Purpose: In previous studies, we have shown that the T allele of a specific single-nucleotide polymorphism (SNP) in the Gαs gene (T393C) correlates with increased Gαs expression and hence apoptosis. The T allele was associated with a favorable outcome in a variety of human cancers, e.g., carcinoma of the urinary bladder, kidney, and colorectum.

Experimental Design: The prognostic value of the T393C SNP was evaluated in an unselected series of patients treated with curative intent for oropharyngeal and hypopharyngeal squamous cell carcinomas, including all tumor stages with different therapeutic regimens. Genotype analysis was done using DNA from paraffin-embedded tissue samples from 202 patients (162 men, 40 women) with a median follow-up of 38 months (1-133 months). The various genotypes were correlated with relapse-free and overall survival.

Results:GNAS1 393C homozygous patients displayed a higher risk for disease progression than T393 homozygous patients (hazard ratio CC versus TT, 1.9; 95% confidence interval, 1.1-3.2; P = 0.019). The same genotype effect was observed for overall survival with CC genotypes at higher risk for death compared with TT genotypes (hazard ratio, 1.7; 95% confidence interval, 1.1-2.9; P = 0.015). Multivariate analysis showed that, besides American Joint Committee on Cancer stage, tumor localization, and gender, the T393C polymorphism was an independent prognostic factor for disease progression and death.

Conclusion: The T393C SNP could be considered as a genetic marker to predict the clinical course of patients suffering from oropharyngeal and hypopharyngeal cancer.

Head and neck cancer comprises ∼6% to 7% of all human malignancies (1). Among head and neck cancer, squamous cell carcinoma (SCC) is the most common histologic subgroup, overall (80-85% of oropharyngeal and 97% of hypopharyngeal carcinomas; ref. 2) representing the sixth most frequent cancer worldwide (3). Annually, about 400,000 head and neck SCC (HNSCC) are diagnosed worldwide, most of which are locally advanced at presentation. Some tumor sites, like the hypopharynx (4), imply a particularly bad prognosis among HNSCC due to the lack of obvious symptoms in the early disease stage. Surgery and/or radiochemotherapy are the mainstay of locoregional treatment in these patients (5). For previously untreated advanced stage resectable cancers in the oral cavity, oropharynx, and hypopharynx, the standard therapy regimens of surgery and postoperative radiation therapy result in a 5-year survival rate of only 40% to 50% (6). Because of an already extensive locoregional involvement upon admission, one third of patients with HNSCC have nonresectable tumors (7). Nonresectable advanced tumors are currently treated by combined radiochemotherapy with a 5-year overall survival rate of almost 30% (8). Chemotherapy alone has a modest favorable effect upon outcome; patients with recurrent or metastatic disease receiving chemotherapy show a median survival ranging from 6 to 9 months (9). Poor distant disease control rates in this particular patient group have stimulated the search for more effective systemic cytotoxic therapies, optimized prevention and early detection strategies, as well as the need for more refined prognostic markers. Markers predicting the clinical course and/or survival could have an important influence upon patient management, clinical and therapeutic decision making, as well as the maintenance of the patients' quality of life.

Tumor staging [tumor-node-metastasis stage and American Joint Committee on Cancer (AJCC) stage] and tumor location are still the most significant prognostic factors in HNSCC.

It is possible to predict survival probability by analyzing histologic data from newly diagnosed patients with HNSCC using the Cox regression model; however, the problem with this method is that these data neglect certain genetic factors that should otherwise be taken into consideration (10). Consequently, an enormous amount of data is produced on prognostic factors of outcomes for cancer (11) pointing at HPV-16 status (12), tumor suppressor genes, such as p53 (11, 1315), epidermal growth factor receptor, or angiogenetic markers, like vascular endothelial growth factor receptor (1416). Cytokinetic variables and proliferation activity turned out to be especially interesting predictors (17). Hence, it was shown that primary risk factors, such as advanced clinical stage and increased proliferative activity, correlate positively with increased mitotic activity and decreased apoptotic rate (17, 18). However, until now, there has been no reliable prognostic marker that established itself in daily clinical routine, and it seems rather likely that, finally, the combination of various markers could allow a much better individualized prognostic prediction on the clinical course.

