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Germline variation in DNA damage response may explain variable treatment outcomes from squamous cell carcinoma of the head and neck (SCCHN). Grouping patients according to stage and radiation treatment, we compared SCCHN survival according to ERCC2 A35931C (Lys751Gln, rs13181) and CCND1 G870A (Pro241Pro, rs9344) genotypes.
Recruiting a hospital-based SCCHN case series (all white, 24.7% female, mean age 58.4 years), this treatment outcome cohort study genotyped n=275 stage III-IV cases initially treated with radiation (with or without chemotherapy) and n=80 stage III-IV and n=130 stage I-II cases initially treated without radiation or chemotherapy and used Kaplan-Meier and Cox regression analysis to compare genotype groups according to overall, disease-specific, progression-free, and recurrence-free survival.
ERCC2-35931 AA predicted worse survival in stage III-IV treated with radiation (multiply adjusted hazard ratio (HR) 1.66, 95% confidence interval (CI) 1.15-2.40; HR over the first three follow-up years 1.92, 95% CI 1.28-2.88) and better survival in stage III-IV not treated with radiation (HR 0.26, 95% CI 0.11-0.62). Unassociated with survival in stage III-IV treated with radiation (HR 1.00, 95% CI 0.67-1.51), CCND1-870 GG predicted better survival in stage III-IV not treated with radiation (HR 0.14, 95% CI 0.04-0.50). Survival in stage I-II did not depend on ERCC2 A35931C or CCND1 G870A genotype.
Promoting tumor progression in untreated patients, germline differences in DNA repair or cell cycle control may improve treatment outcome in patients treated with DNA damaging agents.
ERCC2 A35931C may help distinguish advanced stage SCCHN with better outcomes from radiation treatment.
The American Cancer Society expected nearly 50,000 oral cavity, pharynx, and larynx cancer cases in 2010, representing 3.2% of all cancer expected in the United States (1). During 2001-2007, 61% of oral cavity and pharynx cancer and 25% of larynx cancer presented at stages (regional or distant) associated with less than 60% 5-year relative survival (2). Primary treatment for these later stage cancers generally includes surgery and/or radiation, sometimes combined with chemotherapy (3). The less than desired results from these standard treatments motivate the search for predictors of treatment outcome. Based on the notion that tumor cells, as a result of germline genetic differences, may exhibit a different therapeutic response to the DNA damage caused by radiation and chemotherapy, one line of research aims to differentiate disease outcomes according to variation in DNA repair and cell cycle control genes.
In a hospital-based case series containing 485 white patients with squamous cell carcinoma of the head and neck (SCCHN), we examined survival outcomes in relation to stage, treatment, and two polymorphisms, ERCC2 A35931C (Lys751Gln, rs13181) and CCND1 G870A (Pro241Pro, rs9344). ERCC2 A35931C located in exon 23 of ERCC2, a gene that codes for a DNA helicase involved in nucleotide excision repair, produces an amino acid change (Lys→Gln) associated with DNA repair phenotype differences (4-6), with ERCC2 35931C generally associated with lower ERCC2 mRNA levels (6), lower DNA repair efficiency (7), and higher DNA adduct levels (7). CCND1, the gene for a cyclin (cyclin D1) that controls the G1/S cell cycle transition, contains the exon 4 synonymous G870A (Pro→Pro) polymorphism. The CCND1 870A variant promotes alternative splicing of the CCND1 transcript, producing a truncated protein (cyclin D1b) missing exon 5 (8-10). Cyclin D1b lacks two regulatory motifs, a PEST sequence and threonine 286, associated with protein degradation and nuclear export, respectively (10). In addition, cyclin D1b may possess intrinsic cancer-promoting properties (10). Current interest in the survival effects of these two polymorphisms includes not only cancer of the head and neck (11-17), but also cancers at various other sites (18-24).
