Purpose: Early-stage head and neck cancer patients are at high risks for tumor recurrence and secondary primary tumor (SPT) development. We hypothesized that latent genetic instability and proliferation potential may be associated with elevated risks of SPT and recurrence.

Experimental Design: We conducted a nested case-control study within a randomized, placebo-controlled chemoprevention trial in patients with early-stage head and neck cancer. We compared prediagnostic bleomycin-induced chromatid breaks in peripheral blood lymphocyte cultures (as an indicator of latent genetic instability) between 298 cases (patients with SPT/recurrence) and 693 controls (patients without SPT/recurrence). We also determined the joint effects of latent genetic instability and elevated proliferation potential [indicated by serum insulin-like growth factor (IGF) level] in modulating the risk of SPT and recurrence.

Results: In the Cox proportional hazards model, patients with higher mutagen sensitivity (using a cutoff of ≥0.50 breaks per cell) exhibited a significantly increased risk of developing SPT/recurrence [hazard ratio, 1.38; 95% confidence interval (95% CI), 1.02-1.86]. Cases also exhibited significantly higher levels of IGF-I and IGF-binding protein-3 than controls (P = 0.022 and 0.042, respectively). Moreover, there were joint effects between mutagen sensitivity and IGFs in modulating SPT/recurrence risk. Using patients with low IGF-I level and low mutagen sensitivity profile as the reference group, the odds ratios of developing SPT/recurrence for patients with high IGF-I level alone, high mutagen sensitivity alone, and both high IGF-I level and high mutagen sensitivity were 2.85 (95% CI, 0.92-8.82), 3.92 (95% CI, 1.28-11.97), and 6.16 (95% CI, 2.03-18.71), respectively. A similar joint effect was observed for mutagen sensitivity and IGF-binding protein-3 level.

Conclusions: This is the largest prospective study to evaluate mutagen sensitivity as a prognosis marker in head and neck cancer because mutagen sensitivity data were derived from baseline samples drawn before the development of SPT or tumor recurrence. The results also show for the first time that latent genetic instability and elevated proliferation potential jointly elevate the risk of second tumors in early-stage head and neck cancers.

Head and neck cancer (including malignancies in the oral cavity, pharynx, and larynx) will occur with an estimated incidence of 40,500 new cases (22,040 in oral cavity, 8,950 in pharynx, and 9,510 in larynx) and cause ∼11,170 deaths in the United States in 2006 (1). Cigarette smoking and alcohol consumption are the predominant risk factors for head and neck cancer, estimated to be responsible for three fourths of all the cases (2). However, only a small fraction of smokers and drinkers develop these cancers, suggesting that variations in genetic susceptibility may play important roles in cancer etiology. There have been numerous studies supporting this concept, including evidence of increased risk of head and neck cancer in first-degree relatives of cancer probands (36), significant associations of genetic polymorphisms in genes involved in critical cellular functions (carcinogen metabolism, DNA repair, cell cycle control, and apoptosis) with head and neck cancer risk (7, 8), and increased head and neck cancer risk conferred by reduced DNA repair capacity and increased genetic instability (9, 10).

Early-stage head and neck cancer patients are often cured with surgery or radiation. However, local-regional recurrence and secondary primary tumors (SPT) pose major threats to the long-term survival of early-stage head and neck patients. Approximately 10% of early-stage head and neck cancer patients develop local recurrences (1113). SPTs occur in 15% to 25% of patients during the first 5 years after initial diagnosis with a relatively constant rate of about 3% to 6% yearly (1319) and have become the leading cause of mortality in early-stage head and neck cancer patients (2022). Although the term SPT indicates that these tumors and the index (primary) tumors developed independently, recent genetic and biochemical studies have suggested that, in a significant portion of patients, the SPTs and index tumors have a common clonal origin (2326). The risk factors for SPT have been extensively investigated. SPT is not treatment related but is related to the same and/or distinct environmental or genetic factors that cause the index cancers. For example, analogous to the risk of index cancer, smoking and alcohol are unquestionable risk factors for SPT (15, 27, 28). Index tumor site and disease stage also contribute significantly to the risk of SPT and recurrence (13).

Diminished DNA repair capacity has been implicated as a risk factor for a variety of cancers (29). The mutagen sensitivity assay was developed to indirectly measure an individual's DNA repair capacity after in vitro mutagen challenge and may reveal latent genetic instability (30). An earlier pilot study suggested that mutagen sensitivity was a significant predictor of risk for developing SPT in previously untreated early-stage head and neck cancers (31). We have also shown previously from a subset of patients from the current study population that in vitro sensitivity of peripheral blood lymphocytes to bleomycin was associated with risk (10) and recurrence (12) of head and neck cancer. Insulin-like growth factor (IGF)-I is a potent stimulator of normal and tumor cell growth and also has antiapoptotic activities. The major form of circulating IGF-I is complexed with IGF-binding protein-3 (IGFBP-3), which inhibits the mitogenic activity of IGF-I by preventing IGF-I from binding to its receptor. There is considerable interindividual variability in circulating levels of IGF-I and IGFBP-3. Many studies have shown that elevated serum IGF-I levels are associated with increased risks for a variety of cancers (3235). A recent study on a subset of patients with head and neck cancer from the current study population showed that elevated IGF-I levels were associated with a significantly increased risk of subsequent development of SPTs (36).

We therefore hypothesized that an individual's increased intrinsic carcinogen sensitivity (measured by the mutagen sensitivity assay) and proliferation potential (using serum IGF level as an indicator) contribute to elevated risk of SPT/recurrence and, furthermore, that there is a synergistic effect of these two adverse phenotypes. To test these hypotheses, we designed this large nested case-control study and determined the joint effects of mutagen sensitivity and IGF levels on the risk of SPT/recurrence.

