Summary
The modification of risk by either individual or combined risk genotypes of 5 polymorphisms were pronounced in never smokers than in smokers and in SCCOP but not in SCCOC. These findings indicate that the risk of HPV16-associated SCCOP was modified by E2F2 promoter variants, especially in never smokers.
Abstract
Given roles of HPV and genetic factors in cancer risk, we evaluated associations of HPV16 seropositivity and five E2F2 promoter variants with squamous cell carcinoma of oropharynx (SCCOP) and squamous cell carcinoma of oral cavity (SCCOC) risk in a case–control study of 325 patients and 335 cancer-free matched controls. We found that HPV16 seropositivity was significantly associated with SCCOP risk (aOR, 5.4, 95%CI, 3.7–8.9) but not SCCOC (aOR, 0.8, 95%CI, 0.4–1.5), while each E2F2 polymorphism had no significant main effect on SCCOP and SCCOC risk. However, after combining HPV serological status and E2F2 promoter variants together, the modification effect of HPV serology and individual or combined risk genotypes of five polymorphisms on risk was significantly higher among SCCOP than among SCCOC. Furthermore, the stratified analysis by smoking status showed that all such modifying effects aforementioned on SCCOP were more pronounced in never smokers than in smokers. These findings are in agreement with those of previous studies, in which a majority of SCCOP were caused by HPV infection, whereas most SCCOC were found to be caused by smoking and drinking. Taken together, these findings indicate that the risk of SCCOP as opposed to SCCOC associated with HPV16 seropositivity was modified by E2F2 promoter variants either individually or jointly, especially in never smokers.
Introduction
Squamous cell carcinomas of the oropharynx (SCCOP) and oral cavity (SCCOC) are two subgroups of squamous cell carcinoma of the head and neck and is the most common malignant tumor of the oral mucosa worldwide (1,2). In the United States, approximately 40300 new cases will be diagnosed and 7800 deaths will occur from these tumors in 2016 (3). Long-term survival rates have improved only moderately, with high recurrence rates in the past two decades (4). Tobacco smoking and alcohol are crucial risk factors for SCCOP and SCCOC. However, despite decreasing rates of tobacco and alcohol use, the incidence of these tumors, particularly SCCOP and young SCCOC, has increased in recent years (5). This increase can be mainly attributed to the increasing prevalence of human papillomavirus (HPV) infection, especially high-risk HPV type 16 (HPV16) (6). Although HPV infection is major risk factor for SCCOP and SCCOC, only a small portion of individuals with HPV exposure develop SCCOP and SCCOC, suggesting that other risk factors, such as a patient’s genetic factors, may also contribute to risk of SCCOP and SCCOC (7).
The retinoblastoma (RB) tumor suppressor plays a pivotal role in regulating cellular proliferation and the cell cycle. E2F2, a member of the E2F family of transcription factors, is a cell-cycle regulatory protein that interacts with RB family members (RB, p107 and p130) (8). The RB/E2F2 pathway is constantly converted through inactivation of CDKN2A (p16) in SCCOP and SCCOC (9–11). The p16 tumor suppressor gene is one of the crucial RB-dependent regulators that controls cell proliferation at the G1/S checkpoint. Inactivation of RB leads to the release and activation of E2F2 transcription factors. E2F2 initiates transcription required for G1/S progression, which maintains RB-mediated suppression of E2F2 and consequently prevents cell-cycle progression from the G1 to S phase (12).
The malignant transformation potential of oncogenic HPV is attributed to its oncoproteins E6 and E7 (13). HPV E7 protein expression suppresses the RB pathway, which abolishes the requirement for p16 silencing (14). Direct mutations may inactivate or alter RB, but interactions with other proteins, such as HPV E7 oncoprotein and E2F2 protein, can also cause aberrations in RB regulation. Hence, both HPV E7 and E2F2 have critical roles in regulating RB in response to cellular stressors or oncogenic signaling.
No studies have investigated the association between E2F2 promoter polymorphisms and the risk of HPV-associated SCCOP and SCCOC. Previously, we assessed the association between E2F2 promoter polymorphisms and the risk of squamous cell carcinoma of the head and neck, while we did not observe any significant associations partially due to inclusion of mixed tumor sites and without consideration of HPV information (15). However, this study is the first to include HPV information in risk assessment. Because both HPV and E2F2 may play important roles in cell proliferation and cell-cycle regulation through the p16/RB pathway, we hypothesized that E2F2 promoter variants modify the association between HPV16 seropositivity and the risk of SCCOP and SCCOC. To test this hypothesis, we evaluated the effects of interactions between HPV16 serologic status and E2F2 promoter variants on the risk of SCCOP and SCCOC.
Materials and methods
Patients and control samples
Participants were recruited from our ongoing study, which has been previously prescribed (7). Briefly, this study included 325 patients with newly diagnosed and histopathologically confirmed SCCOP and SCCOC and 335 self-reported cancer-free controls recruited between May 1996 and May 2002. The participants with SCCOP and SCCOC were recruited from The University of Texas MD Anderson Cancer Center. Excluded from participation were patients treated with chemotherapy or radiation therapy before recruitment and patients with second primary tumors; with primary tumors of the sinonasal tract, nasopharynx, hypopharynx or larynx; with primary tumors outside the upper aerodigestive tract; with cervical metastases of unknown origin; or with histopathologic diagnoses other than squamous cell carcinoma. Additionally, patients who had received a blood transfusion in the last 6 months or who were receiving immunosuppressive therapy were excluded. Of the 325 non-Hispanic white patients included in the analysis, 137 (42.2%) had primary SCCOC, and 188 (57.8%) had primary SCCOP. Approximately 95% of eligible patients agreed to participate in the study.
For controls, cancer-free participants were recruited from the Kelsey–Seybold Foundation (Houston, TX), a multispecialty practice with multiple clinics, and from cancer-free visitors who accompanied patients with cancer to outpatient clinics at MD Anderson Cancer Center but who were genetically unrelated to the patients. Exclusion criteria for the control group included receiving immunosuppressive therapy, having a history of cancer and having received a blood transfusions in the last 6 months. The 335 cancer-free participants were frequency matched to the patients by age (±5 years), sex, ethnicity and smoking and alcohol consumption. All participants were interviewed by trained interviewers. Each eligible participant provided demographic information (e.g., age, sex, and ethnicity) and risk factor information (e.g., smoking status and alcohol consumption status). The response rate was ~78%.
