 8-oxo-dG (Japan Institute for the Control of Aging, Japan) and transferred to a 96-well ELISA plate precoated with 8-oxo-dG. After overnight incubation at 4 C, the plates were washed 3 times with 250 lL of washing solution (phosphate-buffered saline, pH 7.4; 0.02% Tween 20; and 0.1% bovine serum albumin). Then, 140 lL of horseradish peroxidase-conjugated secondary antibody (goat anti-mouse immunoglobulin G-horseradish peroxidase, Scandinavian Diagnostic Services, Sweden) was added to each well and incubated for 2 hours at room temperature. The wells were washed 3 times with 250 lL of washing solution and finally with phosphate-buffered saline, pH 7.4. Then, 140 lL of tetramethylbenzidine liquid substrate (ICN Biomedicals, Aurora, OH) was added to each well and the wells were incubated for 15 minutes at room temperature; the reaction was terminated by adding 70 lL
OSTEORADIONECROSIS
AND OXIDATIVE STRESS RESPONSE
TABLE 1. Characteristics of the study cohort. Characteristics
Sex Male Female Age at sampling, y (median 1 range) Year completing RT (median 1 range) Smoker at time of sampling Yes No History of alcohol overconsumption Yes No Tumor site Tonsil Tongue Other T classification 1 2 3 4 N classification N0 N1 N2 N3 M classification M0 M1 Dose, Gy (median 1 range) Chemotherapy Yes No Brachytherapy Yes No Years between end of RT and blood sampling (median 1 range) Years from end of RT to ORN onset (median 1 range) Xerostomia after RT Yes No Teeth extraction 2 wk before or any time after RT Yes No Cardiovascular disease (hypertension, myocardial infarction, angina, stroke, bypass surgery) Yes No Diabetes Yes No Serum 8-oxo-dG level (ng/mL) (average 1 95% CI) 5 mGy 2 Gy
Patients with ORN (%)
Patients without ORN (%)
p value
32 (86.5) 5 (13.5) 64 (34–85) 2004 (1995–2009)
29 (78.4) 8 (21.6) 64 (40–84) 2002 (2000–2006)
.54 .81 .027
12 (32.4) 25 (67.6)
9 (24.3) 28 (75.7)
.61
7 (18.9) 30 (81.1)
3 (8.1) 34 (91.9)
.31
17 (45.9) 8 (21.6) 12 (32.4)
29 (78.4) 5 (13.5) 3 (8.1)
.0099
8 (24.2) 14 (42.4) 7 (21.2) 4 (12.1)
9 (24.3) 15 (40.5) 8 (21.6) 5 (13.5)
.99
11 (32.4) 10 (29.4) 11 (32.4) 2 (5.8)
6 (16.2) 11 (29.7) 18 (48.6) 2 (5.4)
.39
34 (100) 0 (0) 68 (54–68)
36 (97.3) 1 (2.7) 68 (50–68)
9 (24.3) 28 (75.7)
3 (8.1) 34 (91.9)
.060
10 (27) 27 (73) 6.5 (
Influence of genetic background and oxidative stress response on risk of mandibular osteoradionecrosis after radiotherapy of head and neck cancer Daniel Danielsson,1,5 Karl Brehwens, PhD,2 Martin Halle, MD, PhD,3 Michal Marczyk, PhD,4 Alice Sollazzo,2 Joanna Polanska, PhD,4 Eva Munck–Wikland, MD, PhD,7 Andrzej Wojcik, PhD,2,6 Siamak Haghdoost, PhD2* 1
Department of Clinical Science, Intervention and Technology, Division of Ear, Nose, and Throat Diseases, Karolinska Institute, Stockholm, Sweden, 2Department of Molecular Bioscience, Centre for Radiation Protection Research, The Wenner–Gren Institute, Stockholm University, Stockholm, Sweden, 3Department of Molecular Medicine and Surgery, Reconstructive Plastic Surgery, Karolinska Institute and Karolinska University Hospital, Stockholm, Sweden, 4Data Mining Group, Institute of Automatic Control, Silesian University of Technology, Gliwice, Poland, 5Department of Oral and Maxillofacial Surgery, Karolinska University Hospital, Stockholm, Sweden, 6Institute of Biology, Jan Kochanowski University, Kielce, Poland, 7Department of Oto-Rhino-Laryngology, Head and Neck Surgery, Karolinska University Hospital and Karolinska Institute, Stockholm, Sweden.