Our group has recently shown that genotypes of the single-nucleotide polymorphism (SNP) T393C in the gene GNAS1, encoding the ubiquitously expressed Gαs subunit of heterotrimeric G proteins, predict the outcome of patients with solid tumors, like urothelial carcinoma (19), sporadic colorectal cancer (20), and clear cell renal carcinoma (21), as well as patients with chronic lymphatic leukemia (22).

Data from in vitro experiments suggest that increased expression of Gαs enhances apoptosis (2325). Patients with the TT genotype showed a prolonged survival compared with patients carrying either TC or CC genotypes. In TT genotypes, Gαs mRNA expression was found to be increased not only in bladder carcinoma tissue (19) but also in human heart and fat cell specimens (26), which may result from altered mRNA stability associated with different genotypes (20). These results prompted us to further investigate whether the T393C polymorphism of the GNAS1 gene may predict the clinical course of HNSCC as well.

The study was strictly done according to the Declaration of Helsinki and approved by the local ethics committee of the University Hospital of Essen.

Patients. The study group consisted of 202 Caucasian patients with SCC of the oropharyngeal or hypopharynx diagnosed and treated in the period 1995 to 2001 at the West German Cancer Center Essen. Relevant clinicopathologic data (AJCC stage, tumor-node-metastasis stage, tumor site, histologic grading, therapeutic regimens, 5-y follow-up, relapse, cause of death) were extracted from the patients' files or collected by telephone interviews with the patients, their relatives, or doctors. Clinical cases which had been defined as clearly palliative (according to the statement of our interdisciplinary tumor board) were excluded.

DNA genotyping. DNA was extracted from paraffin-embedded tissue samples using a commercially available kit (QIAAmp, Qiagen). PCR for the T393C polymorphism was done using the following primers: forward primer 5′-CTCCTAACTGACATGGTGCAA-3′ and reverse primer 5′-TAAGGCCACACAAGTCGGGGT-3′.

After denaturation at 94°C, 35 cycles of DNA amplification were done using Taq PCR Mastermix (Eppendorf) at 94°C for 45 s, 58°C for 40 s, and 72°C for 45 s. The 345-bp PCR products were digested using the restriction enzyme FokI and analyzed on a 2% agarose gel. The unrestricted products (345 bp) represented the TT genotype; the completely restricted products (259 and 86 bp) represented the CC genotype.

Statistical analysis and presentation of data. Disease-free and overall survival were the most important clinical outcome analyzed in the study. Kaplan-Meier plots and the log-rank test for trend were used to retrospectively evaluate the relationship between AJCC stage, tumor localization, T393C genotypes, and outcome from the date of primary diagnosis until relapse or death. Factors commonly accepted as predictors of clinical outcome were tested in univariate analysis. Multivariate models of clinical follow-up were established using clinical and pathologic variables, which were associated with outcome if P value was <0.1 in univariate analysis. A backward stepwise Cox proportional hazard model was applied to calculate hazard ratios, 95% confidence intervals, and P values (27). Contingency tables and the Pearson's χ2 test were used to compare categorical variables using T393C genotypes, as indicated. Because the T393C polymorphism displayed a gene-dose effect (1922), linear ANOVA was used for comparison of parametric continuous variables (e.g., age) and the Kruskal-Wallis test for nonparametric continuous variables. Differences were regarded significant at P value of <0.05. All statistical analysis was done using SPSS 11.0 (SPSS) or Graphpad Prism 4.0 (Graphpad Software). Continuous variables are given as means ± SD.

T393C genotype distributions and correlation with clinical data. Demographic characteristics, genotype, and clinical data at the time of first diagnosis for the whole case group are displayed in Table 1. The mean (±SD) age of the 202 patients (162 males, 40 females) was 58.5 (±10.1) years, and median follow-up time to first therapy was 38 months (range, 1-133 months). The frequency of the C allele in the patient group was 0.54, and this distribution was compatible with the Hardy-Weinberg equilibrium.

Table 1.