Participants who smoked more than 100 cigarettes in their lifetime were defined as ever smokers, and the rest were defined as never smokers, as is standard in epidemiologic studies. Individuals who consumed alcoholic beverages at least once a week for more than 1 year were defined as ever drinkers, and the rest were defined as never drinkers. After providing written informed consent, each participant donated blood (30ml) collected in heparinized tubes. The research protocol was approved by both the Institutional Review Boards of MD Anderson Cancer Center and the Kelsey–Seybold Foundation.
HPV16 serologic testing
We used a standard enzyme-linked immunosorbent assay and HPV16 virus-like particles generated from recombinant baculovirus-infected insect cells to detect antibody against HPV16 capsid protein in the plasma of study participants, as described previously (16,17). Control sera known to be HPV16-positive or HPV16-negative were also tested in duplicate and in parallel with the study samples on each plate. We used the cutoff level for HPV16 seropositivity determined from a standard pooled serum in a previous study (16). Samples within 15% of the cutoff point were tested again twice; those that tested positive three times were considered positive. To exclude potential binding interference by heparin, we treated the plasma samples with heparinase I (43U/ml; Sigma-Aldrich, St Louis, MO) before testing (18). We probed heparinized plasma and serum acquired from three individuals and did not detect discernible differences between the reactions of serum samples and those of the heparinized plasma samples. We also retested 10% of the samples (selected by stochastic choice) and obtained 100% consistent results on the repeat assays.
E2F2 polymorphism genotyping
We extracted genomic DNA from the buffy coat fraction of the blood samples using a QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer’s instructions. To genotype E2F2 promoter polymorphisms, we identified five single nucleotide polymorphisms (SNPs; E2F2-rs6667575, E2F2-rs3218121, E2F2-rs2742976, E2F2-rs3218123 and E2F2-rs3218148) that have minor allele frequencies ≥ 10%, according to a published SNP database from our previous pilot study (15), and included them in the current study.
We genotyped E2F2-rs6667575, E2F2-rs3218121, E2F2-rs2742976, E2F2-rs3218123 and E2F2-rs3218148 as described previously (15) and estimated the results without knowing the samples’ source (i.e., patient or control). A random 10% of the specimens were retested, and the outcomes of retesting were 100% consistent.
Tumor HPV16 status and p16 expression of SCCOP patients
A total of additional 62 SCCOP patients were selected and these patients had tumor HPV16 status and p16 expression clinically available by specific PCR and in situ hybridization and p16 Immunohistochemisrty. The tumor HPV16 status and p16 expression data for SCCOP patients are now part of the patient’s clinical record as the pathology laboratory at MD Anderson classified all SCCOP specimens as a standard clinical practice.
Statistical analysis
The differences between cases and controls in the distributions of HPV16 status and E2F2 genotypes were examined using the chi-squared test. We evaluated the association of HPV16 status and E2F2 genotypes as well as their combination with the risk of patients by computing the odds ratios (ORs) and 95% confidence intervals (CIs) using both univariate and multivariable logistic regression analyses. We also estimated the joint effects of HPV16 serology and E2F2 genotypes on the risk of patients and stratified these effects by smoking status. We assessed the trends in the risk of SCCOP and SCCOC associated with the number of variant alleles of the five polymorphisms.
Logistic regression analysis was also used to assess potential interaction effects by evaluating departures from the model of multiplicative interaction between selected variables. A more-than multiplicative interaction was suggested when OR11 > OR10 × OR01, for which OR11 = OR when both factors were present, OR10 = OR when only factor 1 was present, and OR01 = OR when only factor 2 was present. We assessed the interaction by reporting the P values from the Wald test for testing the coefficients (βE2F2 polymorphism, HPV16 seropositivity) that were different from 0, where the interaction term consisted of the product of the two variables: E2F2 polymorphism and HPV16 seropositivity. To account for the multiple tests of interaction, we used the Benjamini and Hochberg method to cast the false discovery rate-adjusted P values (19). All tests were two-sided, and P values less than 0.05 were considered statistically significant. All statistical analyses were performed with Statistical Analysis System software (version 9.1; SAS Institute, Cary, NC).
Results
Demographics and risk factors for study subjects
Table 1 summarizes the distribution of demographic characteristics and known SCCOP and SCCOC risk factors. HPV16 seropositivity rate was significantly higher in SCCOP patients than in controls (P < 0.001) but not for SCCOC (P = 0.430), and HPV16 seropositivity was significantly associated with an OR of 5.4 for SCCOP (95% CI 3.7–8.9), but with an OR of 0.8 for SCCOC (95% CI 0.4–1.5), after adjusting for patient age, sex, smoking status and alcohol consumption status.
Table 1.
Frequency distribution of demographic and risk factors in patients and cancer-free controls
| Characteristics | All patients (N = 325) | Patients (N = 325) | Controlsa (N = 335) | |
|---|---|---|---|---|
| SCCOP (N = 188) | SCCOC (N = 137) | |||
| N (%) | N (%) | N (%) | ||
| Age (years) | ||||
| <40 | 31 (9.5) | 17 (9.0) | 14 (10.2) | 27 (8.1) |
| 41–55 | 126 (38.8) | 86 (45.8) | 40 (29.2) | 105 (31.3) |
| 56–70 | 119 (36.6) | 64 (34.0) | 55 (40.2) | 154 (46.0) |
| >70 | 49 (15.1) | 21 (11.2) | 28 (20.4) | 49 (14.6) |
| Sex | ||||
| Male | 241 (74.1) | 155 (82.4) | 86 (62.8) | 269 (80.3) |
| Female | 84 (25.9) | 33 (17.6) | 51 (37.2) | 66 (19.7) |
| Ethnicity | ||||
| Non-Hispanic white | 325 (100) | 188 (100) | 137 (100) | 335 (100) |
| Tobacco smoking | ||||
| Ever | 227 (69.8) | 125 (66.5) | 102 (74.4) | 239 (71.3) |
| Never | 98 (30.2) | 63 (33.5) | 35 (25.6) | 96 (28.7) |
| Alcohol drinking | ||||
| Ever | 250 (76.9) | 150 (79.8) | 100 (73.0) | 240 (71.6) |
| Never | 75 (23.1) | 38 (20.2) | 37 (27.0) | 95 (28.4) |
| HPV serology | ||||
| Positive | 100 (30.8) | 87 (46.3) | 13 (9.5) | 42 (12.5) |
| Negative | 225 (69.2) | 101 (53.7) | 124 (90.5) | 293 (87.5) |
aThe controls were frequency matched to all patients on the basis of the factors shown in the table.