We used a cohort study design to test genetic associations with SCCHN treatment outcome. Over a seven year period (February 14, 2000 – February 14, 2008), the University Ear Nose and Throat Specialists practice at the University of Pittsburgh Medical Center consented n=686 20-81 year-old white patients within one year of the diagnosis of a biopsy-verified primary squamous cell carcinoma of the head and neck, used an interviewer administered and standardized questionnaire to collect risk factor information (25), and obtained blood for genotyping. Every case signed an informed consent (IRB#981041) approved by the University of Pittsburgh Institutional Review Board. This case series constituted approximately one-half of all 20-81 year-old white persons treated for a primary SCCHN by the University Ear Nose and Throat Specialists practice. Our analysis excluded 201 cases for the following eight reasons, 1) DNA unavailable for analysis (n=24), 2) ERCC2 and/or CCND1 genotype assay failure (n=21), 3) outcome information unavailable (n=70), 4) enrollment after second or later primary head and neck cancer (n=24), 5) visceral metastasis at diagnosis (n=3), 6) unknown stage at diagnosis (n=6), 7) chemotherapy as primary treatment without radiation therapy (n=6), and 8) radiation (with or without surgery) as primary treatment for stage I or II disease (n=47). Compared with cases used in the current study (n=485), excluded cases (n=201) more often enrolled between 2006 and 2008 and less often enrolled between 2003 and 2005 (p=0.01, Supplemental Table 1). Otherwise, included and excluded cases were similar with respect to sex, age at diagnosis, education, cigarette smoking and alcohol use history, and ERCC2 A35931C and CCND1 G870A genotypes (p>0.15, Supplemental Table 1).
We used Gentra Systems Inc. (Minneapolis, MN) kits to isolate DNA from whole blood, a Thermo Scientific Nanodrop 1000 full-spectrum UV/Vis spectrophotometer to evaluate DNA quantity and quality, and commercial TaqMan® allele discrimination assays run on the ABI 7700 Sequence Detection System (Applied Biosystems, Foster City, CA) to determine ERCC2 A35931C (rs13181) and CCND1 G870A (rs9344) genotypes. Each amplification reaction included negative assay controls and positive control samples with known genotype confirmed by sequencing. Two laboratory technicians independently interpreted each assay. Assays of blind duplicate samples and repeated analysis of a 10% sample set produced completely concordant genotype results.
Using inpatient and outpatient medical records, the Social Security Death Index, and a previously described computer system to control data quality (26), a dedicated and certified tumor registrar abstracted and recorded anatomic subsite, TNM stage, primary treatment, and clinical outcomes. Clinical outcome variables ascertained, classified, and dated included death, death due to head and neck cancer, treatments for head and neck cancer recurrence, progression, or metastasis, and treatments for new primary SCCHN. Recording disease activity (no evidence of disease – NED vs. alive with disease – AOD), the tumor registrar also maintained a catalog of University Ear Nose and Throat Specialists outpatient visits.
Analyses considered one primary endpoint (overall survival) and three secondary endpoints (disease-specific survival, progression-free survival, and recurrence-free survival). The overall survival endpoint calculated survival as time between the treatment start date (date of definitive surgery or date chemotherapy or radiation therapy started for cases treated without surgery) and date of death. The disease-specific survival endpoint calculated survival as time between the treatment start date and date of death due to head and neck cancer. The progression-free and recurrence-free survival endpoints calculated survival as time between the treatment start date and the earliest 1) date of first AOD visit occurring after a NED visit, 2) date of first treatment for head and neck cancer recurrence, progression, or metastasis, 3) date of first treatment for new primary SCCHN, or 4) date of death due to head and neck cancer. For cases not experiencing an endpoint, overall survival analyses censored follow-up on the date of last alive contact. Disease-specific, progression-free, and recurrence-free survival analyses censored follow-up on either the date of death due to known causes unrelated to head and neck cancer or otherwise on the date of last alive contact. Analyses for recurrence-free survival included only cases with a post-treatment NED visit.