Accepted 24 October 2014 Published online 26 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/hed.23903
ABSTRACT: Background. Osteoradionecrosis (ORN) of the mandible is a severe complication of head and neck radiotherapy (RT) treatment, where the impact of individual radiosensitivity has been a suggested explanation. Methods. A cohort of patients with stage II/III ORN was compared to matched controls. Blood was collected and irradiated in vitro to study the capacity to handle radiation-induced oxidative stress. Patients were also genotyped for 8 single-nucleotide polymorphisms (SNPs) in genes involved in the oxidative stress response. Results. A difference in 8-oxo-7,8-dihydro-20 -deoxyguanosine (8-oxodG) levels was found between the patient cohorts (p 5 0.01). The SNP rs1695 in glutathione s-transferase p1 (GSTP1) was also found to be more frequent in the patients with ORN (p 5 .02). Multivariate analysis
of the clinical and biological factors revealed concomitant brachytherapy plus the 2 biomarkers to be significant factors which influense risk of mandibular osteoradionecrosis after radiotherapy of head and neck cancer. Conclusion. The current study indicates that oxidative stress response contributes to individual radiosensitivity and healthy tissue damage caused C 2015 Wiley Periodiby RT and may be predicted by biomarker analysis. V cals, Inc. Head Neck 38: 387–393, 2016
INTRODUCTION
without recurrence of tumor.”3 ORN can occur any time after RT but commonly occurs within the first few years.3 ORN pathogenesis is not completely understood. The incidence of ORN in mandibular bone ranges between 2.6% and 15%,4 but there is data suggesting that the ORN incidence after RT remains constant.5 Symptoms include pain, severe trismus and local infection, often with orocutaneous fistulae. As ORN progresses, it may lead to pathologic fracture of the affected bone requiring extensive surgical procedures. Patient symptoms are often so severe that quality of life is seriously impaired. Treatment ranges from local debridement and sequestrectomy in mild cases to large resections and reconstructive surgery utilizing free tissue transfer in severe cases. An imbalance in the cellular levels of reactive oxygen species (ROS) can aggravate the process of tissue necrosis.6 This implies that the individual capacity to handle oxidative stress could influence the risk of developing late adverse effects. In fact, it has been shown that irradiated tissues suffer from chronic oxidative stress, but the mechanisms behind the pathogenesis are not yet understood.7 A good indicator of cellular oxidative stress is 8oxo-7,8-dihydro-20 -deoxyguanosine (8-oxo-dG), which is produced when ROS react with deoxyguanosine (dG) in DNA or deoxyguanosine triphosphate in the nucleotide
The use of radiotherapy (RT) to treat malignant tumors affects surrounding healthy tissues, and patients often experience acute and delayed normal tissue adverse effects. Tumor doses are prescribed as not to result in more than 5% severe late toxicites.1 Many factors contribute to risk and severity of adverse effects, among which individual susceptibility has recently been highlighted as an important factor. It has been proposed that patientrelated factors could constitute 80% to 90% of the variation seen in the patient response.2 However, there is a paucity of well-defined phenotypes for individual susceptibility to RT. Mandibular osteoradionecrosis (ORN) is a late adverse effect in patients with head and neck cancer. It is defined as “irradiated bone that becomes devitalized and exposed through the overlying skin or mucosa without signs of healing for a period of more than 3 months,
*Corresponding author: S. Haghdoost, Department of Molecular Bioscience, Centre for Radiation Protection Research, The Wenner–Gren Institute, Stockholm University, 106 91 Stockholm, Sweden. E-mail: [email protected] Daniel Danielsson and Karl Brehwens contributed equally to this work.