Main effects of E2F2 variants on risk of SCCOP and SCCOC
Table 2 showed the associations of each individual polymorphism with risk of SCCOP and SCCOC. Overall, there were no any significant effects of any of the five polymorphisms on risk of SCCOP and SCCOC, while E2F2-rs6667575 and E2F2-rs3218121 had a borderline significant minor effect on risk of SCCOP only. The patients with E2F2-rs6667575 GA/AA and E2F2-rs3218121 GA/AA genotypes had a 20–30% increased risk of SCCOP than those with their corresponding GG genotype (aOR, 1.3; 95% CI 1.0–2.2 for E2F2-rs6667575 and aOR 1.2; 95% CI 1.0–1.9 for E2F2-rs3218121), after adjustment for age, sex, smoking status, alcohol use and HPV16 serology. However, such associations were not found for SCCOC.
Table 2.
Associations of HPV16 seropositivity and E2F2 genotypes with risk of SCCOP and SCCOC patients and cancer-free controls
| Variables | Patients (N = 325) | Controls (N = 335) | Adjusted OR (95% CI)a | ||
|---|---|---|---|---|---|
| SCCOP (N = 188) | SCCOC (N = 137) | ||||
| N (%) | N (%) | N (%) | SCCOP | SCCOC | |
| HPV serology | |||||
| _ | 101 (53.7) | 124 (90.5) | 293 (87.5) | 1 | 1 |
| + | 87 (46.3) | 13 (9.5) | 42 (12.5) | 5.4 (3.7-8.9) | 0.8 (0.4–1.5) |
| E2F2 genotypes | |||||
| E2F2-rs6667575 | |||||
| GG | 88 (46.8) | 66 (48.2) | 179 (53.4) | 1.0 | 1.0 |
| GA+AA | 100 (53.2) | 71 (51.8) | 156 (46.6) | 1.3 (1.0–2.2) | 0.9 (0.8–1.9) |
| E2F2-rs3218121 | |||||
| GG | 160 (85.1) | 110 (80.3) | 276 (82.4) | 1.0 | 1.0 |
| GA+AA | 28 (14.9) | 27 (19.7) | 59 (17.6) | 1.2 (1.0–1.9) | 1.1 (0.6–1.8) |
| E2F2-rs2742976 | |||||
| GT/TT | 107 (56.9) | 84 (61.3) | 202 (60.3) | 1.0 | 1.0 |
| GG | 81 (43.1) | 53 (38.7) | 133 (39.7) | 1.2 (0.9–1.8) | 0.9 (0.6–1.3) |
| E2F2-rs3218123 | |||||
| GT/TT | 76 (40.4) | 63 (46.0) | 231 (69.0) | 1.0 | 1.0 |
| GG | 112 (59.6) | 74 (54.0) | 104 (31.0) | 1.1 (0.9–2.2) | 1.0 (0.7–1.7) |
| E2F2-rs3218148 | |||||
| AA | 76 (40.4) | 37 (27.0) | 104 (31.0) | 1.0 | 1.0 |
| AG/GG | 112 (59.6) | 100 (73.0) | 231 (69.0) | 0.6 (0.4–1.1) | 1.1 (0.7–1.8) |
aORs were adjusted for age, sex, smoking and alcohol drinking.
Joint effects of HPV16 seropositivity and E2F2 variants on the risk of SCCOP and SCCOC
As shown in Table 3, compared with individuals with E2F2-rs6667575 GG genotypes and HPV16 seronegativity, the overall risk of SCCOP increased among those with the GA + AA genotype and HPV16 seronegativity (OR, 1.3; 95% CI 0.8–2.1), those with GG genotypes and HPV16 seropositivity (OR 3.6; 95% CI 2.6–11.6), and those with GA + AA genotypes and HPV16 seropositivity (OR 6.6; 95% CI 3.4–12.8). Similarly, increased risk of SCCOP in the different genotype groups was also observed for the other four polymorphisms. However, we did not observe the similar joint modifying effect of HPV16 serpositivity and E2F2 variants on the risk of SCCOC.
Table 3.