Study population. The subjects included in this study were derived from the Retinoid Head and Neck Second Primary Trial that began in November 1991 and closed to new patient accrual in June 30, 1999. The primary end point of this trial was to evaluate whether a daily low dose of 13-cis-retinoic acid (13 cRA) for 3 years prevents SPTs in early-stage head and neck cancer patients who had been successfully treated with surgery or radiation or both. The detailed study design has been published previously (13, 15). Briefly, patients with histologically confirmed stage I or II squamous cell carcinoma of head and neck (including the larynx, oral cavity, or pharynx), who were cancer-free for at least 16 weeks at the time of recruitment, were eligible for randomization. Patients were enrolled from the Radiation Therapy Oncology Group, The University of Texas M. D. Anderson Cancer Center, the Clinical Community Oncology Group, and the Southwest Oncology Group. Patients were assigned to either low-dose (30 mg/d) 13 cRA treatment or the placebo arm with equal probability. The stage (I or II), primary tumor site (larynx, oral cavity, or pharynx), and smoking status (never/former, recent/current smoker) were used as stratifying factors to allocate treatment before randomization. Patients took 13 cRA or placebo for a total of 3 years followed by 4 years of follow-up. Follow-up information on SPTs and tumor recurrence was collected at 3, 6, 9, 12, 16, 20, 24, 28, and 36 months after randomization with additional evaluations at 6-month intervals for 4 years. A SPT was defined using the criteria established by Warren and Gates (37). These were new cancer of a different histologic type, one of identical histologic type occurring >3 years after therapy of the primary tumor or one separated from the initial primary tumor by >2 cm of clinically normal epithelium. The major sites of SPT in this population were lung (29.8%), head and neck (28.0%), prostate (14.2%), and bladder (5.1%). A local recurrence was defined as any tumor of similar histology appearing within 2 cm or 3 years of the primary tumor.

Data collection. Before randomization, participants were given a structured questionnaire that elicited information on sociodemographic factors, clinical information, tobacco exposure, and alcohol consumption. After the interview was completed, blood was drawn into heparinized tubes and delivered by overnight mail to M. D. Anderson Cancer Center to be used in cytogenetic and molecular analyses. The study was approved by all of the relevant review boards and in accordance with an assurance filed with, and approved by, the U.S. Department of Health and Human Services. Written informed consent was obtained for each participant.

Measurement of IGFs. Blood was centrifuged at 3,000 × g for 10 minutes at room temperature to separate the plasma. Collected plasma was stored at −80°C. The levels of IGF-I and IGFBP-3 were determined by an ELISA (Diagnostic Systems Laboratories, Webster, TX) according to the manufacturer's protocol. Cross-reaction of the antibodies with other members of the IGF family is not detected at physiologic concentrations according to the manufacturer. We separated IGF-I from its binding proteins by mixing plasma specimens with acid-ethanol extraction buffer and measured the serum levels of total IGF-I. For IGFBP-3, the specimens were diluted 100-fold in an assay buffer before conducting the ELISA test. All assays for IGF-I and IGFBP-3 were done in duplicate, and the average of the two measurements was used in the data analysis.

Mutagen sensitivity assays. Mutagen sensitivity was measured in vitro in lymphocytes by counting chromatid breaks induced by bleomycin as described previously (10). Briefly, blood cultures were incubated for 3 days and then exposed to bleomycin (0.03 units/mL) for 5 hours. Cells were harvested, and chromatid breaks were scored in 50 metaphases per sample and recorded as the mean number of breaks per cell. Laboratory personnel who read the slides were blinded to the case and control status.

Statistical analysis. Plasma levels of IGF-I and IGFBP-3 as well as bleomycin sensitivity were analyzed as continuous and categorical variables. As continuous variables, the differences between cases (patients with an event) and controls (patients without an event) were calculated with a two-sample Student's t test for demographic variables and mutagen sensitivity and a Wilcoxon rank sum test for IGFs (which are not normally distributed). As categorical variables, high levels of IGFs and mutagen sensitivity have been associated with increased SPT risk. The median value of plasma IGF-I and IGFBP-3 in controls was used as a cutoff point for high or low IGFs. A cutoff point determined by classification and regression tree analysis was applied to categorize each subject as either in the high or in the low bleomycin sensitivity stratum. The recursive partitioning process in classification and regression tree analysis does not depend on any underlying distributional assumptions and allows for nonlinear relations between predictive factors and outcomes. The cutoff points obtained correspond to the lowest cross-validation error rates. Univariate and multivariate Cox proportional hazards models were applied to calculate hazard ratios (HR) and corresponding 95% confidence intervals (95% CI) and analyze the effect of mutagen sensitivity and other variables on the development of SPT/recurrence. The joint effects between IGFs and bleomycin sensitivity were estimated by stratified analysis in which we formed joint strata of IGFs and bleomycin sensitivity values. All Ps were two sided. Associations were considered statistically significant at P < 0.05.

Table 1 summarizes the characteristics of 991 early-stage head and neck cancer patients available for analyses. An event (recurrence or SPT) occurred in 298 patients (referred to as cases) and 693 patients were event-free (referred to as controls). The median duration between blood draw and event (recurrence or SPT) development was ∼26 months (range, 0-92 months). The cases were significantly older than the controls (mean age, 62.6 ± 10.3 versus 60.3 ± 10.9 years; P = 0.003). Gender was not significantly associated with event development (P = 0.709). Compared with controls, cases included more patients with oral (32.55% versus 28.14%) and pharyngeal cancers (13.09% versus 9.09%) but fewer patients with laryngeal cancers (54.36% versus 62.77%). The cases had a significantly higher percentage of stage II patients (40.60%) than the controls (32.32%; P = 0.012). Compared with controls, cases were more likely to be current smokers (41.95% versus 35.79%) and current drinkers (52.35% versus 46.03%) and less likely to be never smokers (11.07% versus 14.14%) and never drinkers (15.44% versus 21.65%). Among smokers, the cases smoked more cigarettes daily (28.6 ± 16.6 versus 26.3 ± 15.8; P = 0.048) for a longer duration (37.4 ± 13.9 versus 34.8 ± 13.8 years; P = 0.005) than the controls, and consequently, the mean pack-years was significantly greater for the cases than the controls (55.2 ± 39.4 versus 47.6 ± 35.2; P = 0.010). The mean number of chromatid breaks per cell was higher in the cases (0.86 ± 0.44) than in the controls (0.82 ± 0.46; P = 0.083) but only of borderline statistical significance. Radiation and 13 cRA chemoprevention did not have an obvious effect on SPT/recurrence, whereas surgery provided some benefit in reducing the occurrence of SPT/recurrence (P = 0.076). Because the majority of the participants were Caucasians, our subsequent analyses were limited to this group.

Table 1.