T393C genotype distribution with regard to tumor site, demographic characteristics, primary therapy, tumor stage (AJCC), and histologic grading in 202 patients with oropharyngeal/hypopharyngeal SCC

TotalTTTCCCP
n (%), total 202 48 (23.8) 89 (44.1) 65 (32.2)  
Hypopharyngeal SCC 58 13 (22.4) 21 (36.2) 24 (41.4)  
Oropharyngeal SCC 127 31 (24.4) 61 (48.0) 35 (27.6)  
Overlapping (oropharynx-hypopharynx) 17 4 (23.5) 7 (41.2) 6 (35.3) 0.436 
Mean age ± SD (y) 58.5 ± 10.1 58.0 ± 10.1 59.5 ± 10.7 57.3 ± 9.4 0.641 
Median follow up, mo (range) 38 (1-133) 57 (8-118) 40 (6-133) 24 (1-108) 0.017* 
Gender (male/female) 162/40 41/7 66/23 55/10 0.160 
Smoking, n (%) 177 (87.6) 44 (24.9) 73 (41.2) 60 (33.9) 0.143 
Pack years 47.1 ± 23.6 44.2 ± 24.5 48.1 ± 22.0 47.8 ± 25.0 0.561 
Primary therapy oropharyngeal SCC      
    Surgery ± RT 49 10 (20.4) 28 (57.1) 11 (22.4)  
    Primary RT 74 19 (25.7) 32 (43.2) 23 (31.1)  
    Others§ 2 (50.0) 1 (25.0) 1 (25.0) 0.895 
Primary therapy hypopharyngeal SCC      
    Surgery ± RT 3 (33.3) 4 (44.4) 2 (22.2)  
    Primary RT 49 10 (20.4) 17 (34.7) 22 (44.9) 0.210 
AJCC stage      
    I 3 (42.9) 4 (57.1) 0 (0)  
    II 15 4 (26.7) 8 (53.3) 3 (20.0)  
    III 21 5 (23.8) 8 (38.1) 8 (38.1)  
    IVA 130 33 (25.4) 54 (41.5) 43 (33.1)  
    IVB 26 3 (11.5) 14 (53.8) 9 (34.6)  
    IVC 0 (0) 1 (33.3) 2 (66.7) 0.030 
Grade      
    1 3 (42.9) 2 (28.6) 2 (28.6)  
    2 131 32 (24.4) 50 (38.2) 49 (37.4)  
    3-4 61 11 (18.0) 37 (60.7) 13 (21.3) 0.727 
TotalTTTCCCP
n (%), total 202 48 (23.8) 89 (44.1) 65 (32.2)  
Hypopharyngeal SCC 58 13 (22.4) 21 (36.2) 24 (41.4)  
Oropharyngeal SCC 127 31 (24.4) 61 (48.0) 35 (27.6)  
Overlapping (oropharynx-hypopharynx) 17 4 (23.5) 7 (41.2) 6 (35.3) 0.436 
Mean age ± SD (y) 58.5 ± 10.1 58.0 ± 10.1 59.5 ± 10.7 57.3 ± 9.4 0.641 
Median follow up, mo (range) 38 (1-133) 57 (8-118) 40 (6-133) 24 (1-108) 0.017* 
Gender (male/female) 162/40 41/7 66/23 55/10 0.160 
Smoking, n (%) 177 (87.6) 44 (24.9) 73 (41.2) 60 (33.9) 0.143 
Pack years 47.1 ± 23.6 44.2 ± 24.5 48.1 ± 22.0 47.8 ± 25.0 0.561 
Primary therapy oropharyngeal SCC      
    Surgery ± RT 49 10 (20.4) 28 (57.1) 11 (22.4)  
    Primary RT 74 19 (25.7) 32 (43.2) 23 (31.1)  
    Others§ 2 (50.0) 1 (25.0) 1 (25.0) 0.895 
Primary therapy hypopharyngeal SCC      
    Surgery ± RT 3 (33.3) 4 (44.4) 2 (22.2)  
    Primary RT 49 10 (20.4) 17 (34.7) 22 (44.9) 0.210 
AJCC stage      
    I 3 (42.9) 4 (57.1) 0 (0)  
    II 15 4 (26.7) 8 (53.3) 3 (20.0)  
    III 21 5 (23.8) 8 (38.1) 8 (38.1)  
    IVA 130 33 (25.4) 54 (41.5) 43 (33.1)  
    IVB 26 3 (11.5) 14 (53.8) 9 (34.6)  
    IVC 0 (0) 1 (33.3) 2 (66.7) 0.030 
Grade      
    1 3 (42.9) 2 (28.6) 2 (28.6)  
    2 131 32 (24.4) 50 (38.2) 49 (37.4)  
    3-4 61 11 (18.0) 37 (60.7) 13 (21.3) 0.727 

Abbreviations: RT, radiotherapy; RCT, radiochemotherapy; R(C)T, radiotherapy or radiochemotherapy.