Association of HPV16 seropositivity and E2F2 genotypes on risk of SCCOP and SCCOC patients and cancer-free controls
| HPV16 status | E2F2 genotypes | Patients(N = 325) | Controls | Adjusted OR (95% CI)a | ||
|---|---|---|---|---|---|---|
| SCCOP (N = 188) | SCCOC (N = 137) | (N = 335) | SCCOP | SCCOC | ||
| N (%) | N (%) | N (%) | ||||
| E2F2-rs6667575 | ||||||
| − | GG | 49 (26.1) | 59 (43.1) | 161 (48.1) | 1.0 | 1.0 |
| − | GA + AA | 52 (27.7) | 65 (47.4) | 132 (39.4) | 1.3 (0.8–2.1) | 1.4 (0.9–2.1) |
| + | GG | 48 (25.5) | 6 (4.4) | 24 (7.2) | 3.6 (2.6–11.6) | 0.7 (0.3–1.9) |
| + | GA + AA | 39 (20.7) | 7 (5.1) | 18 (5.3) | 6.6 (3.4–12.8) | 1.1 (0.4–2.8) |
| E2F2-rs3218121 | ||||||
| − | GG | 88 (46.8) | 97 (70.8) | 242 (72.2) | 1.0 | 1.0 |
| − | GA + AA | 13 (6.9) | 27 (19.7) | 51 (15.2) | 0.7 (0.4–1.4) | 1.2 (0.7–2.1) |
| + | GG | 15 (8.0) | 0 (0) | 8 (2.4) | 2.8 (1.3–11.6) | — |
| + | GA + AA | 72 (38.3) | 13 (9.5) | 34 (10.2) | 5.6 (3.5–9.2) | 1.0 (0.5–2.0) |
| E2F2-rs2742976 | ||||||
| − | GT + TT | 44 (23.4) | 52 (38.0) | 112 (33.4) | 1.0 | 1.0 |
| − | GG | 57 (30.3) | 72 (52.6) | 181 (54.0) | 0.7 (0.5–1.2) | 0.9 (0.6–1.5) |
| + | GT + TT | 37 (19.7) | 1 (0.7) | 21 (6.3) | 3.9 (2.0–7.5) | 0.1 (0.0–0.8) |
| + | GG | 50 (26.6) | 12 (8.7) | 21 (6.3) | 5.6 (3.0–10.4) | 1.4 (0.6–3.1) |
| E2F2-rs3218123 | ||||||
| − | GT + TT | 61 (32.4) | 66 (48.2) | 90 (26.9) | 1.0 | 1.0 |
| − | GG | 40 (21.3) | 58 (42.3) | 203 (60.5) | 0.3 (0.2–0.5) | 0.4 (0.2–0.6) |
| + | GT + TT | 51 (27.1) | 8 (5.8) | 14 (4.2) | 2.0 (1.1–3.6) | 0.9 (0.3–2.3) |
| + | GG | 36 (19.2) | 5 (3.7) | 28 (8.4) | 5.0 (2.5–9.9) | 1.2 (0.1–1.6) |
| E2F2-rs3218148 | ||||||
| − | AA | 43 (22.9) | 32 (23.4) | 92 (27.4) | 1.0 | 1.0 |
| − | AG + GG | 58 (30.9) | 92 (67.2) | 201 (60.0) | 0.6 (0.4–1.0) | 1.3 (0.8–2.1) |
| + | AA | 54 (28.7) | 8 (5.8) | 30 (9.0) | 3.5 (2.0–6.3) | 0.7 (0.3–1.8) |
| + | AG + GG | 33 (17.5) | 5 (3.6) | 12 (3.6) | 5.6 (2.6–12.1) | 1.4 (0.4–4.4) |
| E2F2 combined genotypes | ||||||
| − | Low-risk groupb | 23 (12.2) | 25 (18.2) | 106 (31.6) | 1.0 | 1.0 |
| − | Medium-risk group | 65 (34.6) | 73 (53.3) | 167 (49.8) | 1.8 (1.1–3.1) | 1.8 (1.1–3.1) |
| − | High-risk group | 13 (6.9) | 26 (19.0) | 20 (6.0) | 2.9 (1.3–6.8) | 5.3 (2.5–11.1) |
| + | Low-risk groupb | 60 (31.9) | 8 (5.8) | 32 (9.6) | 5.1 (3.2–15.6) | 1.1 (0.4–2.7) |
| + | Medium-risk group | 16 (8.5) | 5 (3.7) | 8 (2.4) | 9.6 (3.6–25.7) | 2.7 (0.8–9.2) |
| + | High-risk group | 11 (5.9) | 0 (0) | 2 (0.6) | 21.3 (4.4–103.9) | — |
aORs were adjusted for age, sex, smoking and alcohol drinking.
bLow-risk group (used as the reference group): individuals carrying 0–1 genotype; medium-risk group: individuals carrying 2–3 genotypes; and high-risk group: individuals carrying 4–5 genotypes.
Joint effect of HPV16 seropositivity and combined E2F2 risk genotypes on risk of SCCOP and SCCOC
To investigate the modifying effects of the combined genotypes of the five polymorphisms on the risk of SCCOP and SCCOC associated with HPV16 serological status, we used the individuals in the low-risk group (0–1 combined risk genotype) with HPV16 seronegativity as the comparison group (Table 3). The risk of SCCOP increased in the medium-risk group (2–3 combined risk genotypes) with HPV16 seronegativity (OR 1.8; 95% CI 1.1–3.1); the high-risk group (4–5 combined risk genotypes) with HPV16 seronegativity (OR 2.9; 95% CI 1.3–6.8); the low-risk group with HPV16 seropositivity (OR 5.1; 95% CI 3.2–15.6); the medium-risk group with HPV16 seropositivity (OR 9.6; 95% CI 3.6–25.7); and the high-risk group with HPV16 seropositivity (OR 21.3; 95% CI 4.4–103.9). However, no significant joint modifying effect of combined E2F2 risk genotypes of 5 polymorphisms and HPV16 seropositivity on risk of SCCOC was found (Table 3).
Joint effects of HPV16 seropositivity and E2F2 variants on risk of SCCOP and SCCOC stratified by smoking status
We stratified the joint effects of HPV16 serology status and E2F2 variants on SCCOP and SCCOC risk by smoking status (Table 4). Overall, for each polymorphism, the SCCOP risk for patients with HPV16 seropositivity was much higher in never smokers than in ever smokers. However, the similar associations were not found for patients with SCCOC (Table 4). Furthermore, we did not detect a significant interaction between HPV16 seropositivity and most of E2F2 variants on the risk of SCCOP, partially due to the small sample sizes in each of these subgroups. Although the interaction between the E2F2-rs3218121 polymorphism and HPV16 seropositivity on the risk of SCCOP was significant in the never smokers, this interaction was not significant after adjusting for multiple comparisons.
Table 4.