Distribution of selected variables by SPT/recurrence status

VariablesSPT/recurrence (N = 298)No SPT/recurrence (N = 693)P*
Age    
    Mean (SD) 62.6 (10.3) 60.3 (10.9) 0.003 
Ethnicity    
    White 272 (91.28) 631 (91.05)  
    Hispanic 7 (2.35) 27 (3.90)  
    Black 16 (5.37) 26 (3.75)  
    Oriental 3 (1.01) 8 (1.15)  
    Native American 0 (0.00) 1 (0.14) 0.544 
Sex    
    Male 236 (79.19) 556 (80.23)  
    Female 62 (20.81) 137 (19.77) 0.709 
Smoking status    
    Never 33 (11.07) 98 (14.14)  
    Former 140 (46.98) 347 (50.07)  
    Current 125 (41.95) 248 (35.79) 0.136 
    Ever 265 (88.93) 595 (85.86) 0.191 
Years smoked    
    Mean (SD) 37.4 (13.9) 34.8 (13.8) 0.005 
No. cigarette/day    
    Mean (SD) 28.6 (16.6) 26.3 (15.8) 0.048 
Pack-year    
    Mean (SD) 55.2 (39.4) 47.6 (35.2) 0.010 
Alcohol    
    Never 46 (15.44) 150 (21.65)  
    Former 96 (32.21) 224 (32.32)  
    Current 156 (52.35) 319 (46.03) 0.055 
    Ever 252 (84.56) 543 (78.35) 0.024 
Site    
    Larynx 162 (54.36) 435 (62.77)  
    Oral 97 (32.55) 195 (28.14)  
    Pharynx 39 (13.09) 63 (9.09) 0.029 
Stage    
    I 177 (59.40) 469 (67.68)  
    II 121 (40.60) 224 (32.32) 0.012 
Radiotherapy    
    No 71 (23.83) 182 (26.30)  
    Yes 227 (76.17) 510 (73.70) 0.413 
Surgery    
    No 198 (66.44) 418 (60.49)  
    Yes 100 (33.56) 273 (39.51) 0.076 
13 cRA treatment    
    Yes 146 (48.99) 349 (50.36)  
    No 152 (51.01) 344 (49.64) 0.693 
VariablesSPT/recurrence (N = 298)No SPT/recurrence (N = 693)P*
Age    
    Mean (SD) 62.6 (10.3) 60.3 (10.9) 0.003 
Ethnicity    
    White 272 (91.28) 631 (91.05)  
    Hispanic 7 (2.35) 27 (3.90)  
    Black 16 (5.37) 26 (3.75)  
    Oriental 3 (1.01) 8 (1.15)  
    Native American 0 (0.00) 1 (0.14) 0.544 
Sex    
    Male 236 (79.19) 556 (80.23)  
    Female 62 (20.81) 137 (19.77) 0.709 
Smoking status    
    Never 33 (11.07) 98 (14.14)  
    Former 140 (46.98) 347 (50.07)  
    Current 125 (41.95) 248 (35.79) 0.136 
    Ever 265 (88.93) 595 (85.86) 0.191 
Years smoked    
    Mean (SD) 37.4 (13.9) 34.8 (13.8) 0.005 
No. cigarette/day    
    Mean (SD) 28.6 (16.6) 26.3 (15.8) 0.048 
Pack-year    
    Mean (SD) 55.2 (39.4) 47.6 (35.2) 0.010 
Alcohol    
    Never 46 (15.44) 150 (21.65)  
    Former 96 (32.21) 224 (32.32)  
    Current 156 (52.35) 319 (46.03) 0.055 
    Ever 252 (84.56) 543 (78.35) 0.024 
Site    
    Larynx 162 (54.36) 435 (62.77)  
    Oral 97 (32.55) 195 (28.14)  
    Pharynx 39 (13.09) 63 (9.09) 0.029 
Stage    
    I 177 (59.40) 469 (67.68)  
    II 121 (40.60) 224 (32.32) 0.012 
Radiotherapy    
    No 71 (23.83) 182 (26.30)  
    Yes 227 (76.17) 510 (73.70) 0.413 
Surgery    
    No 198 (66.44) 418 (60.49)  
    Yes 100 (33.56) 273 (39.51) 0.076 
13 cRA treatment    
    Yes 146 (48.99) 349 (50.36)  
    No 152 (51.01) 344 (49.64) 0.693 
*

P was calculated from Wilcoxon rank sum test, χ2, Fisher's exact test.

Smokers only.

We next calculated the HRs using the Cox proportional hazards model (Table 2). A cutoff of 0.5 chromatid breaks per cell was chosen by the recursive partitioning procedure to dichotomize the subjects into two groups of high and low bleomycin sensitivity. Overall, high bleomycin sensitivity was associated with a statistically significantly elevated risk for developing SPT/recurrence (adjusted HR, 1.38; 95% CI, 1.02-1.86). This risk was more evident in younger (HR, 1.84; 95% CI, 1.10-3.08) than in older patients (HR, 1.09; 95% CI, 0.75-1.58), in women (HR, 2.17; 95% CI, 1.05-4.46) than in men (HR, 1.24; 95% CI, 0.89-1.72), in former smokers (HR, 1.66; 95% CI, 1.03-2.68) than in current smokers (HR, 1.18; 95% CI, 0.76-1.81), and in lighter smokers (HR, 1.63; 95% CI, 0.98-2.69) than in heavy smokers (HR, 1.20; 95% CI, 0.80-1.80). Interestingly, the elevated risk was more evident in patients who had surgery (HR, 1.89; 95% CI, 1.15-3.12) than those who did not (HR, 1.12; 95% CI, 0.76-1.63) and in patients who did not receive radiotherapy (HR, 2.07; 95% CI, 1.21-3.55) than those who did (HR, 1.19; 95% CI, 0.82-1.72).

Table 2.

Associations between bleomycin sensitivity and SPT/recurrence risk by selected variables