*

Kruskal-Wallis test.

Surgery ± RT = Surgery or Surgery + R(C)T.

Primary RT = RCT, RT, RCT + salvage surgery, RT + salvage surgery.

§

Others = R(C)T + surgery (DÖSAG scheme), palliative CT.

Genotypes and allele frequencies of the 202 patients were comparable with those of healthy White blood donors; details of this control group have been published previously (26), thus arguing against an association of T393C genotypes with an increased susceptibility for oropharyngeal/hypopharyngeal SCC.

We found no genotype association with age at time of first diagnosis. Moreover, genotypes were not significantly associated with different tumor localization and primary therapy (Table 1). However, genotypes were significantly associated with the AJCC stage at time of first diagnosis (Table 1), and the CC genotype was significantly associated with higher AJCC stages (III-IVC), whereas the TT genotype carriers were more frequently found in the lower stages (I-II). This observation became even more apparent when AJCC stages were grouped by T393C alleles (Table 2). Here, a P value of 0.0215 could be calculated for an association of T393C alleles and AJCC stages.

Table 2.

T allele and C allele frequencies within the group of 202 patients with regard to tumor stages according to AJCC

AJCC stagenTCfT (%)fC (%)
10 71.4 28.6 
II 15 16 14 53.3 46.7 
III 21 18 24 42.9 57.1 
IV A 130 120 140 46.2 53.8 
IV B 26 20 32 38.5 61.5 
IV C 16.7 83.3 
AJCC stagenTCfT (%)fC (%)
10 71.4 28.6 
II 15 16 14 53.3 46.7 
III 21 18 24 42.9 57.1 
IV A 130 120 140 46.2 53.8 
IV B 26 20 32 38.5 61.5 
IV C 16.7 83.3 

Abbreviations: fC, frequency of the C allele; fT, frequency of the T allele.

T393C genotype and clinical follow up. During follow up, 100 patients (48.8%) experienced a relapse of the disease and 133 (64.6%) died. To confirm that our sample was representative for patients with oropharyngeal/hypopharyngeal SCC, we calculated Kaplan-Meier curves for relapse-free and overall survival, depending on AJCC stage and localization of the tumor. Relapse-free survival and overall survival were significantly dependent upon AJCC stage and tumor localization (Fig. 1A-D), and computed values were compatible with published data (4, 8, 9, 28).

Fig. 1.

A-D, relapse-free survival and overall survival Kaplan-Meier curves for 202 patients with oropharyngeal/hypopharyngeal SCC based on the AJCC stage and localization of the tumor.

Fig. 1.

A-D, relapse-free survival and overall survival Kaplan-Meier curves for 202 patients with oropharyngeal/hypopharyngeal SCC based on the AJCC stage and localization of the tumor.

Close modal

Additionally, we observed a significant genotype-dependent treatment-free interval with an apparent gene-dose effect (Fig. 2A; P = 0.014): GNAS1 393C homozygous patients displayed a higher risk for disease progression than T393 homozygous patients (CC versus TT: hazard ratio 1.9, 95% confidence interval 1.1-3.2, P = 0.019) with TC genotypes showing intermediate values. Proportions of 5-year progression-free intervals were 60.0% for TT, 52.4% for TC, and 40.7% for CC genotypes, respectively, suggesting a gene-dose effect (Fig. 2A).

Fig. 2.

Relapse-free survival (A) and overall survival (B) Kaplan-Meier curves for 202 patients with oropharyngeal/hypopharyngeal SCC based on T393C genotypes.

Fig. 2.

Relapse-free survival (A) and overall survival (B) Kaplan-Meier curves for 202 patients with oropharyngeal/hypopharyngeal SCC based on T393C genotypes.