Analyses used sex, age on treatment start date, primary head and neck tumor subsite, AJCC diagnostic stage group (27), and treatment category (no radiation or chemotherapy, radiation therapy combined with platinum chemotherapy, and radiation therapy with or without non-platinum chemotherapy) to define analytic cohorts and to adjust genotype associations with clinical outcome. Analyses grouped subsites into four categories, 1) lip and oral cavity, 2) oropharynx (including soft palate, base of tongue, vallecula, and tonsil), 3) larynx (ICD-O-3 C32.0 through C32.9), and 4) other (including nasal cavity and sinus, nasopharynx, hypopharynx, and undetermined primary subsite). A previous publication defines cigarette smoking and alcohol use history and describes procedures used to calculate lifetime cigarette and alcohol dose exposures (25).
We based inferences on three cohorts, one cohort of primary interest (a stage III-IV cohort exposed to radiation, with or without chemotherapy, as part of initial treatment) and two cohorts of secondary interest (stage I-II and stage III-IV cohorts, neither exposed to radiation or chemotherapy as part of initial treatment). We used the product-limit (Kaplan-Meier) method to estimate survival functions and the log-rank test with a Wilcoxon weight (the number at risk ni at failure time ti) to compare survival differences according to genotype. We evaluated overall survival differences under dominant and recessive genetic models, selected the model that produced the most statistically significant survival difference in our cohort of primary interest, and based statistical inferences on a Bonferroni-corrected p-value = 0.0125 = 0.05/4 (for two polymorphisms evaluated under two models). Using SAS 9.2 (PROC PHREG), we fit proportional hazards (Cox) models to control genotype-outcome associations for potential confounders and applied a log-likelihood ratio test to genotype by co-factor interaction terms to screen for effect modification. To detect departures from the proportional hazards assumption, we examined Schoenfeld residual plots and tested the statistical significance of terms representing the time dependent interaction between genotype and the natural logarithm of survival time. If results suggested non-proportional hazards, we examined Wald-type test statistics from single variable and multiple variable piecewise-constant time-varying coefficients (Gray’s) models (28, 29) to confirm the statistical significance of outcome differences according to genotype.
Table 1 summarizes the three study cohorts, which represent SCCHN categorized according to stage at diagnosis and use of radiation as part of initial treatment. Over all cohorts, key characteristics include 24.7% female, mean age 58.4 years (standard deviation 10.7 years), 13.0% positive history of cancer, 76.7% history of cigarette smoking, mean 44.3 pack-years among ever smokers (standard deviation 30.0 pack-years), 86.2% history of alcohol use, and median 16,400 lifetime total drinks among ever drinkers (3500 to 39,900 interquartile range – IQR), an amount corresponding to 1.5 drinks/day for 30 years. Male sex, younger age, and an oropharyngeal subsite primary tumor location characterized stage III-IV radiation-treated cases, the cohort of main interest.
Among cases from all three cohorts combined, ERCC2-35931 AA, AC, and CC genotypes appeared in 39.2%, 44.7%, and 16.1% (C minor allele frequency 0.384, standard error 0.016) and CCND1-870 GG, GA, and AA genotypes in 27.8%, 49.3%, and 22.9% (A minor allele frequency 0.475, standard error 0.016). Neither genotype distribution violated Hardy-Weinberg equilibrium (p>0.2). In a white control group described elsewhere (25), but not otherwise mentioned in this manuscript, ERCC2-35931 AA, AC, and CC genotypes appeared in 36.4%, 46.5%, and 17.0% (C minor allele frequency 0.403, standard error 0.012, n=834) and CCND1-870 GG, GA, and AA genotypes in 28.6%, 48.2%, and 23.2% (A minor allele frequency 0.473, standard error 0.012, n=838). Whether evaluated under additive, dominant, or recessive genetic models, ERCC2 A35931C and CCND1 G870A case and control genotype differences were not statistically significant (p>0.3).