VariablesSPT/recurrence, n (%)No SPT/recurrence, n (%)Adjusted HR (95% CI)*P
Overall breaks/cell     
    <0.50 55 (18.46) 194 (27.99)   
    ≥0.50 243 (81.54) 499 (72.01) 1.38 (1.02-1.86) 0.036 
Age     
    <62     
        <0.5 18 (14.63) 103 (30.29)   
        ≥0.5 105 (85.37) 237 (69.71) 1.84 (1.10-3.08) 0.02 
    ≥62     
        <0.5 37 (21.14) 91 (25.78)   
        ≥0.5 138 (78.86) 262 (74.22) 1.09 (0.75-1.58) 0.665 
Sex     
    Male     
        <0.5 46 (19.49) 153 (27.52)   
        ≥0.5 190 (80.51) 403 (72.48) 1.24 (0.89-1.72) 0.209 
    Female     
        <0.5 9 (14.52) 41 (29.93)   
        ≥0.5 53 (85.48) 96 (70.07) 2.17 (1.05-4.46) 0.036 
Ethnicity     
    White     
        <0.5 53 (19.49) 179 (28.37)   
        ≥0.5 219 (80.51) 452 (71.63) 1.33 (0.98-1.81) 0.07 
    Other     
        <0.5 2 (7.69) 15 (24.19)   
        ≥0.5 24 (92.31) 47 (75.81) 2.08 (0.45-9.62) 0.349 
Smoking status     
    Never     
        <0.5 6 (18.18) 27 (27.55)   
        ≥0.5 27 (81.82) 71 (72.45) 1.55 (0.63-3.86) 0.342 
    Former     
        <0.5 20 (14.29) 88 (25.36)   
        ≥0.5 120 (85.71) 259 (74.64) 1.66 (1.03-2.68) 0.038 
    Current     
        <0.5 29 (23.20) 79 (31.85)   
        ≥0.5 96 (76.80) 169 (68.15) 1.18 (0.76-1.81) 0.461 
Pack-year     
    Light (<40)     
        <0.5 19 (17.43) 86 (29.55)   
        ≥0.5 90 (82.57) 205 (70.45) 1.63 (0.98-2.69) 0.059 
    Heavy (≥40)     
        <0.5 30 (19.23) 81 (26.64)   
        ≥0.5 126 (80.77) 223 (73.36) 1.20 (0.80-1.80) 0.381 
Alcohol     
    Never     
        <0.5 9 (19.57) 37 (24.67)   
        ≥0.5 37 (80.43) 113 (75.33) 1.39 (0.66-2.92) 0.39 
    Ever     
        <0.5 46 (18.25) 157 (28.91)   
        ≥0.5 206 (81.75) 386 (71.09) 1.35 (0.97-1.87) 0.076 
Site     
    Larynx     
        <0.5 31 (19.14) 109 (25.06)   
        ≥0.5 131 (82.86) 326 (74.94) 1.19 (0.80-1.76) 0.4 
    Oral     
        <0.5 16 (16.49) 73 (37.44)   
        ≥0.5 81 (83.51) 122 (62.56) 2.17 (1.25-3.76) 0.006 
    Pharynx     
        <0.5 8 (20.51) 12 (19.05)   
        ≥0.5 31 (79.49) 51 (80.95) 1.03 (0.42-2.56) 0.948 
Stage     
    I     
        <0.5 37 (20.90) 139 (29.64)   
        ≥0.5 140 (79.10) 330 (70.36) 1.37 (0.95-1.97) 0.096 
    II     
        <0.5 18 (14.88) 55 (24.55)   
        ≥0.5 103 (85.12) 169 (75.45) 1.47 (0.86-2.50) 0.156 
Radiotherapy     
    No     
        <0.5 19 (26.76) 81 (44.51)   
        ≥0.5 52 (73.24) 101 (55.49) 2.07 (1.21-3.55) 0.008 
    Yes     
        <0.5 36 (15.86) 113 (22.16)   
        ≥0.5 191 (84.14) 397 (77.84) 1.19 (0.82-1.72) 0.354 
Surgery     
    No     
        <0.5 35 (17.68) 99 (23.68)   
        ≥0.5 163 (82.32) 319 (76.32) 1.12 (0.76-1.63) 0.573 
    Yes     
        <0.5 20 (20.00) 94 (34.43)   
        ≥0.5 80 (80.00) 179 (65.57) 1.89 (1.15-3.12) 0.012 
13 cRA     
    Yes     
        <0.5 25 (17.12) 99 (28.37)   
        ≥0.5 121 (82.88) 250 (71.63) 1.42 (0.91-2.21) 0.121 
    No     
        <0.5 30 (19.74) 95 (27.62)   
        ≥0.5 122 (80.26) 249 (72.38) 1.33 (0.88-2.00) 0.183 
VariablesSPT/recurrence, n (%)No SPT/recurrence, n (%)Adjusted HR (95% CI)*P
Overall breaks/cell     
    <0.50 55 (18.46) 194 (27.99)   
    ≥0.50 243 (81.54) 499 (72.01) 1.38 (1.02-1.86) 0.036 
Age     
    <62     
        <0.5 18 (14.63) 103 (30.29)   
        ≥0.5 105 (85.37) 237 (69.71) 1.84 (1.10-3.08) 0.02 
    ≥62     
        <0.5 37 (21.14) 91 (25.78)   
        ≥0.5 138 (78.86) 262 (74.22) 1.09 (0.75-1.58) 0.665 
Sex     
    Male     
        <0.5 46 (19.49) 153 (27.52)   
        ≥0.5 190 (80.51) 403 (72.48) 1.24 (0.89-1.72) 0.209 
    Female     
        <0.5 9 (14.52) 41 (29.93)   
        ≥0.5 53 (85.48) 96 (70.07) 2.17 (1.05-4.46) 0.036 
Ethnicity     
    White     
        <0.5 53 (19.49) 179 (28.37)   
        ≥0.5 219 (80.51) 452 (71.63) 1.33 (0.98-1.81) 0.07 
    Other     
        <0.5 2 (7.69) 15 (24.19)   
        ≥0.5 24 (92.31) 47 (75.81) 2.08 (0.45-9.62) 0.349 
Smoking status     
    Never     
        <0.5 6 (18.18) 27 (27.55)   
        ≥0.5 27 (81.82) 71 (72.45) 1.55 (0.63-3.86) 0.342 
    Former     
        <0.5 20 (14.29) 88 (25.36)   
        ≥0.5 120 (85.71) 259 (74.64) 1.66 (1.03-2.68) 0.038 
    Current     
        <0.5 29 (23.20) 79 (31.85)   
        ≥0.5 96 (76.80) 169 (68.15) 1.18 (0.76-1.81) 0.461 
Pack-year     
    Light (<40)     
        <0.5 19 (17.43) 86 (29.55)   
        ≥0.5 90 (82.57) 205 (70.45) 1.63 (0.98-2.69) 0.059 
    Heavy (≥40)     
        <0.5 30 (19.23) 81 (26.64)   
        ≥0.5 126 (80.77) 223 (73.36) 1.20 (0.80-1.80) 0.381 
Alcohol     
    Never     
        <0.5 9 (19.57) 37 (24.67)   
        ≥0.5 37 (80.43) 113 (75.33) 1.39 (0.66-2.92) 0.39 
    Ever     
        <0.5 46 (18.25) 157 (28.91)   
        ≥0.5 206 (81.75) 386 (71.09) 1.35 (0.97-1.87) 0.076 
Site     
    Larynx     
        <0.5 31 (19.14) 109 (25.06)   
        ≥0.5 131 (82.86) 326 (74.94) 1.19 (0.80-1.76) 0.4 
    Oral     
        <0.5 16 (16.49) 73 (37.44)   
        ≥0.5 81 (83.51) 122 (62.56) 2.17 (1.25-3.76) 0.006 
    Pharynx     
        <0.5 8 (20.51) 12 (19.05)   
        ≥0.5 31 (79.49) 51 (80.95) 1.03 (0.42-2.56) 0.948 
Stage     
    I     
        <0.5 37 (20.90) 139 (29.64)   
        ≥0.5 140 (79.10) 330 (70.36) 1.37 (0.95-1.97) 0.096 
    II     
        <0.5 18 (14.88) 55 (24.55)   
        ≥0.5 103 (85.12) 169 (75.45) 1.47 (0.86-2.50) 0.156 
Radiotherapy     
    No     
        <0.5 19 (26.76) 81 (44.51)   
        ≥0.5 52 (73.24) 101 (55.49) 2.07 (1.21-3.55) 0.008 
    Yes     
        <0.5 36 (15.86) 113 (22.16)   
        ≥0.5 191 (84.14) 397 (77.84) 1.19 (0.82-1.72) 0.354 
Surgery     
    No     
        <0.5 35 (17.68) 99 (23.68)   
        ≥0.5 163 (82.32) 319 (76.32) 1.12 (0.76-1.63) 0.573 
    Yes     
        <0.5 20 (20.00) 94 (34.43)   
        ≥0.5 80 (80.00) 179 (65.57) 1.89 (1.15-3.12) 0.012 
13 cRA     
    Yes     
        <0.5 25 (17.12) 99 (28.37)   
        ≥0.5 121 (82.88) 250 (71.63) 1.42 (0.91-2.21) 0.121 
    No     
        <0.5 30 (19.74) 95 (27.62)   
        ≥0.5 122 (80.26) 249 (72.38) 1.33 (0.88-2.00) 0.183 
*