Joint effect of HPV16 seropositivity and E2F2 genotypes on risk of SCCOP and SCCOC patients and cancer-free controls stratified by smoking status
| HPV16 status | E2F2 genotypes | Patients (N = 325) | Controls | Adjusted OR (95% CI)a | ||
|---|---|---|---|---|---|---|
| SCCOP (N = 188) | SCCOC (N = 137) | (N = 335) | ||||
| N (%) | N (%) | N (%) | SCCOP | SCCOC | ||
| Never smokers | ||||||
| E2F2-rs6667575 | ||||||
| − | GG | 13 (20.6) | 14 (40.0) | 53 (55.2) | 1.0 | 1.0 |
| − | GA + AA | 14 (22.2) | 18 (51.4) | 35 (36.5) | 1.7 (0.7–4.3) | 2.2 (0.9–5.5) |
| + | GG | 17 (27.0) | 2 (5.7) | 5 (5.2) | 19.8 (5.6–69.9) | 1.1 (0.2–7.8) |
| + | GA + AA | 19 (30.2) | 1 (2.9) | 3 (3.1) | 28.4 (6.8–118.6) | 2.3 (0.1–41.0) |
| E2F2-rs3218121 | ||||||
| − | GG | 22 (34.9) | 25 (71.4) | 73 (76.0) | 1.0 | 1.0 |
| − | GA + AA | 5 (7.9) | 7 (20.0) | 15 (15.6) | 1.3 (0.4–4.2) | 1.8 (0.6–5.7) |
| + | GG | 28 (44.5) | 3 (8.6) | 7 (7.3) | 17.8 (6.3–50.4) | 1.1 (0.2–5.7) |
| + | GA + AA | 8 (12.7) | 0 (0) | 1 (1.1) | 25.1 (2.8–222.6) | — |
| E2F2-rs2742976 | ||||||
| − | GT/TT | 15 (23.8) | 11 (31.4) | 29 (30.2) | 1.0 | 1.0 |
| − | GG | 12 (19.0) | 21 (60.0) | 59 (61.4) | 1.5 (0.2–2.4) | 0.9 (0.4–2.3) |
| + | GT/TT | 17 (27.0) | 0 (0) | 4 (4.2) | 7.1 (3.0–44.0) | — |
| + | GG | 19 (30.2) | 3 (8.6) | 4 (4.2) | 12.9 (3.4–49.5) | 2.0 (0.3–13.4) |
| E2F2-rs3218123 | ||||||
| − | GT/TT | 10 (15.9) | 27 (77.1) | 32 (33.3) | 1.0 | 1.0 |
| − | GG | 17 (27.0) | 5 (14.3) | 56 (58.3) | 1.2 (0.1–2.8) | 0.3 (0.1–0.8) |
| + | GT/TT | 24 (38.1) | 3 (8.6) | 5 (5.2) | 5.6 (2.9–31.3) | 1.1 (0.2–6.9) |
| + | GG | 12 (19.0) | 0 (0) | 3 (3.2) | 11.7 (2.7–51.5) | — |
| E2F2-rs3218148 | ||||||
| − | AA | 7 (11.1) | 8 (22.9) | 24 (25.0) | 1.0 | 1.0 |
| − | AG/GG | 20 (31.7) | 24 (68.6) | 64 (66.7) | 1.2 (0.4–2.9) | 0.9 (0.3–2.6) |
| + | AA | 11 (17.5) | 1 (2.9) | 3 (3.1) | 8.2 (3.5–89.1) | 1.3 (0.1–11.6) |
| + | AG/GG | 25 (39.7) | 2 (5.6) | 5 (5.2) | 18.8 (4.9–71.8) | 0.6 (0.0–7.4) |
| Ever smokers | ||||||
| E2F2-rs6667575 | ||||||
| − | GG | 36 (28.8) | 45 (44.1) | 108 (45.2) | 1.0 | 1.0 |
| − | GA + AA | 38 (30.4) | 47 (46.1) | 97 (40.6) | 1.2 (0.7–2.0) | 1.2 (0.7–2.1) |
| + | GG | 31 (24.8) | 4 (3.9) | 19 (7.9) | 4.7 (2.3–9.4) | 0.6 (0.2–1.9) |
| + | GA + AA | 20 (16.0) | 6 (5.9) | 15 (6.3) | 4.0 (1.8–8.7) | 1.1 (0.4–3.0) |
| E2F2-rs3218121 | ||||||
| − | GG | 66 (52.8) | 72 (70.6) | 169 (70.7) | 1.0 | 1.0 |
| − | GA + AA | 8 (6.4) | 20 (19.6) | 36 (15.1) | 0.6 (0.3–1.3) | 1.2 (0.6–2.3) |
| + | GG | 44 (35.2) | 10 (9.8) | 27 (11.3) | 4.1 (2.3–7.2) | 1.0 (0.4–2.20 |
| + | GA + AA | 7 (5.6) | 0 (0) | 7 (2.9) | 2.6 (0.9–7.9) | — |
| E2F2-rs2742976 | ||||||
| − | GT/TT | 32 (25.6) | 38 (37.3) | 83 (34.7) | 1.0 | 1.0 |
| − | GG | 42 (33.6) | 54 (52.9) | 122 (51.1) | 0.8 (0.5–1.4) | 1.0 (0.6–1.6) |
| + | GT/TT | 20 (16.0) | 1 (1.0) | 17 (7.1) | 2.8 (1.3–6.1) | 0.1 (0.0–1.0) |
| + | GG | 31 (24.8) | 9 (8.8) | 17 (7.1) | 4.5 (2.2–9.3) | 1.4 (0.5–3.4) |
| E2F2-rs3218123 | ||||||
| − | GT/TT | 44 (35.2) | 46 (45.1) | 58 (24.2) | 1.0 | 1.0 |
| − | GG | 30 (24.0) | 46 (45.1) | 147 (61.5) | 0.3 (0.2–0.5) | 0.4 (0.2–0.6) |
| + | GT/TT | 27 (21.6) | 5 (4.9) | 9 (3.8) | 3.8 (1.6–8.9) | 0.8 (0.2–2.7) |
| + | GG | 24 (19.2) | 5 (4.9) | 25 (10.5) | 1.3 (0.6–2.6) | 0.3 (0.1–0.7) |
| E2F2-rs3218148 | ||||||
| − | AA | 36 (28.8) | 24 (23.5) | 68 (28.4) | 1.0 | 1.0 |
| − | AG/GG | 38 (30.4) | 68 (66.7) | 137 (57.3) | 0.5 (0.3–0.9) | 1.5 (0.8–2.6) |
| + | AA | 22 (17.6) | 3 (2.9) | 9 (3.8) | 4.4 (1.8–10.6) | 1.5 (0.4–6.3) |
| + | AG/GG | 29 (23.2) | 7 (6.9) | 25 (10.5) | 2.1 (1.1–4.2) | 0.8 (0.3–2.1) |
| E2F2 combined | ||||||
| Genotypes | ||||||
| Never smokers | ||||||
| − | Low-risk groupb | 3 (4.8) | 4 (11.4) | 33 (34.4) | 1.0 | 1.0 |
| − | Medium-risk group | 19 (30.2) | 22 (62.9) | 49 (51.0) | 3.8 (1.0–14.3) | 3.3 (0.9–11.4) |
| − | High-risk group | 5 (7.9) | 6 (17.1) | 7 (7.3) | 11.0 (1.9–63.2) | 12.7 (2.1–76.8) |
| + | Low-risk groupb | 25 (39.7) | 1 (2.9) | 4 (4.2) | 42.3 (12.5275.0) | 2.2 (0.3–18.0) |
| + | Medium-risk group | 4 (6.3) | 2 (5.7) | 2 (2.1) | 70.4 (4.8–723.1) | 9.3 (0.2–386.5) |
| + | High-risk group | 7 (11.1) | 0 (0) | 1 (1.0) | 98.8 (4.9–677.9) | — |
| Ever smokers | ||||||
| − | Low-risk groupb | 20 (16.0) | 21 (20.6) | 73 (30.5) | 1.0 | 1.0 |
| − | Medium-risk group | 46 (36.8) | 51 (50.0) | 118 (49.4) | 1.4 (0.8–2.6) | 1.6 (0.9–3.0) |
| − | High-risk group | 8 (6.4) | 20 (19.6) | 14 (5.9) | 2.1 (0.7–5.6) | 5.2 (2.2–12.5) |
| + | Low-risk groupb | 35 (28.0) | 6 (5.9) | 26 (10.9) | 4.7 (2.3–9.7) | 0.8 (0.3–2.3) |
| + | Medium-risk group | 11 (8.8) | 4 (3.9) | 7 (2.9) | 6.2 (2.1–18.7) | 3.4 (0.8–14.3) |
| + | High-risk group | 5 (4.0) | 0 (0) | 1 (0.4) | 15.8 (1.7–144.3) | — |
aORs were adjusted for age, sex, smoking and alcohol drinking.