Among all cases and among stage III-IV radiation-treated cases only, ERCC2 A35931C and CCND1 G870A genotypes were statistically independent of enrollment year, sex, age, prior history of cancer, smoking and alcohol histories, pack-years, and lifetime total drinks (p>0.05; Supplemental Table 2). As shown in Table 1, ERCC2 A35931C genotype distributions in the three study cohorts were statistically indistinguishable from each other. However, CCND1 G870A genotype distributions differed (p=0.04, Table 1), with relative paucity of 870A homozygotes among stage I-II cases. This latter finding corresponds to the lower proportion of stage I-II disease observed among all CCND1 870A homozygous cases (Supplemental Table 3).
The Figure shows overall survival, separately for each cohort, according to ERCC2 A35931C genotype and CCND1 G870A genotype. The radiation-treated stage III-IV cohort contained 275 cases, including 155 censored before death a median 4.3 years (IQR 3.2-6.0 years) after the start of treatment. The stage III-IV cohort not treated with radiation contained 80 cases, including 46 censored before death a median 4.3 years (IQR 2.6-6.8 years) after the start of treatment. The stage I-II cohort contained 130 cases, including 103 censored before death a median 3.8 years (IQR 2.1-5.6 years) after the start of treatment.
In stage III-IV cases, the effect of genotype depended on treatment. In cases treated with radiation, overall survival was worse in ERCC2 35931A homozygotes (AA) than in cases with at least one 35931C allele (AC+CC; p=0.0013) and statistically indistinguishable between CCND1 870G homozygotes (GG) and cases with at least one 870A allele (GA+AA; p=0.49). In cases not treated with radiation, however, overall survival was better in ERCC2 35931A homozygotes than in cases with at least one 35931C allele (p=0.0013) and also better in CCND1 870G homozygotes than in cases with at least one 870A allele (p=0.0089). In stage I-II cases, overall survival was independent of genotype. Although levels of statistical significance varied with the number of events, as shown in Table 2, analyses of genotype associations with disease-specific, progression-free, and recurrence-free survival were consistent in direction with the associations observed with overall survival.
Table 3 shows results from multiple variable models of overall survival. Controlling for sex, age, subsite, stage, platinum treatment, and CCND1 G870A genotype, ERCC2 35931A homozygosity remained a statistically significant predictor of poor overall survival in radiation-treated stage III-IV SCCHN (hazard ratio (HR) 1.66, 95% confidence interval (CI) 1.15-2.40). Controlling for sex, age, subsite, and stage, ERCC2 35931A and CCND1 870G homozygosity both remained statistically significant predictors of better overall survival in stage III-IV SCCHN not treated with radiation (ERCC2 A35931C AA vs. AC+CC: HR 0.26, 95% CI 0.11-0.62; CCND1 G870A GG vs. GA+AA: HR 0.14, 95% CI 0.04-0.50). Screening procedures suggested a non-proportional ERCC2 A35931C hazard over time in relation to overall survival for radiation-treated stage III-IV SCCHN. However, ERCC2 A35931C genotype remained a statistically significant predictor in a multiple variable piecewise-constant time-varying coefficients model (Table 3) and in a model that truncated follow-up three years post entry (HR 1.92, 95% CI 1.28-2.88; Supplemental Table 4). In stage III-IV cases treated with radiation, interaction tests did not identify statistically significant differences in the ERCC2 A35931C polymorphism survival effect in relation to head and neck cancer subsite, stage, platinum chemotherapy, or CCND1 G870A genotype.
Finally, in subgroup analyses restricted to persons without personal history of cancer, ERCC2 35931A homozygosity persisted as statistically significant predictors of poor overall survival in stage III-IV SCCHN treated with radiation (n=241, log-rank p-value=0.0006) and better overall survival in stage III-IV SCCHN not treated with radiation (n=69, log-rank p-value=0.0072).