Cox hazards model adjusted by age, sex, ethnicity, pack-years, alcohol, stage, site, and treatment when appropriate.

To evaluate the joint effects of bleomycin sensitivity and IGFs on the risk of SPT/recurrence, we measured the plasma levels of IGF-I and IGFBP-3 in a matched subset of patients (107 cases and 94 controls; Table 3). Controls were randomly chosen from the entire pool of patients who did not develop SPT/recurrence and were frequency matched to the cases on age (±5 years), sex, ethnicity, year of randomization, and length of follow-up. The cases exhibited significantly higher levels of IGF-I (mean, 160 ng/mL) and IGFBP-3 (mean, 3,164 ng/mL) than the controls (mean, 142 and 2,852 ng/mL; P = 0.022 and 0.042, respectively). The molar ratios of IGF-I/IGFBP-3 were not significantly different between the cases and controls (P = 0.957). In this subset of patients, the mean number of chromatid breaks per cell was significantly higher in the cases (0.76 ± 0.42) than in the controls (0.66 ± 0.48; P = 0.009). The joint effects of bleomycin sensitivity and IGF-I or IGFBP-3 on the risk of developing SPT/recurrence were next assessed (Table 4). The mean values of IGF-I and IGFBP-3 levels in controls were used as cutoff points to dichotomize the subjects into high and low IGF-I or IGFBP-3 groups. Using patients with low IGF-I and low bleomycin sensitivity as the reference group, the adjusted odds ratios (OR) of developing SPT/recurrence for patients with high IGF-I level alone, high mutagen sensitivity alone, and both high IGF-I level and high mutagen sensitivity were 2.85 (95% CI, 0.92-8.82), 3.92 (95% CI, 1.28-11.97), and 6.16 (95% CI, 2.03-18.71), respectively. A similar joint effect was observed for bleomycin sensitivity and IGFBP-3 level. Compared with patients with low IGFBP-3 and low bleomycin sensitivity, patients with high IGFBP-3 level alone (OR, 3.49; 95% CI, 1.02-11.86), high mutagen sensitivity alone (OR, 5.39; 95% CI, 1.56-18.63), and both elevated IGFBP-3 level and mutagen sensitivity phenotypes (OR, 7.07; 95% CI, 2.06-24.19) exhibited significantly elevated risks of SPT/recurrence.

Table 3.

IGF-I and IGFBP-3 levels and bleomycin sensitivity in a subset of cases and controls

VariablesnMean (SD)P*
IGF-I (ng/mL)    
    Event 107 160.0 (69.9)  
    No event 94 142.0 (69.6) 0.022 
IGFBP-3 (ng/mL)    
    Event 107 3,164 (1,161)  
    No event 94 2,852 (1,110) 0.042 
Molar ratio    
    Event 107 0.22 (0.19)  
    No event 94 0.21 (0.11) 0.957 
Bleomycin    
    Event 107 0.76 (0.42)  
    No event 94 0.66 (0.48) 0.009 
VariablesnMean (SD)P*
IGF-I (ng/mL)    
    Event 107 160.0 (69.9)  
    No event 94 142.0 (69.6) 0.022 
IGFBP-3 (ng/mL)    
    Event 107 3,164 (1,161)  
    No event 94 2,852 (1,110) 0.042 
Molar ratio    
    Event 107 0.22 (0.19)  
    No event 94 0.21 (0.11) 0.957 
Bleomycin    
    Event 107 0.76 (0.42)  
    No event 94 0.66 (0.48) 0.009 
*

Kruskal-Wallis test for mean difference.

Event = SPT/recurrence.

Molar ratio = IGF-I/IGFBP-3 molar ratio.

Table 4.