bLow-risk group (used as the reference group): individuals carrying 0–1 genotype; medium-risk group: individuals carrying 2–3 genotypes; and high-risk group: individuals carrying 4–5 genotypes.
Joint effect of HPV16 seropositivity and combined E2F2 risk genotypes on risk of SCCOP and SCCOC stratified by smoking status
To explore the modifying effects of the combined genotypes of the five polymorphisms on the risk of SCCOP and SCCOC associated with HPV16 by smoking status, we used the individuals in the low-risk group with HPV16 seronegativity as the comparison group (Table 4). For never smokers, the risk of SCCOP increased among individuals in the medium-risk group with HPV16 seronegativity (OR 3.8; 95% CI 1.0–14.3); the high-risk group with HPV16 seronegativity (OR 11.0; 95% CI 1.9–63.2); the low-risk group with HPV16 seropositivity (OR 42.3; 95% CI 12.5–275.0); the medium-risk group with HPV16 seropositivity (OR 70.4; 95% CI 4.8–723.1); and the high-risk group with HPV16 seropositivity (OR 98.8; 95% CI 4.9–677.9). For ever smokers, the risk of SCCOP increased among individuals in the medium-risk group with HPV16 seronegativity (OR 1.4; 95% CI 0.8–2.6); the high-risk group with HPV16 seronegativity (OR 2.1; 95% CI 0.7–5.6); the low-risk group with HPV16 seropositivity (OR 4.7; 95% CI 2.3–9.7); the medium-risk group with HPV16 seropositivity (OR 6.2; 95% CI 2.1–18.7); and the high-risk group with HPV16 seropositivity (OR 15.8; 95% CI 1.7–144.3). However, for both never smokers and ever smokers, the significant modifying effects of combined E2F2 risk genotypes on the risk associated with HPV16 seropositivity were not observed for patients with SCCOC (Table 4).
As shown in Table 4, because the difference in the tumor’s HPV status between patients with SCCOP and SCCOC may result from different etiologies at these two different sites, the modifying effect of individual or combined E2F2 risk genotypes for five polymorphisms on the association between HPV16 seropositivity and the risk of SCCOP and SCCOC stratified by smoking status was more pronounced for SCCOP as opposed to SCCOC.
Genotype–phenotype correlation of E2F2 polymorphisms
To further characterize the potentially functional relevance of E2F2 promoter polymorphisms, using E2F2-rs6667575 as an example, we performed correlation analyses between tumor p16 expression and genotypes of E2F2-rs6667575 polymorphism among a subset of 62 SCCOP patients since these patients had tumor HPV16 status and relative p16 expression data clinically available. Among 62 SCCOP tissue specimens, we found that 50 patients had HPV16-positive tumors and 12 patients had HPV16-negative tumors. In HPV-positive patients, for E2F2-rs6667575 polymorphism, 26 were GG genotype and 24 were GA or AA genotypes. In HPV-negative patients, five were GG genotype and seven were GA or AA genotypes. As shown in Figure 1, in tumor HPV16-positive patients, the E2F2-rs6667575 GA/AA genotypes had a significantly higher p16 expression than E2F2-rs6667575 GG genotype (P = 0.036), while in HPV16-negative cases, the p16 expression was not significantly different between E2F2-rs6667575 GA/AA and GG genotypes (P = 0.169) (Figure 1).
Figure 1.
Comparison of p16 expression in tumor tissue specimens among SCCOP patients with different genotypes of E2F2-rs6667575 promoter polymorphism.
Discussion
We found that the five E2F2 promoter polymorphisms, individually or in combination, significantly modified the risk of HPV16-associated SCCOP. Moreover, our results suggested that the combined effect of HPV16 seropositivity and individual or combined E2F2 variants on the risk of SCCOP was higher in never smokers than in ever smokers, while the similar modifying effects were not found for the patients with SCCOC. To our knowledge, this is the first study of E2F2 gene/virus interaction and SCCOP risk.