KEY WORDS: serum 8-oxo-7,8-dihydro-20 -deoxyguanosine (8-oxodG), glutathione s-transferase p1 (GSTP1) mutation, oxidative stress, radiotherapy, rs 1695, head and neck, osteoradionecrosis
HEAD & NECK—DOI 10.1002/HED
MARCH 2016
387
DANIELSSON ET AL.
pool.8 The 8-oxo-dG is excised from the DNA by the OGG1 protein belonging to the base excision repair pathway.9 Oxidized deoxyguanosine triphosphate is removed by the nucleotide pool sanitization mechanism10 by which it is dephosphorylated to 8-oxo-7,8-dihydro-20 -deoxyguanosine monophosphate (by MTH1) and further to 8-oxo-dG by 8-oxo-7,8-dihydro-20 -deoxyguanosine monophosphatase and subsequently excreted to the extracellular matrix.11 Thus, the levels of stress induced extracellular 8-oxo-dG, both under in vitro and in vivo conditions, can be regarded as a marker of the capacity to handle oxidative stress.12 Elevated levels of oxidative stress as measured by urinary and/or cellular 8-oxo-dG were observed in patients with radiosensitivity syndromes, including ataxia telangiectasia and Fanconi anemia.13,14 Indeed, in a recent study, it was shown that patients with breast cancer who developed skin reactions to RT had a lower level of in vitro radiation-induced 8-oxo-dG in blood serum and urine than patients without side effects.15 Possibly, 8-oxo-dG levels could be affected by low penetrance, high frequency point mutations (singlenucleotide polymorphisms [SNPs]) in genes involved in the oxidative stress response. In the present study, we compared the levels of radiation-induced 8-oxo-dG in serum in a unique patient group receiving treatment for stage II/III ORN with that of a closely matched control group. The patients were also genotyped for 8 SNP in genes involved in the oxidative stress response. Results from these endpoints were analyzed in conjunction with clinical data using multivariate analysis and an ORN risk model was constructed. The results suggest that reduced levels of 8-oxo-dG and the SNP rs1695 in glutathione s-transferase p1 (GSTP1) are associated with a risk of developing ORN.
MATERIALS AND METHODS This retrospective study was conducted with permission from the Stockholm Regional Ethical Committee, according to the Declaration of Helsinki.
Study cohort Approximately 280 cases of patients with head and neck cancer undergo treatment at the Karolinska University Hospital in Stockholm yearly. Treatment modalities include surgery, external RT or RT in combination with brachytherapy, and/or chemotherapy. Patients who develop ORN are referred to the Department of Oral and Maxillofacial Surgery at the Karolinska University Hospital for treatment. In 2008, according to clinic records, a total of 64 patients could be found with the diagnosis ORN. All patients alive were asked to participate in the study. Thirty-seven accepted, 16 were already deceased, and 11 declined. There were no significant differences between the patients who participated or declined. With assistance of the Stockholm Regional Cancer Center, 37 patients with head and neck cancer (controls) were recruited. The controls were matched for tumor site, irradiation dose, TNM classification and sex. The inclusion criterion was the absence of ORN over a time period between RT and recruitment, equal or longer than for the matched case. ORN stage was classified according to 388
HEAD & NECK—DOI 10.1002/HED
MARCH 2016
Schwartz and Kagan.16 All patients diagnosed with ORN were classified as stage II or III. In the ORN(1) group, 29 patients had dental extractions identified as a direct cause to onset of ORN and for 8 patients the cause was unknown. In the ORN(-) group, 24 patients had dental extractions either associated with infection screening before RT or any time post-RT. Nine patients had not had any teeth extracted post-RT and in 4 cases there were no data available. The patients were given a median tumor dose of 68 Gy delivered in 2 Gy fractions, 5 times per week. A few patients received brachytherapy and/or chemotherapy. A summary of the relevant cohort characteristics is presented in Table 1. Based on a 3D dose planning system, the highest accepted dose in the mandible was 100% 6 5% for patients in both groups and the dose plans were approved by the responsible radiotherapists, oncologists, and physicists to ensure no over dosage to the mandible.