Close modal

The same genotype effect was found for overall survival (Fig. 2B). CC genotypes were at higher risk for death compared with TT genotypes (hazard ratio 1.7, 95% confidence interval 1.1-2.9, P = 0.015), and 5-year survival proportions were 51.3% for TT, 44.7% for TC, and 36.8% for CC. As we observed a significant association of GNAS1 genotypes with the AJCC stage at time of first diagnosis, we investigated whether genotypes of the T393C polymorphism showed an interaction with disease stage or whether they were independently associated with disease progression and overall survival. Cox proportional hazard models for factors being associated with outcome in univariate analysis proved that the T393C polymorphism was indeed an independent risk factor for progression (Table 3) and death (Table 4). Hazard ratios for patients with CC genotype ranged from 1.6 to 1.8 for relapse-free survival and overall survival compared with homozygous 393T allele carriers.

Table 3.

Multivariate Cox proportional hazard model for progression in 202 patients with oropharyngeal/hypopharyngeal SCC

VariablenHazard ratio95% confidence intervalP
AJCC     
    I-II 22 1*   
    III 21 0.9 0.3-2.7 0.824 
    IVA 130 1.8 0.8-4.3 0.163 
    IVB + IVC 29 3.2 1.3-8.1 0.015 
Tumor location     
    Oropharynx 127 1*   
    Hypopharynx 58 1.8 1.1-2.8 0.010 
    Overlapping 17 1.7 0.9-3.3 0.123 
Gender     
    Female 40 1*   
    Male 162 2.0 1.1-3.9 0.019 
T393C     
    TT 48 1*   
    TC 89 1.3 0.8-2.3 0.310 
    CC 65 1.8 1.1-3.0 0.049 
VariablenHazard ratio95% confidence intervalP
AJCC     
    I-II 22 1*   
    III 21 0.9 0.3-2.7 0.824 
    IVA 130 1.8 0.8-4.3 0.163 
    IVB + IVC 29 3.2 1.3-8.1 0.015 
Tumor location     
    Oropharynx 127 1*   
    Hypopharynx 58 1.8 1.1-2.8 0.010 
    Overlapping 17 1.7 0.9-3.3 0.123 
Gender     
    Female 40 1*   
    Male 162 2.0 1.1-3.9 0.019 
T393C     
    TT 48 1*   
    TC 89 1.3 0.8-2.3 0.310 
    CC 65 1.8 1.1-3.0 0.049 

NOTE: Variables with significant results from the univariate model were included in the analysis.

*

Reference.

Table 4.

Multivariate Cox proportional hazard model for tumor-related death in 202 patients with oropharyngeal/hypopharyngeal SCC

VariablenHazard ratio95% confidence intervalP
AJCC     
    I-II 22 1*   
    III 21 0.9 0.4-2.4 0.839 
    IVA 130 2.1 1.1-4.2 0.042 
    IVB + IVC 29 2.7 1.2-6.1 0.014 
Tumor location     
    Oropharynx 127 1*   
    Hypopharynx 58 1.9 1.3-2.8 0.002 
    Overlapping 17 1.6 0.9-2.9 0.116 
Gender     
    Female 40 1*   
    Male 162 2.2 1.3-3.7 0.003 
T393C     
    TT 48 1*   
    TC 89 1.2 0.7-1.9 0.452 
    CC 65 1.6 1.1-2.6 0.046 
VariablenHazard ratio95% confidence intervalP
AJCC     
    I-II 22 1*   
    III 21 0.9 0.4-2.4 0.839 
    IVA 130 2.1 1.1-4.2 0.042 
    IVB + IVC 29 2.7 1.2-6.1 0.014 
Tumor location     
    Oropharynx 127 1*   
    Hypopharynx 58 1.9 1.3-2.8 0.002 
    Overlapping 17 1.6 0.9-2.9 0.116 
Gender     
    Female 40 1*   
    Male 162 2.2 1.3-3.7 0.003 
T393C     
    TT 48 1*   
    TC 89 1.2 0.7-1.9 0.452 
    CC 65 1.6 1.1-2.6 0.046 

NOTE: Variables with significant results from the univariate model were included in the analysis.

*

Reference.