Binding of E7 to phosphorylated RB leads to activation of E2F2, which induces cell-cycle progression and p16 expression (20) as shown in Figure 2. Elevated expression of the oncogene E7 is important for further progression of the disease and inactivation of cellular tumor suppressor protein Rb. Thus, it is biologically plausible that E2F2 promoter polymorphisms might affect E2F2 expression, which, in turn, could affect p16 expression level, thereby influencing cancer susceptibility. So far, no studies on functional relevance of these E2F2 promoter variants have been reported. Since expression of p16 is regulated by E2F2 through Rb-mediated pathways and these E2F2 polymorphisms are within the functional regions of the E2F2’s promoter, we speculated that these E2F2 polymorphisms may have potentially functional effect on expression levels of p16, leading to inter-individual differences in susceptibility to risk of HPV16-associated SCCOP. Indeed, in this study, we used the most significant E2F2-rs6667575 polymorphism as an example for functional characterization and found that the variant genotypes of this E2F2-rs6667575 polymorphism significantly modified expression of p16 in HPV16-positive patients but not in HPV16-negative cases in SCCOP tumor specimens. While the functional relevance of the E2F2-rs6667575 polymorphism has not yet been elucidated, our results might partially suggest a functional correlation between this polymorphism and p16 expression, which may provide preliminary evidence of biological plausibility for the observed association in this study. As shown in Figure 2, individuals with E2F2-rs6667575 GA/AA had significantly higher p16 expression than those with GG genotypes in HPV16-positive SCCOP patients only, indicating that in HPV-associated SCCOP patients, binding of HPV16 E7 to Rb might lead to release of transcription factor E2F2. Genetic variations, such as E2F2-rs6667575 polymorphism, may thus affect promotion of cell cycle progression and induction of p16 expression, thereby affecting susceptibility to SCCOP.
Figure 2.
The putative roles of E2F2-rs6667575 polymorphism in the p16INK4a/Rb/E2F2 pathway in HPV-positive cancers. Binding of HPV16) E7 oncoprotein to Rb, resulting in the release and activation of E2F transcription factors that induce cell cycle progression and p16 expression. E2F2-rs6667575 polymorphism might affect p16 expression and cell cycle control, leading to different susceptibility to cancer (e.g. SCCOP).
Previously, we found that E2F2 variants did not affect risk of squamous cell carcinoma of the head and neck, which is consistent with findings from previous studies on other cancers (15,21,22). Thus far, the only case–control study of E2F1/E2F2 polymorphisms and the risk of head and neck cancers found no significant association (15). However, that study did not take HPV and tumor sites into consideration for association of E2F1/E2F2 polymorphisms with risk of head and neck cancers. Our current study is the first to investigate the association of E2F2 SNPs with HPV16-associated SCCOP and SCCOC risk and the combined effects of E2F2 variant genotypes and smoking status on HPV16-associated SCCOP and SCCOC risk.
The finding of a synergistic effect of E2F2 polymorphisms on the risk of SCCOP associated with HPV16 seropositivity is consistent with the idea that HPVs and E2F2 may act synergistically through the RB pathway in the development of SCCOP. These five E2F2 polymorphisms are located in the E2F2 promoter region, which could destroy transcriptional regulation factors, leading to its altered transcription and expression (23). Alternatively, these polymorphisms likely are also in linkage disequilibrium with other tagSNPs that mark functional or disease-causing variants in the genome (24).
Smoking affects the risk of developing both SCCOP and SCCOC and can be a major confounder when evaluating other risk factors including genetic factors. To minimize this confounding effect on such association studies, stratified analysis by smoking status, including ever smokers and never smokers, should be considered. Such analyses, which may further help accurately evaluate these populations HPV infection, appears fundamental in SCCOP, while genetic predisposition may play a more relevant role for SCCOC. Indeed, in this study, after stratifying the participants by smoking status for each polymorphism, we found that the modifying effect of E2F2 variants on the risk of SCCOP associated with HPV16 seropositivity was higher in never smokers than in ever smokers, and the size of this modification differed between the subgroups by smoking status. Therefore, these five E2F2 polymorphisms may play a role in the development of SCCOP associated with HPV16 among never smokers in the general population. Thus, from these findings, we suggest that when assessing the modifying effects of E2F2 variants on the risk associated with HPV16 seropositivity, smoking status might also need to be taken into account.
We also found that the modifying effects of E2F2 variants and HPV16 seropositivity were pronounced in patients with SCCOP only but not in those with SCCOC. These results are in line with the notion that HPV infection is found more frequently in SCCOP than in SCCOC. Our finding also supports those of previous studies, in which most SCCOP were caused by HPV infection, whereas most SCCOC were caused by smoking (17,25–27). The finding that the modifying effects of E2F2 polymorphisms on the association between HPV16 seropositivity and the risk of SCCOP was higher in never smokers than ever smokers may further support a role for E2F2 polymorphisms in the development of SCCOP associated with HPV rather than SCCOC associated with non-HPVs. However, these findings need to be further investigated in studies with large sample sizes.
While our study had a relatively large sample size and minimized potential confounding factors, there were several limitations. On one hand, selection bias cannot be ruled out because this was a hospital-based case–control study, and the controls may not represent the same population as the patients. On the other hand, the stratified analyses included a limited number of individuals in each subgroup; thus, our results could be chance findings and should be confirmed in larger studies. Moreover, because our study included only non-Hispanic white participants, these results might not be generalizable to other ethnicities. Lastly, because HPV16 seropositivity indicates previous HPV exposure but is not specific to HPV16, the results might not reflect the tumor’s actual HPV16 status, leading to some misclassifications (false-negatives for cases). However, the use of serologic status allowed us to include a cancer-free control group.
Taken together, the modifying effect of E2F2 promoter variants on cancer risk was particularly pronounced in never smokers and never drinkers, and for SCCOP as opposed to SCCOC. Smoking could be a major confounding factor for evaluation of associations between HPV and genetic polymorphisms and risk of SCCOP and SCCOC, and stratified analysis by smoking is necessary for such association studies. However, for further analysis of gene/environment or gene/gene interactions and for more accurate estimates of SCCOP risk, future functional and molecular epidemiologic studies with larger sample sizes in different populations are needed. Since the RB/E2F2 pathway involves many genes that may interact with HPV oncoproteins, future studies should investigate the effects of HPV status on multiple genes in this pathway. Additionally, future studies are needed to clarify the mechanisms behind the interactions between HPV16 seropositivity and E2F2 variants in the development of SCCOP.
Funding
The University of Texas MD Anderson Cancer Center start-up funds to E.M.S., the National Institutes of Health Head and Neck Specialized Program of Research Excellence Career Development Award (P50CA097007 to E.M.S.), The University of Texas MD Anderson Cancer Center Institutional Research Grant to E.M.S., the National Institutes of Health (ES 011740 and CA131274 to Q.W.), the Clinician Investigator Award (K12 CA88084 to E.M.S.), the National Institutes of Health Cancer Center Support, The University of Texas MD Anderson Cancer Center (CA 16672) and National Institutes of Health grants (CA135679 to G.L., CA133099 to G.L., and CA186261-01A1).