In our cohort of main interest, radiation-treated stage III-IV SCCHN, we observed worse overall survival in 102 patients homozygous for ERCC2 35931A than in 173 patients with at least one ERCC2 35931C allele (p=0.0013; Figure). ERCC2 35931A homozygosity predicted not only worse overall survival, but also worse disease-specific (p=0.02) and progression-free survival (p=0.03; Table 2). The ERCC2 A35931C genotype overall survival effect appeared limited to the first three years of follow-up, after which time the product-limit estimated risks of death were 48% (standard error 5%) in ERCC2 35931A homozygotes and 30% (standard error 4%) in persons with at least one ERCC2 35931C allele, a 1.6-fold difference. Adjustment for CCND1 genotype, sex, age, head and neck cancer subsite, stage, and platinum chemotherapy did not change this association (unadjusted HR 1.65, 95% CI 1.15-2.38; adjusted HR 1.66, 95% CI 1.15-2.40, Table 3). Finally, our statistical screens for effect modification in stage III-IV cases treated with radiation could not reject the null hypothesis of equivalent ERCC2 A35931C genotype survival effects in CCND1 870G homozygotes vs. persons with at least one CCND1 870A allele or in oropharyngeal vs. non-oropharyngeal, in stage III vs. stage IV, or in platinum-treated vs. non-platinum-treated SCCHN.
Two hypotheses could explain these results. The presence of at least one ERCC2 35931C allele could result in the development of tumors that respond more favorably to treatment. Alternatively, the presence of two ERCC2 35931A alleles could simply result in the development of tumors with greater malignant potential. However, relatively poor survival with ERCC2-35931 AA does not appear to represent a feature of SCCHN more generally. Most notably, for stage III-IV cancer not treated with radiation or chemotherapy, overall survival was better, not worse, in 32 patients with ERCC2-35931 AA than 48 patients with ERCC2-35931 AC or CC (p=0.0024; Figure). These results (the ERCC2 35931C allele associating with better survival in those exposed and worse survival in those unexposed to DNA damaging treatment) concur with observations in esophageal cancer either treated or not treated with cisplatin (18). A simple explanation for a treatment’s influence follows. Ineffective DNA damage response due to germline polymorphisms in DNA repair or cell cycle control genes may promote the death of tumor cells unable to repair the DNA damage caused by radiation and, thereby, render these tumor cells more vulnerable to radiation therapy. Absent radiation, however, ineffective DNA damage response may actually promote tumor cell genetic instability and consequently tumor progression. The general notion that ERCC2 35931C signifies reduced DNA repair (6, 7) is consistent with the better survival we observed in radiation-treated stage III-IV cases with at least one ERCC2 35931C allele.
A CCND1-870 GG survival benefit also appeared to depend on treatment, with survival difference only observed in stage III-IV disease not treated with radiation (Figure). Stage I-II disease occurred less often in cases with CCND1-870 AA than in cases with CCND1-870 GG or GA (Supplemental Table 3), an occurrence that explains the relatively low frequency of the CCND1-870 AA genotype in our stage I-II cohort (Table 1). To the extent that the CCND1 870A allele may contribute to malignant potential, the association observed between CCND1-870 AA and advanced stage among all cases complements the association observed between CCND1-870 GG and better survival in stage III-IV not treated with radiation.
A literature search located only two published studies, both included in a 2008 review (12), of ERCC2 A35931C and head and neck cancer outcome. Reporting in 2005, Gal et al. (11) determined ERCC2 A35931C genotype for n=276 18-65 year-old western Washington residents (71% male, mean age 54 years, 93% white) with oral squamous cell carcinoma (25% oropharynx, 60% treated with radiation, stage distribution not specified) incident between 1988-1995. Although ERCC2-35931 CC genotype tended to predict lower risk of subsequent second primary SCCHN (adjusted HR 0.17, 95% CI 0.02-1.29), ERCC2 A35931C genotype did not predict overall or disease-specific survival, with survival results said to be similar for persons who received radiotherapy as part of initial treatment. Reporting in 2006, Quintela-Fandino et al. (16) determined ERCC2 A35931C genotype for n=103 Spanish patients (94% male, median age 60 years) treated between 1988-1995 for primary or relapsed stage IV SCCHN (48% oropharynx, 18% oral cavity, 16% larynx, 18% other). Patients, chemotherapy naïve, received platinum-based induction chemotherapy with radiation in 25%. Patients carrying the 35931C allele experienced better survival than patients homozygous for the 35931A allele (p=0.0012), a result in agreement with our observations in stage III-IV treated with radiation (with or without chemotherapy).