We have recently shown that different genotypes of the T393C SNP in the gene GNAS1 are significantly associated with the clinical outcome of patients with carcinomas of the urinary bladder, kidney, and colorectum (1921). Patients carrying the TT genotype showed a prolonged survival compared with patients carrying either TC or CC genotypes. This occurrence was suggested to be mediated by an increase in the apoptotic rate in TT genotypes, potentially due to an enhanced Gαs mRNA expression compared with TC and CC genotypes which exhibit altered mRNA stability (20).

The results of the present study also show a significant correlation of the genotypes of the T393C SNP with the clinical outcome in a series of 202 HNSCC. The homozygous CC genotype was significantly associated with a decreased overall survival, as well as relapse-free survival, when compared with 393T allele carriers. In multivariate analysis, including AJCC stage, tumor location, and gender as covariates, the T393C SNP was shown to be an independent prognostic factor for clinical outcome. As in other carcinomas (1921), the T393C SNP per se does neither represent a risk factor for the development of HNSCC nor it is related to the most prominent etiologic risk factor, namely tobacco smoking. The C allele frequency was, however, significantly related to the HNSCC tumor stage at the time of diagnosis and thus associated with an aggressive tumor growth (Table 2).

When detected early, the 5-year survival rate of HNSCC is 75%; however, when the majority of patients initially diagnosed with metastatic disease are included, the 5-year survival rate decreases to ∼35% (13). Genotyping of the T393C SNP may thus be proposed as a tool to further select people with an increased risk to develop HNSCC (tobacco and/or alcohol abusers) who would benefit from shorter screening intervals to detect HNSCC at an earlier disease stage. Additionally, genotyping of the T393C SNP offers the possibility to identify patients with lower risk of recurrence who would benefit from resection alone followed by reconstructive surgery and those who require additional treatment, such as radiotherapy.

Due to the fact that the outcome of HNSCC is still very unfavorable, much effort is devoted to research on new multimodal treatment strategies (610, 28) and also to the detection of new prognostic biomarkers (4, 11, 1318, 2931).

The GNAS1 TT genotype is associated with increased Gαs mRNA expression in different tissues (20), which led us to hypothesize that the T393C exchange itself could have an effect upon mRNA stability. Indeed, we found that the T393C polymorphism influences GNAS mRNA folding (20), which could have a major effect upon mRNA stability and Gαs expression. Determinants of mRNA stability have been described in the coding region of some other genes (3234). Different in vitro experiments suggest that increased expression of Gαs is associated with enhanced apoptosis (2325). The second messenger cyclic AMP, which is generated subsequently to the activation of Gαs, seems to play a major role in this proapoptotic process. Cyclic AMP can augment or suppress extracellular signal-regulated kinase activity, depending on the cell type. As an example, Raf-1 is inhibited by protein kinase A, which is one of the downstream effectors of cyclic AMP (35). A recent report has shown that cyclic AMP blocks Raf-1 activity in different cell types, resulting in suppression of the oncogenic activity of Raf-1 (36). Subsequently, Raf-1 inhibition by antisense nucleotide treatment induced potent antiproliferative effects in tumor cell lines. In addition, depletion of Raf-1 by treatment with ansamycins induced growth inhibition and apoptosis of tumor cells (37, 38). This process could be of particular importance in TT genotypes, in which Gαs expression is increased. There are already the first drugs, such as the ansamycin-based heat shock protein 90 inhibitor 17-AAG or the novel dimeric ansamycin EC-5 in clinical studies. These drugs aim for a down-regulation of c-Raf-1 and, thereafter, the resulting antitumor activity in HNSCC (39). It would, therefore, be of interest to investigate whether patients with specific T393C genotypes would especially benefit from such novel anticancer drugs due to synergistic or complementary effects, which could substantially contribute to increase efficacy and safety of such new drugs.

In summary, it can be stated that the genotypes of the T393C SNP of the G-protein gene may represent a promising “tumor host”–derived marker for the clinical management of patients with HNSCC. Although the genotypes of this SNP are themselves not associated with a particular risk for the development of HNSCC, carriers of the C allele with known risk factors (tobacco and/or alcohol abuse) may benefit from regular screening for HNSCC.

Grant support: IFORES program of University of Duisburg/Essen, Germany.

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.

We thank Karl Worm (Institute of Pathology and Neuropathology) for his experienced assistance with the DNA extraction and Crista Collins for revising the manuscript.

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