Joint effects of bleomycin sensitivity and IGF levels

IGF-IBleomycinSPT/recurrenceNo SPT/recurrenceORAdjusted OR*P
Low Low 23 Reference Reference  
High Low 19 3.11 (1.07-9.02) 2.85 (0.92-8.82) 0.07 
Low High 32 24 4.38 (1.62-11.88) 3.92 (1.28-11.97) 0.016 
High High 50 28 5.87 (2.24-15.39) 6.16 (2.03-18.71) 0.001 
       
IGFBP-3
 
Bleomycin
 
SPT/recurrence
 
No SPT/recurrence
 
OR
 
Adjusted OR*
 
P
 
Low Low 21 Reference Reference  
High Low 20 21 4.00 (1.26-12.65) 3.49 (1.02-11.86) 0.046 
Low High 39 26 6.30 (2.11-18.82) 5.39 (1.56-18.63) 0.008 
High High 43 26 6.95 (2.34-20.66) 7.07 (2.06-24.19) 0.002 
IGF-IBleomycinSPT/recurrenceNo SPT/recurrenceORAdjusted OR*P
Low Low 23 Reference Reference  
High Low 19 3.11 (1.07-9.02) 2.85 (0.92-8.82) 0.07 
Low High 32 24 4.38 (1.62-11.88) 3.92 (1.28-11.97) 0.016 
High High 50 28 5.87 (2.24-15.39) 6.16 (2.03-18.71) 0.001 
       
IGFBP-3
 
Bleomycin
 
SPT/recurrence
 
No SPT/recurrence
 
OR
 
Adjusted OR*
 
P
 
Low Low 21 Reference Reference  
High Low 20 21 4.00 (1.26-12.65) 3.49 (1.02-11.86) 0.046 
Low High 39 26 6.30 (2.11-18.82) 5.39 (1.56-18.63) 0.008 
High High 43 26 6.95 (2.34-20.66) 7.07 (2.06-24.19) 0.002 

NOTE: Low and high IGF level was determined by using their median values as a cutoff point; low and high bleomycin sensitivity was determined by using 0.5 breaks/cell as a cutoff point.

*

Adjusted by age, sex, ethnicity, pack-years, alcohol, stage, site, and treatment.

We have previously reported that bleomycin-induced mutagen sensitivity was a significant predictor of SPT or recurrence in a subset of the current study population (12, 31). In this study, we confirmed this finding with substantially larger sample size and using SPT and recurrence as the combined outcome event. To our knowledge, this is the largest prospective study to evaluate mutagen sensitivity as a prognosis marker in head and neck cancer, and the data confirmed that high mutagen sensitivity was associated with significantly elevated SPT/recurrence risk in early-stage head and neck cancer patients. In addition, we showed for the first time the joint effects between elevated IGF levels (high proliferative potential) and the mutagen sensitivity phenotype (latent genetic instability) in the risk of developing SPT/recurrence.

Genetic instability may contribute to a high genetic heterogeneity in a tumor and hence contribute to poor clinical outcome (38). Charuruks et al. (39) showed a statistically significant increment of genetic instability in terms of normalized chromosome index and polysomy index in recurrent primary tumors and SPTs of head and neck cancer patients compared with tumors without recurrence and SPT, suggesting that genetic instability might be a potential molecular marker for risk assessment of recurrence and SPT in head and neck tumorigenesis. Likewise, in vitro mutagen sensitivity, reflecting latent genetic instability, may also be a biomarker for tumor recurrence and SPT in head and neck cancer patients. As shown in our previous small-scale studies (12, 31) and confirmed in this large-scale study, high mutagen sensitivity indeed confers an increased risk of developing recurrence and SPT in head and neck cancer patients. The large sample size allowed us to do stratified analyses based on age, gender, and smoking status. The elevated risk was more evident in women than in men, in younger cases than in older cases, in former smokers than in current smokers, and in lighter smokers than in heavy smokers. These observations are consistent with previous studies. We have shown that the association between benzo(a)pyrene diol epoxide (a tobacco carcinogen) sensitivity and head and neck cancer risk was higher in former than in current smokers and in younger than in older patients. In a case-control lung cancer study, younger cases, women, and lighter smokers exhibited the lowest DNA repair capacity and the highest cancer risk among their subgroups (40). The theory is that exposed individuals with susceptible phenotypes might develop primary or secondary tumors earlier and with less carcinogen exposure than those with more resistant phenotype. Genetic differences in risks for primary or secondary tumors tend to be smaller at high doses of carcinogens. For example, the current smokers in this population were generally heavy smokers; therefore, the risk of smoking might overpower a weaker genetic risk factor, which explains why former smokers and light smokers with bleomycin-sensitive phenotypes exhibited higher risks than current and heavy smokers with similar sensitive phenotypes. Therefore, our data, obtained from the largest phenotypic assays to date, strengthened the notion that mutagen sensitivity constitutes a susceptible phenotype not only for cancer risk but also for SPT/recurrence.

Several studies have shown that IGF-I is a stimulator of cell proliferation and inhibitor of apoptosis and that elevated serum IGF-I levels are associated with increased risks for a variety of cancers (3235). We recently reported that elevated IGF-I levels were associated with a significantly increased risk of SPT in a subset of patients from the current study population (36). The data in this study were consistent with previous observations. More significantly, for the first time, we found that there is a joint effect between mutagen sensitivity and serum IGF-I level in modulating SPT/recurrence risk. Biologically, IGF level and mutagen sensitivity reflect two distinct yet interacting cellular functions (i.e., cell proliferation and DNA repair). Cells with deficient DNA repair plus uncontrolled proliferation will lead to a disproportionate increase in cells with damaged DNA. With a higher rate of cell turnover, there is increased likelihood for propagation of genetic errors. A previous study has shown that IGF-I and mutagen sensitivity jointly increased lung cancer risk (41). In this study, we found that IGF-I and mutagen sensitivity jointly increased risks of secondary tumors. These observations are consistent and biologically plausible.

Although the positive association between IGF-I level and various cancer risks has been consistently reported in the literature (3235), the relationship between IGFBP-3 level and cancer risk remains controversial. Some studies showed that elevated serum levels of IGFBP-3 were associated with either increased or decreased cancer risk (34, 35, 4247), whereas other studies did not detect an association between serum levels of IGFBP-3 and cancer risk (35, 48). The dual function of IGFBP-3 may explain these contradictory results. Circulating IGFBP-3 modulates the amount of bioavailable free IGF, thereby preventing their binding to IGF-I receptor and suppressing cell proliferation (49, 50). IGFBP-3, in this respect, inhibits IGF-I activity and may play a protective role against cancer risk. On the other hand, IGFBP-3 can also enhance IGF activity by presenting and slowly releasing IGF-I for receptor interactions while protecting the receptor from down-regulation by high IGF-I exposure (50). In this case, IGFBP-3 may serve as a risk factor for cancer. In this current study, the high risk of developing an SPT/recurrence conferred by higher level of IGFBP-3 may be explained by the dual function of IGFBP-3 in regulating IGF-I.