Measurement of extracellular 8-oxo-7,8-dihydro-20 deoxyguanosine Morning blood was collected by venipuncture in 3 tubes without anticoagulant (Vacutainer; Sarstedt, N€umbrecht, Germany), allowed to coagulate, and the samples were then kept on ice. Within a few hours, 2 of the samples were irradiated on ice with either 5 mGy (15 mGy/h) or 2 Gy (0.40 Gy/min) from a 137Cs source. All 3 samples were subsequently kept in a 37 C water bath for 20 minutes followed by 40-minute incubation in a humidified tissue culture incubator (37 C; 5% CO2). After incubation, the samples were put on ice for 15 to 20 minutes, followed by centrifugation at 250 3 g, 5 C, for 20 minutes in order to separate the coagulate and serum. This experimental design has also been used in our previously published studies.12,15,17,18 All samples were coded and analyzed blind. The 8-oxodG was measured using enzyme-linked immunosorbent assay (ELISA), as described previously.12,17 The ELISA kit was provided by Health Biomarkers Sweden AB. Briefly, 800 lL blood serum was purified using a C18 solid phase extraction column (Varian, CA), as described previously.19 This step is necessary to remove products other than 8-oxo-dG that could cross-react with the monoclonal antibody. The purified samples were freeze-dried, reconstituted, and the filtration step was repeated. The final volume was adjusted to 600 lL. Then, 90 lL of the purified sample were mixed with 50 lL of primary antibody against 8-oxo-dG (Japan Institute for the Control of Aging, Japan) and transferred to a 96-well ELISA plate precoated with 8-oxo-dG. After overnight incubation at 4 C, the plates were washed 3 times with 250 lL of washing solution (phosphate-buffered saline, pH 7.4; 0.02% Tween 20; and 0.1% bovine serum albumin). Then, 140 lL of horseradish peroxidase-conjugated secondary antibody (goat anti-mouse immunoglobulin G-horseradish peroxidase, Scandinavian Diagnostic Services, Sweden) was added to each well and incubated for 2 hours at room temperature. The wells were washed 3 times with 250 lL of washing solution and finally with phosphate-buffered saline, pH 7.4. Then, 140 lL of tetramethylbenzidine liquid substrate (ICN Biomedicals, Aurora, OH) was added to each well and the wells were incubated for 15 minutes at room temperature; the reaction was terminated by adding 70 lL
OSTEORADIONECROSIS
AND OXIDATIVE STRESS RESPONSE
TABLE 1. Characteristics of the study cohort. Characteristics
Sex Male Female Age at sampling, y (median 1 range) Year completing RT (median 1 range) Smoker at time of sampling Yes No History of alcohol overconsumption Yes No Tumor site Tonsil Tongue Other T classification 1 2 3 4 N classification N0 N1 N2 N3 M classification M0 M1 Dose, Gy (median 1 range) Chemotherapy Yes No Brachytherapy Yes No Years between end of RT and blood sampling (median 1 range) Years from end of RT to ORN onset (median 1 range) Xerostomia after RT Yes No Teeth extraction 2 wk before or any time after RT Yes No Cardiovascular disease (hypertension, myocardial infarction, angina, stroke, bypass surgery) Yes No Diabetes Yes No Serum 8-oxo-dG level (ng/mL) (average 1 95% CI) 5 mGy 2 Gy
Patients with ORN (%)
Patients without ORN (%)
p value
32 (86.5) 5 (13.5) 64 (34–85) 2004 (1995–2009)
29 (78.4) 8 (21.6) 64 (40–84) 2002 (2000–2006)
.54 .81 .027
12 (32.4) 25 (67.6)
9 (24.3) 28 (75.7)
.61
7 (18.9) 30 (81.1)
3 (8.1) 34 (91.9)
.31
17 (45.9) 8 (21.6) 12 (32.4)
29 (78.4) 5 (13.5) 3 (8.1)
.0099
8 (24.2) 14 (42.4) 7 (21.2) 4 (12.1)
9 (24.3) 15 (40.5) 8 (21.6) 5 (13.5)
.99
11 (32.4) 10 (29.4) 11 (32.4) 2 (5.8)
6 (16.2) 11 (29.7) 18 (48.6) 2 (5.4)
.39
34 (100) 0 (0) 68 (54–68)
36 (97.3) 1 (2.7) 68 (50–68)
9 (24.3) 28 (75.7)
3 (8.1) 34 (91.9)
.060
10 (27) 27 (73) 6.5 (