Acknowledgements
The authors thank Margaret Lung, Kathryn L. Tipton, Liliana Mugartegui and Angeli Fairly for their help with participant recruitment; Li-EWang for laboratory management; John T. Schiller and Karen Adler-Storthz for their help with establishing the HPV serology methods; and Maude Veech, Diane Hackett and Jill Delsigne for scientific editing.
Conflict of Interest Statement: None declared.
Glossary
Abbreviations
- CI
confidence interval
- E2F2
E2F transcription factor 2
- HPV
human papillomavirus
- HPV16
human papillomavirus type 16
- OR
odds ratio
- SCCOP
squamous cell carcinoma of oropharynx
- SCCOC
squamous cell carcinoma of oral cavity
- SNP
single nucleotide polymorphisms
- RB
retinoblastoma
References
- 1. Safdari Y., et al. (2014) Recent advances in head and neck squamous cell carcinoma--a review. Clin. Biochem., 47, 1195–202. [DOI] [PubMed] [Google Scholar]
- 2. Lee G., et al. (2002) Characterization of novel cell lines established from three human oral squamous cell carcinomas. Int. J. Oncol., 20, 1151–1159. [PubMed] [Google Scholar]
- 3. Siegel R.L., et al. (2016) Cancer statistics, 2016. CA. Cancer J. Clin., 66, 7–30. [DOI] [PubMed] [Google Scholar]
- 4. Vokes E.E., et al. (1993) Head and neck cancer. N. Engl. J. Med., 328, 184–194. [DOI] [PubMed] [Google Scholar]
- 5. Gillison M.L. (2007) Current topics in the epidemiology of oral cavity and oropharyngeal cancers. Head Neck, 29, 779–792. [DOI] [PubMed] [Google Scholar]
- 6. Fakhry C., et al. (2006) Clinical implications of human papillomavirus in head and neck cancers. J. Clin. Oncol., 24, 2606–2611. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Chen X., et al. (2010) HPV seropositivity synergizes with MDM2 variants to increase risk of oral squamous cell carcinoma. Cancer Res., 70, 7199–208. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Polager S., et al. (2008) E2F - at the crossroads of life and death. Trends Cell Biol., 18, 528–535. [DOI] [PubMed] [Google Scholar]
- 9. Stransky N., et al. (2011) The mutational landscape of head and neck squamous cell carcinoma. Science, 333, 1157–1160. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10. Mountzios G., et al. (2014) The mutational spectrum of squamous-cell carcinoma of the head and neck: targetable genetic events and clinical impact. Ann. Oncol., 25, 1889–1900. [DOI] [PubMed] [Google Scholar]
- 11. Chen S.J., et al. (2015) Ultra-deep targeted sequencing of advanced oral squamous cell carcinoma identifies a mutation-based prognostic gene signature. Oncotarget, 6, 18066–18080. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Ku B.M., et al. (2016) The CDK4/6 inhibitor LY2835219 has potent activity in combination with mTOR inhibitor in head and neck squamous cell carcinoma. Oncotarget, 7, 14803–14813. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Münger K., et al. (2002) Human papillomavirus immortalization and transformation functions. Virus Res., 89, 213–228. [DOI] [PubMed] [Google Scholar]
- 14. Rothenberg S.M., et al. (2012) The molecular pathogenesis of head and neck squamous cell carcinoma. J. Clin. Invest., 122, 1951–1957. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15. Lu M., et al. (2012) Combined effects of E2F1 and E2F2 polymorphisms on risk and early onset of squamous cell carcinoma of the head and neck. Mol. Carcinog., 51 (suppl. 1), E132–E141. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Kirnbauer R., et al. (1994) A virus-like particle enzyme-linked immunosorbent assay detects serum antibodies in a majority of women infected with human papillomavirus type 16. J. Natl. Cancer Inst., 86, 494–499. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Dahlstrom K.R., et al. (2003) Human papillomavirus type 16 infection and squamous cell carcinoma of the head and neck in never-smokers: a matched pair analysis. Clin. Cancer Res., 9, 2620–2626. [PubMed] [Google Scholar]
- 18. Alcantara F.F., et al. (1999) Heparin in plasma samples causes nonspecific binding to histones on Western blots. J. Immunol. Methods, 226, 11–18. [DOI] [PubMed] [Google Scholar]
- 19. Benjamini Y., et al. (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. Roy. Stat. Soc. Ser. B, 57, 289–300. [Google Scholar]
- 20. Wittekindt C., et al. (2012) Basics of tumor development and importance of human papilloma virus (HPV) for head and neck cancer. GMS Curr. Top. Otorhinolaryngol. Head Neck Surg., 11, Doc09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21. Cunningham J.M., et al. (2009) Cell cycle genes and ovarian cancer susceptibility: a tagSNP analysis. Br. J. Cancer, 101, 1461–1468. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22. Justenhoven C., et al. (2009) Polymorphic loci of E2F2, CCND1 and CCND3 are associated with HER2 status of breast tumors. Int. J. Cancer, 124, 2077–2081. [DOI] [PubMed] [Google Scholar]
- 23. Sun T., et al. (2007) A six-nucleotide insertion-deletion polymorphism in the CASP8 promoter is associated with susceptibility to multiple cancers. Nat. Genet., 39, 605–613. [DOI] [PubMed] [Google Scholar]
- 24. Wang M., et al. (2010) A novel XPF -357A>C polymorphism predicts risk and recurrence of bladder cancer. Oncogene, 29, 1920–1928. [DOI] [PubMed] [Google Scholar]
- 25. Gillison M.L., et al. (2000) Evidence for a causal association between human papillomavirus and a subset of head and neck cancers. J. Natl. Cancer Inst., 92, 709–720. [DOI] [PubMed] [Google Scholar]
- 26. Lingen M.W., et al. (2012) Validation of LOH profiles for assessing oral cancer risk. Cancer Prev. Res. (Phila)., 5, 1075–1077. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27. Bussu F., et al. (2013) HPV infection in squamous cell carcinomas arising from different mucosal sites of the head and neck region. Is p16 immunohistochemistry a reliable surrogate marker? Br. J. Cancer, 108, 1157–1162. [DOI] [PMC free article] [PubMed] [Google Scholar]