In our analyses, we combined SPT and recurrence as a single end point to increase sample size and power. In addition, it is difficult to clearly distinguish SPT and recurrent tumor biologically because recent genetic and biochemical studies have suggested that, in a significant portion of patients, the SPTs and index tumors have a common clonal origin (2326). We also did the same analyses separating these two events. The results were very similar to what we presented here with combined events. Higher bleomycin sensitivity conferred borderline significantly increased risks for SPT or recurrence (data not shown). We did not elaborate the effect of tumor characteristics and smoking status on SPT and recurrence, which have been presented in detail previously (13, 15).

There are several strengths to this study. This is the largest prospective study of mutagen sensitivity to date. There is no selection bias because the population is a well-defined population of early-stage head and neck cancer patients and biological samples were obtained before an event developed, which suggests that the observations in this study were more likely to be the contributor rather than the effect of the event. A major limitation of the study is the limited number of subjects with serum IGF values and a one-time point measurement of IGF levels, which may be subjected to temporal confounders and laboratory variability. However, Kaaks et al. (46) reported that blood samples drawn 0.75 to 4.75 years apart produced highly correlated values for both IGF-I (r = 0.87) and IGFBP-3 (r = 0.73). Additional studies are warranted to validate the joint effects between IGF and mutagen sensitivity.

In conclusion, this prospective study confirmed that high mutagen sensitivity was associated with significantly elevated SPT/recurrence risk in early-stage head and neck cancer patients. In addition, this is the first study to find a joint effect between elevated IGF levels and mutagen sensitive phenotype in elevating SPT/recurrence risk. By combining these two pathways, we may be able to improve our ability to define high-risk populations of SPT/recurrence in early-stage head and neck cancer patients, which may have clinical implications for patient surveillance and chemoprevention.