Four other publications mention CCND1 G870A-dependent outcomes in SCCHN, with reports from 1) Germany (n=194, (14)), 2) Portugal (n=66, (15)), 3) India (n=165, (17)), and 4) the U.S. (n=698, (13)). Each study observed worse overall and/or disease-free survival in relation to CCND1 870G. These studies did not examine variation in effect in relation to both stage and treatment. In contrast, we observed better, not worse, overall, progression-free, and recurrence free survival in relation to CCND1-870 GG, but only within a small subgroup, stage III-IV not treated with radiation.
Study limitations include non-comparable stage III-IV cohorts, one treated with radiation and another not treated. Radiation treatment can not be regarded as a random exposure. In our study population, for example, more recent enrollment period, very late stage (stage IV), and oropharyngeal subsite characterized the radiation-treated cohort (Table 1). Although we can observe significantly better survival in radiation-treated than non-treated patients with at least one ERCC2 35931C allele and significantly worse survival in radiation-treated than non-treated patients homozygous for with ERCC2 35931A (Supplemental Figure), the non-comparability of radiation-treated and non-treated cohorts precludes isolation and measurement of the specific effects of radiation treatment. Nevertheless, our results suggest that radiation, if effective, is either more effective or only effective in patients with at least one ERCC2 35931C allele.
Other study limitations arise as a consequence of 1) case loss secondary to missing data, 2) uncertainty regarding disease-specific, progression-free, and recurrence-free survival outcomes, 3) incomplete knowledge of the extent or amount of radiation or chemotherapy delivered, 4) unknown human papilloma virus (HPV) tumor status, 5) sample size, and 6) polymorphism selection. Many factors contributed to case loss. However, case loss as a whole appeared to be random with respect to genotype and other prognostic variables (Supplemental Table 1). Moreover, our inclusion and exclusion criteria defined three distinct and relatively homogeneous cohorts that, together, encompass most of head and neck cancer clinically. Our primary analysis used a hard endpoint, death due to any cause. But, one would expect genetic factors interacting with treatment to affect in particular the cancer relevant outcomes, disease-specific, progression-free, and recurrence-free survival. Results with these latter outcomes corroborated overall survival results (Table 2). However, determination of causes of death and ascertainment of cancer progression events from medical records entail subjectivity and introduce variability. Our reliance on one research-dedicated and certified tumor registrar may have produced more reliable information about these difficult to measure secondary endpoints. More importantly, outcome determinations occurred in a manner blind to genotype. We documented radiation and type of chemotherapy started as part of primary treatment, but not the amount of radiation or doses of chemotherapy actually completed. This limitation prohibited study of genetic effect according to treatment intensity. We lacked information about HPV tumor status, a favorable prognostic factor usually observed in oropharyngeal cancer (30). However, the genotype effects we observed on overall SCCHN survival were not only statistically equivalent in oropharyngeal and non-oropharyngeal radiation-treated stage III-IV SCCHN, but also observed in analyses restricted to oral cavity cancer (data not shown). Although our study included many cases (n=485), sample size limited our ability to compare genotype effects between important subgroups, in particular radiation-treated stage III-IV subgrouped according to platinum chemotherapy. Finally, we studied only two polymorphisms in two genes. However, these genetic variants were chosen because of their known functional significance. Fully useful approaches to outcome prediction or treatment selection may require more complete genetic characterization of DNA repair and cell cycle control pathways (12, 16, 31).
Though genetic determinants of head and neck cancer outcome and treatment response remain poorly understood, we find that ERCC2 A35931C and, perhaps, CCND1 G870A when combined with information about stage and treatment, may predict outcomes in advanced stage SCCHN and help clinicians tailor treatment selection.
This study received funding from NIH/NIDR P60 DE13059 and NIH/NCI 1P50CA097190.