Grant support: National Cancer Institute grants CA86390, CA52051, CA097007, and CA106541.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1
Jemal A, Siegel R, Ward E, et al. Cancer statistics, 2006.
CA Cancer J Clin
2006
;
56
:
106
–30.
2
Blot WJ, McLaughlin JK, Winn DM, et al. Smoking and drinking in relation to oral and pharyngeal cancer.
Cancer Res
1988
;
48
:
3282
–7.
3
Goldgar DE, Easton DF, Cannon-Albright LA, Skolnick MH. Systematic population-based assessment of cancer risk in first-degree relatives of cancer probands.
J Natl Cancer Inst
1994
;
86
:
1600
–8.
4
Foulkes WD, Brunet JS, Sieh W, Black MJ, Shenouda G, Narod SA. Familial risks of squamous cell carcinoma of the head and neck: retrospective case-control study.
BMJ
1996
;
313
:
716
–21.
5
Yu GP, Zhang ZF, Hsu TC, Spitz MR, Schantz SP. Family history of cancer, mutagen sensitivity, and increased risk of head and neck cancer.
Cancer Lett
1999
;
146
:
93
–101.
6
Li X, Hemminki K. Familial upper aerodigestive tract cancers: incidence trends, familial clustering, and subsequent cancers.
Oral Oncol
2003
;
39
:
232
–9.
7
Lazarus P, Park JY. Metabolizing enzyme genotype and risk for upper aerodigestive tract cancer.
Oral Oncol
2000
;
36
:
421
–31.
8
Sturgis EM, Wei Q. Genetic susceptibility—molecular epidemiology of head and neck cancer.
Curr Opin Oncol
2002
;
14
:
310
–7.
9
Cheng L, Eicher SA, Guo Z, Hong WK, Spitz MR, Wei Q. Reduced DNA repair capacity in head and neck cancer patients.
Cancer Epidemiol Biomarkers Prev
1998
;
7
:
465
–8.
10
Wu X, Gu J, Hong WK, et al. Benzo[a]pyrene diol epoxide and bleomycin sensitivity and susceptibility to cancer of upper aerodigestive tract.
J Natl Cancer Inst
1998
;
90
:
1393
–9.
11
Leemans CR, Tiwari R, Nauta JJ, van der Waal I, Snow GB. Recurrence at the primary site in head and neck cancer and the significance of neck lymph node metastases as a prognostic factor.
Cancer
1994
;
73
:
187
–90.
12
Spitz M, Lippman S, Jiang H, et al. Mutagen sensitivity as a predictor of tumor recurrence in patients with cancer of the upper aerodigestive tract.
J Natl Cancer Inst
1998
;
90
:
243
–5.
13
Khuri FR, Kim ES, Lee JJ, et al. The impact of smoking status, disease stage, and index tumor site on second primary tumor incidence and tumor recurrence in the head and neck retinoid chemoprevention trial.
Cancer Epidemiol Biomarkers Prev
2001
;
10
:
823
–9.
14
Haughey BH, Gates GA, Arfken CL, Harvey J. Meta-analysis of second malignant tumors in head and neck cancer: the case for an endoscopic screening protocol.
Ann Otol Rhinol Laryngol
1992
;
101
:
105
–12.
15
Do KA, Johnson MM, Doherty DA, et al. Second primary tumors in patients with upper aerodigestive tract cancers: joint effects of smoking and alcohol (United States).
Cancer Causes Control
2003
;
14
:
131
–8.
16
Cooper JS, Pajak TF, Rubin P, et al. Second malignancies in patients who have head and neck cancer: incidence, effect on survival, and implications based on the RTOG experience.
Int J Radiat Oncol Biol Phys
1989
;
17
:
449
–56.
17
McDonald S, Haie C, Rubin P, Nelson D, Divers LD. Second malignant tumors in patients with laryngeal carcinoma: diagnosis, treatment, and prevention.
Int J Radiat Oncol Biol Phys
1989
;
17
:
457
–65.
18
Gluckman JL, Crissman JD. Survival rates in 548 patients with multiple neoplasms of the upper aerodigestive tract.
Laryngoscope
1983
;
93
:
71
–4.
19
Winn DM, Blot WJ. Second cancer following cancers of the buccal cavity and pharynx in Connecticut, 1935-1982.
Natl Cancer Inst Monogr
1985
;
68
:
25
–48.
20
Vikram B. Changing patterns of failure in advanced head and neck cancer.
Arch Otolaryngol
1984
;
110
:
564
–5.
21
Lippman SM, Hong WK. Second malignant tumors in head and neck squamous cell carcinoma: the overshadowing threat for patients with early-stage disease.
Int J Radiat Oncol Biol Phys
1989
;
17
:
691
–4.
22
Larson JT, Adams GL, Fattah HA. Survival statistics for multiple primaries in head and neck cancer.
Otolaryngol Head Neck Surg
1990
;
103
:
14
–24.
23
Bedi GC, Westra WH, Gabrielson E, Koch W, Sidransky D. Multiple head and neck tumors: evidence for a common clonal origin.
Cancer Res
1996
;
56
:
2484
–7.
24
Scholes AGM, Woolgar JA, Boyle MA, et al. Synchronous oral carcinomas: independent or common clonal origin?
Cancer Res
1998
;
58
:
2003
–6.
25
Califano J, Leong PL, Koch WM, Eisenberger CF, Sidransky D, Westra WH. Second esophageal tumors in patients with head and neck squamous cell carcinoma: an assessment of clonal relationships.
Clin Cancer Res
1999
;
5
:
1862
–7.
26
Tabor MP, Brakenhoff RH, Ruijter-Schippers HJ, et al. Multiple head and neck tumors frequently originate from a single preneoplastic lesion.
Am J Pathol
2002
;
161
:
1051
–60.
27
Wynder EL, Mushinski MH, Spivak JC. Tobacco and alcohol consumption in relation to the development of multiple primary cancers.
Cancer
1977
;
40
:
1872
–8.
28
Day GL, Blot WJ, Shore RE, et al. Second cancers following oral and pharyngeal cancers: role of tobacco and alcohol.
J Natl Cancer Inst
1994
;
86
:
131
–7.
29
Berwick M, Vineis P. Markers of DNA repair and susceptibility to cancer in humans: an epidemiologic review.
J Natl Cancer Inst
2000
;
92
:
874
–97.
30
Hsu TC, Spitz MR, Schantz SP. Mutagen sensitivity: a biologic marker of cancer susceptibility.
Cancer Epidemiol Biomarkers Prev
1991
;
1
:
83
–9.
31
Spitz MR, Hoque A, Trizna Z, et al. Mutagen sensitivity as a risk factor for second malignant tumors following malignancies of the upper aerodigestive tract.
J Natl Cancer Inst
1994
;
86
:
1681
–4.
32
Chan JM, Stampfer MJ, Giovannucci E, et al. Plasma insulin-like growth factor-I and prostate cancer risk: a prospective study.
Science
1998
;
279
:
563
–6.
33
Hankinson SE, Willett WC, Colditz GA, et al. Circulating concentrations of insulin-like growth factor-I and risk of breast cancer.
Lancet
1998
;
351
:
1393
–6.
34
Yu H, Spitz MR, Mistry J, Gu J, Hong WK, Wu X. Plasma levels of insulin-like growth factor-I and lung cancer risk: a case-control analysis.
J Natl Cancer Inst
1999
;
91
:
151
–6.
35
Ma J, Pollak MN, Giovannucci E, et al. Prospective study of colorectal cancer risk in men and plasma levels of insulin-like growth factor (IGF)-I and IGF-binding protein-3.
J Natl Cancer Inst
1999
;
91
:
620
–5.
36
Wu X, Zhao H, Do KA, et al. Serum levels of insulin growth factor (IGF-I) and IGF-binding protein predict risk of second primary tumors in patients with head and neck cancer.
Clin Cancer Res
2004
;
10
:
3988
–95.
37
Warren S, Gates O. Multiple primary malignant tumors. A survey of the literature and a statistical study.
Am J Cancer
1932
;
16
:
1358
–414.
38
Chang JY, Komaki R, Sasaki R, et al. High mutagen sensitivity in peripheral blood lymphocytes predicts poor overall and disease-specific survival in patients with stage III non-small cell lung cancer treated with radiotherapy and chemotherapy.
Clin Cancer Res
2005
;
11
:
2894
–8.
39
Charuruks N, Shin DM, Voravud N, Ro JY, Hong WK, Hittelman WN. Genetic instability and the development of recurrence of primary tumor and second primary tumor during head and neck tumorigenesis.
J Med Assoc Thai
1996
;
79
:
S49
–55.
40
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
2000
;
92
:
1764
–72.
41
Wu X, Yu H, Amos CI, Hong WK, Spitz MR. Joint effect of insulin-like growth factors and mutagen sensitivity in lung cancer risk.
J Natl Cancer Inst
2000
;
92
:
737
–43.
42
Vadgama JV, Wu Y, Datta G, Khan H, Chillar R. Plasma insulin-like growth factor-I and serum IGF-binding protein 3 can be associated with the progression of breast cancer, and predict the risk of recurrence and the probability of survival in African-American and Hispanic women.
Oncology
1999
;
57
:
330
–40.
43
el Atiq F, Garrouste F, Remacle-Bonnet M, Sastre B, Pommier G. Alterations in serum levels of insulin-like growth factors and insulin-like growth-factor-binding proteins in patients with colorectal cancer.
Int J Cancer
1994
;
57
:
491
–7.
44
Giovannucci E, Pollak MN, Platz EA, et al. A prospective study of plasma insulin-like growth factor-1 and binding protein-3 and risk of colorectal neoplasia in women.
Cancer Epidemiol Biomarkers Prev
2000
;
9
:
345
–9.
45
Probst-Hensch NM, Yuan JM, Stanczyk FZ, Gao YT, Ross RK, Yu MC. IGF-1, IGF-2, and IGFBP-3 in prediagnostic serum: association with colorectal cancer in a cohort of Chinese men in Shanghai.
Br J Cancer
2001
;
85
:
1695
–9.
46
Kaaks R, Toniolo P, Akhmedkhanov A, et al. Serum C-peptide, insulin-like growth factor (IGF)-I, IGF-binding proteins, and colorectal cancer risk in women.
J Natl Cancer Inst
2000
;
92
:
1592
–600.
47
London SJ, Yuan JM, Travlos GS, et al. Insulin-like growth factor I, IGF-binding protein 3, and lung cancer risk in a prospective study of men in China.
J Natl Cancer Inst
2002
;
94
:
749
–54.
48
Toniolo P, Bruning PF, Akhmedkhanov A, et al. Serum insulin-like growth factor-I and breast cancer.
Int J Cancer
2000
;
88
:
828
–32.
49
Kelley KM, Oh Y, Gargosky SE, et al. Insulin-like growth factor-binding proteins (IGFBPs) and their regulatory dynamics.
Int J Biochem Cell Biol
1996
;
28
:
619
–37.
50
Clemmons DR. Insulin-like growth factor binding proteins and their role in controlling IGF actions.
Cytokine Growth Factor Rev
1997
;
8
:
45
–62.